THE ISOLATION, FRACTIONATION AND ELECTROPHORETIC CHARACTERIZATION OF THE GLOBULINS OF MUNG BEAN ( PHASEOLUS AUREUS) By Stephen Sung Tsing Djang A THESIS Submitted to the School o f Graduate S tu d ies of Michigan S ta te C ollege o f A gricu lture and Applied Science in p a r t ia l f u lf illm e n t o f th e requirements f o r th e degree o f DOCTOR OF PHILOSOPHY Department o f Chemistry 1951 ProQuest Number: 10008244 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10008244 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 //-A 5 ' - /. v i - " fl%U'A ' ' ACKNOWLEDGMENT To Prof* C. D. B all A ppreciation i s g r a te fu lly extended fo r h is stim u latin g guidance and h e lp fu l c r itic is m in th e accomplishment o f t h is work. To Dr. H. A. L ille v ik His valuable a s sista n c e and advice in t h is work i s lik e w ise deeply appreciated* To Michigan S tate C ollege For granting the opportunity to carry out t h is study the author i s g r a te fu l. 288379 CONTENTS I. II* III. INTRODUCTION............................................................................................. HISTORICAL . .................................................................................... Page 1 5 EXPERIMENTAL................................................................................................. 15 A. M a t e r ia l ............................................................ 15 B. I s o l a t i o n ............................................................................. 22 a. E x t r a c t i o n ..................................................................... 1. 22 The Fundamental R ela tio n sh ip s Between the S o lu b ilit y o f Mung Bean P rotein and the Nature and Concentration o f S olven ts . . . 2. 22 The Study o f Some Factors which E ffe c t the P e p tiza tio n o f Mung Bean P r o t e i n .......................42 (a) Time and Sample-Solvent R atios MS-S-R”43 (b) P a r tic le S ize and Sample-Solvent R a t i o s .................................. (c ) 47 Mechanical Shaking, H an d -stirrin g and S u ccessive E x t r a c t io n s ................................ 5 1 (d) Temperature and O il-F ree and Non-Oil Free Condition of the Samples. . . . b. 56 P r e c i p i t a t i o n ...........................................................................6 l 1. D eterm ination o f P rotein N itrogen - NonP ro tein N itrogen Ratio and the AlbuminG lobulin R a t i o .............................................................. 62 2 . Demonstration o f the E ffe c t o f D ilu tio n upon the Sedim entation Time o f Globulin F ra ctio n ..........................................................64 3. P ro tein Sedim entation from 0 .4 m KaCl Ex­ tr a c t o f Mung Bean Meal a t Various Hydro­ gen Ion Concentrations * ........................................ 68 4. Suggested Procedure fo r the I s o la t io n o f th e G lobulin from Mung Bean Meal . . . . . 5 . A Q u an titative Study o f the Procedure . . F r a c tio n a t io n .............................. 1. JO J1 75 The C onstruction o f a R otating Outside Liquid D i a l y z e r ..........................................................75 2 . A p p lication o f the Slow Stepwise D ilu tio n Procedure and R otating Outside Liquid D ia ly s is fo r the P u r ific a tio n o f Mung Bean Globulin .................................................................. 81 3* Determ ination o f the Molar Concentration o f Sodium Chloride a t which the Gg F raction P r e c ip ita te s from the Extract w ith the In­ d ir e c t, Slow, Stepwise D ilu tio n Procedure. 4. 82 Determ ination o f the Molar Concentration o f Sodium Chloride a t which Gg P r e c ip ita te s w ith th e D irect D ilu tio n M e t h o d .......................83 5* F r a ctio n a tio n o f Gj from Gg-free D ia ly sa te 87 Page 6* I n v e stig a tio n o f F raction G^ o f Globulin in a Gg- and G^-free D i a l y s a t e ........................... 90 7. A Q u an titative A p p lication o f the Proce­ dure fo r F ra ctio n a tio n o f Mung Bean G lobulins ................................... . . . . . . . 93 $6 d...........................P u r i f i c a t i o n ............. 1. P u r ific a tio n o f the P r e c ip ita te Gg . . . . 96 2. R esolu tion o f the Gg F r a c t i o n ........................... 97 3. F ra ctio n a tio n o f th e R esolved Products o f G g ............................................................................... 98 4. U sing S p e c ific Molar Concentrations o f Sodium Chloride fo r P u r i f i c a t i o n ....................... 99 C.A n alysis ..................................................................................102 a. E le c t r o p h o r e s is ...... 102 1. P r i n c i p l e .................................................................... 102 2. A p p a r a t u s .................................................................... 104 3* B uffer E ffe c ts . . . . . 4. The Preparation o f B uffers o f D esired pH ................................... 105 and Io n ic S t r e n g t h ................................................... 107 5. O perational Procedure in a Complete E lectro p h o retic A n alysis ................................... I l l b* E lectro p h o retic A n alysis o f Mung Bean G lo b u lin .........................................................................114 IV. SUMMARY........................ AND CONCLUSION.................................................. 126 V. LITERATURE CITED......................................................................................137 VI. Page ADDENDA....................................................................................................... l 4 l A. A New Apparatus fo r Freezing-Dehydration and Tech­ nique fo r High Vacuum M a n ip u la tio n .................................. B. 142 A High Temperature Bath Made from Aluminum Shavings. 158 1 INTRODUCTION Separation o f p ro te in s from b io lo g ic a l systems and t h e ir p u r ific a ­ tio n as chemical substances have been the concern o f b io lo g ic a l chem ists fo r over a century ( l ) . Data are a v a ila b le on the p u r ifie d p r o te in s o f many o f the common seeds o f commercial importance. I t i s su rp risin g th at l i t t l e i s known about the p r o te in s o f Mung bean, Phaseolus aureus, an econom ically important crop in A s ia tic cou n tries which has become an ac­ knowledged crop in the U nited S ta te s . te d to : The o b ject o f t h is work was devo­ 1) a study o f the development o f a method in order to produce and reproduce homogeneous fr a c tio n or fr a c tio n s , and 2) p a r tic u la r care o f the products to keep them a s n early a s p o s s ib le in th e ir n atu ral s t a t e . The preparation o f undenatured p ro tein s i s in many r esp ects a s p e c ia l a rt. The procedures when developed are g en e r a lly sim ple but the ch oice o f the b est co n d itio n s from among the v a r ie ty o f p o s s i b i l i t i e s i s d i f f i ­ c u lt , and every step in v o lv es rigorous a tte n tio n to d e t a il in order to m aintain the p ro tein in an undenatured state* The la rg e dimensions and unique stru ctu re o f th ese h ig h ly organized p ro te in m olecules render them p a r tic u la r ly l a b i l e . Exposure to h e a t, to high a c id or a lk a lin e c o n d itio n s, or to co n d itio n s o f low d ie le c t r ic constant b rin gs about changes in the s p a tia l arrangements o f the r e a c tiv e groups which a l t e r , and may com pletely d estroy, n a tiv e p r o p e r tie s. The phenomenon c a lle d denaturation may be noted a s a change in s o lu b ilit y , a s a change in m olecular shape, a s a change in chemical r e a c t iv it y or o f immunological s p e c i f i c i t y . The s p a tia l r e la t io n s between fr e e r e a c tiv e 2 groups o f th e n a tiv e p ro tein may he d is to r te d or destroyed ( 2) t ( 3) , ( 4 ) . The procedure used fo r the separation o f Mung hean g lo b u lin s was e x tr a c tio n o f uniform ly ground meal w ith sodium ch lo rid e s o lu tio n . The e x tra c t was d ilu te d in conform ity w ith g lo b u lin preparations w ith d is ­ t i l l e d water and th e p r e c ip ita te d p ro tein was thus ob tain ed . Separation o f the p r e c ip ita te from th e so lv en t was accom plished by ordinary c e n tr i­ fu g a tio n . The exposure o f p ro tein to the la r g e a ir in te r fa c e s o f the Sharpies c en tr ifu g e was avoided. The foaming produced by Sharpies cen­ t r ifu g e s in d ic a te s denaturation o f p r o te in . Since th e Mung bean g lo b u lin s are in so lu b le a t low con cen tration s o f n eu tra l s a lt s o lv e n t, th e ad ju st­ in g o f th e pH o f th e ex tra ct fo r p r e c ip ita tio n was not n ecessa ry . With t h i s procedure a b u ffer was not u sed fo r the e x tr a c tio n and an a c id was not employed fo r the a d ju stin g o f the pH o f the ex tra ct fo r p r e c ip ita tio n . P r o tein su b -typ es, such a s g lu t e lin s and g lo b u lin s , when they occur to­ geth er are r e a d ily separated sin c e the g lo b u lin s d is s o lv e in d ilu t e s a lt s o lu tio n s whereas th e g lu t e lin s are in so lu b le in a l l n eu tral s o lv e n ts . The p r o te in which p r e c ip ita te d was regarded a s g lo b u lin , whereas th e a l­ bumins remained in so lu tio n ( 5) , ( 6* ) , ( 7)» (8 ) . I t was very important to d iscover th at the Mung bean g lo b u lin s have ♦P rotein o f th e wheat and other cerea l seeds contain a sim ila r complement o f p r o te in s , i . e . , g lu te n in , g lia d in , g lo b u lin and albumin. The g lu te n in and g lia d in make up the b u lk ( 90$) o f the p ro tein o f cerea l seed s, where­ a s th e seeds o f d icotyledonous p la n ts in general con tain g lo b u lin s as th e ir p r in c ip a l p ro tein components. 3 t h e ir h ig h e st s o lu b ilit y in 0.4-M NaCl and are in so lu b le a t th e concentra­ tio n o f 0.08M NaCl* This s o lu b le -in so lu b le range perm its a p u r ific a tio n scheme o f th e g lo b u lin s by repeated d isp ersio n in 0 .4 m N a d s o lu tio n and p r e c ip ita tio n a t a con cen tration o f 0.08M by d ilu tio n w ith d i s t i l l e d water* The s o lu b le -in so lu b le range o f p ro tein must be determined before fr a c tio n a tio n or p u r ific a tio n can be accomplished* The aim o f t h is study was the is o la t io n o f the p ro tein in a s ta te c lo s e to i t s s ta te in nature* The s t a b i l i t y and s o lu b ilit y o f p ro tein are u su a lly grea ter in d ilu te s a lt so lu tio n s than in water or in d ilu te a c id s or b ases ( 9 ) , (10)* Mung bean g lo b u lin s , which were h ig h ly so lu b le in tile d ilu te NaCl s o lu tio n , were a ls o h ig h ly p ro tected from denaturat io n because th e d ie l e c t r i c constant o f th e medium was high* That the s t a b i l i t y o f p r o te in s i s g rea ter in s o lu tio n s o f greater d ie le c t r i c constant has lon g been recogn ized (1 0 ). With t h is procedure the globu­ l i n s are brought to a r e la t iv e ly in e r t s o lid s ta t e by d ilu tin g a s rap id ly as p o s s ib le to 0.Q8M NaCl, They are m aintained in so lu b le a t low tempera­ tu re , and in t h is s ta te r e a c tiv e groups are p rotected from each other (p r o te in -p r o te in rea ctio n ) and from the enzymes f o r which they are the su b stra te (11)* In the course o f th e study o f the p u r ific a tio n o f the is o la t e d pro­ t e in by d ia ly s is , th e need fo r a rapid method o f m em brane-equilibration d ilu tio n became apparent. was constructed* A r o ta tin g 11o u tsid e -liq u id " d ia ly s is apparatus The sep aration and p u r ific a tio n o f Mung bean g lo b u lin was undertaken w ith the m em brane-equilibration procedure. With th e in ­ d ir e c t slow step -w ise d ilu tio n technique the fr a c tio n a tio n o f g lo b u lin k was made p o s s ib le . E xtensive e le c tr o p h o r e tic s tu d ie s were made fo r the c h a r a c te r iz a tio n o f the t o t a l g lo b u lin as w ell a s th e is o la t e d fr a c tio n s . 5 HISTORICAL That c e r ta in p ro te in s were so lu b le in s a lin e so lu tio n s was f i r s t observed by Denis in 1S59 (12)# by H oppe-Seyler. This observation was la t e r confirmed Denis noted th a t when sodium ch lorid e e x tr a c ts were made o f both animal and v egetab le t is s u e s , c e r ta in p r o te in m a teria ls were d issolved * During th e l a s t decade o f the n in eteen th century Osborne (13) did th e grea ter p art o f h is ou tstanding work on the is o la t io n o f th e p ro tein s o f seed s. He demonstrated a remarkable a p p recia tio n o f the fundamental r e la tio n s h ip between the s o lu b ilit y o f th e p ro tein and the s a lt con ten t, th e a c id it y , and th e temperature o f th e solven t and brought about h is sep aration s o f th e p ro tein components o f the e x tr a c ts from seed by w ell conceived m anipulations o f th ese fa cto rs* In 1902 (lh ) h e pu blished one paper on th e b a sic character o f the p ro te in m olecule in which he showed th a t e d e s tin , a ty p ic a l seed g lo b u lin , en ters in to io n ic re a c tio n s w ith a c id s to form true s a lts * In 1905 (15) k® pu blished a s o lu b ilit y curve o f e d e stin in s a lt s o lu tio n s which showed both the ascending limb o f the s a lt in g - in and the descending limb o f the s a ltin g -o u t e ffe c ts * These were p ion eerin g attem pts to form ulate the p r in c ip le s of s o lu b il it y upon which a l l o f h is p ro tein is o la t io n stu d ie s had been predicated* In IS96 Osborne and Canqpbell (16) stu d ied s ix d is t in c t p ro teid s (g lo b u lin s) from ten d iffe r e n t seeds* In IS97 Osborne and Campbell reported on stu d ie s o f the p ro teid s o f $ lu p in e seed ( 1 7 ), the ca sto r hean ( I S ), th e sunflower seed (1 9 ), the cow pea (20) and th e w hite podded adzuki hean (21)* D ilu te s a lt solu ­ t io n ex tra cte d very l i t t l e g lo b u lin m aterial from lu p in meal hut la rg e qu an tities; were obtained w ith stronger s o lu tio n s . I t was suggested th a t the two g lo b u lin fr a c tio n s recovered by d ia ly s is and d ir e c t d ilu ­ t io n may have been th e same but th a t th ere was a probable combination o f some so r t between the g lo b u lin and other c o n stitu e n ts o f th e seed* Comparisons o f th e c a sto r bean, sunflower seed and the hemp seed g lo b u lin s , which show sim ila r com position and a property o f b ein g p a r tly in s o lu b le and p a r tly so lu b le in a saturated s o lu tio n o f sodium ch lo r id e , le d to fu rth er study o f the c a sto r bean seed g lo b u lin s . They found th a t a d d itio n o f a small q u antity o f a c id caused such changes in the ca sto r bean fr a c tio n th a t had been so lu b le in saturated s a lt s o lu tio n so th a t i t behaved much l i k e the in so lu b le f r a c tio n s . I t was th e op in ion o f th ese authors th at the sunflower contained a s i t s p r in c ip a l p ro tein ( c a lle d p roteid by them) the g lo b u lin e d e stin , but th a t which they recovered was mixed w ith h elia n th o ta n n ic a c id from which they did not succeed in b ringing about a complete sep aration . The c h ie f p r o te in o f the cow pea was found to be a g lo b u lin that c lo s e ly resembled th e legum inin o f the pea and v etch . F ra ctio n a l pro­ cedures in v o lv in g r e d is s o lv in g in brine and p r e c ip ita tio n by d ir e c t d ilu tio n and d ia ly s is d isc lo s e d a second g lo b u lin which resembled p h a seo lin . The com position o f th e g lo b u lin extracted from adzuki beans was found to be id e n tic a l to th a t obtained from the white bean, Phaseolus v u lg a r is . 7 Later in the same year Osborne (22) reported r e s u lt s o f fu rth er in v e s tig a tio n as to the amount and p ro p erties o f th e p ro tein s o f th e maise k e r n e l. One ‘e a te r -e x tr a c te d p r o te in , p r e c ip ita te d by d ia l y s is , he in d i­ ca ted a s a g lo b u lin named mays in . Prolonged d ia ly s is y ie ld e d y e t another sm all f r a c tio n which resembled edestin* In 1S9S Osborne and Campbell r e in v e s tig a te d th e p r o te in c o n s titu e n ts o f th e pea ( 23) and v etch (24) a f te r repeated fr a c tio n a l p r e c ip ita tio n o f th e g lo b u lin s from th e seeds o f the horse bean ( 25) and l e n t i l (26) r e s u lte d in two fr a c tio n s , one o f which coagulated a t 100°C. When the p r e v io u sly reported legumin o f the pea was separated from contam inating v i c i l i n , they found th a t i t c lo s e ly resembled th e legumin o f v etch . A ca refu l comparison o f th e rea ctio n s and p ro p erties o f th e p ro tein s o f th e pea, l e n t i l , h orse bean and v etch were reported in a separate paper ( 27) • Legumin i s a g lo b u lin which d isso lv e d r e a d ily in s a lin e s o lu tio n and was p r e c ip ita te d therefrom e ith e r by d i a l y s i s , d ilu tio n or c o o lin g . V ic ilin was a g lo b u lin they found a s so c ia te d w ith legumin in the pea, l e n t i l and horse bean but none was obtained from v e tch . In s a lt s o lu tio n i t was the more so lu b le g lo b u lin which f a c t made sep aration o f th e two p o s s ib le . This f r a c tio n was com pletely coagu lated a t 100°C. Two v a r ie t ie s o f soy beans were stud ied by Osborne and Campbell (2 8 ). They named th e c h ie f p ro te in co n stitu en t g ly c in in , a g lo b u lin somewhat sim ila r to legumin* The more so lu b le g lo b u lin , which resembled p h a seo lin in com position, remained in the supernatant a f t e r g ly c in in was removed. Osborne and H arris ( 29) were the f i r s t to study pure p r o te in s in r e la t io n to th e ir s o lu b ilit y in so lu tio n s o f d iffe r e n t s a l t s . They 8 in v e s tig a te d the a c tio n o f s a lt s on the g lo b u lin e d e stin obtained from hemp seed . C hlorides o f monovalent b a se s, sodium and potassium and caesium, were found to have very n early the same so lv en t power. The d iv a le n t b a ses, barium, strontium , calcium and magnesium d iss o lv e d approx­ im ately tw ice a s much e d e stin a s the monovalent c h lo r id e s. The s o lu b ilit y in general was observed to be dependent on the nature o f the m etal, the d iv a len t m e ta llic ch lo rid es were found to have more d isp ersin g power. Lithium ch lo rid e proved to be the only excep tion; i t s so lv en t power was much l e s s than th at o f th e other monovalent c h lo r id e s. The e f f e c t o f s u lf a t e on th e s o lu b ilit y o f the g lo b u lin was very sim ila r to th a t o f th e corresponding ch lo rid es o f the d iv a len t m eta ls. During th e years 1916 - 1927 Jones and Johns togeth er and w ith other in v e s tig a to r s stu d ied th e p r o tein s from a number o f se e d s. Two g lo b u lin s , canavalin and concanavalin, were is o la t e d from th e jack bean by Jones and Johns (3 0 ). F r a ctio n a tio n o f the sodium ch lorid e e x tr a c ts w ith v a r i­ a b le sa tu ra tio n s o f ammonium s u lfa te produced the two glob u lin s* Johns and Jones ( 31) ex tra cted a ir -d r ie d o i l - f r e e peanut meal w ith a 10 percent sodium ch lo rid e s o lu tio n a t sev era l d iffe r e n t temperatures and found th a t th e y ie ld o f p ro tein obtained was not apparently tempera­ tu re dependent. A major g lo b u lin fr a c tio n was obtained by p a r tia l sat­ u ra tio n o f the e x tra ct w ith ammonium s u lfa te and a minor fr a c tio n by com plete sa tu ra tio n o f th e f i l t r a t e . The p r in c ip a l p ro tein was recovered from the v e lv e t bean by Johns and Finks (3 2 ). The g lo b u lin was obtained by d ia ly s is o f a sodium ch lo rid e ex tr a c t in parchment bags suspended in running w ater. 9 Johns, Finks and Gersdorff (33) reported th a t th e p r in c ip a l p ro tein o f the coconut endosperm was a g lo b u lin . d ia ly s is fo r 7 The g lo b u lin was recovered hy 10 days o f a ten percent sodium ch lo rid e e x tr a c t. Johns and Waterman (3*0 stu d ied the p ro tein s o f th e Mung bean, Phaseolus aureus Roxburgh. They found a f i v e percent sodium ch lo rid e s o lu tio n th e most e f f e c t i v e e x tr a c ta n t. Part o f the p ro tein m aterial was removed from the e x tra c t by heat coagu lation a t 40°C ., and two other fr a c tio n s were p r e c ip ita te d a t high temperatures ( 71° C . and 100°C. respec­ t iv e ly ) which in d ica ted an albumin and two g lo b u lin f r a c tio n s . The glob­ u l i n fr a c tio n was fu rth er separated by v a r ia tio n s in sa tu ra tio n o f ammon­ ium s u lf a t e . The g lo b u lin o f th e cohune nut was is o la t e d by Johns and G ersdorff (35)* E x tra ctio n experim ents were made w ith d iffe r e n t con cen tration s o f sodium ch lo rid e in w ater, with 70 percent a lco h o l and w ith one percent h yd roch loric a c id and e x tr a c tin g fo r one hour in each c a se . Ten percent sodium ch lo rid e was found to ex tra ct the maximum amount o f p r o te in . D ia ly s is o f th e ex tr a c t y ie ld e d a higher percentage o f g lo b u lin than p a r tia l sa tu ra tio n w ith ammonium s u lf a t e . The so lu b le s a l t s occurring n a tu r a lly in th e lim a bean seed were found by Jones e t a l . ( 36) to be s u f f ic ie n t to d is s o lv e 15*15 percent o f th e p r o te in when water was the ex tra c ta n t. This was p r a c t ic a lly as much a s was ob tain ed by a th ree percent sodium ch lo rid e s o lu tio n which was determined as the most e f f i c i e n t concentration o f the s a l t . Two g lo b u lin s were separated by fr a c tio n a l p r e c ip ita tio n . In a study o f th e p ro tein s of wheat bran by Jones and Gersdorff (37) 10 a g lo b u lin was one o f th e th ree recovered* Bran ground to pass a ^40 mesh s ie v e was found to be a s e f f e c t iv e o f ex tr a c tio n a s th a t ground to pass a 100 mesh s ie v e . Seven g lo b u lin p reparations were recovered from 4 per­ cent sodium ch lo rid e e x tr a c ts by variou s methods but d ir e c t d i a ly s is a g a in st water and a c id if ic a t io n with carbon d ioxid e gas follow ed by d ilu t e a c e t ic a c id was productive o f th e la r g e s t q u an tity o f g lo b u lin . By d ia ly z in g or by a d d itio n o f ammonium s u lfa te to s a lin e e x tr a c ts o f w hite r ic e flo u r , J o ses and Gersdorff (33) were ab le to I s o la t e a pro­ t e i n fr a c tio n which c o n siste d o f two g lo b u lin s, coagu latin g a t 7^-° and 90°C. Ammonium s u lf a t e fr a c tio n a tio n was not p o s s ib le sin c e both p r e c ip i­ ta te d too c lo s e ly to g eth er so fr a c tio n a l h eat coagu lation was the means by which they were separated. The p r in c ip a l p r o te in o f th e seed of th e s il v e r maple was is o la t e d by Anderson (39) and found to be a g lo b u lin . I t was ex tra cte d w ith ten percent sodium ch lo rid e and p r e c ip ita te d from th e ex tr a c t w ith saturated ammonium s u lf a t e . Gortner, Hoffman and S in c la ir (40) were the f i r s t to emphasize the e f f e c t o f the p a r tic u la r ion o f various s a lt so lu tio n s on th e p e p tiz a tio n o f p r o te in s . Samples o f wheat flo u r were stu d ied a t alm ost constant hydrogen io n con cen tration s w ith d iffe r e n t s a lt s and a marked ly o tr o p ic e f f e c t was observed. I t was evident th at the hydrogen io n con cen tration per se could not have been p layin g the major r o le , and th at such p ep tiza­ tio n d iffe r e n c e s a s th ey observed must be a ttr ib u te d to some oth er fa c to r . They found a d e f in it e ly o tr o p ic e f f e c t o f th e order KF < KOI < O r < KI* The same r e la t iv e order h eld fo r a l l the s a l t s stu d ied . In con clu sion 11 th ey remarked th a t "there i s a ly o tr o p ic s e r ie s o f io n ic e f f e c t s in an aqueous sy sten o f p ro tein and s a l t s , and th at th e se e f f e c t s are due to p r o p e r tie s o f th e anion and the ca tio n o f th e s a lt and are measurable even a t a constant hydro gen -ion co n cen tra tio n .N Bishop (Hi) observed th at the y ie ld o f n itrogen a s s a lt so lu b le from b a rley flo u r was in crea sed when the p a r t ic le s iz e was decreased. Staker (4 2 ), in a study o f th e p e p tiz a tio n o f seed p r o te in , thought th a t whenever p o s s ib le samples should be ground to pass through a 100 mesh siev e* C r y s ta llin e g lo b u lin was prepared from seeds o f pumpkin and squash by Vickery e t a l . (4-3). c h lo rid e s o lu tio n . A ir-d ried meals were extracted w ith warm sodium Albumin was removed by h eat coa g u la tio n . By d ilu tin g w ith fou r volumes o f warm water (60°C .) g lo b u lin p r e c ip ita te d when the s o lu tio n came to room tem perature. By fa rth er d ilu tio n w ith co ld water and on standing in a c o ld room another fr a c tio n o f g lo b u lin p r e c ip ita te d in c r y s ta ls . Quensel (44) ex tra cted b a rley meal with NaCl b u ffered w ith phosphate to pH 7*0. F ra ctio n a l p r e c ip ita tio n o f th e ex tra ct wa3 accom plished w ith s o lid ammonium s u lf a t e . The g lo b u lin s were separated from th e albumins, low m olecular and p o ly -d isp e rsed m aterial by d isp ersio n o f th e p r e c ip i­ t a t e in NaCl s o lu tio n and subsequent d ia ly s is a g a in st d i s t i l l e d w ater. The method o f c h a r a cter iza tio n o f the g lo b u lin components was based on th e u ltr a c e n tr ifu g a tio n sedim entation co n sta n ts, which confirmed the presence o f fou r g lo b u lin s . Vassel (45) Iso la te d two g lob u lin s, lin in and co n lin in , from lin se e d H7 TABLE XXXXI. ELECTROPHORETIC MOBILITY CALCULATIONS G lobulin F raction Sun No. EE & r2 (17) I l4 115 116 120 121 119 122 3.28 3.90 4 .3 6 4.61 7.15 7.48 7.72 2.67 2.07 2.16 l.l4 0.85 1.65 2.62 2.40 1.93 2.03 1.09 0.78 1.59 2.94 <*2(14) 124 128 126 127 136 130 129 3.27 3.85 4 .4 4 4.78 7.27 7.52 7.75 2.19 2.21 1 .5 2 0.9 2 1.9 6 2.27 1.69 ^ (ll) 97 96 99 105 104 3.34 3.80 4 .4 0 6.15 6.63 h(oi) 137 138f 139 140 152 142 153 3.32 3.90 4.21 4.52 7.30 7.50 7.78 4 kc cro ss a t 1®C. se c tio n area o f c e l l (cm) ^2 ^1 Ascend­ Descend­ in g in g (cm) (cm) R R e s is t­ ance o f p ro tein so lu tio n in ohms 0 .3 0 .3 o*3 0 .3 0 .3 0 .3 0 .3 .0048937 .0048937 .0048937 .0048937 .0048937 .0048937 .0048937 80 76 68 69 72 72.5 74.5 2.02 2.19 1.45 0.95 1.71 2.45 2.14 0 .3 0 .3 0 .3 0 .3 0 .3 0 .3 0 .3 .0048937 .0048937 .0048937 .0048937 .0048937 .0048937 .0048937 81 72.3 65 69.5 78 77 62.7 2.91 2.80 i;2 i 1.87 2.77 2.60 2.73 1.07 2.06 2.85 0 .3 0 .3 0 .3 0 .3 0 .3 .0048937 .0048937 .0048937 .0048937 .0048937 62.6 70 53.5 74 78 2.64 1.75 1.43 1 .1 4 1.65 2.27 1.91 2.42 1.67 l.4 i 1.32 1.67 2.29 1.82 0 .3 0 .3 0 .3 0 .3 0 .3 0 .3 0 .3 .0048937 .0048937 .0048937 .0048937 .0048937 .0048937 .0048937 7b.5 70.3 64 68.5 5 8 .4 74.5 6O.5 . 13 was d isp ersed in normal sodium a c e ta te a t 4o°C ., c r y s ta ls formed a s the temperature rev erted to room temperature* The i s o e l e c t r i c p oin t o f t h is fr a c tio n , pH 5*5, was determined by titr a tio n * C r y sta llin e g lo b u lin was recovered from fr e sh tomato Ju ice by Car­ p enter (U g). The Ju ice was concentrated to a syrup (35-40 percent) by fr e e z in g out th e water* I t was then sub jected to d ia ly s i s and an amor­ phous p r e c ip ita t e formed in the cellophane sac* The g lo b u lin was d is s o l­ ved in molar sodium ch lo rid e a t *jO°C. and when f iv e volumes o f water a t 50° were added, p r e c ip ita tio n occurred* A fter standing a t 0°G. f o r 48 hours, the g lo b u lin was found to be c r y s ta llin e * A ca refu l study o f the lit e r a t u r e i s important in order th at we b u ild f o r th e fu tu re on th e broad foundation o f p a st ex p erien ce. p resen t i t i s s t i l l n ecessary to tr e a t p lan t p ro tein s a s a g r o u p * At The n ext phase o f in v e s tig a tio n may rev ea l how they can be trea te d a s in d i­ v id u a ls, th at i s , as s p e c if ic substances* The M ung bean, Phaseolus aureus, is a summer annual legume, belonging to the field bean family. I t is a native of Asia and was known in the United State as early as 1835 under the name of Chicksaw pea, but it has not been until recent years th a t it has become an acknowledged crop, rapidly gaining in popularity. The M ung bean seed is about one half the size of soybean seed. I t is globose or oblong in shape and most varieties are green in color, but others are yellow, brown and marble black. Photograph by courtesy of the Corncli Seed Co., Saint Louis, Missouri 15 THE MATERIAL The seeds* used in th ese experiments were ground to p ass through a 60 mesh sieve* A fter g rin d in g, the meal was thoroughly mixed so th at uniform a liq u o ts could he obtained* The meal was analyzed fo r m oisture, ash, t o ta l n itrogen , lip id e and crude f ib e r , a s d escrib ed in th e methods o f a n a ly sis (3 0 ), w ith some m o d ifica tio n s as noted below* f o r the m oisture determ ination 10 gram samples were d ried a t 101°G« to constant weight w ith a Brabender's m oisture te ste r * I t was found th at the ground meal contained 8*20 percent water (Table 1 and fig u r e l ) . In the determ ination o f the ash content (5 l)» 5*000 gram samples o f meal were put in to each o f four weighed and marked p o reela in c r u c ib le s. The samples were charred over an open flame under a hood, and then trans* fer re d to an e l e c t r i c furnace and heated a t 650°C. u n t il a l l carbon was oxidized* The c r u c ib le s and con ten ts were cooled in a d esic c a to r and then weighed* The average ash content fo r the four samples was 3*098 p ercent (Table 2 ) . ♦The seed s were sup p lied by cou rtesy o f th e Johnston Seed Company, Enid, Oklahoma* The sample used was Johnston Jumbo type o f th e 19I+S crop and had not been p rocessed in any way and were in t h e ir natural s ta t e when received* The seeds were b rig h t in color and f u l l y matured* 16 TABLE I DETERMINATION OF MOISTURE CONTENT OF 60 MESH MUNG BEAN MEAL AND TOOLE BEANS WITH BRABENEER MOISTURE TESTER AT 101°C. Time in Minutes 10 20 ?° 4o 50 60 70 so 90 100 120 1*10 160 ISO 200 220 2*10 260 280 300 320 3lK) 360 3S0 400 420 44o 460 480 500 520 540 560 580 600 64o 700 10 gm. Meal 10 gm. Meal 10 gm. Whole Bean 10 gm. Whole B $ Water Loss $> Water Loss $ Water Loss $ Water Loss 5-3 7*1 7*5 7.8 s .o s .l 8.2 8 .2 8 .2 8 .2 5 .6 7 .3 7 .6 S.O S .l 8.2 8 .2 8.2 8 .2 8 .2 0.8 1 .4 1 .8 2 .3 2 .6 2.8 3 .2 3 .4 3 .6 3.7 4 .2 4 .5 4.7 5 .0 5 .2 5.45 5.7 5.8 b.0 6 .2 6 .3 6.45 6.55 6.65 fe.75 6.8 6 .9 7 .0 7 .1 7 .2 7.25 7 .3 7 .4 7 .5 7.55 7 .6 7.6 0.7 1 .2 1 .6 2 .2 2 .5 2.8 3 .1 3 .3 3 .5 3 .6 4 .1 4 .4 4 .7 5.0 5 .2 5 .4 5.7 5.S 6 .0 6 .2 6 .3 6 .4 6.5 6 .6 6.7 6.8 6.9 7 .0 7 .1 7 .2 7.25 7 .3 7*4 7.5 7*55 7*6 7.6 Figure 1. 60 mesh meal Percent moisture loss in whole bean 100 200 300 400 Time in minutes 500 600 The effect of grinding Mung bean through 60 mesh size screen upon the rate of drying to constant weight at 101° C as determined by the Brabender’s Moisture Tester. 700 IS TABLE II DETERMINATION OP ASH CONTENT OP MUNG BEAN (A ir Dried) Sample Wt. cr u c ib le gm. 1 2 3 4 22.1080 22.653S 22. 38S6 23.1850 Wt. sample gm. 5*0000 5*0000 5.0000 5.0000 T otal wt. gm. 27.10S0 27.6538 27.3886 28.1850 Wt. c r u c ib le and ash gm. 22.2622 22.8093 22.5441 23.3394 Loss in w t. gm. (Organic m atter) 4.8458 4.8445 4.8445 4.8456 Wt. o f ash 0 . 15^2 0.1555 0.1555 0.1544 3*084 3*11 3*11 3.088 gm. $ o f ash content The t o t a l n itro g en content o f a ir -d r ie d meal was determined by the K jeldahl method (52) • The r e s u lt s obtained from four 2 gram samples were 3*788 percent t o t a l n itrogen o f the a ir -d r ie d meal and 4.127 percent t o t a l n itro g en c a lc u la te d on dry weight o f the samples (Table I I I ) . TABLE I I I TOTAL NITROGEN CONTENT OP MUNG BEAN MEAL Grams Sample K jeldahl T itr a tio n m l. HC1 N 0.1007 io Nitrogen A ir-d ried Sample $ N itrogen C alculated on Dry Wt. B a sis 1. 2.0000 53*75 3*788 4.127 2. 2.0000 53*65 3*781 4.119 3. 2.0000 53*80 3*792 4.131 4. 2.0000 53*80 3*792 4.131 53*75 3*788 4.127 Average 19 The lip id e content o f Mung bean meal was determined. Four 10 gram samples (weighed to w ith in 0 .1 mg.) o f 60 mesh meal were ex tra cted with 125 ml. 95$ ethanol in a Soxhlet E xtractor over the aluminum shaving bath a t 100°C. This was fo llow ed by a 10 hour ex tr a c tio n w ith eth y l eth e r and then another 10 hour e x tra ctio n w ith 125 ml. o f 95$ eth an ol. The combined e x tr a c ts were evaporated to dryness and the resid u e was e x h a u stiv ely ex tra cted w ith petroleum eth er a s described by D i ll (53)* Table IT shows the average value o f 0.809 percent lip id e con ten t. TABLE IV DETERMINATION OF LIPIDE CONTENT OF MUNG BEAN MEAL BY SOXHLET EXTRACTOR USING 10.0 GRAM SAMPLES Sample 2 1 3 4 Wt. o f beaker gm. 41,8234 47.5004 45.7700 50.5056 Wt. b e a k e r /lip id e gm. 41.904$ 47,5816 45.8510 50.5356 Wt. o f lip id e gm. 0.0815 0.0812 0.0810 0.0800 The fib e r content o f Mung bean meal was determined on four 2-gram samples o f a ir -d r ie d meal (Table T ). The procedure (5*0 i s o u tlin e d a s fo llo w s: 1. A th in la y e r o f a sb e sto s was prepared in the bottom o f a Gooch c r u c ib le connected to a f i l t e r f la s k . Water was added to the c r u c ib le to make a uniform and firm la y e r o f a s b e sto s. 2. Two grams o f a ir -d r ie d sample were weighed on a p ie c e o f paper and introduced in to th e c r u c ib le . 3* The sample was washed tw ice w ith 10 ml. h o t 95$ ethanol to re­ move the m oisture and some o f the lip id e . h. The sample was washed once w ith co ld ethanol to bring the temp­ eratu re down to room temperature and washed again tw ice w ith 10 ml* e th y l eth er to remove the major p ortion o f l i p i d e . 20 5 * The c r u c ib le and con ten ts were p laced in a 600 m l. beaker and 200 m l. o f 1.25^ H2S0i|. was added. This was b o ile d g e n tly fo r 30 m inutes. The beaker was covered w ith a round bottomed f la s k which contained water and served as a condenser* 6. At th e end o f 30 m inutes the so lu tio n was c a r e fu lly f i l t e r e d through a 13 cm. p ie c e o f lin e n c lo th f i t t e d in to a 4 inch fun­ n e l. The resid u e was rin se d w ith hot water to wash out the a c id . The f i l t r a t e was discarded* 7* The resid u e was tra n sferred from th e lin e n c lo th in to the 600 ml* beaker u sin g 200 ml. o f 1.25$ UaGH in a wash b o t t le to make the tr a n sfe r . This was b o ile d fo r 30 m inutes with th e r e flu x con­ denser as p rev io u sly d escrib ed. 8 * The m a teria l in th e beaker was f i l t e r e d through the same lin e n c lo th and washed w ith h ot water* 9* The re sid u e was tr a n sferred to a beaker. The con ten ts in the beaker were then tra n sferred in to a Gooch c r u c ib le and washed w ith hot water* 10* The Gooch c r u c ib le and i t s con ten ts were dried fo r 2 hours a t 100°C. then cooled in a d esicc a to r and weighed. 11. The Gooch c ru cib le was ig n ite d f o r 2 hours a t 650°C., cooled in a d e sic c a to r and weighed. The d iffe r e n c e between th e two w eights was crude fib er* TABLE V DETERMIEATIOH OF FIBER OF M03JG BEAU MEAL Sample Wt. Gooch c r u c ib le , a sb e sto s and f ib e r (d ried ) Wt* a f t e r ig n it e d 2 hrs* a t 650°C. Loss in w t. 1 13*1219 2 3 4 12.8734 14.0304 13.2312 13*0332 12. 7S32 13*9434 13.1420 .0887 .0902 .0870 .0888 The average o f the four samples was O.OS87 gm. crude f ib e r . The 21 average percentage o f cru.de fib e r on the a ir -d r ie d sample was 4.435#. The n itr o g e n -fr e e ex tra ct i s composed o f sugars, starch and in la rg e p a rt, m aterial c la ss e d a s p lan t c e llu lo s e and p o ly sa cch rid es. The n itr o ­ g e n -fr e e e x tr a c t was c a lc u la te d by su b tractin g the sum o f w ater, ash, p r o tein , f a t and crude f ib e r o f the sample from 100* Since the fig u r e was determined by c a lc u la tio n in ste a d o f d ir e c t ly , i t in clu d es the cum­ u la t iv e errors o f the o th er determ inations and thus i s not an exact value* From th e previous determ inations the c o n stitu e n ts o f a given sample o f Mung bean meal are as fo llo w s: M oisture 8 *2# Ash 3*0$ P ro tein (N x 6 . 25) 23.69# Lipide 0*91# Crude fib e r 4.^3# N itro g en -free ex tra ct 59-76# 22 EXTRACTION A survey o f th e lit e r a t u r e revealed important inform ation ah out th e a c tio n o f c e r ta in s a l t s on the p e p tiz a tio n of seed p ro tein s ( 29) (40) (42) and some fa c to r s which e f f e c t p e p tiz a tio n ( 31) ( 4 l) (4 6 ). The a u th o r's a tte n tio n has been focu sed on a more system atic in v e s tig a tio n o f t h is f i e l d o f study. I t became apparent th at th e fundamental r e la tio n ­ sh ip between th e s o lu b ilit y o f the Mung bean p ro tein and the nature and amount o f s a lt content o f the so lv en t must f i r s t be stu d ied in d e t a il. E xtraction o f the Mung bean p ro tein , expressed as t o t a l n itro g e n , from (60 mesh) Mung bean meal w ith variou s con cen tration s o f c h lo r id e s, s u lf a t e s , phosphates and carbonates o f sodium and potassium was ca rried out and th e r e s u lt s o f th ese ex tr a c tio n s have been reported in terms o f percent o f t o t a l n itro g en ex tracted and have been p lo tte d a g a in st -pp., th e n eg a tiv e logarithm o f the io n ic stren gth o f th e s o lv e n t. The s a l t s c .p . used fo r e x tr a c tio n are a s fo llo w s: NaCl Na^SOij. NagHPO^ KC1 KgSO^ KgHFO^ NagSO^ KgCO^ One l i t e r o f a one molar s o lu tio n was made o f each o f th e above s a lt s a t 25°C. as sto ck s o lu tio n s . Molar so lu tio n s o f HC1 and NaOH were a lso made fo r purposes o f comparison. Appropriate d ilu tio n s o f th ese molar s o lu tio n s were made a t 25°C. to prepare M/10, M/100 and M/lGOO s o lu tio n s . METHOD.- 1 . F iv e grams o f th e a ir -d r ie d 60 mesh Mung bean meal p rev io u sly d escrib ed was c a r e fu lly weighed and introduced in to a 250 ml. cen trifu g e 23 b o ttle * 2. Twelve g la s s beads were added as a g ita to r s* F if t y ml* o f the d esired con cen tration o f so lv en t were added to the r e a c tio n b o t t l e . The sam ple-solvent weigh t-volume r a tio was thus 1:10* E x tra ctio n c o n siste d o f shaking a t a low speed (120 o s c ill a t io n s per min­ u te) fo r e x a c tly 30 m inutes. S ix b o t t le s were p laced a t one time in a s ix -h o le wooden b lock mounted on the shaking machine.* 3* At the end o f 30 m inutes the b o t t le s were removed from the shaker and imm ediately cen trifu g ed f o r 15 minutes a t 2000 r.p .m . The s o lid m a teria l was packed in the bottom o f the b o t t le s and th e clea r liq u id e x tr a c ts were poured in to K jeldahl f la s k s f o r t o ta l n itrogen determina­ tio n s ( 52 ) . The r e s u lt s obtained from the t o t a l n itrogen determ ination were c a lc u la te d in terms o f percentage o f n itrogen ex tra cted per t o t a l n itr o ­ gen content o f sample. b a sis* C alcu lation s were rep orted on the dry weight In order to determine th e percentage o f n itrogen extra cted per t o t a l n itrogen content o f th e sample, a s e r ie s o f determ inations were made in each case on the ex tra ct and in the case o f were a lso made on the corresponding r e sid u e s. A ll the e x tr a c tio n s were conducted a t room temperature which was approxim ately 25°C. However, the exact room temperature was recorded on th e d ate when the experiment was done. volume a t 25°C. in a water b ath. *Cenco-Meinzer Laboratory Shaker The so lu tio n s were made to 2h The r e s u lt s o f th ese e x tra ctio n are shown in Tables VI through XIX and in Figures 2 through 4 . In the ta b le s the io n ic stren gth i s a ls o expressed as the n e g a tiv e logarithm o f the io n ic stren gth and i s sym bolized by the ex p ressio n pa* 25 TABLE VI SODIUM CHLORIDE AS PEPTIZATION AGENT The sam p le-solven t r a tio was 1:10 (5 gm. o f 60 mesh Mung bean meal was added to 50 ml* o f the s o lv e n t.) E xtraction o f n ro tein was made by shaking fo r 30 m inutes a t 25 C. Molar S o l. NaCl Io n ic Strength 1 .0 •9 .8 .7 •6 *5 .4 *3 .2 *1 *09 .08 .0 7 .0 6 .05 .0 4 .03 .0 2 • 01 .009 •008 .007 .006 .005 .0 0 4 .003 .002 .001 .0009 .000$ .0007 .0006 .0005 .0004 .0003 .0002 .0001 water blank 1 .0 •9 .8 .7 •6 .5 .4 •3 .2 .1 .09 .08 .07 .0 6 .05 .0 4 .03 .0 2 .01 .009 .008 .007 .006 ♦005 .0 0 4 .003 .002 .001 .0009 .0008 .0007 .0006 .0005 .0004 .0003 .0002 .0001 Log o f Io n ic Strength 0 .0 - .046 - .097 - .155 - .222 - .301 - .39S - .523 - .699 -1 .0 0 0 -1 .0 4 6 -1 .0 9 7 -1 .1 5 5 -1 .2 2 2 - 1.301 -1 .3 9 S -1 .5 2 3 -1 .6 9 9 -2 .0 0 0 -2 .0 4 6 -2 .0 9 7 -2 .1 5 5 -2 .2 2 2 - 2.301 -2 .3 9 S -2 .5 2 3 -2 .6 9 9 - 3.000 -3 .0 4 6 -3 .0 9 7 -3 .1 5 5 -3 .2 2 2 - 3.301 -3 .3 9 S -3 .5 2 3 -3 .6 9 9 -4 .0 0 0 IP 0.000 .046 .097 .155 .222 .301 • 39S .523 .699 1.000 1.046 1.097 1.155 1.222 1.301 1.39S 1.523 1.699 2.000 2.046 2.097 2.155 2.222 2.301 2.398 2.523 2.699 3.000 3.046 3.097 3.155 3.222 3-301 3.39S 3.523 3.699 4.000 K jeldahl T itr a tio n 0 . 2027N HC1 42.8 43.3 43.8 44.1 44.7 45.1 45.2 44.5 42.8 38.85 26.7 24.9 23.2 21.9 20.7 19. s 19.0 1 9 .3 19.S5 20.3 20.35 20.45 20.45 20.5 20.5 20.55 20.9 20.9 21.3 21.4 21.6 21.6 a .6 21.6 21.7 21.6 21.6 21.5 0 .6 % N per T otal N a ir d ried >j N per T otal N dry w t. 63*10 63.85 64.59 65.04 65*9^ 66.54 66.69 65.64 63.10 57.19 39.02 36.33 33.79 31.35 30.05 28.71 27.51 27.96 28.78 29.45 29.53 29.68 29.68 29.75 29.75 29.S 3 30.35 30.25 30.95 31.10 31.40 31.40 31.40 31.40 31.55 31.40 31.40 31.23 69.28 69-55 70.36 70.85 71.S3 72.4$ 72.65 71.50 68.74 62.19 42.51 39.5S 36.81 43.69 32.73 31.27 29.97 30.45 31.35 32.08 32.17 32.33 32.33 32.41 32.41 32.49 33*06 33.06 33.71 33.S8 34.20 34.20 34.20 34.20 34.36 34.20 34.20 34.04 26 TABLE VII POTASSIUM CHLORIDE AS PEPTIZATION AGENT The sam ple-sol ren t r a tio was 1:10 (5 gm* o f 60 mesh Mung bean meal was added to 50 m l. o f th e s o lv e n t.) E xtraction o f p ro tein was made hy shaking fo r 3° minutes a t 25°C. Molar S o l. KC1 Io n ic Strength Log o f Ionia Strength 1 .0 •9 .s .7 .6 .5 .4 .3 .2 .1 .09 .o s *07 .0 6 .0 5 .0 4 .0 3 .0 2 .01 .009 .QOS .007 .006 .005 .00** .003 .002 .001 .0009 •OOOS .0007 .0006 .0005 .0004 .0003 .0002 .0001 w ater "blank 1 .0 *9 .8 *7 .6 *5 0 .0 - .046 - *097 - *155 - .2 2 2 - .301 - .398 - *523 - .699 -1 .0 0 0 -1 .0 4 6 -1 .0 9 7 -1 .1 5 5 -1 .2 2 2 - 1.301 -1 .3 9 S -1 .5 2 3 -1 .6 9 9 -2 .0 0 0 -2 .0 4 b -2 .0 9 7 -2 .1 5 5 -2 .2 2 2 - 2.301 -2 .3 9 S -2 .5 2 3 -2 ,6 9 9 -3 .0 0 0 -3 .0 4 6 -3*097 -3 .1 5 5 - 3 .2 2 2 -3 .3 0 1 -3 .3 9 S -3 .5 2 3 -3 .6 9 9 -4 .0 0 0 *3 .2 .1 .09 .OS •07 .0 6 .05 .0 4 .03 .0 2 .01 .009 .008 .007 .006 .005 .0 0 4 .003 .002 .001 .0009 .0008 .0007 .0006 .0005 .0004 .0003 .0002 .0001 Pji 0.000 .046 ..0 9 7 .155 .222 .301 -.398 .523 *699 1.000 1.046 1.097 1.155 1.222 1.301 1.39S 1.523 1.699 2.000 2.046 2.097 2.155 2.222 2.301 2.39S 2.523 2.699 3. GOO 3.046 3.097 3.155 3.222 3.301 3.39S 3.523 3.699 4.000 K jeldahl T itr a tio n 0 . 2072N HC1 41.5 42.3 4 3 .6 4 2.2 4 4 .5 4 4 .2 44.1 43.0 41.85 4 i.g 39.S 38.2 36.1 30.2 25.O 22.8 21.3 1 9 .4 18.5 I 8 .5 1 8 .6 18.8 18.9 19.3 19.6 20.0 20.4 20.8 21.0 21.1 21.1 21.2 a .2 2 1 .3 21.3 2 1 .4 2 1.4 21.5 0 .6 $ N per T otal N a ir d ried >i N per T otal N dry w t. 61.16 62.35 64.30 65-19 65*64 65.19 6 5 .0 4 63.40 61.68 61.60 58.61 56.22 53-08 44.26 36.48 33.19 30.95 28.11 26.76 26.76 26.91 27.24 27.36 27.96 2 8 .4 l 29.OO 29.60 30.20 30.50 30.65 30.65 30.80 30.80 30.95 30.95 31.10 31.10 31.25 66.62 67.92 70.04 71.0 2 71.50 7 1.0 2 70.85 69.06 67*19 68.54 63. S5 61.2 4 57.S2 48.21 39.75 36.16 33.71 32.82 29.15 29.15 29.32 29.6 4 29.80 30.45 30.94 31.59 32.2 4 32.90 33.22 33.39 33.39 33.55 33.55 33.71 33.71 33.S8 33.S8 34.04 27 TABLE VIII SODIUM SULFATE AS PEPTIZATION AGENT The sam p le-solven t r a tio was 1:10 (5 o f 60 mesh Mung heaa meal was added to 50 ml* o f th e s o lv e n t.) E xtraction o f p r o te in was made by shaking fo r 30 minutes a t 2 4 .6 °C. Molar S o l. NagS0ii .oooa .0007 .0006 .0005 .0004 .0003 .0 0 0 2 .0001 water blank 3 .0 22 -7 .4 2 .1 1 .8 1 .5 1 .2 •9 .6 •3 .27 .2 4 .21 .18 .1 5 .1 2 .09 .06 .03 .027 4" CM O. 1 .0 •9 .3 .7 .6 .5 .4 •3 .2 •1 .0 9 .OS .0 7 •06 .0 5 .0 4 .0 3 .0 2 .0 1 .009 .OOS .007 .0 0 6 .005 .0 0 4 .003 .002 .001 .0009 I o n ic Strength .021 .018 .015 .012 .009 .006 .003 .0027 .0024 .0021 .0018 .0015 .0012 .0009 .0006 .0003 Log o f Io n ic Strength pp 0 .477 0.431 0.3 8 0 0.3 2 2 0.255 0 .1 7 6 0.079 - .046 - .2 2 2 - .523 - .569 - .620 - .678 • .7^5 - .8 2 4 - .921 -1 .0 4 6 -1 .2 2 2 -1 .5 2 3 -1 .5 6 9 -1 .6 2 0 -1 .6 7 8 - I .745 -1 .8 2 4 -1 .9 2 1 -2 .0 4 6 -2 .2 2 2 -2 .5 2 3 -2 .5 6 9 - 2.620 -2 .6 7 8 -2 .7 4 5 - 2 .8 2 4 - 2.921 - 3 .0 4 6 - 3.222 -3 .5 2 3 -.4 7 7 -*431 — 380 — 322 -.2 5 5 —176 -.0 7 9 .046 .222 .523 .569 .620 .678 ..7 4 5 .824 .921 1.046 1.222 1.523 1.569 1.620 I .678 1.745 1.824 1.921 2.046 2.222 2.523 2.569 2.620 2.678 2.745 2.824 2.921 3.046 3.222 3.523 K jeldahl T itr a tio n 0.2027N Hca 3 2 .4 3 3 .4 34.55 35.55 3 6 .4 37.9 3 8 .6 3 9 .4 44.75 44.75 4 5 .4 44.85 43.75 43.1 4 2 .4 4 1 .4 38.0 32.6 26.65 2 5 .6 25.2 24.7 24.1 24.0 23*5 23.5 22.3 2 2.0 21.6 21.6 21.5 21.5 21.5 2 1 .4 21.5 21.5 2 1 .4 21.5 0 .6 # N per # N per Total N T otal N a ir d ried dry wt. 51.79 47.55 4 9.0 4 53-42 50.76 55.30 52.26 56.93 58.31 53.53 60.75 55*77 56.82 61*90 58.02 63.20 66.01 71.91 66.01 71.91 66.99 72.97 66.16 72.07 64.52 . 70.28 69.22 63-55 62.50 68.09 66.45 61.01 60.92 55-92 47.85 52.12 42.43 38.95 40.72 37.3S 40.06 36.73 36.00 39.6 3 5.14 32.27 34.98 38.11 3 4.2 4 37.30 3 4.24 37.30 32.44 35.34 33.00 34.85 31.40 34.20 31.40 34.20 34.04 31.25 34.0 4 31.25 34.04 31.25 33.88 31.10 34.0 4 31.25 34.04 31.25 33.88 31.10 34.04 31.25 28 TABLE IX POTASSIUM SULFATE AS PEPTIZATION AGENT The sam ple-3olvent r a tio was 1:10 (5 gm. o f 60 mesh Mung bean meal was added to 50 m l. o f th e s o lv e n t.) E xtraction o f p ro tein was made by shaking f o r 30 m inutes a t 25®C. Molar S o l. KgSOjj, . o o —4 0 .5 .4 .3 .2 .1 .0 9 .08 .0 7 .0 6 .0 5 .0 4 .0 3 .0 2 .01 .009 .008 .0 0 6 .005 .0 0 4 .003 .0 0 2 .001 water blank I o n ic Strength 1 .5 1 .2 0 .9 0 .6 .3 .27 .2 4 .21 .18 .1 5 .1 2 .0 9 .0 6 .03 .027 .0 2 4 .021 .018 .015 .012 .009 .006 .003 Log o f Io n ic Strength Pp. 0.176 0 .079 -0 .0 4 6 - .222 - .523 - .569 - .620 - .678 - .745 - .824 - . 9a -1 .0 4 6 -1 .2 2 2 -1 .5 2 3 -1 .5 6 9 - 1.6 2 0 -1 .6 7 8 -1 .7 4 5 -1 .8 2 4 - 1.921 -2 .0 4 6 -2 .2 2 2 -2 .5 2 3 -.1 7 6 -.0 7 9 .046 .222 .523 .569 .620 .678 .745 .824 .921 1.046 1.222 1.523 1.569 1.620 1.678 i.7*<5 1.8 2 4 1.921 2.046 2.222 2.523 K jeldahl T itr a tio n 0 . 2027N HC1 39.3 4 1 .4 4 3.2 44.9 4 5 .0 4 4.3 4 4 .4 43.9 42.8 4 2.6 40.5 36.8 3 0 .2 24.6 24.9 22,3 22.2 21.5 21.5 21.5 2 1 .4 a .3 21.1 21.0 0 .6 % N per T otal E a ir d ried 7> E per 57.87 61.01 63.65 66.24 66.39 65.34 65.49 64.74 63.IO 62.80 59.66 54.13 44.2 6 35.88 36.33 32.45 32.29 31.25 31.25 31.25 31.10 30.95 30.65 30.50 63.03 66.45 69.34 62.16 6 2 .3 2 71.18 71.34 70.53 68.74 68.41 64.99 58.96 48.21 39.09 39.58 3 5 .3 4 35.18 34.04 34.04 34.0 4 33.88 33.71 33.39 33.22 T otal E Dry w t. 29 TABLE X DISODIUM PHOSPHATE AS PEPTIZATION AGENT The sam p le-solven t r a tio was 1:10 (5 gm. o f 60 mesh Mung bean meal was added to 90 m l. o f th e s o lv e n t.) E xtraction o f p ro tein was made by shaking fo r 30 m inutes a t 25 C. Molar S o l. HagEPOij. 0 .7 •6 •5 .4 .3 .2 .1 .0 9 •OS .0 7 .0 6 .0 5 .0 4 .03 .0 2 .0 1 .009 .008 .007 .0 0 6 .005 .004 .003 .0 0 2 .001 .0009 .0008 .0007 .0006 .0005 .0004 .0003 .0002 .0001 water blank Io n ic Strength Log o f Io n ic Strength 2 .1 1 .8 1 .5 1 .2 .9 .6 .3 .27 .2 4 .21 .18 .15 • 12 .09 •06 .0 3 .027 .024 .021 .018 .015 .012 .009 .006 .003 .0027 .0024 .0021 .0018 .0015 .0012 .0009 .0006 .0003 0 .3 2 2 0.255 0 .1 7 6 0.079 - .046 - .222 - .523 - .569 - .620 - .678 - .745 - .824 - .921 -1 .0 4 6 - 1 .2 2 2 -1 .5 2 3 -1 .5 6 9 -1 .6 2 0 - I . 67S -1 .7 4 5 -1 .8 2 4 -1 .9 2 1 -2 .0 4 6 -2 .2 2 2 -2 .5 2 3 -2 .5 6 9 -2 .6 2 0 -2 .6 7 8 -2 .7 4 5 - 2 .8 2 4 - 2.921 -3 .0 4 6 - 3 .2 2 2 -3 .5 2 3 P4 -0 .3 2 2 -O.255 - 0.1 7 6 -0 .0 7 9 0.0 4 6 .222 .523 .569 .620 .678 •7**5 .824 .921 1.046 1 .2 2 2 1.523 1.569 1.620 1.678 1.745 1.824 1.921 2.046 2 .222 2.523 2.569 2.620 2.678 2.745 2.824 2.921 3.046 3.222 3.523 K jeldahl T itr a tio n 0.1989II HC1 48.8 4 9.0 5 1 .2 5 2 .2 52.3 52.3 52.3 51.8 51.3 51.15 51.1 51.0 5 0 .2 49.8 49.1 4 8 .4 4 8 .2 48.0 4 7 .8 4 7 .4 4 7 .2 45.85 4 2 .4 36.7 29*5 26.1 25.7 25.0 2 4 .4 24.0 24.1 23.7 23.0 2 2,6 22.1 0 .6 f> N per $> N per T otal H T otal H a ir d ried dry wt* 70.72 71.01 74.24 75.71 75.86 75.86 . 75.86 75-12 74.39 74.17 74.10 73*95 72.78 72.19 7 1.16 70.13 69.84 69.55 69.25 68.73 68.37 66.39 61.33 52.99 42.40 37.41 36.82 35.80 34.9 2 3M 3 3 4 .4 8 33.89 32.86 32.28 31.54 77.04 77.36 80.87 82.47 82.63 82.63 82.63 81.83 81.03 8O.79 8O.71 8 0 .1 6 79.28 7 8 .6 4 7 7.5 2 76.40 76.08 75.76 75.44 74.87 74.48 72.32 66.81 57.72 46.19 40.75 40.11 38.99 3 8.0 4 37.40 37.56 36.92 35.80 35.16 34.36 30 TABLE XI DIFOTASSIUM PHOSPHATE AS PEPTIZATION AGENT The sam ple-solvent r a tio was 1:10 (5 gm* o f 60 mesh Mung bean meal was added to 50 m l. o f th e s o lv e n t.) E xtraction o f p ro tein was made by shaking fo r 30 m inutes a t 25 C. Molar S o l. K2HPO4 Io n ic Strength lo g o f Io n ic Strength 0 .7 •6 .5 .4 .3 •2 .1 .09 .0 8 .07 .0 6 .05 .o h .03 .0 2 .0 1 .009 .008 .007 .006 .005 .oo4 .003 .002 .001 .0009 .0008 .0007 .0006 .0005 .0004 .0003 .0002 .0001 water blank 2 .1 1 .8 1 .5 1 .2 •9 .6 ♦3 •27 .2 4 .21 .18 .15 • 12 .09 .0 6 .03 .027 .024 .021 .018 .015 .012 .009 .006 .003 .0027 .0024 .0021 .0018 .0015 .0012 .0009 .0006 .0003 0 .3 2 2 O.255 0 .1 7 6 0.079 - .046 - .222 - .523 - .569 - .620 - .678 - .7 ^ - .8 24 - .921 -1 .0 4 6 -1 .2 2 2 -1 .5 2 3 -1 .5 6 9 - 1.620 -1 .6 7 8 - 1.745 -1 .8 2 4 - 1.921 -2 .0 4 6 -2 .2 2 2 -2 .5 2 3 -2 .5 6 9 - 2.620 -2 .6 7 8 -2 .7 4 5 -2 .8 2 4 - 2.921 -3 .0 4 6 -3 .2 2 2 -3*523 PP — 322 - .2 5 5 -.1 7 6 -.0 7 9 +.046 .222 *523 *569 .620 .678 .745 .8 2 4 •9 a 1.046 1.222 1.523 1*569 1.6 2 0 1.678 1*745 1.824 1.921 2.046 2.222 2.523 2.569 2.620 2.678 2.745 2.824 2.921 3.046 3*222 3.523 K jeldahl T itr a tio n 0 . 1989N HOI 4 6 .2 4 8 .4 50.5 5 1 .4 51*5 51*3 51*2 51.2 50.8 50.2 50.1 49.9 49*3 MS.9 49.0 48.15 47.6 46.7 4 7 .2 46.85 46.9 43*9 38.4 32.1 25.8 25.3 24.8 24.6 24.0 23.6 2 3 .2 22.6 2 2 .4 22.3 22.1 0 .6 # N per T otal N a ir d ried N per T otal N dry wt. 66.91 70.13 73.22 74.54 74.68 74.39 74.24 74.24 73*39 72*78 72.63 72.3 4 71.46 70.87 71.01 69-77 69. U 67.64 68.37 67.86 67*93 63-53 55.46 46.21 36.97 36.2 4 35.50 35.21 34.33 3 3 .7 4 33.16 32.28 31.9S 31*84 31.5 4 77.88 76.MO 79.76 81.19 81.35 81.03 80.87 80.87 79.95 79.28 79.11 78.80 7 7 .S4 77.2 0 77.36 76.00 75.28 73.68 74.48 73.9 2 74.01 69.21 60.41 5 0 .3 4 40.27 39.47 38.67 3S .36 37.39 36.76 36.12 35*16 3 4 .8 4 34.68 36.36 31 TABLE XII SODIUM GfiHBONATE AS PEPTIZATION AGENT The sam p le-solven t r a tio was 1:10 (5 o f 60 mesh Mung Lean meal was added to 30 m l. o f th e s o lv e n t.) E xtraction o f p ro te in was made by shaking fo r 30 m inutes a t 25°C. Molar S o l. NagCOj Io n ic Strength Log o f Io n ic Strength VP 0 .7 •6 •5 .4 .3 *2 .1 .0 9 .08 .0 7 •Ob .0 5 .0 4 .0 3 .0 2 .01 .009 .008 .007 .006 .005 .0 0 4 .003 .002 .001 .0009 .0008 .0007 .0006 *0005 .0004 .0003 .0002 .0001 water blank 2 .1 1 .8 1 .5 1 .2 •9 .6 •3 .27 .2 4 .21 .18 .15 .1 2 .09 .0 6 .03 .027 .024 .021 .018 .015 .012 .009 .006 .003 .0027 .0024 .0021 .0018 .0015 .0012 .0009 .0006 .0003 0 .3 2 2 0.255 0 .1 7 6 0.079 - .046 - .222 - .523 - .569 - .620 - .678 - . 7^ - .824 - .921 -1 .0 4 6 -1 .2 2 2 -1 .5 2 3 -1 .5 6 9 -1 .6 2 0 -1 .6 7 8 -1 .7 4 5 -1 .8 2 4 - 1.921 -2 .0 4 6 -2 .2 2 2 -2 .5 2 3 -2 .5 6 9 -2 .6 2 0 -2 .6 7 8 -2 .7 4 5 -2 .8 2 4 -2 .0 2 1 -3 .0 4 6 -3 .2 2 2 -3 .5 2 3 - .3 2 2 -.2 5 5 - .1 7 6 t .079 i.0 4 6 .222 .523 .569 .620 .678 .745 .824 .921 1.046 1.222 1.523 1.569 1.620 1.678 1.745 1.824 1.921 2.046 2.222 2.523 2.569 2.620 2.678 2.745 2.824 2.921 3.046 3.222 3.523 K jeldahl T itr a tio n 0.1989N HC1 51.4 52.3 52.9 5 3.2 5 3 .4 53*6 5 4 .0 54.1 5 4 .4 5 4 .4 53.S 53.65 53.5 5 3 .4 53.3 53.2 51.9 51.35 51.0 5 0 .6 5 0 .2 49.6 48.8 48.1 40.6 3 8 .4 36.4 3^.5 32.3 3 0 .2 28.6 26.6 25.6 24.2 22.7 0 .6 % N per Total N a ir d ried f> N per Total N dry wt. 7 4.5 4 75.8b 7 6 .7 4 77.13 77.47 77.76 78.35 78.50 78.89 78.89 78.0 6 77.84 77.57 77.42 77.27 77.1S 75.25 74.41 73.90 73.31 72.72 71.90 70.7 2 69.69 58.67 55.46 52.52 49.74 46.51 43.43 41.08 38.1 4 36.68 34.62 32.4 2 81.19 82.63 S3.59 84.07 84.39 84.71 S5.35 85.51 S5.93 S5-93 85 .03 84.79 84.50 84.33 84.17 84.07 82.27 81.0 6 80.51 79 .86 79 .2 2 78.32 77.04 75.92 62.91 60.41 57.22 54.18 50.67 47.31 44.75 41.55 39.95 37.72 35.32 32 TABLE XIII POTASSIUM CARBONATE AS PEPTIZATION AGENT The sam p le-solven t r a tio was Is 10 (5 gm. o f 60 mesh Mung bean meal was added to 50 ®1« o f th e s o lv e n t.) E xtraction o f p ro tein was made byshaking fo r 30 m inutes a t 25° G. Molar S o l. EgCOj Io n ic Strength Log o f Ion ic Strength Pp- 0 .7 .6 •5 .4 •3 .2 .1 .0 9 .OS .0 7 .0 6 .0 5 .0 4 .0 3 .0 2 .01 .009 .008 .007 .006 .005 .004 .003 .002 .001 .0009 .0008 .0007 .0006 .0005 .0004 .0003 .0002 .0001 water blank 2 .1 1 .8 1 .5 1 .2 *9 .6 .3 .27 .2 4 .21 .1 8 .15 .1 2 .09 .0 6 .0 3 .027 .0 2 4 .021 .018 .015 .012 .009 .006 .003 .0027 .0024 .0021 .0018 .0015 .0012 .0009 .0006 .0003 0.322 0.255 0 .1 7 6 0.079 - .046 - .2 2 2 - .523 - .569 - .620 - .678 - .745 - .824 - .9 a -1 .0 4 6 -1 .2 2 2 -1 .5 2 3 -1 .5 6 9 -1 .6 2 0 -1 .6 7 S -1 .7 4 5 -1 .8 2 4 -1 .9 2 1 -2 .0 4 6 -2 .2 2 2 -2 .5 2 3 -2 .5 6 9 -2 .6 2 0 -2 .6 7 S -2 .7 4 5 -2 .8 2 4 - 2.921 -3 .0 4 6 -3 .2 2 2 -3 .5 2 3 — 322 -.2 5 5 - .1 7 6 -.0 7 9 .046 .222 .523 .569 .620 .67S .745 .824 .921 1.046 1.222 1.523 1.569 1.620 1.678 1.745 1.824 1.921 2.046 2.222 2.523 2.569 2.620 2.67S 2.745 2.824 2.921 3.046 3.222 3.523 K jeldahl T itr a tio n 0 . 19S9N HC1 50.0 51.5 53.0 53.1 5 3 .4 53.5 54.0 5 4 .2 5 4 .4 54.3 53.8 53*6 53.3 53.0 52.8 52.6 52.5 5 2 .4 52.0 5 1 .6 5 1 .0 50.1 48.8 47.3 41.65 39.S 38.2 36.5 33.05 31.25 3 0 .4 2 8 .4 26.5 24.8 22.7 0 .6 # N per T otal N a ir d ried $ N per Total N dry wt. 72.48 74.68 76.88 77.03 77.47 77.62 78.35 78.64 78.9 4 78.79 78.06 77-76 77.32 76.88 76.43 76.30 76.15 76.00 75.42 74.83 73-95 72.63 70.72 68.52 60.23 57.51 55.17 52.67 47.61 44.97 43.72 40.79 3S .00 35.50 32.42 78.95 81.35 8 3 .7 6 83.91 84.39 84.55 85*35 85.67 85.99 85.83 85.0 3 84.71 8 4.2 3 83.75 8 3.2 6 83.11 82.95 82.79 82.15 81.51 8O.55 79.11 77-04 74.64 65.61 64.83 60.10 57.38 51.86 48.98 47.63 44.43 41.39 38.67 35.32 33 SABLE XIV SODIUM SULFITE AS PEPTIZATION AGENT The sam p le-solven t r a tio was 1:10 (5 Gm* o f 60 mesh Mung hean meal was added to 50 ml* o f th e s o lv e n t.) E xtraction o f p ro tein was made by shaking fo r 30 m inutes a t 25° C. Molar S o l. Nag SO 1 .0 •9 .8 *7 .6 •5 .4 •3 .2 .1 .0 9 .08 .07 .0 6 .0 5 .0 4 .03 .0 2 .01 .009 .008 .007 .006 .005 .004 .003 ♦002 .001 .0009 ♦0008 .0007 .0006 .0005 .0004 .0003 .0002 .0001 w ater blank Io n ic Strength 3 .0 2 .4 2 .1 1 .8 1 .5 1 .2 •9 .6 .3 .27 .2 4 .21 .18 .15 .1 2 .09 •06 .03 .027 .0 2 4 .021 .018 .015 .012 .009 .006 .003 .0027 .0024 .0021 .0018 .0015 .0012 .0009 .0006 .0003 Log o f Io n ic Strength pji 0.477 O.431 0.3 8 0 0.322 O.255 O.176 0.079 - .046 - .222 - .523 - .569 - .620 - .678 - .745 - .8 2 4 - .921 -1 .0 4 6 - 1.222 -1 .5 2 3 -1 .5 6 9 -1 .6 2 0 -1 .6 7 8 -1 .7 4 5 -1 .8 2 4 - 1.921 -2 .0 4 6 -2 .2 2 2 -2 .5 2 3 - 2.569 -2 .6 2 0 -2 .6 7 8 -2 .7 4 5 -2 .8 2 4 -2 .0 2 1 -3 .0 4 6 -3 .2 2 2 -3 .5 2 3 -.4 7 7 -.4 3 1 -.3 8 0 — 322 -.2 5 5 -.1 7 6 -.0 7 9 .046 .222 .523 .569 .620 .678 .745 .824 .921 1.046 1 .222 1.523 1.569 1.620 1.678 1.745 1.824 1.921 2.046 2.222 2.523 2.569 2.620 2.678 2.745 2.824 2.921 3.046 3.222 3.523 K jeldahl T itr a tio n 0 . 2027N HC1 40.60 43.80 45.85 47.55 49.10 49.60 51.10 51.80 51.75 51.40 51.30 51.10 51.10 50*60 50.60 50.40 50.10 48.70 48.40 47.30 46.40 42.70 39.35 37*65 32.75 29.60 26.40 23.70 23.40 23.15 22.55 22.10 21.70 21.70 21.60 21.60 21.50 21.40 0 .6 $> N per T otal N a ir dried io N per T otal N dry wt. 59.81 64.59 67.66 70.20 72.52 73.27 75.51 76.53 76.48 75.9 6 75.81 75.51 75.51 74.76 74.76 74.46 74.02 71.92 71.47 69. S3 68.48 62.95 57.94 55-40 48.07 43.36 38.58 34.54 34.09 33-71 32.8 2 32.14 31.55 31.55 31.40 31.40 31.33 31.10 65.15 70.36 73.71 76.47 79.00 79.81 82.2 6 83-37 83.31 82.75 82.58 8 2 .2 6 82 .2 6 81.4 4 81.4 4 8 1.1 2 80.63 78.35 77.8 6 76.07 74.60 68.57 63.11 6O.53 52.37 47.23 42.02 37.62 37.13 36.73 35.75 35.02 34.36 34.36 34.20 34.20 34.12 33.88 34 TABLE XV THE DETERMINATION OP NITROGEN IN THE RESIDUE PROM IS OP THE SAMPLES EXTRACTED WITH Na2S0, COMPARED WITH THE CORRESPONDING RESULTS OP NITROGEN IN THE EXTRACTS Molar S o lu tio n Na2S03 0 .9 O.S O.T 0.6 0 .5 0 .4 0 .3 0 .2 0 .1 0.09 0.0s 0 .0 7 0 .0 6 0 .0 5 0 .0 4 0 .0 3 0 .0 2 0.01 K jeldahl T itr a tio n 0.2027N HC1 2 4 .4 2 2 .2 20.6 19.0 1 5 .5 1 6.9 15*9 16.25 16.8 16.8 1 7 .0 17.1 17.45 1 7 .4 17.5 18.1 1 9 .4 19. 5 $ N per T otal N dry wt. Residue $ N per T otal N dry wt. E xtract 1.471 1.335 I .236 1.137 1.106 1.007 0.9 4 6 0.9675 1.001 1.001 1.014 1.020 1.041 2.670 2.797 2.902 2.998 3.029 3.122 3.165 3.162 3*140 3*134 1.03S 1.044 1.082 1.162 1 . I 87 3.122 3.122 3.091 3.091 3.079 3.O6O 2.973 2.955 Average valu e o f percentage o f t o t a l n itro g en in 18 samples 4 .1 3 4 (sum o f average o f columns 3 *®d. 4 ) . O o o O oo o r- \o o oiT> p3]DBj]X3 uaSoaqiu |E] 0 3 jo quacusj O o o protein N from Mung bean. of or pp, is plotted vs amount extracted. by different salts upon the peptization The negative logarithm as contributed (the -log of ionic strength) cS The effect of varying ionic strength pp F ig u re o o o o 'O o m pajDBXjxo uaSojjiu orn jbjoj _jo juaojaj (the -log of ionic strength) This figure shows the effect of ionic strength as contributed by solutions of the salts NaCl, KC1, NaaSOi and K2 SO 1 upon peptization of Mung bean protein at 25° C. pa o if TABEE XVI HYDROCHLORIC ACID AS PEPTIZATION AGENT The sam p le-solven t r a tio was 1:10 ( 5 gm* o f 60 mesh Mung bean meal was added to 50 ml* o f th e s o lv e n t .) E xtraction o f p ro tein was made by shaking fo r 30 m inutes a t 25®C. No. Molar S o lu tio n HC1 1. 2. 3. 4. 5. 6. 7» 8. 9. 10. 1 1. 12. 13. l4 . 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25 . 26. 27. 28. 29. 30. 31. 32. 33* 34. 35. 36. 0 .7 0 .6 0 .5 0 .4 0 .3 0 .2 0 .1 0 .0 9 0 .0 8 0 .0 7 0 .0 6 0 .0 5 0 .0 4 0 .0 3 0 .0 2 0 .0 1 0.009 0.008 0.007 0 .0 0 6 0.0 0 5 0 .0 0 4 0.0 0 3 0.0 0 2 0.001 0.0009 0.0008 0.0007 0.0006 0.0005 0 .0004 0.0003 0.0002 0.0001 fa te r blank K jeldahl T itr a tio n 0.19S9N EC1 3 6 .3 37.1 38.1 3 9 .2 40.5 4 2 .2 43.8 44.3 4 4 .0 4 3 .0 40.1 3 1 .2 l 4 .1 6 .9 6 .8 7 .6 7.S 8 .0 8 .6 8 .8 1 0 .1 1 1 .9 13*1 1 3 .9 1 7 .3 1 8 .0 1 8 .6 1 8 .5 1 9 .6 1 9 .8 2 0 .6 2 1 .1 2 1.3 2 2 .4 2 2.9 0 .6 $ N per Total N a ir d ried $ N per T otal N dry wt. 52.384 53.558 55*024 56.639 5S.547 61.039 63-3S7 64.122 63.682 62.214 57.959 43.431 24.688 9.242 9.095 10.270 10.562 10.857 11.737 12.030 13.93S 16.5SO 18.340 19.515 24.503 25.503 26.410 26.263 27.S75 28.171 29.346 30.078 30.373 31.986 32.721 57.063 58.342 59.939 61.698 63.776 66.491 69.049 69.84§ 69.370 67.771 63.136 47.310 26.893 10.067 9.907 11.187 11.505 11.826 12.785 13.104 15. I 83 18.061 19.978 21.258 26.691 27.780 28.769 28.609 30.365 30.365 31.967 32.764 33.086 34.843 35.643 33 TABLE XVII SODIUM HYDROXIDE AS PEPTIZATION AGENT The sam p le-solven t r a t io was 1:10 (5 g®. o f 60 mesh Mung bean meal was added to 50 » 1 . o f th e s o lv e n t.) E xtraction o f p r o te in was made by shaking f o r 30 m inutes a t 25 C. No. Molar S o lu tio n NaOE 1. 2. 3. 4. 56. 7. S. 9. 10 . 11. 12 . 13. l4 . 15. 16. 17. 18. 19 . 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 0 .1 0 .0 9 0.08 0 .0 7 0 .0 6 0 .0 5 0 .0 4 0 .0 3 0 .0 2 0 .0 1 0.009 0.008 0.007 0 .0 0 6 0.005 0 .0 0 4 0.003 0.002 0.001 0.0009 0.0008 0.0007 0 .0006 0.0005 0.0 0 0 4 0.0003 0.0002 0.0001 water blank K jeldahl T itr a tio n 0.1989H HOI 5 7 .0 5 7 .0 57-0 5 5 .9 55.9 5 4 .8 54.7 5 4 .6 5 4 .6 51.7 51.0 51.0 5 1 .0 50.35 49.0 4 8 .5 46.8 39.1 28.1 27.7 26.95 25.45 24.85 2 4 .0 23.S 23*2 22.55 22.85 21.05 0 .6 $ N per Total H a ir d ried 82.758 82.758 82.75S 81.143 81.143 79.530 79.333 79.235 79.235 74.980 73.952 73.952 73.952 72.99S 71.017 70.285 67.7S7 56.492 40.351 39*764 38.662 36.462 35-582 34.333 34.041 33.161 32.207 32.647 30.007 $> H per Total H dry wt. 90.150 90.150 90.150 88.391 88.391 86.634 86.473 86.212 86.212 81.677 30.557 80.557 80.557 79.513 77*360 76.563 73.842 61.533 43.915 43.316 42.115 39.719 33.760 37.399 37.081 36.123 35.083 35*563 32.687 39 TABLE XVIII HYDROCHLORIC ACID AS PEPTIZATION AGENT 1 .1 7 gm. NaCI ( f i n a l co n cen tration , 0 .4 m) added to each. 5 sample. The sam p le.so lv en t r a tio was 1:10 (5 gm. o f 60 mesh Mung Lean meal was added to 50 ml. o f th e s o lv e n t.) E xtraction o f p ro tein was made by shaking f o r 30 m inutes a t 25°C. No. Molar S o lu tio n HC1 pH o f HC1 Solvent pH o f E xtract K jeldahl T itr a tio n O.1989N HC1 $ N per Total N dry wt. 1. 2. > 4. 5. b. T. S. 9. 10. 11. 12. 13. 1 *1. 15. lb . 17 . IS . 19. 20. 21. 22. 23. 24. 25. 26. 27. 2S. 29 . 30. 31. 32. 333*. 35. 36. 0 .7 0 .6 0 .5 0 .4 0 .3 0 .2 0 .1 0 .0 9 0 . 0s 0 .0 7 0 .0 6 0 .0 5 0 .0 4 0 .0 3 0 .0 2 0 .0 1 0.009 0 . 00s 0.007 0 .0 0 6 0 .0 0 5 0 .0 0 4 0 .0 0 3 0 .0 0 2 0.001 0.0009 0 . 000s 0.0007 0.0006 0.0005 0.0 0 0 4 0.0003 0.0002 0.0001 water blank 0*37 0 .4 3 0 .5 0 0*59 0 .7 0 0 .9 0 1.1 3 1 .2 0 1 .2 3 1 .3 3 1 .4 0 1 .5 0 1 .6 4 1 .7 2 1 .8 7 2.13 2.18 2 .2 2 2.29 2.33 2 .3 8 2.50 2.63 2 .9 0 3 .1 5 3.2 3 3*30 3.41 3 .5 2 3.68 3.88 4 .7 2 5 .8 0 6.50 7.15 7 .1 0 0 .38 0.38 0 .4 2 0 .5 3 0 .6 4 0 .9 0 I .65 1.78 2.00 2.38 2.78 3.20 3.67 4.07 4 .48 5.00 5.06 5.10 5.20 5 .30 5.30 5.42 5.50 5.61 5.71 5.7b 5.7b 5.7b 5.7b 5 .76 5.80 5. so 5.80 5 .82 5 .8 2 5.82 33.4 34.4 35.3 36.1 36.8 37.5 37*7 37*9 3 8 .4 37-5 33.0 30.0 2 7 .4 25.O 23.2 35*8 36.6 38.5 39*8 4 1 .5 40.8 4 2 .4 42.5 43.7 4 4 .2 44.7 4 5 .2 44.8 4 4 .6 44.5 4 4 .4 4 4 .4 4 4 .4 4 4 .4 44.1 0 .6 51.421 54.024 55-461 56.738 57-858 58.977 59.246 59.617 60.415 58.977 51.785 46.989 42.833 38.999 36.119 56.259 57*539 6O.576 62.656 65-372 64.252 66.810 66.970 68.887 69.686 70.487 71.284 70.644 70.325 70.165 70.007 70.007 70.007 70.007 69.528 4o TABLE XIX SODIUM HYDROXIDE AS PEPTIZATION AGENT 1 .1 7 gm* NaCI ( f i n a l co n cen tration , 0 .4 m) added to each 5 g®* sample* The sam ple*solvent r a tio was 1:10 (5 gm. o f 60 mesh Mung Lean meal was added to 50 ml* o f the so lv en t* ) E xtraction o f p ro tein was made ’byshaking fo r 30 m inutes at 25 C. No. Molar S o lu tio n NaOH 1* 2. 3* 4. 5* 6. 7* S. 9. 10. 11. 1 2. 13* 14. 15* 16. 17. IS . 19. 20* 21. 22. 23. 24. 25. 26. 27. 28* 29. 30. 0 .1 0 .0 9 0 .0 8 0 .0 7 0 .0 6 0 .0 5 0 .0 4 0 .0 3 0 .0 2 0 .0 1 0.0 0 9 0.008 0.007 0 .0 0 6 0.0 0 5 0 .0 0 4 0.003 0 .0 0 2 0.001 0.0009 0.0008 0.0007 0 .0006 0.0005 0.0 0 0 4 0.0003 0 .0002 0.0001 water blank pH o f NaOH Solvent pH o f Extract 1 1 .5 1 1 .5 1 1 .5 4 1 1 .5 4 1 1 .5 4 1 1 .5 4 1 1 .5 2 11.50 1 1 .40 11.28 1 1 .2 4 11.18 11.08 1 1 .00 10 .9 0 10.80 10.66 1 0 .62 1 0 .4 2 10.3S 10.30 1 0 .2 0 10 .1 0 10.00 9 .88 9 .67 9 .2 0 8 .2 0 6 .9 2 10,60 10.52 10.38 10.20 1 0 .0 2 9 .80 9.3 0 8 .8 4 8 .1 2 6*73 6.61 6.50 6.36 6.22 6.16 6.03 5*93 5.88 5*82 5*81 5*80 5.80 5 .8 0 5*77 5*74 5.7** 5 .7 * 5 .7 “» 5 . 7^ K jeldahl T itr a tio n 0 . 19S9N HC1 53*4 54.0 5 4 .0 5 4 .4 53*4 52.8 52.55 52.35 51*5 50.9 49.8 50.0 4 8 .4 49*3 47*9 4 7 .4 46.9 46.1 46.0 4 5 .3 4 5 .4 4 5 .4 45.9 45.9 46.1 46.1 46.1 46.1 4 5 .4 0 .6 $ N per Total N dry wt. 84.392 85.350 85.350 85*991 84.392 83.% 3 83.031 82.713 8I .356 80.397 78.638 78.956 76.399 77*839 75*001 74.800 74.002 72.722 72.564 71.445 71.605 71.605 72.1J04 7 2 .4 o4 72.722 72.722 72.722 72.722 71.605 ON < L) U 3 .SP 5 oca x a t 100°C. 43 D. Temperature and o i l - f r e e and n o n -o il fr e e con d ition o f th e samples (A) The D eterm ination o f th e E ffe c t o f th e E xtractin g Time on th e Degree o f P e p tiz a tio n o f N itrogen ($ N per t o t a l N) o f Mung Bean Meal w ith F ive D iffe r e n t Sam ple-solvent R a tio s, h e r e a fte r Termed nS-S-Bn. The 0.4M NaCI s o lu tio n was used as th e p e p tiz in g a g en t. Each ex­ tr a c tio n was made on s ix samples u sin g f i v e d iffe r e n t sam ple-solvent r a t io s and one w ith w ater. NO. 1 . 5*0 gm. sample to 100 ml. 0.4M NaCI No. 2 . 7 .5 gm* sample to 100 m l. 0 .4 m NaCI No. 3- 1 0 .0 gm. sample to 100 ml. 0*4m NaCI No. 4 . 12 .5 gm. sample to 100 ml. 0 .4 m NaCI No. 5* 1 5 .0 gm. sample to 100 ml. 0 .4 m SaCl No. 6. 1 0 .0 gm. sample to 100 ml. water Four e x tr a c tio n p erio d s were employed: per minute) fo r 10, 20, 30 shaking (120 o s c ill a t io n s 4o m inutes a t 25°C. A fter c e n tr ifu g a tio n th e c le a r liq u id s were poured in to E jeld ah l f la s k s fo r t o t a l n itrogen d eterm in ation s. C alcu lation s were made from t it r a t io n data as to: 1 . Mgm. N ex tra cte d from th e sam ples. 2 . Percentage N per a ir -d r ie d samples. 3 . Percentage N per T otal N content o f a ir -d r ie d samples. 4 . Percentage N per T otal N content on dry w eight h a s is . The r e s u lt s o f t h is work are shown in Tahle XX. 44 TABLE XX effect of extracting time on the degree of peptization with fiv e DIFFERENT SAMPLE-SOLVENT RATIOS With, samples 1 -5 100 ml* o f 0.4M NaCl were used and w ith sample No* 6 100 ml. o f d i s t i l l e d water was u sed. Temperature was No. 60 Mesh K jeldahl T itr a tio n Sample Grams O.19S9N HC1 % N per Sample Mgm. N $ N/T N a ir dried f> N/T N dry wt. b a s is 67.26 64.78 61.28 57.25 51.21 30.35 73.26 70.56 66*75 62.36 55.78 33.06 69.67 67.61 65.00 61.95 57.81 31.80 75.89 7 3 .6 4 70.80 67.48 62.97 34.64 70.22 68.60 66.62 64.60 62.22 32.45 76.49 74.72 72.57 70.37 67*77 35.34 70.32 69.30 6S.15 66*80 65.IS 32.70 76.60 75.49 74.23 72.76 7 1.00 35.62 Shaking time 10 minutes . 1 2. 4. 5. 6. 5 .0 7 .5 1 0 .0 1 2.5 1 5 .0 1 0 .0 45.8 6 6 .2 83-5 9 7 .5 104.7 41.35 12.763 18.439 23.258 27.160 29.154 11.519 . 2.55 2.45 2 .32 2.17 1 .94 1 .15 Shaking tim e 20 m inutes 1. 2. 3. 4. 5. 6. 5 .0 7 .5 1 0 .0 1 2 .5 1 5 .0 1 0 .0 4 7 .6 69.1 8 8 .6 105.5 118.2 43.45 13.221 19.245 24.670 29.390 32.911 12.099 2 .6 4 2 .5 6 2 .4 6 2.35 2.19 1.20 Shaking time 30 minutes l. 2. 3. 4. 5. 6. 5 .0 7 .5 1 0.0 12.5 1 5.0 1 0 .0 47.S 5 7 0.1 9 0 .0 110.05 1 2 7 .2 44.25 13.255 19.527 25.285 30.647 35.422 12.316 2.66 2.60 2 .5 2 2.45 2.36 1 .2 3 Shaking tim e iJO minutes 1. 2. 3. 4. 5. 6. 5 .0 7-5 1 0 .0 1 2 .5 1 5 .0 1 0 .0 4 7 .9 70.85 92.9 113.8 133.25 44.55 13.345 19.726 25.865 31.691 31.107 12.411 2 .6 6 2.63 2.58 2.53 2.47 1 .2 4 *5 From Table XX th e r e s u lt s have been c a lc u la te d In terms o f percent­ age n itro g en per t o t a l n itrogen content o f a ir -d r ie d samples (Table XXI). TABLE XXI PERCENTAGE NITROGEN PER TOTAL NITROGEN CONTENT OF AIR-DRIED SAMPLES No. Sampleso lv en t R atio 10 20 30 40 2 3 k 7*5:100 10:100* 12.5:100 1 Min. Min. Min. Min. 5:100 67.2 6 9 .6 7 0 .2 7 0.3 6U.7 6 7 .6 6 8 .6 69.3 61.2 65.0 6 6.6 68.1 57.2 61.9 6 4.6 66.8 5 6 15:100 10:100 water 5 1 .2 57.8 62.2 65.1 30.3 31.8 3 2.4 3 2.7 These experim ents demonstrate th a t in a l l in sta n ces an in crea se e x tr a c tin g time in c re a se s th e amount o f t o t a l n itrogen p ep tized from the sam ples. Of th e f i v e sam ple-solvent r a tio s employed, th e one o f 5:100 gave th e h ig h e st value o f p e p tiz a tio n and th a t o f 15:100 gave the lo w e st. When the S-S-R i s 5:100, time had l e s s in flu en ce upon the p e p tiz a tio n a s shown by an in crea se o f only 3 percent w ith th e in crea se in time from 10 m inutes to 4o m inutes. On the other hand, time had a la r g e r in flu en ce upon th e p e p tiz a tio n when th e S-S-R i s 15:100, in which case th ere was a 14 percent Increase when the same ex ten sio n o f time was employed (Table XXI and Figure 5)* Percent of total nitrogen extracted Figure 5. 60- Curve 1. 50- 5. 7.5 10. 12.5 15. 10. gm gm gm gm gm gm per per per per per per 100 ml 100 ml 100 ml 100 ml 100 ml 100 ml 0.4 NaCl 0.4 NaCl 0.4 NaCl 0.4 NaCl 0.4 NaCl water 40- 6---- 10 20 30 Extraction time in minutes 40 The effect that various sample to solvent ratios have upon extraction time, using 0.4 M NaCl. Mung bean sample was ground to 60 mesh. 47 (B) The Determ ination o f the E ffe c t o f P a r tic le S ize and Time on the Degree o f P e p tiz a tio n . were u sed . Twenty mesh and 40 mesh Mung bean meal samples S ix ty mesh Mung bean meal was p rev io u sly determined. Each e x tr a c tio n was made by shaking (120 o s c ill a t io n s per minute) 5 samples w ith 0.4m EaCl a t 5 d iffe r e n t sam ple-solvent r a t io s (S-S-R ): 5*0:100, 7*5:100, 10:100, 1 2 .5 :1 0 0 and 15 *0:100 and one 10 gm. sample w ith 100 m l. w ater. A fter c e n tr ifu g a tio n the clea r liq u id s were used fo r t o t a l n itr o ­ gen d eterm ination. C alcu lation s from the K jeldahl t it r a t io n data (Table XXII) were expressed in terms o f: 1. Mgm. n itro g en determined from the sample. 2. Percentage o f nitrogen per a ir -d r ie d sample. 3* Percentage o f n itro gen per t o ta l n itrogen content o f a ir -d r ie d sample. 4. Percentage o f n itro gen per t o ta l n itrogen content on dry w eight b a s is . 48 TABLE XXII effect of particle siz e and time on the deghee of peptization WITH FIVE DIFFERENT S-S-B With samples 1-5 100 ml. o f 0.4M NaCl were used and w ith sample 6 100 ml. o f d i s t i l l e d water was u sed . Temperature was 25°C. No* Wgt o f Mgm. N K jeldahl i> N/T N % N/T N f> N Sample a ir T itr a tio n dry wt. Extracted per in Grams O .I989N HC1 Sample dried h a s is Shaking tim e 20 m inutes, 40 mesh Mung hean m eal. 1. 5*0 8.4 9 4 1.69 44.76 30*5 48.75 2. 44.11 45.1 12.556 7*5 48.05 1.67 1 0 .0 1 .6 4 16.430 59-0 43.29 3. 47.15 4. 12.5 71.8 42.15 19.996 1.59 45.91 1 .5 4 1 5 .0 8 3 .2 40.70 5. 23.170 44.33 10.289 27. l l 6. 1 0 .0 36.9 1 .0 2 29.51 Shaking time 30 m inutes, 4o mesh Mung hean meal. 4?.22 1. 5 .0 8.81? 31.6 I .76 ^9.25 44.42 12.644 1.68 48.38 2. 4 5 .4 7 .5 43.68 10*0 47.58 16.578 1.65 59*5 34. 72.8 42.76 20.286 1 .6 2 1 2 .5 46.57 41.48 45. 1s 1 5 .0 8 4 .8 23.615 5. 1 .5 7 6. 10.00 1.1 0 11.063 29.15 31.75 39*7 Shaking time 4o m inutes, 40 mesh Mung hean meal. 8 .8 2 4 1.7 6 46.50 1. 5*0 50.65 31*7 13.122 46.10 50.21 2. 1 .7 4 7*5 47 .1 45.64 17.254 1 .7 2 1 0 .0 49.52 6 2 .0 3. 4. 44.80 48.80 12.5 21.254 1.70 76*3 44.15 1.67 48.09 1 5 .0 90.25 5. 25.135 4 1 .3 6. 1 0 .0 1.15 11.511 33.03 30.33 Shaking time 20 m inutes, 20 mesh Mung hean meal. 25.68 5 .0 1. 27.97 4.873 0.97 17*5 24.80 27.01 2. 0 .9 4 25.35 7.059 7*5 23.52 0.89 25.62 8.926 1 0 .0 32.05 3. 24.19 0 .8 4 22.21 4. 1 2 .5 10.537 37*8 22.54 20.70 0 .7 8 4 2 .3 15*0 11.785 55.818 0 .5 8 20.9 1 0 .0 16.69 6. 15.33 Shaking time 30 m inutes. 20 mesh Mung hean m eal. 26.28 28.62 4.987 1. 5*0 0 .99 17*9 O.96 27.60 2. 25.34 7*5 25*9 7^3 24.49 0 .9 2 26.67 1 0 .0 9 .2 9 4 33*35 3. 23.40 0.88 25.50 11.106 4. 1 2 .5 39*9 24.01 O.83 22.05 4 5 .1 1 5 .0 12.553 5. 17.81 19.40 0.65 1 0 .0 6. 6.759 24.25 hean meal* 20 mesh Mung Shaking time 40 m inutes, 33.02 30.32 1 .15 5.753 5 .0 20.65 1* 29.44 3 2.06 1.11 8.380 30.1 2. 7*5 31.10 1.08 10.835 28.55 1 0 .0 38.9 3* 30.07 13.184 1 .05 27.79 1 2 .5 4. 47.35 28.46 26.13 14.876 0.99 1 5 .0 53.*» 5. 26.80 24.61 9.340 0.93 1 0 .0 33.55 b. 49 From Table XXII we can c a lc u la te the e f f e c t o f p a r t ic le s iz e and tim e o f e x tr a c tio n in terms o f percentage (Table XXIII). TABLE XXIII EFFECT OF PARTICLE SIZE AND TIME OF EXTRACTION IN TERMS OF PERCENTAGE No. 1 S-S-R 5:100 2 7 *5:100 4 3 10:100 5 12. 5:100 6 15:100 10:100 water 20*7 40.7 57.8 15*3 27.I 31. s 22.0 41.5 62*2 17.8 29.1 32*4 26.1 4 4 .1 b5*l 2 4.6 30.3 32*7 E xtraction time 20 minutes 20 Mesh 40 Mesh 60 Mesh 2 5 .6 4 4 .7 6 9 .6 24.8 44.1 67*6 23.5 43.2 65*0 22.2 42.1 61*9 E xtraction time 30 minutes 20 Mesh 40 Mesh 60 Mesh 2 6.2 4 5 .2 7 0 .2 24.5 43.6 6 6 .6 25*3 4 4 .4 6 8 .6 2 3 .4 42.7 64.6 E xtraction time 40 minutes 20 Mesh 40 Mesh 60 Mesh 30*3 46*5 7 0 .3 2 9 .4 4 6 .1 69*3 28.5 4 5.4 68.1 27.8 44.8 66.8 This work in v o lv ed th ree v a ria b le s: l ) sam ple-solvent r a tio and 3) the p a r t ic le s iz e , 2) ex tra c tio n tim e. the I t i s eviden t that the p a r t ic le s iz e a f f e c t s th e degree o f p ep tiza tio n to a g rea ter ex ten t than e ith e r th e S-S-R or th e e x tr a c tio n tim e. The over the 60 mesh s iz e gave about 20 percent g rea ter y ie ld 4o mesh s iz e which in turn gave about o f n itrogen 20 percent g r e a te r y ie ld o f n itro g e n over th e 20 mesh s iz e . When u sin g 3:100 as the low est S-S-R, th ere was a decrease in y ie ld o f n itro g e n when th e S-S-R was increased* An in c r e a se in e x tr a c tin g time y ie ld o f n itr o g e n . produced a The r e s u lt s are p lo tte d in s lig h t in crea se in the Figure 6. Figure 6. This figure shows the effect of the particle size, time and sample - solvent ratio on the degree of peptization of Mung bean meal in 0.4 M NaCl. extracted Sample sizes are indicated by: O for 60 mesh □ for 40 mesh Percent of total nitrogen O for 20 mesh Curves in each case number­ ed 1, 2, 3, 4 and 5 represent 5, 7.5, 10, 12.5, and 15 grams respectively of sample per 100 ml 0.4 M NaCl. All curves numbered 6 represent 10 grams of sample per 100 ml water. 30- 20 - 20 30 Extraction time in minutes 40 51 (0 ) Comparative E f f e c ts o f Mechanical Shaking and Hand S tir r in g on the Amount o f N itrogen P ep tized from Mung Bean Meal. Mung hean meal (60 mesh) was extra cted w ith O.hM NaCl a t f iv e sampleso lv e n t r a t io s (s e e Tahle XXII). o f d i s t i l l e d w ater. Sample No. 6 was 10 gm. o f meal to 100 ml. S ix samples were ex tra cted a t one tim e fo r 30 m inutes a t 25°C. A fter the f i r s t e x tr a c tio n was made from the s ix samples, th e c le a r liq u id s were poured in to E jeld ah l f la s k s fo r n itrogen d eterm ination. r e sid u es were saved fo r a second e x tr a c tio n , The f i v e 100 m l. p o rtio n s o f O.^M NaCl were added to th e resid u es o f Nos. 1 -5 , and 100 ml. o f d i s t i l l e d water was added to th e No. 6 resid u e fo r th e second e x tr a c tio n . A th ird e x tr a c tio n was conducted in the same manner. The n itro g en content o f a l l e x tr a c ts was determined and reported in terms o f percent o f n itro g en per t o t a l n itrogen on a ir -d r ie d h a sis and shown in Tahle XXIV, XXV, XXVI and fig u r e 7* 52 TABLE XXIV DETERMINATION OP NITROGEN CONTENT OP THREE SUCCESSIVE EXTRACTIONS OP MUNG BEAN MEAL SAMPLES (A) With Mechanical Shaking fo r 30 m inutes a t 25°C. No* Sample 60 Mesh Solvent 100 ml. K jeldahl T itr a tio n 0.1969N HC1 Mgm. N $ N per Sample $ N/T 1 a ir d ried 1 s t E xtraction 1* 5*0 2. 7*5 10*0 3. 4. 12*5 15*0 5. 6* 1 0 .0 *4m NaCl N u II n II n w n water 49.1 7 0 .2 92.25 116.1 13S.0 4 4 .0 13.672 19.547 25.6SS 32.329 38.427 12.252 2 .73 2 .60 2 .56 2.58 2 .5 6 1 .2 2 6 4.5 4 61.52 6 0.6 2 61.04 60.1*5 28.91 2nd E xtraction 1. 5*0 2. 7*5 10 .0 0 3. 4* 12*5 15*0 5. 6* 1 0 .0 *4m NaCl n H N n II n H n water S .l 11.5 l 4 .0 I S .2 21.1 3 0 .4 2.255 3.202 3.926 5.067 5.875 8.465 0 .4 5 0 .4 2 0 .39 0.1*0 0.39 0 .8 4 1 0 .6 4 10.08 9 .2 5 9 .5 6 9 .2 3 19*97 3rd E xtraction 1. 5*0 2. 7*5 1 0 .0 3. 4. 12*5 15*0 5* 6. 1 0 .0 •4 m NaCl ii n n H H it it ii water 2 .0 3 .5 4.S 6 .0 7*1 17.O 0.557 0 .9 7 4 1 .3 3 6 1.670 1.977 4.733 0.11 0 .1 2 0 .13 0 .1 3 0 .1 3 0.47 2 .6 2 3 .0 4 3*13 3 .1 3 3 .09 11.1 6 16.9 28.95 41.0 49.9 62.1 60.8 9.705 8.061 11.416 13.895 17.292 16.930 0 .9 4 1.0? 1 .1 4 1.11 1.15 1 .6 9 22.21 25.35 26.93 26.22 27.19 39.96 R esidues l. 5 .0 2. 7 .5 1 0 .0 3. 4. 12.5 1 5 .0 5. 6. 1 0 .0 53 TABLE XXV DETERMINATION OP NITROGEN CONTENT OF THREE SUCCESSIVE EXTRACTIONS OP MUNG SEAN MEAL SAMPLES (B) With Hand S tir r in g O ccasion ally fo r 30 m inutes. Temperature 25°C. No. Sample 60 Mesh 1 s t . E xtraction 1. 5 .0 2. 7-5 1 0 .0 3. 4. 1 2 .5 1 5 .0 5. 0. 1 0 .0 2nd. E x tra ctio n 1. 5 .0 2. 7 .5 1 0 .0 3. 4. 1 2 .5 1 5 .0 5. 6. 1 0 .0 3rd. E x tra ctio n 1. 5 .0 2. 7-5 1 0 .0 3. 4. 1 2 .5 1 5 .0 5. 6. 1 0 .0 R esidues 1. 5 .0 2. 7-5 1 0 .0 3. 4. 1 2 .5 1 5 .0 5. 6. 1 0 .0 Solvent 100 ml. .4M NaCl « n n 11 11 it n 11 water .4 m NaCl 11 11 it ti n it H It water K jeldahl T itr a tio n 0.1989N HC1 n it ti 11 ii It R water # N per Sample N/T I a ir d ried 48.7 7 0 .2 9 2 .4 112.4 133.5 43.5 13.561 19.547 25.729 31.298 37.174 12.113 2.71 2.60 2.57 2.50 2.47 1.21 63-99 61.49 60.69 59*06 58.^7 28.57 6 .2 10.8 1 6 .0 2 3.2 29.3 29.O 1 .7 2 6 3.007 4.455 6.460 S . 158 8.075 0 .3 4 0 .40 0 .4 4 0.51 0 .5 4 0.80 8 .i4 9.^3 10.50 12.17 12.81 19.04 4 .3 5 .9 8 .2 9 .2 1 4 .6 O.919 1.197 1.643 2.283 2. 56I 4.065 0.18 0.15 0 .1 6 0.18 0 .1 7 0.40 4.31 3.75 3 .87 4.29 4.01 9.58 17.9 28.85 37.9 4 6 .4 56.3 65*1 4 .984 s . 033 10.553 12.920 15.677 18.127 0.99 1.07 1.05 1.03 1 .0 4 1.81 23.50 25.27 24.89 24.37 24.65 42.75 •4 m NaCl i» Mgm. N 54 TABLE XXVI DETERMINATION OE NITROGEN CONTENT OE THREE SUCCESSIVE EXTRACTIONS OE MONO BEAN MEAL SAMPLES No* S -S -l 1 s t Ext* N 2nd E xt. % N 3rd E xt. %N Residue N Recovery f N 22.21 25.35 26.93 26.22 27.19 39.96 100.01 99.99 99.93 99.95 99.36 100.00 With Hand S tir r in g O ccasion ally fo r 30 minutes a t 8 .1 4 5:100 4.31 63-99 61.49 7*5:100 9.43 3.75 10:100 60.69 10.50 3.37 12.17 4.29 12. 5:100 59.06 12.81 15:100 4.01 53.47 1 9.0 4 28.57 10:100 9*53 25° C. 23.50 25.27 24.89 24.37 24.65 42.72 99.94 9 9.9 4 99.95 99.39 99.94 99.91 ir^^o With Mechanical Shaking f o r 30 m inutes a t 25°C. 2.62* 10.64* 5:100 64.54* 3 .0 4 10.08 7*5:100 61.52 10:100 60.62 9.25 3.13 12. 5*100 61.04 9.56 3.13 60.45 15:100 9.23 3 .09 28.91 10:100 11.16 19.97 w r-f oo <4 iH cvt A. 1. 2. 3. 4. 5. 6. itaoo B. 1. 2. 3. 4. 5. 6. * A ll N v a lu es are reported as $ N o f t o t a l N ( a ir -d r ie d sam ples). N itrogen determ ination was made on a l l residues* Figure 7. 60 Q i w U *T3 50 Extraction mechanical hand E S g 40 c* 2nd ( residue of 1st) < $> 3rd ( residue of 2nd) <§> (S o 30 c § N/T dry wt 'basis 25°C,f 10 non-O.F. 1. 2. 10 non-O.F. 10 non-O.F. 3. M. 9.Q2 O.F. 9*92 O.F. 5* 6. 9 .9 2 O.F. .MM .1M .05M .Mm .1M . 05M 73 .2 *7*5 33-6 77.5 M9.S 35.8 23.332 13*193 9.332 a . 526 13.832 9 . 9M3 53.57 3M.76 2M.5S 56.71 36.MM 26.19 58.35 37.86 26.78 61.78 39-69 28.53 35° 0, I. 2. 3* M. 5. 6. 10 non-O.F. 10 non-O.F. 10 non-O.F. 9 .9 2 O.F. 9 .9 2 O.F. 9 .9 2 O.F. .Mm .1M .05M .Mm .1M •05M 73*3 55 .2 40.1 76.2 56.6 M1.0 20.359 i5.3&> 11.138 21.165 15.721 11.388 5 3 .6M M0.M8 29. 3M 55.76 Ml. M2 30.00 5S.M3 MM.10 31.96 6 0 .7M M5.12 32.68 45°C » 10 non-O.F. 1. 10 non-O.F. 2. 10 non-O.F. 3. M. 9 .9 2 O.F. 9 .9 2 O.F. 5. b. 9 .9 2 O.F. .Mm .1M .05M .MM .1M .05M 72. 68 5b.S M2.S 7M.M 5 7.0 Ml.S 20.23s 15.776 11.888 20.665 15.832 11.610 53.32 Ml.5b 31.32 5M.MM Ml. 71 30.58 58.08 45.27 3M.12 59.31 M5-M3 33.32 .Mm .1M . 05M .Mm .1M .05M bb.O 51. M 39.3 b6.7 51.1 36.2 18.332 lM.315 10.952 18.526 1^.193 10.05M M8.30 37.71 28.85 MS.81 37.39 26. M9 52.61 Ml. 08 31.M3 53.17 M0.73 28.85 55°C3-7 1 2 .5 99.7 0 .3 6 6,97 1:3 0 .1 -1 .0 0 0 3S.9 48.5 12.5 99-9 0 .8 0 6.99 1 :4 0.08 - 1.097 50.5 36.7 12.5 99.7 1.37 6.97 1:5 0.066 -1 .1 7 4 55.0 32.5 1 2.5 100.0 1.69 7 .0 0 1 :6 0.057 -1 .2 4 4 56 .0 31.5 12.5 100.0 1 .7 2 6.96 1:7 0 .0 5 -1 .3 0 1 57.5 29.7 12.5 99.7 1*93 6.97 1:8 0 .0 4 4 -1 .3 5 S 58.0 29.2 12.5 99.7 1 .9 8 6.97 1 :9 o .o 4 -1 .3 9 S 58 .0 29.5 12.5 100.0 1 .9 6 7 .0 0 1:10 0 .0 3 6 -1 .4 4 4 57.5 30.0 12.5 100.0 1.85 6 .8 4 Figure 9. Volume of Dilution 1:5 • Protein N precipitated by dilution O Protein N remaining in solution N P N remaining in solution Percent of total nitrogen a 1 O O' .A. -0.7 - 0.8 -0.9 - 1.0 - 1.1 - 1.2 -1.3 -1.4 log of Molarity This figure shows the amount of protein precipitated and that which remained in solution when the salt concentration of a 0.4 M NaCl extract of Mung .bean meal was diminished by dilution. -1.5 62 d ilu tio n a p a r t ia l p r e c ip ita tio n occurred which appeared a s a w hite clou d , in e e s throughout th e liq u id * TJith th e 1:3 d ilu tio n there was on ly a sm all amount o f p r o te in m atter flo c c u la te d and the p r e c ip ita tio n e v id e n tly was incomplete* Prom cy lin d er Xoy 4 through No* 10 the p r e c ip ita tio n with d iffe r e n t d ilu tio n s apparently was complete and the supernatant liq u id s were water clear* As a consequence o f th ese r e s u lt s the 1 :4 d ilu tio n was employed f o r th e p r e c ip ita tio n o f t o t a l p ro tein from 0*^11 NaCl ex tra ct o f Mung hean meal (F igure 1 0 ). (C) P ro tein Sedim entation from 0.4M NaCl E xtract o f Mung Bean Meal a t Various Hydrogen Ion Concentrations* The purpose o f t h is experiment was to demonstrate sedim entation o f p r o te in by v a r ia tio n in hydrogen ion con cen tration . For t h is work a d ilu tio n was found n ecessa ry th at should n ot he the optimum fo r the pre­ c ip it a t io n o f p r o te in s as p rev io u sly shown. By experiment i t was found th a t a d ilu tio n r a tio o f 1:5 appeared to he l e s s a ffe c te d hy sim ple d ilu ­ t io n and could he used to g iv e inform ation regarding the in flu en ce o f hydrogen ions* The procedure and r e s u lt s are in d ica ted in Tahle XXXI* This experiment demonstrated th a t in four cases* C ylinders Nos. 2 , 4 , 10 and 1 5 , th e p r e c ip ita t e s appeared to he more compact as in d ic a ­ te d hy th e sm aller volumes (Figure 11)* This method o f p r e c ip ita tio n was not used hy t h is in v e s tig a to r fo r th e i s o la t io n o f Mung hean g lo b u lin sin c e the a d d itio n o f a c id for a d ju stin g the pH causes denaturation o f the p ro tein . When the p ro te in i s suspended in a very d ilu te s a lt s o lu tio n , i t i s more e a s ily denatured Figure 10. Photographed after 15 minutes 1 2 3 4 5 6 7 8 9 Photographed after 45 minutes 10 2 3 10 4 5 6 7 8 9 Photographed after 75 minutes Visual observation of the gravitational sedimentation rate of M ung bean protein extract when the salt concentration was diminished by dilution. The numbers beneath the cylinders indicate the volumes with which 10 ml portions of 0.4 M NaCl extract was diluted. Figure 11. Visual observation of the effect of H-ion concentration (pH) on the manner of precipitation from a 0.4 M NaCl extract of Mung bean protein diluted to 0.06 M . The numbers beneath the cylinders identify the data in the preceding table. 70 by a very sm all amount o f a cid than when dispersed in a concentrated s a lt so lu tio n ( 6 l ) . TABLE XXXI SEDIMENTATION OP PROTEIN BY VARIATION OP HYDROGEN ION CONCENTARATION S e r ie s o f 100 ml* C ylinders pH 1. 2. 3. 4. 5. 6. 7S. 9. 10. 11. 12. 6 .2 6 .0 5 .8 5*6 5*^ 5*2 5 .0 4.S 4 .6 4 .4 4 .2 4 .0 3 .8 3 .6 3 .* 3 .2 3-0 2.S 2 .6 2 .4 1?* i4 . 15* 16. 17* IS . 19. 20. (D) m l. Water w ith pH Adjusted 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 55 ml. E xtract Reading in ( 0 . MM NaCl) ml. o f Ppt. added a f te r 12 h rs. 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 46 20 54 49 53 52 51 50 44 53 52 52 46 ^7 52 0 0 0 P inal pH 6.17 5.8S 5*7^ 5 .8 4 5.75 5*74 5.7^ 5.73 5 .7 4 5.82 5.7^ 5-72 5.72 5.58 5.52 5.30 4.S2 4.31 3 .6 4 3 .0 6 Suggested Procedure fo r the I s o la tio n o f the Globulin from Mung le a n Meal* Prom th e r e s u lt s o f the previous experiments have shown th e follow ­ in g necessary procedure: 1. The so lv e n t. A 0.4M NaCl so lu tio n was used* 2. S o lid -s o lv e n t r a tio (S-S-R) 1:10. 3* Three su c c essiv e e x tra ctio n s were employed* 4. E xtraction tim e. The most s a tis fa c to r y r e s u lts were obtained 71 w ith one h o a r 's ex tra c tio n a t 25°C. w ith occasion al hand s t ir r in g . 5* The removal o f the resid u e s. The residu e was removed by cen­ tr ifu g a tio n fo r 15 minutes a t 2000 r.p .m .. b. The p r e c ip ita tio n o f p rotein from extract with 1 :4 d ilu tio n . Tor each volume o f e x tr a ct four volumes o f water were added and the t o t a l p ro tein p r e c ip ita te d . Trom p reviou s a n a ly s is Mung bean p ro tein p r e c ip ita te d with 1 :4 d iliu t io n showed: (E) 50.50 percent n itro g en per ex tra cta b le n itrogen as g lo b u lin n itrogen 37*00 percent n itrogen per ex tra cta b le n itrogen as albumin n itrogen 1 2 .50 percent n itrogen per ex tra cta b le n itrogen as N P N. A Qa&ntative Study o f the Procedure Involved. A q ualitative study o f the procedure ju s t o u tlin ed was carried out. a. E x traction . A mixture o f 100 grams of Mung bean meal (SO mesh) «t> and 1000 ml. o f 0.4M NaCl s o lu tio n (S-S-R 1:10) were extracted fo r one hour at 25°C. w ith o cca sio n a l hand s tir r in g . fo r 15 m inutes to remove the resid u e. The ex tra ct was cen trifu ged The residu e (I*) was re~extracted tw ice and a l l the e x tr a c ts (I I ) were combined. ml. added O .tyL NaCl P ir s t E xtraction m l. C entrifugate C ollected 1000 860 Second E x tra ctio n (resid u e of 1 s t) 860 850 Third E x tra ctio n (resid u e of 2nd) 850 SbO A volume o f 860 m l. o f cen trifu g a te were c o lle c te d from the f i r s t e x tr a c tio n so 860 ml. o f 0.4M NaCl were added to the resid u e fo r the * See Plow Sheet - Pigure 12. 72 second e x tr a c tio n , g iv in g 850 ml, o f cen trifu g a te c o lle c te d from t h is second e x tr a c tio n and 850 ml* o f 0.4M NaCl were then added to the r e s i­ due fo r the th ir d e x tr a c tio n from which 860 ml, were c o lle c te d . Thus the s o lid -s o lv e n t r a tio was kept in each case a t is 10, b. P r e c ip ita tio n o f p ro tein . The combined cen trifu g a te ( I I ) ( in 0 .4 m N&Cl) was d ilu te d w ith four volumes o f d i s t i l l e d water (1 :4 ra tio * )* The f in a l con cen tration o f the d ilu te d ex tra ct was 0.08M NaCl* c ip it a t io n o f p ro tein occurred in s ta n tly . The pre­ The p r e c ip ita te was allow ed to stand fo r a few hours, u su a lly over n ig h t, to allow a more complete p r e c ip ita tio n , and was then separated from th e liq u id by c en trifu g a tio n . c. a t 25°C. P u r ific a tio n . The p r e c ip ita te ( I I I ) was d isso lv e d in 0 .4 m NaCl With g e n tle hand s t it r i n g , ten minutes were needed to com pletely d is s o lv e the p r e c ip ita te . tr ifu g e d . The dispersed p ro tein so lu tio n was then cen­ The resid u e was designated as (V) and the cen trifu g a te as (V I). A la rg e q u a n tity o f in so lu b le m aterial separated by c e n tr ifu g a tio n , in d i­ c a tin g a globulin-bound (G-b) (V) substance, or p rotein m atter which i s not d isp ersa b le in d ilu te neu tral s a l t . The c le a r cen trifu g a te (VI) was d ilu te d w ith four volumes o f d i s t i l l e d water and p r e c ip ita tio n occurred * The a d d itio n o f water to the ex tra ct was conducted w ith a la rg e funnel w ith th e stem extended by g la s s tubing. The lower end o f the tube was immersed under the surface o f the e x tr a c t. The time required fo r sed i­ m entation o f the p ro tein was three to four hours a t 6°C* 73 in sta n tly * A fter standing fo r four hours, th e p r e c ip ita te (VII) was separated from th e supernatant (V III) by cen trifu g a tio n . The process o f p u r ific a tio n ( c .) was repeated u n t il no more Gb substance could be removed* The amount o f g lo b u lin obtained from 100 grams o f 60 mesh Mung bean meal by th ree su c c e ssiv e ex tra ctio n s with 0.4M NaCl and p r e c ip ita ­ te d by 1 :4 d ilu tio n was then determined. The p r e c ip ita te (VII) which was freed from globulin-bound substance and albumin was f i r s t d isso lv ed in 300 ml. o f 0.4M NaCl so lu tio n and two 10 ml. a liq u o ts were taken fo r K jeldahl n itro g en determ ination. The t o ta l g lo b u lin nitrogen from 100 gm. o f Mung bean meal (a ir -d r ie d ) was found to be 1.405b gm. This weight represented 37»01 percent o f the t o ta l n itrogen content o f 100 gm. o f Mung bean meal or 49.45 percent o f the t o t a l ex tra cta b le n itrogen . 7* Figure 12 FLO SHEET Sample - Mung beam meal (60 mesh) Solvent - O.^M NaCl, MS-S-BM 1:10 E xtraction time - 1 hour at 25°C ., hand s tir r e d E xtract was separated from resid u e by cen trifu g a tio n 1 (I) Residue 1 (I I ) C entrifugate P r e c ip ita tio n by 1 :4 d ilu tio n Allowed to stand overnight Centrifuged 1------------------------------------- C. (III) P r e c ip ita te D isso lv ed in 0.4M N ad Cen trifu g ed ____________ (V) Residue (Gb)* r ~ ---------------------------(VII) P r e c ip ita te (g lo b u lin ) (IV) C entrifugate discarded* ~l (VI) C entrifugate P r ecip ita ted by 1:4 d ilu tio n Allowed to stand overnight Centrifuged (n n ) C entrifugate discarded* A - e x tr a c tio n , B - p r e c ip ita tio n , C - p u r ific a tio n . * The c e n tr ifu g a te s IV and VIII contain albumin fr a c tio n s and were d is­ carded. The resid u e V was th e globulin-bound (Gb) substance in so lu b le in 0.4M NaCl. 75 FRACTIONATI ON AND FURTHER PURIFICATION OF GLOBULIN In the previous experiments the pur i f i ca tio n procedure was carried out "by repeated p e p tiz a tio n in 0.4M NaCl and p r e c ip ita tio n hy d ir e c t d ilu tio n a t a 1 :4 r a t io . in form. The r e s u ltin g product was w hite hut amorphous Since th e substance obtained was not c r y s t a llin e in form, oth er means than sim ple r e c r y s ta lliz a t io n had to be employed fo r p u r ific a tio n and p o s s ib le fr a c tio n a tio n . I t was observed th a t when 100 ml. o f p ro tein so lu tio n (0.4M NaCl) were d ir e c t ly d ilu te d w ith 400 m l. d i s t i l l e d water dropwise from a bur­ e t t e , then allow ed to stand fo r 12 hours a t b°C., f in e c r y s ta ls formed as noted by exam ination under a m icroscope. Thus, slow d ilu tio n , as might be expected, encouraged the form ation o f cry sta ls* An e f f e c t iv e d ia ly z in g apparatus fo r p r e c ip ita tio n and p u r ific a tio n o f p ro tein m aterial was constructed in t h is laboratory and i s shown in Figure 13* A g la s s tank o f seven l i t e r s cap acity (IS in . x 6 in .) was p laced in an aluminum tray which was centered and fa sten ed by screws to a p u lle y s ix in ch es in diam eter. The p u lley was secured to the a x le o f th e stand o f a cork boring machine by two s e t screw s. system was obtained from a fr a c tio n a l horsepower to th e a x le by means o f p u lle y s . were used to connect th e p u lle y s . Drive fo r the H .P.) motor coupled Round le a th e r b e lts in . diameter) A four p u lle y arrangement served to reduce th e motor speed from 1750 r.p.m . to 35 r.p .m . a t the a x le . The d ia ly z in g membrane was th ree inch f l a t width cellophane tubing IS in ch es in le n g th . The top o f the tube was fa sten ed to a rin g 1 J>fk in ch es 76 In diameter* The "base o f the tube was secured to an open-mouthy f l a t - bottomed g la s s b o t t le 1 3 f b in ch es a t top and 2 inches in depth* The top rin g r e ste d in an E-shaped metal frame which was attached to the rin g stand* The apparatus was designed fo r slow, step -w ise, in d ir e c t d ilu tio n in order to ob tain the d esired p r e c ip ita te d and c r y s ta llin e forms o f the glob u lin s* The amount o f d i s t i l l e d water added to the tank from a res­ er v o ir p laced above th e tank was co n tro lled by a stopcock. The appara­ tu s a lso could be used to remove the e n tir e s a lt content from a p ro tein s o lu tio n by continuous a d d itio n o f water to the tank from the reserv o ir and removal from the tank by siphoning u n t il the d ia ly sin g water was fr e e o f ch lo rid e io n s . And fu rth er, t h is apparatus could be employed to reduce a s o lu tio n o f a known concentration to a lower d esired concen­ t r a tio n . C a lcu lation s showing th e reduction o f concentration are shown in Tables XXXII and XXXIII. I t i s w e ll known th at p ro tein so lu tio n s th at are rap id ly d ilu te d d ir e c t ly by d i s t i l l e d water produce an amorphous form o f p r e c ip ita te which adsorbs many im p u r itie s. Slow d ir e c t d ilu tio n produced c r y s ta ls but the p rocess was d i f f i c u l t to c o n tr o l. The slow in d ir e c t d ilu tio n device ju s t d escribed overcame th ese d i f f i c u l t i e s . The p ro tein so lu tio n in the membrane was suspended in the tank containing s a lt s o lu tio n o f the same con cen tration as the so lu tio n in the membrane. The slow addi­ tio n o f d i s t i l l e d water to the d ia ly zin g liq u id in the tank prevented d ir e c t con tact o f d i s t i l l e d water to the membrane. gave the proper r a te o f d ilu tio n . The membrane i t s e l f Best r e s u lt s were obtained when the Figure 13. Rotating Outside Liquid Dialyzer is TABLE XXXII DILUTION TABLE "1000" m l. s o l. j la r it y 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 1000 0 .4 0.35 0 .3 4 0 .3 3 0 .3 2 0 .3 1 0 .3 0 0 .2 9 0 . 2S 0 .2 7 0 .2 6 0.25 0 .2 4 0 .2 3 0 .2 2 0 .2 1 0 .2 0 0 .1 9 0 .1 8 0.17 0 .1 6 0 .1 5 0 .1 4 0 .1 3 0 .1 2 0 .1 1 0 .1 0 0 .0 9 0.08 0 .0 7 0 .0 6 0 .0 5 0 .0 4 0 .0 3 0 .0 2 ml. o f Water Added 142.8 2 9 .4 30.3 31.2 32.2 33.3 3 4.4 35.7 37.0 3 8 .4 40.0 41.6 4 3 .4 4 5 .4 4 7 .6 50.0 5 2.6 55-5 58.8 b2 .5 66.6 71.4 76.9 83.3 90.9 100.0 111.1 125.0 142.8 16b. 6 200.0 250.0 333.3 500.0 1000.0 T otal Volume f in a l Cone. 1142.8 1029.4 1030.3 1031.2 1032.2 1033.3 1034.4 1035.7 1037.0 1038.4 1040.0 1 0 4 l.b 1043.4 1045.4 1047.b 1050.0 1052.6 1055.5 1058.8 1062.5 1066.6 1071.4 1076.9 IO83.3 1090.9 1100.0 1111.1 1125.0 1142.8 1166.6 1200.0 1250.0 1333.3 1500.0 2000.0 0.35 0 .3 4 0.33 0 .3 2 0.31 0 .3 0 0 .29 0.28 0.27 0 .2 6 0.25 0 .2 4 0.23 0 .2 2 0.21 0 .2 0 0 .1 9 0.18 0.17 0 .1 6 0.15 0 .l4 0.13 0 .1 2 0.11 0.1 0 0.09 0.08 0.07 0 .0 6 0.05 0 .0 4 0.03 0 .0 2 0.01 79 TABLE XXXIII DILUTION TABLE "lOOOx" Ml. Sol to be D ilu ted M olarity 1000 1142.8 1176.4 1212.1 1250.0 1290.3 1333.3 1379.3 1428.5 1481.4 153S .4 1600.0 1666.6 1739.0 1818.0 1904.8 2000.0 2105.2 2222.2 2352.9 2500.0 2666.6 2857.1 3076.9 3333.3 3636.3 4ooo.o 4444.4 5000.0 571^.3 6666.7 8000.0 0 .4 0 .3 5 0 .3 4 0 .3 3 0 .3 2 0 .3 1 0 .3 0 0 .2 9 0 .2 8 O.27 0 .2 6 0 .2 5 0 .2 4 0 .2 3 0 .2 2 0 .2 1 0 .2 0 0 .1 9 0 .1 8 0.17 0 .1 6 0.15 0 .l4 0 .1 3 0 .1 2 0 .1 1 0 .1 0 0 .0 9 0 .0 8 0 .0 7 0 .0 6 0 .0 5 Ml. o f Water Added 142.8 33*6 35.7 37.8 40.3 43.0 46.0 4 9 .2 52.9 57.0 61.6 66.6 7 2 .4 79.0 86.8 95.2 105.2 117.0 130.7 147.1 166.6 190.5 219.8 256.4 303.0 363.7 4 4 4 .4 555.6 714.3 952.4 1333.3 2000.0 Total Volume F in a l Gone. 1142.8 1176.4 1212.1 1250.0 1290.3 1333.3 1379.3 1428.5 1481.4 1538.4 1600.0 1666.6 1739.0 1818.0 1904.8 2000.0 2105.2 2222.2 2352.9 2500.0 2666.6 2857.1 3076.9 3333.3 3636.3 4000.0 4444.4 5000.0 5714.3 6666*7 8000.0 10000.0 0.35 0 .3 4 0.3 3 0 .3 2 0.31 0.30 0.29 0.28 0 .2 7 0 .2 6 O.25 0 .2 4 0 .2 3 0 .2 2 0 .2 1 0 .2 0 0.19 0.18 0.17 0 .1 6 0.15 0 .l4 0.13 0 .1 2 0.11 0.10 0.09 0.08 0.07 0 .0 6 0.05 0 .0 4 so p o s it io n o f th e membrane was e c c e n tr ic , thus crea tin g a g e n tle a g ita tio n o f the d ia ly z in g water when th e tank was r o ta te d . Another important p o in t was to f i l l th e membrane no more than th ree-fo u rth s f u l l . When immersed in the tank, th e s o lu tio n in the membrane rose to the le v e l o f the out­ sid e d ia ly z in g w ater. Ehis in creased the in te r fa c e between so lu tio n and the cellophane membrane. When the tank was ro ta ted , both the d ia ly z in g water and the so lu tio n were g e n tly a g ita te d . I f the membrane was com pletely f i l l e d , th ere was l i t t l e or no movement in s id e the membrane. Movement in s id e the membrane aid ed in e s ta b lish in g an equilibrium more promptly w hile s t i l l m aintaining the proper rate o f d ia ly s is . SI APPLICATION OP THE SLOW STEPWISE DILUTION PROCEDURE AND ROTATING OUTSIDE LIQUID DIALYSIS FOR THE PURIFICATION OF MUNG BEAN GLOBULIN A 300 ml. p ortion o f a preparation o f Mung "bean p ro tein extra cted w ith 0 .4 m NaCl, p r e c ip ita te d By 1 :4 d ilu tio n and red isso lv e d in O.Uil NaCl was placed i n cellophane tubing. The d ia ly z e r was suspended in the d ia ly z in g tank which was a ls o h a lf f u l l o f O.lJM NaCl s o lu tio n . The le v e l o f the s o lu tio n in the tank was then adjusted to the le v e l o f the p ro tein s o lu tio n in the cellophane tube* The in d ir e c t, slow , step -w ise d ilu tio n was begun by in trod ucin g d is ­ t i l l e d water from the r e se r v o ir in to th e d ia ly z in g tank by means o f g la s s tu bin g. hour. The flo w was c o n tr o lle d by a stopcock to th a t o f 500 ml. per The water l e v e l o f the tank slow ly rose to the s ix l i t e r mark. The d ia ly z in g water l e v e l was m aintained a t the s ix l i t e r mark by means o f siphoning. The d ia ly z in g water le v e l could be ad justed to any heigh t by r eg u la tin g the le n g th o f the siphon tube. The d ia ly s is o f th e 300 ml. p ro tein s o lu tio n , covered w ith to lu en e, was allow ed to proceed ov ern igh t. At th at time two d is t in c t la y e r s o f p r e c ip ita te were observed in th e bottom o f the cellophane membrane. The lower f r a c tio n was f a in t y e llo w ish in co lo r and the upper p ortion was a snowy white* f l u f f y m a te ria l. T e n ta tiv e ly the symbol G j was assign ed to the m a teria l in the upper la y e r and Gg to th a t o f the lower la y e r . The two p o rtio n s were separated by pouring the contents o f the c e llo ­ phane tube in to a beaker. The upper la y e r or G^ poured out but the lower la y e r o f Gg was g e la tin o u s m aterial and firm ly packed in the bottom o f 82 th e tu b e. To fu rth er p u rify the Gg fr a c tio n the g e la tin o u s m aterial was r e d is ­ so lv ed in 300 ml* o f 0.*(M NaCl s o lu tio n . to remove any in so lu b le m a te ria l. The so lu tio n was cen trifu g ed The s o lu tio n was o f b lu ish op alescen ce. The c l a r i f i e d p ro tein so lu tio n was placed in th e d ia ly z in g cellophane tube and d i a l y s i s proceeded as p reviou sly d escrib ed . When an equal amount o f water had been added to the tank, Gg was r e p r e c ip ita te d . t h is p o in t the approximate NaCl concentration was 0.2M. At I t was observed th a t th e d ia ly s a te appeared cloudy w ith w hite f l u f f y m aterial suspended throughout. DETERMINATION OF THE MOLAR CONCENTRATION OE SODIUM CHLORIDE AT WHICH THE Gg FRACTION PRECIPITATES FROM THE EXTRACT A fr e sh 0.4M NaCl e x tra ct was prepared according to the p reviou sly d escribed standardized procedure. The c e n tr ifu g a te was d ilu te d with four volumes o f d i a t i l l e d water and allow ed to stand four hours u n t il the pre­ c ip it a t io n was com plete. t io n . The p r e c ip ita tio n was separated by cen trifu g a ­ The p r e c ip ita te was red isp ersed w ith 0.4M NaCl, s t ir r in g u n t il i t was com pletely d is s o lv e d . The m aterial was cen trifu g ed to remove the in so lu b le m a teria l or globulin-bound substance (Gb). The c l a r if ie d solu ­ t io n in 0.4M NaCl contained the t o t a l g lo b u lin and was used fo r the deter­ m ination o f th e molar con cen tration a t which Gg p r e c ip ita t e s . The molar con cen tration o f NaCl at which Gg p r e c ip ita te d from the s o lu tio n con tain in g t o t a l g lo b u lin s was determined by in d ir e c t, slow , s te p -w ise d ilu tio n procedure w ith the d ia ly z in g apparatus. For t h is work a 350 m l. p o rtio n o f 0.4M NaCl s o lu tio n con tain in g th e t o t a l g lo b u lin s 83 was p laced in th e cellophane tube and 2650 ml. o f 0.4M NaCl so lu tio n were p laced in th e tank. film o f to lu e n e . The p r o te in s o lu tio n was covered w ith a th in There was, th erefo re , a t o t a l o f 3000 ml. o f 0.414 NaCl s o lu tio n i f both the so lu tio n in s id e and th at o u tsid e the cellophane tube were con sid ered. The in d ir e c t, slow , step -w ise d ilu tio n procedure was carried out by adding to the tank from a separatory funnel a measured volume o f water a t a speed o f 100 m l. per hour w hile th e tank was r o ta tin g . measured volume o f water added was 482.6 m l.. The f i r s t This volume was used so th a t the f in a l c a lc u la te d concentration o f NaCl in the tank and tube would be 0.35M. A fter the 4-82.6 ml. were added, the tank and tube were ro ta ted fo r a p eriod o f time to a llo w the m aterial on both s id e s of the membrane to reach eq u ilib rium . t io n . I t was noted th a t there was no sig n o f p r e c ip ita ­ Therefore a second measured volume o f water was added. ume o f 100.8 m l. brought the f in a l con cen tration to 0.34-M. lowed fo r equilibrium to be reached. This v o l­ Time was a l­ Since there was not sig n o f precip ­ i t a t i o n , fu rth er d ilu tio n w ith water was continued in measured volumes w ith time allow ed to e s ta b lis h equilibrium a fte r each a d d itio n . o f the tank was continuous throughout the e n tir e procedure. N otation The r e s u lt s showing each a d d itio n , the f in a l concentration o f NaCl and v is u a l obser­ v a tio n made during th e experiment were recorded in Table XXXIV. I t was observed th a t the d ialysat.e began to cloud when the c a lc u l­ a ted con cen tration was O.23M NaCl and mass p r e c ip ita tio n occurred a t .22M. Since the clo u d in ess o f the d ia ly sa te not only p e r s is te d but in creased a t low er co n cen tra tio n s, i t was somewhat confusing as to what d e f in it e 84 s a l t con cen tration Gg com pletely p recip ita ted * Farther means were sought fo r determ ination o f th e p r e c ise NaCl concentration a t which Gg p re cip ita ted * TABLE XXXIV DETERMINATION OF THE MOLAR CONCENTRATION OF NaCl AT WHICH Gg PRECIPITATES ( in d ir e c t , slow , step -w ise d ilu tio n ) T otal Vol. o f S o l. in Tank and Tube 3000 3428.6 3529.4 3636.3 3750.© 3871.0 4000.0 4137*9 4285.7 4444.4 4615.4 4800.0 5000.0 5 217.4 5454.5 5714.3 M olarity o f NaCl 0 .4 O.3 5 0 .3 4 0*33 0*32 0*31 0*30 0 .2 9 0.28 0 .2 7 0 .2 6 0.2 5 0 .2 4 0 .2 3 0 .2 2 0.21 m l. Water Added 48 2 .6 100.8 106.9 113.6 121.0 129.0 137*9 147.8 158.7 170.9 184.6 200.0 217.3 237.1 259.7 285.7 Total Volume F inal Cone. 3428.6 3529.^ 3636.3 3750.0 3371.0 4000.0 4137-9 4285.7 4444.4 4615.4 4800.0 5000.0 5217.4 5454.5 571^.3 6000.0 0 .3 5 0.3 4 0 .3 3 0 .3 2 0.31 0 .3 0 0.29 0.28 0.27 0 .2 6 0.25 0 .2 4 0.2 3 0 .22 0.21 0 .2 0 V isual Observation no p p t. no p p t. no ppt. no ppt. no p pt. no p p t. no p p t. no p p t. no pp t. no p p t. no ppt. no p p t. s lig h t clouding p p t. occurred clou d in ess F lo c c u la tio n complete This fu rth er study o f the proper molar concentration was conducted w ith a new batch o f g lo b u lin preparation from which the globulin-bound sub­ stance was removed. again employed* The d ir e c t , slow , step -w ise d ilu tio n method was To each o f the fo llo w in g s e r ie s o f nine 100 ml* grad­ uated cy lin d e rs a 50 ml. p o rtion o f g lo b u lin so lu tio n in 0.4M NaCl was added. Varied volumes o f d i s t i l l e d water were added to each cylin d er from a b u rette dropwise u n t il the d esired approximate concentration was reached in each in sta n c e . The so lu tio n s were g e n tly s tir r e d and stored 85 a t 6 °0. fo r 12 hoars* The r e s u lt s as recorded in Table XXXV were observed* TABLE XXXV DETERMINATION OF THE MOLAR CONCENTRATION 0? NaCl AT WHICH &2 PRECIPITATES (D irect D ilu tio n Method) rlinder 1. 2. 3. 4. 5. 6. 7. 8. ■9. Ml. G lobulin in 0.4M NaCl 50 50 50 50 50 50 50 50 50 Ml. Water Added 12.5 16.65 21.43 26.9 33.3 40.9 50.0 61.1 75.0 F in al M olarity 0.3 2 0.30 0.28 0 .2 6 0 .2 4 0*22 0 .2 0 0.18 0 .1 6 Approximate Amt* in Ml. a f te r 12 hr s . a t 6°C* no ppt* no pp t. no pp t. no ppt. incom plete p pt. 2 .8 3 .0 4 .0 1 0.0 The c h a r a c te r is tic s o f the system in each o f the nine cy lin d ers are d escrib ed below* C ylinders Nos. 1 through 4 were a lik e in having no sign o f p r e c ip i. ta tio n or clou d in ess* In c y lin d e r No* 5 there was incom plete p r e c ip ita tio n . There was no sharp d iv id in g l i n e or boundary between p r e c ip ita tio n and supernatant. In c y lin d e r No. 6 dense p r e c ip ita tio n occurred which appeared as a fa in t y e llo w , o p a lescen t m aterial* The volume o f p r e c ip ita te was o about 2 .8 ml. a f t e r standing 12 hours a t 6 C. In cy lin d er No. 7 p r e c ip ita tio n appeared, somewhat w hiter than in No. 6. Cylinder No. 8 produced a p r e c ip ita te which appeared w hiter than No. 7 and was la r g e r in volume. I t was fu rth er n oticed th a t there was a very sm all amount o f w hite f l u f f y p r e c ip ita tio n on top o f the p rin cip a l 86 p r e c ip it a t e . This d id not occur in No. 7* In c y lin d e r No. 9 p r e c ip ita tio n was complete and was uniform ly w h itish . I t had th e la r g e s t volume (10 m l.) and was id iite r than No* 7* The supernatant was the c le a r e s t o f any in the s e t . The in d ic a tio n s from t h is study of the molar concentration a t which Gg p r e c ip ita te s could he summarized in the fo llo w in g manner. The p r e c ip i­ t a t e in cy lin d er No. 6 a t the concentration o f 0.22M which ex h ib ited f a in t y ello w colored , op a lescen t m aterial was Gg which had been is o la t e d by slow , step -w ise continuous d ilu tio n . but apparently dense. The volume, 2 .8 m l., was small Cylinder No. 9 a t m olarity o f 0.1b had the la r g e s t volume o f p r e c ip ita te but e n tir e ly l o s t the Gg c h a r a c te r is tic appearance. T herefore, 0.16M NaCl did not seem a d v isa b le fo r fr a c tio n a tio n o f Gg. In both cy lin d er No. 7 8114 No. 8 the p r e c ip ita tio n s were probably com­ posed o f Gg fr a c tio n but they had been contaminated w ith other glo b u lin f r a c tio n s . Nrom the above ob servation s, i t appeared that Gg in pure fr a c tio n p r e c ip ita te d b est at 0 .2 2 molar concentration o f NaCl. 87 FRACTIONATION OF G^ FROM G^-FREE DIALYSATE The d ia ly s a te or supernatant from which G^ was is o la t e d by the in ­ d ir e c t, slow , step -w ise d ilu tio n procedure when te s te d by the u se o f the a d d itio n o f t r ic h lo r o a c e tic a cid and by fu rth er d ilu tio n to 0.08M NaCl was found to con tain a su b sta n tia l q uan tity o f in so lu b le p ro tein m atter. I t was d ecided that in order to d etect or is o la t e the fu rth er fr a c tio n or fr a c tio n s , both (1) d ir e c t and (2) in d ir e c t, slow, step -w ise d ilu tio n procedures should be fo llo w e d . ( l) D ir e c t, slow , step -w ise d ilu tio n procedure was employed fo r d e te c tin g the molar con cen tration of NaCl at which G^ p r e c ip ita te d from the supernatant. In to each o f ten 100 ml. graduated cy lin d ers was added a 50 oil* p o rtio n o f the G^-free supernatant. Various amounts o f d i s t i l l e d water were added to each cy lin d er from a b u rette dropwise to secure a d esired d ilu tio n fo r each. The so lu tio n s were g e n tly s tir r e d throughout th e e n tir e period o f d ilu tio n and then were allow ed to stand fo r 12 hours a t 6°C. The r e s u lt s obtained are presented in Table XXXVI. TABLE XXXVI DETERMINATION OF THE MOLAR CONCENTRATION OF NaCl AT WHICH G-z ERECIFITATES (D ir e c t, slow, ite p -w is e d ilu tio n ) Cylinder . 1 2. 3. 4. 5. 6. 7* S. 9. 10. D ia ly sa te Ml. Water 0.2M NaCl ml. Added 50 2.6 50 5*5 50 8.S 50 1 2 .5 50 16.6 50 2 1 .4 50 26. S 50 33*3 50 40-9 50 5 0.0 F in al M olarity 0 .19 0.1S 0.17 0 .1 6 0.15 0 .1 4 0 .1 3 0 .1 2 0.11 0 .1 0 Visual Observation no pp t. no p pt. no p p t. no p p t. ho p pt. no p pt. no p p t. no ppt. p p t. occurred p p t. occurred sg I t was observed th at heavy p r e c ip ita tio n o f G-j occurred a t 0»11 and 0 .1 0 m o la r ity . The p r e c ip ita te appeared almost white w ith a y ello w ish tin g e . (2) For fu rth er study the in d ir e c t, slow, step -w ise d ilu tio n pro­ cedure was employed. A 350 ml. p ortion o f the d ia ly sa te (0.22M NaCl) was placed in th e d ia ly z e r and 1468 m l. o f 0.22M NaCl were added to the tank. The d ilu tio n procedure was the same as th at fo r the p r e c ip ita tio n o f Gg. Two hours were allow ed fo r e s ta b lish in g equilibrium a f t e r the a d d itio n o f th e c a lc u la te d amount o f water. R otation o f the tank was continuous throughout the e n tir e procedure. The ca lcu la ted volumes o f w ater used and the f in a l molar concentrations o f NaCl in each case are recorded in Table XXXVII* TABLE XXXVII DETERMINATION OE THE MOLAR CONCENTRATION OF NaCl ; AT WHICH 0 , PRECIPITATES (I n d ir e c t, slow, step -w ise d ilu tio n ) T otal Vol. o f S o l. in Tank and Tube 3000.0 3142.g 3300.0 3473.7 3666.6 3SS2.3 4125.0 4400.0 4714.3 5076.9 5500.0 NaCl M olarity 0 .2 2 0 .2 1 0 .2 0 0.19 0 .1 8 0 .1 7 0 .1 6 0.15 0 .l4 0 .1 3 0 .1 2 Water Added m l. 142.8 157.2 173.7 192.9 215.7 242.7 275.0 31* . 3 362.6 423.1 500.0 Total Volume m l. F in a l NaCl Cone. M olarity 3142.8 3300.0 3^73.7 3666.6 3882.3 4125.0 4400.0 4714.3 5076.9 5500.0 6000.0 0.21 0 .2 0 0.19 0.18 0.17 0 .1 6 0 .15 0 .1 4 0 .13 0 .1 2 0.11 V isual O bservations no p p t. no p pt. no p p t. no p p t. no p p t. no p p t. no p pt. no ppt. no p p t. no p p t. p r e c ip ita tio n occurred The p r e c ip ita tio n was allow ed to stand in the d ia ly zer overn igh t. 89 At the end o f t h i s period the p r e c ip ita tio n was complete. The p r e c ip ita te was very easy to separate from the supernatant as i t was packed in th e hot tom o f the d ia ly se r and remained there when the supernatant was poured off* The p r e c ip ita te was very so lu b le in 0.4M NaCl and gave a c le a r s o lu tio n without stirrin g * From the above data i t may be concluded th a t the NaCl molar concentration a t which c ip it a t e s i s 0.11* pre­ 90 INVESTIGATION OF FRACTION \ OF GLOBULIN IN A Gg- AND G,-FRSE DIALYSATE V Itli. th e is o la t io n o f Gg and G^ from the t o t a l p ro tein m ixture, the supernatant from which Gg and G^ were removed was s t i l l thought to con­ ta in u n p r ec ip ita ted p ro tein m a teria l. Since the molar concentration o f NaCl a t which Gg p r e c ip ita te d was 0 .2 2 and fo r G^, 0*11, i t was thought th a t inasmuch as the so lu tio n s t i l l contained p ro tein m a teria l, as in d i­ cated by th e t r ic h lo r o a c e tic p r e c ip ita te , t h is p ro tein might be p r e c ip i­ ta te d as th e r e s u lt o f a fu rth er reduction in the NaCl concentration. The combined supernatant fr e e o f Gg and G^ was used fo r the in ves­ t ig a t io n o f s t i l l another fr a c tio n whose symbol has been designated a s G^. The is o la t io n o f G^ was carried out with the in d ir e c t, slow, step ­ w ise d ilu tio n procedure. The d ilu tio n s and observations were in d ica ted in Table XXXVIII. TABLE XXXVIII DETERMINATION OF THE MOLAR CONCENTRATION OF NaCl AT WHICH PRECIPITATES (I n d ir e c t, slow , stepuw ise d ilu tio n ) T otal V ol. Tank and Tube 3000.0 3300.0 3666*7 4125.0 NaCl M olarity Ml. Water Added Ml. Total Vol. Final Cone. Molar V isual O bservations 0 .1 1 0 .1 0 0.09 0 .0 s 300.0 366.7 458.3 589.2 3300.0 3666.7 4125.0 471^.2 0 .10 0.09 0.08 0.07 no p p t. no p p t. no p p t. p r e c ip ita tio n The G^ f r a c tio n was p r e c ip ita te d when the molar concentration o f NaCl reached 0.07M. I t was a white p r e c ip ita te with a f a in t y ello w ish 91 t in g e . I t was e a s ily separated from the supernatant "by pouring o f f the liq u id ; th e Gjj, remaining in the tube undisturbed. The molar con cen tration o f NaCl a t which p r e c ip ita te d from the supernatant was fu rth er stu d ied by employing the d ir e c t, pour-in d ilu tio n procedure. The G^ is o la t e d from the above experiment was d isso lv e d in 100 ml. o f O.Mji NaCl s o lu tio n . A 10 ml. p ortion o f s o lu tio n was p ip etted in to each of e ig h t 100 m l. c y lin d e r s. water were made a s fo llo w s: Various r a tio s o f d ilu tio n w ith In cy lin d er No. 1, 1:1; No. 2 had 1 :2 d ilu ­ t io n and so on u n t il w ith cylin d er No. 8 the r a tio was 1 :8 . The general procedure and r e s u lt s are tab u lated in Table XXXIX. TABLE XXXIX THE SEPARATION OF A GLOBULIN FRACTION (GO BY THE FURTHER DILUTION OF THE NaCl SOLUTION Cylinder 1. 2. 3* 4. 56. 7* 8. G lobulin0.4M NaCl S o l. ml. 10 10 10 10 10 10 10 10 Water Added ml. 10 20 30 40 50 6o 70 80 Amt. Ppt. 10 min. Amt. Ppt. 20 min. _ — +4+ 44+ +4 4 - - 44+4 4444 + 4+ ++ 4 Mass p r e c ip ita tio n o f G^ was observed a t 1 :4 and 1:5 d ilu tio n . At both d ilu tio n s the p r e c ip ita te was firm and was c le a r ly separated from th e supernatant f lu i d in 15 - 20 m inutes. With greater d ilu tio n s (1 :6 and 1 : 7 ) , a d e f in it e p r e c ip ita te was formed a f te r 20 minutes but i t s sep aration from the supernatant was not c le a r -c u t in comparison w ith 1 :4 and 1:5 d ilu tio n s . t l i n g took p la c e . With a 1:8 d ilu tio n clou d in ess occurred but no s e t­ 92 The v a r ia tio n in the character o f the ahove p r e c ip ita te s in d ic a te d a d iffe r e n c e in the in s o lu b ilit y o f g lo b u lin s a t d iffe r e n t low concentra­ tio n s o f NaCl s o lu tio n . A 0.4M NaCl so lu tio n when d ilu te d w ith four volumes o f d i s t i l l e d water (1:4 ) has approximately a concentration o f 0.08M and the 1:5 r a tio low ers the concentration to 0.06M. The molar con cen tration o f NaCl a t which p r e c ip ita te d was d eter­ mined to be between 0.08 and O.ObM when th e d ir e c t d ilu tio n procedure was used and 0 . 07M when the in d ir e c t, slow, step -w ise d ilu tio n method was employed* The supernatant from which fu rth e r. had been removed was stu d ied s t i l l Further reduction o f the s a lt concentration of the supernatant was ca rried out w ith the in d ir e c t, slow , step -w ise d ilu tio n method. d ia ly s a te appeared cloudy but no p r e c ip ita tio n occurred. The The d ia ly s is Was fu rth er ca rried to the p oin t where there were p r a c tic a lly no ch lorid e io n s but no p r e c ip ita tio n occurred. con tain er and stored a t 6°C. no p r e c ip ita tio n . The d ia ly sa te was tran sferred to a There was an in crease in clou d in ess but 93 A QUANTITATIVE APPLICATION OF TEE PROCEDURE FOR FRACTIONATION OF MUNG BEAN GLOBULINS The procedure p rev io u sly described f o r the separation o f Mung bean g lo b u lin s , Gg, Gj, G^ -was sub jected to a q u a n tita tiv e study. Mung bean meal ex tr a cte d w ith 0.4M NaCl so lu tio n and freed o f globulin-bound sub­ stance was used fo r t h is q u a n tita tiv e work. A p o rtio n o f t h is so lu tio n was analyzed fo r t o t a l n itrogen and found to con tain 0.004671 grams o f n itro g en per m l. Separation o f Gg. The 300 ml. o f 0.4M N ad so lu tio n con tain in g t o t a l g lo b u lin s and a t o t a l o f 1.4014 grams o f nitrogen were placed in a clea n cellophane membrane and 27OO ml. o f 0.4M NaCl so lu tio n were in ­ troduced in to th e d ia ly z in g tank. T otal volume o f the so lu tio n s in the and tank were 3000 m l. o f 0.4M NaCl. The in d ir e c t, slow, step -w ise d i­ lu tio n procedure was employed fo r the fr a c tio n a tio n o f the g lo b u lin Gg from the t o t a l g lo b u lin m ixture. Gg was p rev io u sly determined to pre­ c ip it a t e a t 0.22M NaCl, th erefore 2454 ml. o f d i s t i l l e d water were added to the tank in order to b rin g the t o t a l volume to 5454 m l. and to have a f in a l con cen tration o f 0.22M NaCl. D is t il le d water was added a t a slow r a te , two drops per second, from a separatory funnel extended with g la s s tubing so th a t the lower end was immersed in the d ia ly z in g water in the tank. Twelve hours were allow ed fo r completion o f p r e c ip ita tio n and sed im en tation . The Gg fr a c tio n c o lle c te d in the bottom o f the b o t t le which was a tta ch ed to the end o f the cellophane membrane. The fr a c tio n , Gg, formed a s e m i-so lid p a ste and was easy to separate from th e superna­ ta n t by d ecan tation o f th e l a t t e r . The supernatant was cen trifu ged and 9^ th e e n tir e Gg fr a c tio n was c o lle c t e d and d isso lv e d in 250 ml. o f 0.4M NaCl. Two 10 m l. a liq u o ts were taken fo r K jeldabl n itrogen determ ination. T otal n itro g en content o f the Gg fr a c tio n was 0.5 7 2 grams or 40.8 percent o f the t o t a l g lo b u lin o f Mung bean. F r a ctio n a tio n o f Gj. The supernatant from which Gg was removed was made to 300 m l. volume w ith 0 .2 2M NaCl so lu tio n and placed in a clean cellop h an e membrane. A volume o f 2,700 ml. o f 0.22M NaCl d ia ly z in g water was introduced in to the tank. was 3,000 m l. o f 0.22M NaCl. The volume o f so lu tio n s in tube and tank A volume of 3»000 ml. o f d i a t i l l e d water was added to the tank to bring the t o t a l volume to 6000 ml. and to a f in a l con cen tration o f 0.11M NaCl. o f th e p r e c ip ita tio n . Twelve hours were allow ed fo r com pletion The fr a c tio n , Gj, was p a s t e - lik e in form and was separated by d ecan tation . The supernatant was cen trifu ged . The p r e c ip i­ t a te c o lle c t e d was combined w ith the G^ and was d isso lv ed in 250 m l. o f O.MM NaCl s o lu tio n . d eterm ination. Two 10 ml. a liq u o ts were taken fo r K jeldahl n itrogen N itrogen content o f the G^ fr a c tio n was 0.58658 gm. or 49 percent o f the t o t a l g lo b u lin o f Mung bean. F ra ctio n a tio n o f G4 * The supernatant from which G^ had been removed was made to 300 ml, volume w ith 0.11M NaCl so lu tio n and placed in a clean cellophane membrane. p laced in the tank. out a s b e fo r e . A volume o f 27OO ml. o f 0.11M NaCl so lu tio n was The in d ir e c t, slow , step -w ise d ilu tio n was carried A volume o f 1 ,7 1 4 ml. o f d i s t i l l e d water was added to th e tank to b rin g th e t o t a l volume to 4714 ml. and the f in a l concentra­ t io n to 0 . 07M NaCl. Twelve hours were allow ed fo r the com pletion o f pre­ c ip it a t io n o f Gjj, which was separated from the supernatant by cen trifu g a ­ t io n . The amount o f G4 obtained was small and was d isso lv ed in 100 ml. 95 o f 0.4M NaCl. Two 10 ml. a liq u o ts were used fo r K jeldahl n itrogen deter­ m ination and th e n itro g en content o f the G^ fr a c tio n was 54*65 or 3*9 percent o f th e t o t a l g lo b u lin o f Mung bean. The summation o f th e q u a n tita tiv e fr a c tio n a tio n i s shown in Table XXXX and th e Flow Sheet in Figure 14. TABLE XXXX NITROGEN DISTRIBUTION IN VARIOUS FRACTIONS OF MUNG BEAN GLOBULINS F raction Mgm. N Percentage o f Total g2(22) 573*39 40.95 G3 (U ) 687*37 49.09 0^(07) 54.70 3*90 85.94 6.06 Undetermined Figure l4 FLOW SHEET The fr a c tio n a tio n o f Mung bean g lo b u lin was accomplished by the in ­ d ir e c t , slow , step -w ise d ilu tio n method and the use o f th e r o ta tin g out­ s id e liq u id d ia ly 2er* 300 ml. o f t o t a l g lo b u lin in 0.4M NaCl D ilu ted to 0.22M NaCl P r e c ip ita tio n o f Gg occurred Centrifuged I----------------- Gh rGy T Gfy , Supernatant D ilu ted to 0.11M NaCl P r e c ip ita tio n o f Gj occurred Centrifuged 4 --------------------------------------------------------------- — Supernatant D ilu ted to 0.07M XaCl P r e c ip ita tio n o f Gfy occurred Centrifuged ----------------------- — I' Supernatant 96 PURIFICATION OF FRACTIONATED MUNG BEAN GLOBULINS There i s no sharp d iv id in g l in e between t h is procedure and th at o f f r a c tio n a tio n . Since the purpose o f t h is work was the preparation o f s in g le g lo b u lin components, i t was necessary to remove sm all q u a n titie s o f oth er fr a c tio n s and substances which were a sso c ia te d with or adsorbed by th ese fr a c tio n a te d glob u lins* PURIFICATION OF THE PRECIPITATE Gg* was d isp ersed in 0.4m NaCl* For p u r ific a tio n , the Gg fr a c tio n A fter the p r e c ip ita te was com pletely d is­ so lv ed , th e s o lu tio n was cen trifu ged to remove tra c e s o f in so lu b le mate­ For r e -p r e c ip ita tio n o f the G2 fr a c tio n , the so lu tio n was in tro ­ r ia l. duced in to cellophane tubing and the concentration was lowered to 0.22M NaCl by membrane e q u ilib r a tio n d ilu tio n . The p r e c ip ita te formed in the tubing and was separated from the supernatant by cen tr ifu g a tio n . The supernatant was discarded* On rep ea tin g th e p u r ific a tio n process i t was observed th at there was a red u ction in th e quantity o f the Gg fr a c tio n . produced co n sta n tly dim inishing q uantity o f Gg. Further p u r ific a tio n By low ering th e ma x imum p r e c ip ita tio n con cen tration o f Gg to 0.20M and la t e r to 0.18M NaCl, approxim ately the o r ig in a l amount o f p r e c ip ita te was obtained* This change in p r e c ip ita tin g concentration le d to fu rth er in v e s t i­ g a tio n . A Gg f r a c tio n was sub jected to e lectro p h o retic a n a ly s is and found to be composed o f two d is t in c t components which co—p r e c ip ita te d to g eth er a t 0.22M NaCl, see Figure 15* Throu^a repeated d is s o lu tio n and p r e c ip ita tio n , w ith removal o f tra c e s o f in so lu b le dark m aterial 97 from th e m ixture by c e n tr ifu g a tio n , the maximum p r e c ip ita tin g concen­ t r a tio n o f Gg was lowered to 0.18M NaCl. I t was evident th at the Gg fr a c tio n contained c e r ta in substances other than th e two g lo b u lin com­ ponents and a l l were c o -p r e c ip ita te d together at 0.22M NaCl. A fter re­ p e a tin g the d isp e r s io n -p r e c ip ita tio n o f Gg and removing a sm all quantity o f the dark substance, the maximum p r e c ip ita tin g concentration was low­ ered to 0.1SM NaCl. Such a complex form ation may be expected to a f f e c t the s o lu b i l i t y o f the in d iv id u a l components. This suggested to the author th a t a more e f f e c t iv e method should be sought for the removal o f the dark colored substance. RESOLUTION OF THE Gg FRACTION. The Gg fr a c tio n was d isp ersed in 0.4m NaCl and p laced in cellophane tubing which was suspended in a r o ta tin g o u tsid e liq u id d la ly z er con tain in g d i s t i l l e d w ater. The volume o f d is ­ t i l l e d water was ten tim es g rea ter than that o f the p ro tein so lu tio n in th e cellophane tu b in g. In comparison with the p rev io u sly described in ­ d ir e c t , slow , step -w ise m embrane-equilibration d ilu tio n procedure, t h is procedure may be designated as d ir e c t, rapid m em brane-equilibration. P r e c ip ita tio n occurred in the cellophane tubing w ith in 15 minutes but the r o ta tio n o f the tank was continued fo r two hours. In two hours o f d ia ly z in g equ ilib rium was not reached but t h is was to permit the sed i­ m entation o f a dark, dense p r e c ip ita te w hile a w hite f lu f f y p r e c ip ita te was separated from th e dark m aterial by d ecantation. The dark p r e c ip i­ t a te was sub jected to d is s o lu tio n fo r a second time and a sim ila r phen­ omenon occurred. As more w hite f lu f f y m aterial went in to suspension, l e s s o f the dark m aterial was l e f t in the tube. The process o f r e s o lu tio n 98 was rep eated u n t il l i t t l e or none o f the w hite m aterial formed* r e s o lu tio n p rocess was then considered com plete. The The f in a l resid u e o f the Gg fr a c tio n was d isso lv e d in 0.4M NaCl so lu tio n and became very dark, b lu ish -p u r p le in co lo r but was not stu d ied fu rth er. TRACTIONATION OF THE RESOLVED PRODUCTS OF Gg. Nhen two volumes of d is ­ t i l l e d water were added to th e t o t a l c o lle c t io n of decantations o f the re so lv e d product o f th e Gg fr a c tio n , the p ro tein p r e c ip ita te d im m ediately. T his was allow ed to stand overnight a t 4°C. The p ro tein was separated from th e water by c e n tr ifu g a tio n and th e p r e c ip ita te was r e d isso lv e d in O.^-M NaCl. A fter the p r e c ip ita te was com pletely d isp ersed , i t was cen­ tr ifu g e d to remove in so lu b le p a r t ic le s . tio n a tio n . The so lu tio n was ready fo r fr a c ­ The c l a r i f ie d p ro tein so lu tio n was p laced in a cellophane membrane which was immersed in NaCl so lu tio n o f the same concentration (O.^iM) in th e d ia ly z in g tank. The in d ir e c t, slow, step -w ise d ilu tio n procedure was used w ith r o ta tin g o u tsid e liq u id d ia l y s is . The measured volume o f d i s t i l l e d water was added to the O.hM NaCl so lu tio n in the d ia ly z in g tank a t two drops per second. I t was noted th at in c ip ie n t p r e c ip ita tio n occurred a t 0.1SM NaCl and then the max imum p r e c ip ita tio n occurred a t 0.17M. not d ilu te d fu r th e r . by c e n tr ifu g a tio n . The con cen tration o f the so lu tio n in the tank was The p r e c ip ita te was separated from the supernatant This fr a c tio n was designated as ^2 ( 17) . Since th e supernatant from which $ 2 ( 17) 193,8 s t i l l contained an ap p reciab le q u an tity o f p ro tein (tr ic h lo r o a c e tic a cid t e s t ) , fu rth er fr a c tio n a tio n fo r the r esid u a l p ro tein was continued. The c le a r super­ natant from which ®2 ( 17) 19as removed was placed in cellophane tubin g. 99 A measured volume o f d i s t i l l e d water was added in order to bring the con cen tration o f the d ia ly se r to th at o f 0.1&M NaCl. not occu r. The con cen tration o f the so lu tio n was fu rth er lowered to 0.15M and l a t e r to O.l^M* NaCl. P r e c ip ita tio n did The maximum p r e c ip ita tio n occurred at O.l^M This p r e c ip ita te was recovered by cen trifu g a tio n and designated a s ®2(lU)* suPerBa‘t ant from which the ^ ( l ^ ) 1jas remove<* was a c le a r s o lu tio n and was te ste d with tr ic h lo r o a c e tic a c id . I t contained a sm all amount o f p r o te in but was not stu d ied fu rth er. USING SPECIFIC MOLAR CONCENTRATIONS OF SODIUM CHLORIDE FOR PURIFICATION. The fr a c tio n a tio n and p u r ific a tio n procedure p reviou sly employed fo r the is o la t io n o f g lo b u lin fr a c tio n s were accomplished by ad ju stin g them to th e ir maximum p r e c ip ita tin g concentrations "m .p.c.". The r e s u ltin g pro­ d ucts so fa r as the homogenity o f the p rotein was concerned were freed o f g ro ss contam ination on ly. I t was necessary to u se both the maximum p r e c ip ita tin g con cen tration "m.p.c." and the in c ip ie n t p r e c ip ita tin g con cen tration " i.p .c ." to con trol the p r e c ip ita tio n o f each fr a c tio n in order to elim in a te tra c e contamination or to secure the d esired s ta te o f p u r ity . The p u r ific a t io n o f th e 82( 17 ) fr a c tio n was accomplished by su b ject­ in g i t to th e procedures in v o lv in g d e f in it e molar concentrations o f sodium c h lo r id e . 1. The d e ta ile d account i s described below* The 82 ( 17 ) f r a c t*on was dispersed in 0.4M NaCl. A fter complete d isp e r sio n , th e s o lu tio n was cen trifu ged and the volume measured. 2. A fter the cellophane tubing was rin sed w ith 0.4M NaCl s o lu tio n , th e p r o te in s o lu tio n was placed th e r e in . The p ro tein so lu tio n was covered 100 w ith to lu e n e . She cellophane tubing was immersed in the d ia ly zin g tank co n ta in in g 0.4M NaCl s o lu tio n . The t o t a l volume o f O.hM NaCl s o lu tio n in th e tubing and tank measured ex a ctly 1000 ml. 3* The in d ir e c t, slow, step -w ise m embrane-equilibration d ilu tio n procedure was ca rried ou t. Follow ing the c a lc u la tio n s o f Table XXXIII, d i s t i l l e d water was added to the d ia ly z in g tank a t a ra te o f two drops per second from th e reserv io r to the bottom o f the d ia ly zin g tank through g la s s tu b in g. H. The tank was rotated throughout the d ilu tio n p rocess. A fter the ca lc u la te d volume o f water (1222 m l.) was added, the t o t a l volume was 2222 ml. and the f in a l s a lt concentration was 0.1SM. The in c ip ie n t p r e c ip ita tio n o f fr a c tio n &>(17) occurre^ a t t h is concen­ t r a tio n . N o ta tio n o f the d ia ly zer continued fo r eigh t hours u n t i l equi­ lib riu m was reached. 5. The cloudy formation o f p r e c ip ita te which appeared a t the in c ip ­ ie n t p r e c ip ita tio n con cen tration was removed by cen tr ifu g a tio n . The cla r­ i f i e d c e n tr ifu g a te was returned to the o r ig in a l cellophane tube and the d ilu tio n procedure continued* b* The c a lcu la te d volume o f water (130 m l.) was added slow ly as b efore u n t il th e volume ro se to 2352 ml. and the f in a l concentration was lowered to 0.17M. R otation o f the d ia ly zer continued fo r eig h t hours to e s ta b lis h eq u ilib rium . The p r e c ip ita te i&ich formed in the tubing was tra n sferred to a 250 ml. cen trifu g e tube. 15 m inutes, the supernatant was decanted. A fter cen trifu g in g for This p u r ifie d 1,3,8 sto red in a d eep -freeze u n it . The above procedure was follow ed fo r the p u r ific a tio n o f the other 101 f r a c tio n , &2 (lh )» ®3 ( 11) and ®ty(o7 )* ®ie *n c iP*ent p r e c ip ita tin g con­ c e n tr a tio n s o f the Gg(14 )* ®3 ( n ) %.(o7 ) ^rac*io n s were 0.15» 0.12 and 0.08M r e s p e c tiv e ly and the maximum p r e c ip ita tin g concentrations were 0 .1 ^ , 0 .1 1 and 0.07M. The ca lcu la te d volumes o f water fo r d ilu tio n o f each s a lt con cen tration are given in Table XXXIII. 102 TEE ELECTROPHORETIC ANALYSIS OE MUNG BEAN GLOBULINS The knowledge that charged p a r t ic le s in so lu tio n m igrate in an e l e c t r i c f i e l d has le d to the development o f one o f the most powerful t o o ls fo r c h a ra cterizin g p ro tein s and numerous carbohydrates. E lectro­ p h o retic a n a ly s is has provided one o f the few physico-chem ical c r it e r ia o f p r o te in homogeneity. I t i s e s s e n tia l that one he fa m ilia r with the p r in c ip le s o f the moving boundary method as used in the T is e liu s e le c ­ tr o p h o re sis apparatus (6 2 ,6 3 ,6 4 and 65) to be ab le to evalu ate c r i t i c a l l y th e data which appear in the lit e r a t u r e . I f a p r o tein so lu tio n b uffered at any given pH i s placed in a U -c e ll and pure b u ffer so lu tio n i s c a r e fu lly layered over i t , the p ro tein w ill m igrate in to th e b u ffer toward one o f the electro d es when d ir e c t current i s passed through th e s o lu tio n . With a s in g le p ro tein , a l l the m olecules w i l l move at the same ra te so that sharp boundaries w ill be m aintained between the p r o te in and the b u ffe r . With a s o lu tio n containing n -p ro tein components, m igrating a t d iffe r e n t speeds, n-boundaries w ill be formed soon a f t e r th e current has been s ta r te d . In the ascending limb o f the U -c e ll the f a s t e s t moving p ro tein w ill form a boundary a g a in st the b u ffer, the next f a s t e s t m igrating p ro tein w ill form one a g a in st the f a s t e s t pro­ t e in , e t c . Movement o f the p ro tein m olecules may be follow ed by observ­ in g the boundaries. I f the p r o te in in s o lu tio n i s on the a lk a lin e sid e o f i t s is o e le c ­ t r i c p o in t, and hence i s n eg ative in charge, the m igration w ill be toward 103 th e anode (+) term in al. I f the p ro tein i s on the a cid sid e o f i t s is o ­ e l e c t r i c p o in t, the o p p o site i s tr u e . I f the p ro tein i s at i t s is o e le c ­ t r i c p o in t, no m igration w ill take place* At any s p e c if ic pH, temperature and s a lt concentration , the d ista n ce (d) moved by a g iv en p ro tein boundary per u n it o f time ( t) w ill depend upon the p o te n tia l gradient ( F ). For a given p o te n tia l g ra d ien t, the r a te o f m igration d /t w i l l be c h a r a c te r is tic fo r each in d iv id u a l protein* The p o te n tia l gradient may be ca lcu la ted by the expression below: i F I ---ak where F * the p o te n tia l g ra d ien t, i = current (amp*), a • the cross sec­ t io n a l area o f the U - c e ll and k s the con d u ctivity of the b u ffer or pro­ t e in s o lu tio n . The speed o f m igration, or e lectro p h o retic m o b ility ( u ) , may be d efin ed as the d ista n ce moved in centim eters per second under a p o te n tia l grad ien t o f 1 v o lt/cm . a t a c e r ta in pH, in a d e f in it e b u ffer o f a d efined d dak cm u (c m /v o lt-se c) - - — * ---------- ( ---- -— — — —) tF it se c/v o lt/cm io n ic stren g th . For the determ inations o f e lectro p h o retic m o b ility , i t i s n ecessary to measure a cc u r a te ly 1 ) the d istan ce moved by the p ro tein boundary, 2) the tim e in seconds, 3 ) the current p assin g through (ampere), 4) the con­ d u c tiv ity o f the so lu tio n (k a kc/ r ) , and 5) the cross s e c tio n a l area o f ? the c e l l in cm • 10^ APPARATUS The ele c tr o p h o r e tic a n a ly sis o f Mung bean g lo b u lin was conducted in t h is lab oratory w ith the new compact T is e liu s e lectro p h o resis appar­ a tu s* . This apparatus, based on Longsworth's scanning m o d ifica tio n o f the Toepler sc h lie r e n method, has been described by Moore and White (6 6 ). This apparatus has th e valuable fea tu res o f the T is e liu s method y e t avoid s i t s disadvantages o f la rg e s iz e and d if f ic u lt y o f operation. An e le c tr o p h o r e sis c e l l o f the type developed by T is e liu s i s used to contain the sample and in which the boundaries are formed. I t has a cap acity fo r two m l, o f s o lu tio n , the o p tic channels have dimensions o f 2 mm. in w idth, 15 mm. along the o p tic path and 50 nun* h eig h t. This c e l l has e x c e lle n t dimensions fo r e le ctro p h o retic a n a ly sis because i t i s so nar­ row th at a r e l a t i v e l y la rg e amount o f heat may be generated in i t by th e passage o f current without causing convection, p erm itting higher f i e l d stren g th and more rapid a n a ly s is . The a c tu a l measurement o f the p rotein d istr ib u tio n can b e st be made by Longsworth* s m o d ifica tio n o f the Toepler sch liere n method. The c e l l and i t s con ten ts are illu m in ated w ith p a r a lle l lig h t and the d ev ia tio n s o f the rays caused by the r e fr a c tiv e index grad ien ts are observed. In t h is scanning method the c e l l i s photographed on a moving photographic p la t e . Both p la te and k n ife edges are driven sim ultaneously by a small motor th a t moves the p la te a t r ig h t an gles to the motion o f the k n ife edges and o f th e l i g h t beam, g iv in g p ic tu r e s sim ila r to those in Figure 15 . * Manufactured by Perkin Elmer. 105 BUFFER. EFFECTS Since the charge and th e magnitude o f charge o f a p ro tein m olecule depends upon th e surrounding rea c tio n ( 67)* i t i s necessary th a t e le c tr o ­ p h o resis experim ents he ca rried out in s u ita b le b u ffers in order to ob­ ta in comparable r e s u lt s . The b u ffer chosen as a so lv en t should have a high b u ffer ca p a city i t s e l f so th a t th e p ro tein b u ffer cap acity i s r e la ­ t i v e l y reduced. This w ill r e s u lt in fewer boundary anom alaties ( 68) . A b u ffer w ith a low s p e c if ic conductance i s d esira b le in order to reduce the d isturb an ces due to the h eatin g e f f e c t o f the cu rren t. The genera­ tio n o f h eat r e s u lt s from f r i c t io n o f th e ion s p assin g through the solu­ tio n and i s r e la te d to the speed o f m igration o f the ion s (6 9 ). Both b u ffe r ca p a city and conductance in crease w ith the concentration o f b u ffer s a l t s , and i t has been p oin ted out that because o f t h is in co m p a tib ility a compromise must be made (67)* Since b u ffer cap acity does not depend upon io n ic m o b ilit ie s , b u ffer s a lt s the io n s o f which have low m o b ilitie s should be s e le c te d . B u ffer so lv e n ts fo r use in the electr o p h o r e tic a n a ly sis o f human plasma and serum have been stu d ied a t len gth by Longsworth (6 4 ). The r e s u lt s o f th ese experim ents show that in r e so lv in g power none o f the b u ffe r s are superior, to the d ie th y l barbiturate (veronal) s o lu tio n at pH 8 . 6. The d iffe r e n c e s in r e so lv in g power o f three d iffe r e n t b u ffers a t the same io n ic stren gth and p o te n tia l gradient on normal human plasma were stu d ied by Longsworth. The p attern s obtained in the b a rb ita l buf­ f e r o f pH 8 .6 w ith io n ic stren gth o f 0 .1 showed the components w ell 10b separated from each other and the peaks sharp and w ell d efin ed , Where­ a s , th e same sample separated in a carbonate b u ffer o f pH 9*9 P 0 ,1 , and a phosphate b u ffer o f pH 7»7 P 0 .1 showed th at the r e s o lv in g power o f th ese two b u ffe rs was l e s s s a tis fa c to r y w ith human plasma. E crse plasma was a ls o examined in the same b u ffers as those used in the above work. In co n tra st w ith human plasma, t h is m aterial gave a more s a tis fa c t o r y p a ttern in the phosphate than in the d ie th y l-b a r b itu r a te b u ffe r . Karon (70) reported on borate and g ly c in e b u ffer th at were pre­ pared according to Olark; veronal b u ffer according to Longsworth, and an ammonia b u ffer prepared by adding 0 .2 molar ammonia to 0 .1 molar hy­ d ro ch lo ric a c id s o lu tio n . Whereas th ese b u ffers were s a tis fa c to r y s o l­ v en ts fo r peanut p r o te in , on ly the g ly c in e b u ffer was s a tis fa c t o r y as a so lv en t fo r co tto n seed p r o te in . A b u ffer composed o f 0 .2 mole o f ethylam ine and 0 .1 mole o f veronal in a l i t e r o f so lu tio n having a pH o f 1 0 .7 fcad most d e sir a b le c h a r a c te r is tic s fo r cotton seed p r o te in . I t was thefcety suggested (64) th at the proper solven t fo r the a n a ly sis o f the p r o te in o f a g iv en type v a r ie s w ith the sp e c ie s and should be determined exp erim en tally. 107 THE PREPARATION OE BUFFERS OF DESIRED pH AND IONIG STRENGTH Tables have "been prepared "by Cohn (71) and hy Green (72) from which th e m olecular r a t io s o f s a lt to acid can he obtained in preparing phos­ phate and a c e ta te b u ffe r s o f constant io n ic strength and varying pH or con stan t pH and varying io n ic stren g th . For a l l the b u ffer m ixtures in e le c tr o p h o r e tic work in t h is study, the Henderson-Hasselbach equation was used fo r c a lc u la tio n o f the pK v a lu e s. A l i s t o f pK valu es o f the more common a c id s used fo r b u ffer m ixtures i s as fo llo w s: Acid pK value A c e tic 4.73 B a rb itu ric 7*90 O acodylic b.20 G lycine 2.35» Phosphoric 6*77 (second d iss o c ia tio n ) 9-77 The fo llo w in g are examples o f sev era l types o f b u ffer problems that a r is e in p r a c tic e . 108 (A) The p reparation o f a sodium a c e ta te b u ffer o f pE 4 .7 and io n ic stren g th o f O .l* In order to secure t h is b u ffer valu e one must c a lc u la te the pH value o f a c e t ic a c id , s a lt / a c i d , 3)* 2 ). l). c a lc u la te th e r a tio o f c a lc u la te m olarity o f sodium a c e ta te and m olarity o f a c e t ic acid* 1 ). Since the io n iz a tio n constant K o f a c e t ic a c id equals 1*86 x 10"5 10-5 .................. lo g o f 1 .8 6 -5 .0 0 0 0 0.2695 -^■*7305 the lo g o f io n iz a tio n K pK s th e n eg a tiv e lo g o f th e io n iz a tio n K or - ( - 4 .7 3 ) or 4,73 2 ). To c a lc u la te the r a tio o f s a lt /a c id , Henderson-Hasselbach equation i s u sed. pH a pK ♦ lo g s a lt /a c id or 4 .7 0 * ^ .73 + lo g s a lt /a c id or - 0 .0 3 * lo g s a lt /a c id p o s it iv e valu e o f - 0 .0 3 i s 9 .97 - 10 a n t ilo g o f 9*97 i s 0.933 0 .9 3 3 i s th e r a tio o f s a lt /a c id 3 ). To c a lc u la te M o f sodium a c e ta te and M o f a c e t ic a c id . The io n ic stren g th o f u n ivalen t compounds i s equal to the molar co n cen tra tio n . Since wealc a cid does not contribute appreciably to th e io n ic stren g th , the s a lt concentration had to be 0.1M in order to g iv e an io n ic strength o f 0 .1 . 0.933 s a lt r a tio 0.933 = ——— ------1 .000 a cid r a tio z a c id 0 .9 3 3 x a c id s a lt ♦ a c id (0 .9 3 3 a cid ) 1 .9 3 3 a c id » a s a lt * s a lt a 0.1M ♦ a cid = 0.1M 0.1M 0 .1 0 0 _ 0.0517M a c id 1 .933 “ 0 . 0 1<83M s a lt 109 (B) The p reparation o f a sodium phosphate b u ffer o f pH 7*0 and io n ic stren g th o f 0 . 1 . The pKg o f phosphoric a cid » b*77 1) • To c a lc u la te the r a tio o f s a lt /a c id . pH = pK ♦ lo g s a lt /a c id 7 .0 - 6.77 ♦ lo g s a lt /a c id O.2 3 s lo g s a lt /a c id a n t ilo g o f 0 .2 3 i s 1*7 1 .7 i s th e r a tio o f s a lt /a c id 2) * To fin d M concentration o f s a lt and o f a c id . The sodium phosphate b u ffer a t pH 7*0 i s d iss o c ia te d as fo llo w s: Na2HK>4------> Na+ * NaHFOif Na+ 4 HP0^““ NaHgKfy------> Ha+ + HgFOif , j i = 0*5 { CV2 s u b s titu te x fo r a cid concentration , 1 . 73c fo r s a lt concentration 0 .1 « 0 .5 (1-7*) U 2) ♦ (1*7*) ( l 2) ♦ (1*7=0 ( 22) (x) ( l 2 ) 4. ( * ) ( l 2) 0 .2 » 1 .7 x 4 1.73C ♦ 6 .Sx ♦ x ♦ x 0 .2 = 12. 2x x = 0 .2 1 2 . 2x = 0.0164 M o f HaHgPOij. and 1 .7 x 0.0164 = 0.027S M o f NagHFOij. g iv e io n ic stren gth o f ©.1 M a t pH 7 .0 A l i s t o f common b u ffers o f 0 .1 io n ic stren gth (Hardt) com position & 1 . 7s 0.02N HC1 t 0.08N NaCl 3 .0 5 0 .1 N HC1 ♦ 0.5N g ly c in e 3 .6 2 0 .2 N HAc ■». 0.02N NaAc ♦ 0.08N NaCl 3.91 0 .1 N HAc ♦ 0.02N NaAc + O.OSN NaCl 0 .2 N HAc * 0 .1 N NaAc 0.15N HAc ♦ 0 .1 N NaAc ^ .64 0 .1 IT HAc + 0 .1 N NaAc 5 .3 3 0.02N HAc 5.^2 0 .1 N HCac ♦ 0.02N NaCac + 0.08N NaCl 5-65 0.01N HAc ♦ 0 .1 N NaAc 6.1 2 0.02N HCac ♦ 0.02N NaCac ♦ O.OSN NaCl 6.79 O.OO^N HCac* 0.02N NaCac + 0.08 N NaCl 7 .S 3 0.02N HV + 0.02N NaV 8 .6 0 0.02N HV ♦ 0 .1 N NaV 0 .1 N NaAc ♦ O.OSN NaCl 10.28 0.02N g ly c in e ♦ 0 .1 N NaOH 10.88 0.125H g ly c in e t 0 .1 S NaOH 11.81 0 .1 IT g ly c in e ♦ 0 .1 N NaOH Ac s a c e ta te Cac * cacod ylate V s d i e th y l-b a r b itu ra t s I ll OPERATIONAL PROCEDURE IN A COMPLETE ELECTROPHORETIC ANALYSIS PREPARATORY 1. 2. 3» E lectro p h o retic c e l l s should he cleaned in D reft and roast he dry b efo re u sin g . Preparation o f sample fo r e le c tr o p h o r e tic a n a ly sis : The p ro tein i s d isp ersed in 10 ml. o f the b u ffer in a 50 ml. beaker. The con­ ce n tra tio n o f p ro tein should be 0 .5 to 1 p ercen t. Transfer the p r o te in to cellophane tubing and d ia ly ze in 1000 ml. o f the same b u ffe r fo r two hours w ith r o ta tin g ou tsid e liq u id d ia l y s is . Turn on th e current o f power supply fo r electro p h o r e sis apparatus two hours b efore i t i s to be used. ASSEMBLY OP CELL 4. 5. Urease th e co n ta ctin g c e l l p la te s with sp e c ia l g rea se. Do not g rea se too c lo s e to the channel. Leave a bare rectangu lar area extending 3 mm. around each channel. I t i s necessary to grease o n ly one o f th e two con tactin g su rfa ces. Assemble bottom and cen ter se c tio n togeth er with rotary motion to o b ta in a lea k -p ro o f s e a l. Assemble the top se c tio n in th e same manner. MEASURING- THE RESISTANCE OP BUFFER AND PROTEIN SOLUTION 6. The c o n d u c tiv ity c e l l i s suspended in an ic e bath in a 1000 ml. beaker. Transfer 2 ml. so lu tio n (b u ffer f i r s t and then p rotein) to c o n d u ctiv ity c e l l w ith a hypodermic syrin ge. In order to prevent th e form ation o f a i r bubles in the c e l l , the n eedle o f th e syringe must touch the bottom o f the c e l l . Allow only enough so lu tio n to f i l l th e c e ll* A constant reading may be obtained a f te r standing fo r a period o f tim e. FILLING CELL 7. S. 9. Transfer 10 ml. o f d ia ly zed p ro tein in to a small tube and mark "Protein” and stopper. O v e r f ill bottom s e c tio n o f c e l l with p rotein so lu tio n . a . To do t h is u se a lo n g n eedle syrin ge. b. Syringe i s in se r te d through lefth a n d channel. c . To prevent th e trapping o f a ir , t ip the whole assembly to the l e f t w hile l e t t i n g so lu tio n flow g en tly in to the bottom s e c tio n . F i l l l e f t cen ter o f c e l l w ith p ro tein and rig h t center w ith b u ffer, a . To do t h is the cen ter and top se c tio n s must s h if t to the righ t to seg reg a te channels from bottom s e c tio n s . 112 b. c. 10. 11 . 12. 13* Over f i l l the l e f t cen ter c e l l w ith p ro tein s o lu tio n . Remove ex cess p ro tein so lu tio n in the r ig h t channel "by a long need le syrin ge and r in se 3 tim es w ith "buffer. d. O v e r fill the r ig h t center c e l l with b u ffe r . e* How, keep the cen ter s e c tio n in segregated p o s itio n and push top s e c tio n to the l e f t . Clamp firm ly w ith spring clamp. f . Remove excess p ro tein so lu tio n from top l e f t c e l l and rin se 3 tim es w ith b u ffe r . g . f i l l both s id e s o f top s e c tio n with b u ffer to the le v e l o f the b u ffer tube arms. Connect b u ffer tu b es. In se r t e le c tr o d e s. F i l l b u ffe r tubes w ith b u ffer so lu tio n to S f l O f u l l lea v in g l/lO space fo r KC1 s o lu tio n . I n je c t w ith a syrin ge 10 ml. o f 1 /3 saturated KC1 so lu tio n in to each e le c tr o d e c a p illa r y w hile the connecting gate o f the top sec~ t io n i s open to a llo w b u ffer so lu tio n to flow through f r e e ly . PLACING THE CELL IN THE ELECTROPHORETIC APPARATUS l4 . 13* 16 . 17« 18. 19. P lace the e n tir e u n it o f c e l l in the bath chamber o f the apparatus and clamp i t to th e bottom. Connect the le a d s to e le c tr o d e s. F i l l bath with i c e . Pour cold water over ic e and f i l l the bath to w ith in an inch o f overflow p ip e. Close the bath w ith p la te . Turn on s t ir r e r sw itch . Twenty m inutes are required fo r reaching uniform temperature of 0°C. b ath. ASSEMBLY OF THE COMPENSATOR 20. 21. 22. F i l l the syrin ge w ith 7 ®1» o f b u ffer, elim in ate a ir bubbles and mount on the compensator and turn on the motor. Make sure th at the sm all d ro p lets o f b u ffe r are formed from the nBedle p o in t. The n eed le p o in t i s immersed in a beaker containing b u ffer. I n se r t th e n eed le through a h ole to the lefth an d b u ffer b o t t le . Do not a llo w n eed le to touch the e le c tr o d e . Turn on the compensator fo r 15 seconds and turn o f f . STARTING BOUNDARY 23. 24. 25 . 26. Push down the cen ter g ate fo r th e c e l l to segregate the channels. S h ift the cen ter s e c tio n o f c e l l in to a lig n ed p o s itio n by turning the r ig h t hand s h if t in g rod. Turn on compensator, Min ” p o s itio n , to allow a g e n tle flow o f buf­ fe r in to the le fth a n d b u ffer tube u n t il boundaries are brought in to view . Photograph the s ta r tin g p o in ts , i f d esire d . 113 27. Turn on cu rren t, "normal", and record the time o f s ta r tin g .* 28* Adjust th e current to approximately 2 watts* (Y olts E tim es m i l l i amps I ■ w a tts .) 29 . Time required fo r th e development of f u l l m igration o f charged par­ t i c l e s v a r ie s from 1 - 3 hours and depends upon the d iffe r e n t m aterials* TO MAKE EXPOSURE 30. 31* 32. 33. 3^. 35. 36, 37* 38. ; , 39* Turn o f f current and record tim e. Focus the c e l l on screen in te n s e ly by a d ju stin g the rig h t hand knob. Mask the image w ith m etal p la te and a lig n the s l i t with the ascend­ in g c e l l (to th e r ig h t .) Tarn o f f l i g h t . Take the p ictu r e o f ascending l in e . a . Expose the p attern by scan from *4- to 8 .5 mark. Again, fo cu s the c e l l on screen in te n s e ly by ad ju stin g the rig h t hand knob. Mask th e image w ith m etal p la te and a lig n the s l i t with the descend­ in g l i n e (to the l e f t . ) Turn o f f l i g h t . Take th e p ictu r e o f descending l i n e . a . Expose the p attern by scan from 8 .5 - 1^» Develop the n eg a tiv e . a . Develop ^ minutes in D19 i& t o ta l dark* b . Hypo fo r 10 to 15 m inutes. c . Wash 1 hour. Ilk ELECTROPHORETIC ANALYSIS OF MUNG BEAN GLOBULINS TOTAL GLOBULINS. As a s ta r tin g p o in t, i t was considered ad v isa b le to u se th e b u ffe r s th a t D anielson (73) used in h is e le c tr o p h o r e tic stu d ie s o f pea p r o te in s . However the a c e ta te b u ffer of pH 3.72 w ith 0.2M NaCl would not d is s o lv e the t o ta l g lo b u lin s a t 0°C. TUhen the a c e ta te b u ffer was made to pH 3*19 a^d 0.2M NaCl was added, the p ro tein was so lu b le a t 0°C. and th e e le c tr o p h o r e sis a n a ly sis was conducted. B u ffers other than a c e t a te , th e phosphate b u ffer o f pH 7*5 &&d the borate b u ffer o f pH 8 .3 0 (a s prepared by D an ielson ), were a lso employed fo r d isp ersin gth e t o t a l g lo b u lin . None o f th ese b u ffers was a s a tis fa c to r y solven t fo r the sep a ra tio n o f t o t a l Mung bean g lo b u lin in electro p h o resis a n a ly s is . An NEj-ECl b u ffe r , however, o f pH 9*26, as prepared by Irv in g (7*0, was sup erior in r e so lv in g power in comparison with those mentioned above. The various components were w e ll separated from each other and the peaks were sharp and w ell d efin ed . The ele c tr o p h o r e tic pattern s o f t o ta l Mung bean p r o tein s w ith variou s b u ffers are shown in Figure 15 and 16. A l a t e r experiment showed th at v e r o n a l-c itr a te b u ffer, prepared according to S tan ley (7 5 ), was not s a tis fa c to r y fo r the Mung bean glob­ u lin s even though i t had proved to be a superior b u ffer fo r whey p r o te in s. A comparison o f two typ es o f photographic film was made. The con- t r a s t p ro cess panchromatic film which was recommended fo r electr o p h o r e tic record in g, was compared w ith con trast process ortho film . I t was found th at th e la t e r was th e more d e sir a b le film fo r t h is work sin ce i t produced much sharper and smoother l i n e s . 115 THE PRECIPITATE 62( 22)* P r e c ip ita te 6^(22) ira's d isp ersed 1° 10 m1, o f NHy-ECl "buffer o f pH 9 .2 6 and was d ialyzed in 1000 ml. o f the same "buffer fo r two hours in a r o ta tin g , ou tsid e liq u id d ia ly z e r . A fter equi­ lib riu m was reached, the p ro tein was subjected to electr o p h o r e tic a n a ly sis* The duration o f t h is experiment was 5185 seconds, at p o te n tia l gradien t o f 5.75 v o l t s per c n f T h e photographic record showed two peaks a s can be seen in Figure 15. THE FRACTION Gg(l7) * *n electro p h o resis of 62( 17) one ma*n Pea^ was o b tain ed a s shown in F igures 15 ©rid 17* Seven electr o p h o r e tic an alyses were conducted, four o f which were made w ith a c e ta te b u ffers o f pH's 3*2S, 3 . 90, 4*36 and 4.61 and th ree o f which were made w ith phosphate b u ffe r s o f pH’ s 7«15» 7*48, e le c tr o p h o r e s is . ®2(17) was 4omogeneous ^ 7 7*78* The r e s u lts o f th ese measurements are shown in Table XXXXI and Figure 21, from which a determ ination o f the is o e l e c t r ic point of gave th e value o f pH 5*4. THE FRACTION 62(^4) • *n seven e le c tr o p h o r e tic an alyses o f t h is fr a c tio n one main peak was obtained. Four a c e ta te b u ffers o f pH 3*27$ 3»85» 4 .4 4 and 4.7S and th ree phosphate b u ffers o f pH 7*27 •7*52 and 7*75 were The &2(l4) ^rac<;l on homogeneous by e le c tr o p h o r e sis. used. The m o b ility measurements made w ith th ese seven pH values in d ica ted th a t the is o e le c ­ t r i c p oin t o f t h is fr a c tio n was 5»7» THE FRACTION G ^(n)* (See Figure 18 and Table XXXXI.) The e le c tr o p h o r e tic m ob ility determ ination of t h is f r a c tio n was made w ith both a c e ta te and phosphate b u ffers a t f iv e pH v a lu es: 3 .3 4 , 3 . 80 , 4 .4 0 , 6.15 and 6. 63. A ll p attern s showed one peak, th e r e fo r e i t was a homogeneous f r a c tio n . The pH m o b ility curve o f 6^ ( n ) 116 was a str a ig h t l i n e from which the is o e le c t r ic poin t o f pH 5*0 was thus determ ined. (See Figure 19 and Table XXXXI). THE FRACTION G j^q^. a n a ly se s . This fr a c tio n gave one peak in ele c tr o p h o r e tic M ob ility determ ination o f ^4.(07) was a*a<^® a c e ta te and phosphate b u ffer s with pH valu es! 3 . 32, 3 . 9 0 , 4 .2 1 , 4 .5 2 , 7 .3 0 , 7 . 5 O and 7 . 7 8 . The r e s u lt s o f th e measurements are shown in Table XXXXI and F igure 20 from which a determ ination o f the is o e l e c t r ic point o f 0*4(07) gave the v alu e o f 5 *15* RECONSTITUTION OF MUNG BEAN GLOBULINS. An e le ctro p h o retic study o f re­ c o n s titu tio n o f p u r ifie d Mung bean g lo b u lin fr a c tio n s was conducted. Approximately 0 .^ percent o f each o f the ^ ( 17) * ^2( 14)» ^3 (11) and ®4(07) was u sed . The concentration o f t o t a l p rotein o f the mixture was determined to contain I .70 percent of p rotein (N x 6. 25) . A 10 ml. por­ t io n o f the p ro tein mixture was d ialyzed in 2000 m l. of NH^HCl b u ffer o f pH 9 .2 6 . A fter two hours d ia ly s is the f in a l pH o f the b u ffer was 9*0. An e le c tr o p h o r e tic p attern showed four peaks, two o f which were separate s in g le peaks and two were peaks that overlapped as can be seen in Figure 15. 117 TABLE XXXXI. Globulin F raction ELECTROPHORETIC MOBILITY CALCULATIONS R R e s is t­ ance o f p ro tein s o lu tio n in ohms Run No. IS Ilk 115 116 120 121 119 122 3.28 3.90 4 .3 6 4.61 7.15 7*48 7.7S 2.67 2.07 2.16 l.l4 O.85 1.65 2.62 2.40 1.93 2.03 1.09 0.7S 1.59 2.94 0 .3 0 .3 0 .3 0 .3 0 .3 0 .3 0 .3 .0048937 .0048937 .0048937 .0048937 .0048937 .0048937 .0048937 80 76 68 69 72 72.5 74*5 ° 2 (l4 ) 124 128 126 127 136 130 129 3.27 3.85 4 .4 4 4 .78 7.27 7 .52 7.75 2.19 2.21 1 .5 2 0 .9 2 I .96 2.27 1.69 2.02 2.19 1.45 0.95 1.71 2.45 2.14 0 .3 0 .3 0 .3 0 .3 0*3 0 .3 0 .3 .0048937 .0048937 .0048937 .0048937 .0048937 .0048937 .0048937 81 72.3 65 69*5 78 77 62.7 ^ (ll) 97 96 99 105 104 3.34 3 . so 4.40 6.15 6.63 2.91 2.80 1.21 1.87 2.77 2.60 2.73 1.07 2.06 2.85 0 .3 0 .3 0 .3 0 .3 0 .3 .0048937 .0048937 .0048937 .0048937 .0048937 62.6 70 53*5 7* 78 ^(07) 137 13S 139 l4o 152 142 153 3-32 3.90 4.21 4.52 7.30 7.50 7.7S 2 .6 4 1.75 1.43 1 .1 4 1.65 2.27 1.91 2.42 1.67 l.4 i 1.32 1.67 2.29 1.82 0 .3 0 .3 0 .3 0 .3 0 .3 0 .3 0 .3 .0048937 .0048937 .0048937 .0048937 .0048937 .0048937 .0048937 76*5 70*3 64 68.5 5 8 .4 7^*5 6O.5 *2(17) ^1 ^2 Ascend­ Descend­ in g in g (cm) (cm) <1 *c cro ss a t 1°C. s e c tio n area o f c e l l (cm) 118 Of MUNG BEAK GLOBULIN FRACTIONS Ka i Conduct­ ance o f p ro tein so lu tio n a t 1°C. dlq k .006117 .006439 .007196 .007092 .006797 .006750 .006568 t fs i qk it Current as amp­ eres Time in secon< .004900 .003998 .004663 .002425 .001733 .003341 .005163 .0100 . 0100' •Ol4o .01 *K) .0140 .0118 .0140 7200 9000 10800 7376 3657 7226 8000 72.00 90.00 151.00 103.26 51.20 85.26 112.00 .00604-1 .OO6768 .007528 .007041 .006274 .006355 .007800 .003969 .004487 .003433 .001943 .003689 .004328 .003955 .0100 .0 l 4 l .0142 .0158 .0 l4 l .0 l4 l .o i4 i 7200 7067 9007 6000 7200 7200 6000 .007817 .006991 .009147 .006613 .OO6274 .006824 .005827 .003320 .003710 .005213 .0160 .0155 .0168 .0150 .0150 ♦006397 .006961 .007646 .007149 .OOS379 .006568 .008088 .005066 .003654 .003280 .002443 .004l4S .004473 .004635 .0102 .0121 • 0 l4 l .0152 .0142 .0 l 4 i .0142 h or dLqk p otcu iiial u—u——7" *10-5 i1 0 -5 5.449 5*176 6.485 6.580 6.865 5*827 7*105 6.805 4.443 3.084 2.349 3*385 3.918 4.609 6.805 4.442 3.084 2.349 3-385 3.918 4.609 72.00 99*64 127.90 94.80 101.52 101.52 84.60 5.517 6.944 6.287 7.480 7.491 7.395 6.025 5.513 4.503 2.684 2.050 3.634 4.263 4.674 5.513 4.503 2.684 2.050 3.634 4.263 4.674 9110 10960 10114 7289 7196 145.76 169.88 169.91 109.47 107.94 6.822 7.390 6.122 7.560 7-969 4.682 3.457 1.954 3.393 4.830 4.682 3.457 1.954 3*393 4.830 7200 7200 7200 7200 7200 7244 7200 73*80 87.12 101.52 109.44 101.52 102.14 101.52 5.341 5.794 6.147 7.087 5.649 7.156 5.852 6.865 4.194 3.231 2.232 4.056 4.379 4.533 6.865 4.194 3.231 2.232 4.056 4.379 4.533 v o lt cm A Figure 15. FRACTIONATION SCHEME OF TOTAL GLOBULINS EXTRACTED FROM MUNG BEAN Purified extract in 0.4 NaCl m 1. Membrane-equilibration diluted t o 0 .2 2 2. Precipitate separated by centrifugation _ I Supernatant (0.22 m) m Precipitate G 2 (22 ) 1. Membrane-equilibration diluted to 0.11 M 2. Precipitate separated by centrifugation 1. Peptized in 0.4 m NaCl 2. Centrifuged clear 3. Membrane-equilibration diluted to 0.17 m 4. Precipitate separated by centrifugation Supernatant (0.17 Fraction m) 1. Membrane-equilibration diluted to 0.14 m 2 . Precipitate separated by centrifugation Supernatant (0.14 m ) (not further studied) Supernatant ( 0 .1 1 G 2 (22) Gatm Fraction G 2 (14) Fraction G 3 (11) m) I Membrane-equilibration diluted to 0.07 m 2 . Precipitate separated by centrifugation _____________ G 2 (14) I • Fraction G 4 (07) Supernatant (0.07 m ) (not further studied) Fractions G 2 ( 1 7 ) + G 2 ( i 4 ) + G a o i ) + G reconstituted in ca equal amounts i W DATA ON ELECTROPHORETIC PATTERNS* Buffer Fraction pH Total 9.10 ammonia 9.26 ammonia acetate 3.90 acetate 3.27 3.34 acetate acetate 3.90 9.00 ammonia G 2 (22) G 2 (17) G 2 (14) GaOi) G 4 (07) Recons. P (total) Time secs. 0.20 0.20 0.21 0.21 0.21 0.21 0.20 6500 5185 9000 7200 9110 7200 3600 ( * 0.5 magnification) Potential Concn gradient percent v cm-' 1.55 4.95 2.53 6 75 0.78 4.66 0.25 5.51 6 82 0.55 0.43 5.20 1.70 7.81 G4«*) k 11 Reconstituted ELECTROPHORETIC PATTERNS OF TOTAL PROTEINS FROM MUNG BEAN IN THE PRECIPITATE OF A 0.4 M NaCl EXTRACT DILUTED 1 :4 ASCENDING Figure 16. DECENDING pH 3.19; acetate buffer-*- 0 .2 m NaCl; u = 0.21; 7200 secs; 4.33 volts cm."1; concentration 0.77 per cent. pH 7.50; phosphate buffer; u = 0.03; 1800 secs; 17.05 volts cm.*1; concentration 0.75 p e r c e n t. pH 8.30; borate buffer; u = 0.003; 334 secs; 29.93 ivolts cm r1; concentration 0.77 p ercen t. pH 9.10; ammonia buffer; u = 0.2; 6500 secs; 5.47 volts cm."1; concentration 1.55 p ercen t. Figure 17. ELECTROPHORETIC PATTERNS OF THE FRACTION G 2 (17) A purified fraction of the total globulins extracted with 0.4 m NaCl from Mung bean meal by: (1) membrane-equilibration dilution of the extract to 0.22 m NaCl thus precipitating a fraction called G 2 (22 ) , and (2) repeptization of G 2 (22 ) in 0.4 m NaCl followed by similar dilution to 0.17 m NaCl. This yielded the precipitate designated as fraction G 2 (17 ) . ASCENDING DESCENDING pH 3.28; acetate buffer 4 0.2 m NaCl; u =* 0.21; 7200 secs; 5.44 volts cmr1; concentration 0.78 percen t. - pH 4.36; acetate buffer 4- 0.2 m NaCl; u —0.21; 10800 secs; 6.51 volts cm.-1; concentration 0.78 p ercent pH 7.48; phosphate buffer 4 0.2 m NaCl; u = 0.23; 7200 secs; 5.82 volts cm.-1; concentration 0.68 per cent H----------------------------- 1 f~--------------------------- pH 7.78; phosphate buffer 4- 0.2 m NaCl; u ^ 0 .2 3 ; 8000 secs; 7.10 volts cm.J ; concentration 0.60 p ercen t. Figure 18. ELECTROPHORETIC PATTERNS OF THE FRACTION G2 A purified fraction of the total globulins of Mung bean meal obtained by membraneequilibration dilution of the supernatant fiom precipitate G 2 (17) to 0.14 m NaCl. This yielded the precipitate designated as fraction G i ( u ) . ASCENDING |4 ------------------------------, DESCENDING \----------------------------- H pH 3.27; acetate buffer + 0.2 m NaCl; u « 0.21; 7200 secs; 5.51 volts cmr'; concentration 0.25 percen t. H------------------------------1 I----------------------------- H pH 4.44; acetate buffer 4- 0.2 m NaCl; u - 0.21; 9000 secs; 6.28 volts cm r1; concentration 0.28 percent. pH 7.27; phosphate buffer 4- 0.2 m NaCl; u = 0.23; 7200 secs; 7.49 volts cm r1; concentration 0.35 percent. pH 7.75; phosphate buffer 4- 0.2 m NaCl; u - 0.23; 6000 secs; 6.02 volts cmr1; concentration 0.34 per cent. Figure 19. ELECTROPHORETIC PATTERNS OF THE FRACTION G 3 (n) A purified fraction of the total globulins of Mung bean meal obtained by membraneequilibration dilution of the supernatant from precipitate G 2 (22 ) to 0 .1 1 m NaCl. This yielded the precipitate designated as fraction Gs(ii). ASCENDING DECENDING pH 3.34; acetate buffer +■ 0.2 m NaCl; u = 0.21; 9110 secs; 6.82 volts cm r1; concentration 0.55 per cent. pH 3.80; acetate buffer +- 0.2 m NaCl; u = 0.21; 10960 secs; 7.60 volts cm.'1; concentration 0.74 per cent. h — -------------------------1 l----------------------------- h pH 6.15; phosphate buffer 4- 0.2 m NaCl; u = 0.23; 7296 secs; 7.56 volts cm r1; concentration 0.33 per cent. pH 6.63; phosphate buffer + 0.2 m NaCl; u = 0.23; 7196 secs; 7.96 volts cm."1; concentration 0.62 per cent. Figure 20. ELECTROPHORETIC PATTERNS OF THE FRACTION G 4 <07> A purified fraction of the total globulins of Mung bean meal obtained by membraneequilibration dilution of the supernatant from precipitate G 3 (ii) to 0.07 M NaCl. This yielded the precipitate designated as fraction G 4 (07) . ASCENDING DECENDING pH 3.32; acetate buffer +■ 0.2 m NaCl; u » 0 . 2 1 ; 7200 secs; 6.82 volts cm.-1; concentration 0.43 per cent. U ----------------------------- 1 I----------------------------- M pH 4.21; acetate buffer + 0.2 m NaCl; u=«0.21; 7200 secs; 6.14 volts cm.-1; concentration 0.43 p ercen t. f - ---------------------------- 1 I---------------------------- H 7.05; phosphate buffer 4- 0.2 m NaCl; u — 0.23; 7 2 0 0 secs; 7.71 volts cm r1; concentration 0.43 percent. pH H----------------------------- 1 I----------------------------- M 7.78; phosphate buffer + 0.2 m NaCl; u = 0.23; 7 2 0 0 secs; 5 .8 5 volts cm r1; concentration 0.36 p ercent. pH Figure 21. pH — MOBILITY CURVES OF THE PURIFIED FRACTIONS OF TOTAL GLOBULINS FROM MUNG BEAN u 8 Fraction G in* 7 6 5 4 3 2 1 0 j. 1 2 -2 3 4 5 6 J. u T 9 4 0 T 7 T 8 I9 U 9 T 4 T 0 6 T 7 T 8 r9 8 7 Fraction G 4(ot> Fraction G um 6 5 4 3 2 1 0 1 2 3 4 5 6 pH pH 126 SUMMARY AND CONCLUSION R ela tio n sh ip s Between the S o lu b ility o f Mung Bean P r o tein and th e Nature and Concentration o f S olven ts The r e s u lt s obtained from th e study are shown in Tables VI through XIV and F igure 2 . A* The s a l t s o f strong a c id s and b a ses, such as those o f ch lorid e and s u lf a t e , showed n ea rly the same e ffe c tiv e n e s s in p ep tizin g approxim ately equal amounts (72 percent o f the t o t a l n itrogen on a dry weight b a sis ) from Mung bean m eal. The s a l t s o f phosphates, carbonate and s u l f i t e a ls o showed p a r a lle l e f f e c t iv e n e s s in p ep tiz in g n early equal amounts, S3 to S3 percent o f th e t o t a l n itro g en from the same samples. B. row. The range I t was noted very c lo s e to 0.4M. o f io n ic strength o f the n eu tral so lv e n ts was very narth a t the h ig h e st p ep tizin g power o f NaCl s o lu tio n was On th e oth er hand th e range o f io n ic stren g th o f so lu ­ t io n s o f phosphate, carbonate and s u l f i t e was much broader. C. Since th e d e f in it io n o f g lo b u lin r e fe r s to the s o lu b il it y in neu­ t r a l s a lt s o lu tio n , i t i s obvious th at the maximum amount o f Mung bean g lo b u lin p ep tized by ch lo r id e s and s u lf a t e s was 72 p ercen t. The q uestion a r is e s as to what kind o f p r o te in (beyond the 72 percent) i s p ep tized by th o se a lk a lin e s o lv e n ts (phosphate, carbonate and s u l f i t e ) . Since t h is study was devoted to g lo b u lin s , oth er p ro tein s were not stu d ied in d e t a il . However, th e se data support the p ro p o sitio n th at there i s a v a r ia tio n in th e nature or kind o f p r o te in p ep tized by these two groups o f s o lv e n ts . D. I t was noted th a t the sodium ch lorid e so lv en t y ie ld e d a curve w ith 127 a ste e p s lo p e , part o f ifaich was n ea rly v e r t ic a l. The s ig n ific a n c e o f t h i s typ e o f curve has "been p oin ted out p rev io u sly in th a t i t not only i l l u s t r a t e s the s o lu b i l i t y "behavior o f Mung bean p ro tein in t h i s so lv en t but i t a ls o r e v e a ls a scheme by which the p ro tein might be is o la t e d by low erin g th e con cen tra tio n o f the s o lu te by d ilu tio n . F actors th a t In flu en ce the Degree o f P e p tiza tio n o f P rotein A. Table XXI and Figure 5 demonstrate the e f f e c t o f time and sample- so lv e n t r a t io s and show th a t in a l l in sta n ces an in crea se in ex tr a c tin g tim e in crea sed the amount o f t o t a l n itro g en p ep tized from th e samples. Of th e f i v e sam ple-solvent r a t io s employed, the one o f 5s 100 gave th e high­ e s t v a lu e o f p e p tiz a tio n and th a t o f 15:100 gave the lo w e st. When the S-S-R was sm all (5 :1 0 0 ), time had l e s s in flu en ce upon p e p tiz a tio n . On th e other hand when the S-S-R was la rg e (1 5 :1 0 0 ), tim e had more in flu en ce upon p e p tiz a tio n . B. Table XXIII and Figure 6 showed th at the p a r t ic le s iz e a f fe c te d th e degree o f p e p tiz a tio n to a great e x te n t. The 60 mesh s iz e gave about 20 p ercen t g rea ter y ie ld o f n itro g en over the 40 mesh s iz e which in turn gave about 20 percent g r ea ter y ie ld o f n itrogen over the 20 mesh s iz e . C. Table XXVI and Figure 7 show no appreciable d iffe r e n c e between the m echanical and hand s t ir r in g . There was a great d iffe r e n c e between th e f i r s t e x tr a c tio n and the second and th ir d e x tr a c tio n s . When the S-S-R was 1 0:100, 60 p ercent o f the n itrogen was p ep tized by th e f i r s t extrac­ t io n , 9 percent by th e second and 3 percent by the f in a l e x tr a c tio n w ith 27 p ercen t o f the n itro g en remaining in th e resid u e. 128 D. The e f f e c t o f temperature upon the degree o f p e p tiz a tio n from o i l f r e e and n o n -o il fr e e samples i s somewhat complex. From Table XXVIII and F igu re & i t appears th at o i l f r e e samples g en e r a lly y ie ld e d higher v a lu e s than th e n o n -o il fr e e samples except th at w ith lower con cen tration s and h ig h er tem peratures rev erse r e s u lt s were observed. Using n o n -o il f r e e samples th e temperature o f e x tr a c tio n was shown to have l i t t l e or no in flu e n c e upon the degree o f p e p tiz a tio n below U5°C. Above **5°C. there was a decrease in th e degree o f p e p tiz a tio n in both th e o i l f r e e and the n o n -o il f r e e sam ples. The downward slop e o f the curves in a l l in sta n ces above 45°C. may be due to th e h eat coagu lation o f some o f the p ro tein m a te r ia l. When u sin g low con cen tration s of s a l t , i t seems th at the pres­ ence o f o i l in th e natural sample gave some p ro tectio n a gain st heat coag­ u la t io n o f p r o te in a t higher tem peratures. The P r e c ip ita tio n o f P rotein as E ffected by D ilu tio n Figure 10 shows th e sedim entation ra te o f Mung bean g lo b u lin when a s e r ie s o f ten d iffe r e n t r a te s o f d ilu tio n was made. A. I t wa3 in te r e s t in g to note the p r e c ip ita tin g behavior o f Mung bean g lo b u lin when one volume o f 0.4M NaCl ex tra ct was d ilu te d w ith four volumes o f d i s t i l l e d water ( f in a l concentration 0.08M); the ra te o f sed­ im en tation was f a s t e r and the p r e c ip ita te was more compact than in any oth er d ilu tio n s in th e s e r ie s . B. The s o lu b i l i t y range o f Mung bean g lo b u lin was thus concluded to be between O.M-M NaCl a s th e maximum s o lu b ilit y con centration and 0.08M NaCl a s th e p r e c ip ita t in g con cen tration . 129 0* I h is le a d s to a con sid eration o f th e g lo b u lin behavior a t d ilu ­ t io n s g re a te r than 1 :4 . In Figure 10 i t was shown th a t a s th e d ilu tio n r a t io s were in crea sed (to th e Number 10 C7lin d e r ) t the volume o f the pre­ c i p i t a t e s were correspondingly la r g e r . Since th e amount o f p ro tein in a l l c y lin d e r s was the same, why did the volume o f the p r e c ip ita te in crea se a s th e d ilu tio n r a tio increased? I t i s obvious th at under such co n d itio n s th e hydration i s g rea ter and probably the amount o f adsorption o f albumin i s a ls o g r e a te r . This i s supported by data in Table XXX. Therefore, a g r ea te r d ilu tio n fo r p r e c ip ita tin g g lo b u lin must not be employed because i t may le a d to e x c e ssiv e o c clu sio n o f other p ro tein s (albumin) from the s o lu tio n . D. The removal o f albumin from g lo b u lin was accom plished by the d ilu tio n method. There i s no e x is tin g method o f sep aration o f albumin from g lo b u lin which i s superior to p r e c ip ita tio n o f the g lo b u lin by d ilu ­ tio n , le a v in g the albumin in the so lu tio n . E. From th e above d isc u ssio n i t w i ll be seen th at the p r e c ip ita tio n e f f e c t e d by th e 1 :4 d ilu tio n rep resen ts the g lo b u lin fr a c tio n o f Mung bean. Hence Table XXX ( d ilu t io n 1:4) rep resen ts the more accurate fra c­ tio n a l v a lu es: g lo b u lin n itrogen 50*5 p ercent, albumin n itrogen 36*7 percent and N.P.N. 1 2 .5 p ercen t. The globulin/album in r a tio was I .37 and th e p r o te in n itro g e n /n o n -p ro tein n itrogen r a tio was 6.97* These r a t io v a lu e s may be used a s a means o f ch a ra cteriza tio n o f the sp e c ie s from which th e p r o te in s are secured. 130 C onditions E s se n tia l fo r the I s o la tio n o f and Upon Which the C haracterization o f Mung Bean G lobulins Depend The is o la t io n o f p ro te in s in th e ir natural co n d itio n i s s t i l l in a s t a t e o f development. The nature and the q u an tity o f p ro tein p ep tized from d iffe r e n t sources has varied from ex tra ct to ex tra ct sin ce much de­ pends upon the te ch n ic s employed. In order to e s ta b lis h a method by which the r e s u lt s o f t h is study can be reproduced, the con d ition s essen ­ t i a l fo r the preparation o f Mung bean g lo b u lin s must be considered. 1. Nature o f so lv en t: A so lu tio n o f n eu tral s a lt should be u sed. 2. Io n ic stren g th o f so lv en t: should be used fo r p e p tiz a tio n . A so lu tio n o f maximum io n ic stren gth However, a so lu tio n o f io n ic stren gth above th e maximum must be avoided sin ce e x c e ssiv e con centration causes o c c lu sio n o f p r o te in s other than g lo b u lin . C ontrariw ise, so lu tio n s o f low er than p r e c ip ita tio n con cen tration should be avoided to prevent den atu ration o f p ro tein . 3. S o lu b ilit y range: A narrow s o lu b ilit y range req u ires l e s s d ilu tio n to e f f e c t p r e c ip ita tio n . 4. P a r tic le s iz e o f sample: The sm aller the p a r t ic le s iz e , the g r ea ter the y i e l d . 5. Sam ple-solvent ra tio* e x tr a c tio n . A la rg e r a tio r e s u lt s in an i n e f f ic i e n t On the other hand, a small r a tio g iv e s e f f i c i e n t e x tr a c tio n but may r e s u lt in a volume o f ex tra ct which i s d i f f i c u l t to handle when d ilu te d fo r p r e c ip ita t io n . Therefore, an e f f i c i e n t and convenient S—S-E must be chosen. b. E x tra ctio n tim e: A period o f time that i s o f lon g duration 131 must be avoid ed to prevent contam ination, denaturation, growth o f micro-* organism s and ferm en tation . A period o f tim e th a t i s too short r e s u lt s in i n e f f i c i e n t e x tr a c tio n . 7* E x tra ctio n temperature: a lly e ffic ie n t. Room temperature o f 20 to 25°C. i s usu­ Higher temperatures are apt to cause denaturation . On th e contrary low temperature, which might appear d e sir a b le , decrease the r a te o f p e p tiz a tio n and may r e s u lt in p r e c ip ita tio n o f p ep tized p r o te in . 8. A g ita tio n : O ccasional hand s t ir r in g was as e f f e c t iv e fo r extrac­ t io n as m echanical s t ir r in g . High speed and v ib r a tio n a l a g ita tio n may cause d en aturation . 9. S u ccessiv e ex tra ctio n : This i s th e only way to secure an ex­ h a u stiv e e x tr a c tio n o f a sample. 10 . Separation o f resid u e: most d e s ir a b le . The ordinary standard c en trifu g e seems A Sharpies cen trifu g e should not be employed fo r t h is type o f work. 11. P r e c ip ita tio n : TShen p r e c ip ita tio n i s e ffe c te d by d ilu tio n , water must be added to the ex tra ct a t a slow ra te w ith continuous m ixing. 12. Removal o f other p ro tein s: This can be achieved by repeated d isp e r s a l o f the p r e c ip ita t e in s a lt so lu tio n follow ed by p r e c ip ita tio n by d ilu tio n . R otatin g O utside Liquid D ia ly s is Apparatus The development o f the r o ta tin g o u tsid e liq u id d ia ly s is apparatus f o r rap id membrane e q u ilib r a tio n d ilu tio n proved to be e f f i c i e n t fo r pre­ c i p it a t io n , fr a c tio n a tio n and p u r ific a tio n o f p ro tein . The e s s e n t ia l char­ a c t e r i s t i c s o f t h is type o f d ia ly z e r are: 1) the container which r o ta te s 132 a t a speed o f b 2 r .p .m ., 2) the cellophane tu.hi.ng only two th ir d s f u l l w ith p r o te in s o lu tio n in ste a d o f com pletely f i l l e d to th e l e v e l o f the d ia ly z in g w ater, 3) the cellophane tubing suspended e c c e n tr ic a lly in th e liq u id in th e tank in order to in crease a g ita tio n o f the e n tir e sys­ tem when th e con tain er i s in motion* As a r e s u lt o f th e movement, the d iffu s a b le io n s adjacent to both s id e s of the membrane w all are co n sta n tly removed w ith a change o f con cen tration and thus e q u ilib r a tio n i s e ffe c te d in a sh o r ter tim e. Some o f the s p e c if ic a p p lic a tio n s o f t h is type o f d ia ly z e r are: 1* P r e c ip ita tio n o f p ro tein by in d ir e c t d ilu tio n was accomplished when a measured volume o f p ro tein so lu tio n was p laced in the cellophane tubing and immersed in a measured volume o f d i s t i l l e d water in the di&ly z in g tank. The p r o te in p r e c ip ita te d when the d esired con cen tration o f th e so lv en t was reached. In d irect d ilu tio n fo r p r e c ip ita tin g p ro tein p reven ts denaturation o f th e p ro tein by avoiding d ir e c t con tact w ith an ex cess amount o f water. 2. An in d ir e c t, slow step w ise membrane e q u ilib r a tio n d ilu tio n tech­ n ic w ith r o ta tin g o u tsid e liq u id d ia ly s is was employed fo r fr a c tio n a tio n and p u r ific a t io n o f p r o te in . This was accomplished when the p ro tein in O.hM NaCl s o lu tio n in the cellophane tubing was suspended in the same con cen tra tio n o f NaCl s o lu tio n in the d ia ly zin g tank. The t o t a l volume o f both were measured and then a measured volume o f water was slow ly added to the tank* o b tain ed . When eq u ilib rium was reached, a d esired con cen tration was The Mung bean g lo b u lin was separated in to i t s component fra c­ t io n s by t h is te c h n ic . 133 3* This d ia ly z e r can a lso he used fo r continuous d ia ly s is to re­ move a l l d iffu s a b le io n s. Continuous change o f d ia ly z in g mater i s con­ ducted in a very convenient way. The F ra ctio n a tio n o f Mung Bean Globulin and the I s o la tio n o f Gg, and G^ U sing th e membrane e q u ilib r a tio n d ilu tio n procedure fo r low ering th e s a l t ion con cen tration o f the p ro tein so lu tio n , the f i r s t fr a c tio n a l p r e c ip ita t io n o f Mung bean g lo b u lin occurred a t 0.22M NaCl. i t a t e was d esign ated as fr a c tio n p r e c ip ita te 02( 22 )* from th e p r e c ip ita t e ®2(22) was The cen­ was fu rth er d ilu te d to 0.07M NaCl and a th ird p r e c ip ita t e was obtained. 64(07)* c ® ntrifugate sim ila r ly to 0.11M NaCl and y ie ld e d a p r e c ip ita t e which was designated as fr a c tio n tr if u g a t e o f th e p r e c ip ita te This precip­ This fr a c tio n was design ated a s The n itro g en d is tr ib u tio n in terms o f percentage o f t o t a l glob­ u lin in th ese fr a c tio n s are 40.95t **9*09 and 3*90 ^4 ( 07 ) r e s p e c tiv e ly . G2 ( 22)» G3 ( l l ) and The e le ctro p h o retic p a ttern s o f th ese fr a c tio n s showed th a t the 62( 22) con^a ^ne^ two components whereas the o th ers were s in g le components. Development o f a D isso lu tio n Process for R esolving the G^ Complex The p r e c ip ita t e ®2(22) W&S th e d is s o lu tio n te c h n ic . to fu rth er fr a c tio n a tio n by I t was f i r s t p ep tized in a small volume o f 6.4M NaCl s o lu tio n in order to keep both the p ro tein concentration and the d i e l e c t r i c constant o f the medium h igh . This tech n ic decreased the in te r a c tio n o f components o f d iffe r e n t sp e cie s and other charged substances and th e decom position o f any complex was p o s s ib le under such an environment, 134 I t was found th a t th e fr a c tio n a l p r e c ip ita te , @2(22)* was a corD3P^ex two g lo b u lin components and some pigmentary m aterial which were hound to g eth er and c o -p r e c ip ita te d a t the concentration o f 0.22M NaCl. Such complex form ation may "be expected to a f f e c t the s o lu b i lit y o f th e in d i­ v id u a l components. The d is s o lu tio n procedure fo r the decom position o f such a complex was sim ple to perform but d i f f i c u l t to p e r fe c t. Since the is o la t io n o f each o f the s in g le components from a p ro tein mixture was the primary o b je c t o f t h is study, con sid erab le time was devoted to t h is problem. The fr a c tio n a l p r e c ip ita te ®2(22) was P©P*ized in 0.4M NaCl was introduced in to the cellophane tubing which was then suspended in d i s t i l l e d water in the con tain er. The volume o f d i s t i l l e d water was ten tim es g r ea te r than th at of the p ro tein so lu tio n in the cellophane tubin g. P r e c ip ita tio n occurred in the cellophane tubing w ith in 15 m inutes, but a p erio d o f two hours was allow ed fo r sedim entation o f th e f a s t p recip ­ i t a t i n g components. The f a s t p r e c ip ita tin g components, pigmentary sub­ stan ce combined w ith p r o te in , were c o -p r e c ip ita te d and appeared a s dark, dense m a teria l in th e bottom of the tubing. The true g lo b u lin appeared a s w hite f l u f f y p r e c ip ita t e (amorphous in form) which was suspended throughout the supernatant and was separated from the dark p r e c ip ita te by d eca n ta tio n . The d is s o lu tio n procedure was repeated w ith th e dark p r e c ip ita t e u n t il a l l th e g lo b u lin was relea sed from the pigmentary mater­ ia l. Further fr a c tio n a tio n o f the d is s o lu tio n product r e s u lte d in one component p r e c ip ita tin g a t 0.17M NaCl and designated a s ®2(l7)» 135 other p r e c ip ita tin g a t O.llfld NaCl and c a lle d p a tte rn s showed th at the ®g(x7) an<1 * E lectrop h oretic ^ 2(l4) were ®lnS le components* Gs® of, Magi mom P r e c ip ita tio n Concentration and In c ip ien t P r e c ip ita tio n C oncentration to Control the P o r ific a tio n o f the Globulin F raction s The fr a c tio n a tio n and p u r ific a tio n procedure p reviou sly employed fo r th e i s o la t io n o f g lo b u lin fr a c tio n s were accomplished by a d ju stin g them to th e ir maximum p r e c ip ita tio n concentration "m .p.c.". The r e s u lt ­ in g produ cts, so fa r as the homogenity of th e p ro tein was concerned, were fr e e d o f g ross contamination on ly. I t was n ecessary to u se s p e c if ic molar co n cen tration s o f the so lv e n t, both the maximum p r e c ip ita tio n co n cen tra tio n and the in c ip ie n t p r e c ip ita tio n con cen tration " i . p . c . 11, to co n tro l th e p r e c ip ita tio n o f each fr a c tio n and elim in ate tra ce con­ tam ination and secure the d esired s ta te o f p u rity . The maximum p r e c ip ita tio n concentrations o f the Mung bean g lo b u lin f r a c tio n s ®2(l7)» ^ (lU )* &3 ( l l ) 811,1 ^ ( 07 ) Were c a r e fu lly determined to be 0 .1 7 , 0 .1 ^ , 0 .1 1 and 0.07M NaCl r e s p e c tiv e ly . Their in c ip ie n t p recip ­ i t a t i o n co n cen tra tio n s were accord in gly 0 .1 3 , 0 .1 5 , 0 .1 2 and 0.08M NaCl. By means o f e le c tr o p h o r e tic a n a ly s is , th e p attern s show th a t four in d iv id u a l s in g le g lo b u lin components have been is o la t e d in supposedly undenatured s t a t e from Mung bean m eal. The problems of g lo b u lin in d iv id ­ u a l i t y , g lo b u lin d e f in it io n and g lo b u lin is o la t io n have been stu d ied c r i t ­ i c a l l y and th e author has attem pted to fo llo w the ad vice of Osborne in k eep in g h im se lf from a l l preconceived notion s a s to what c o n s titu te s ortho­ dox or non-orthodox te c h n ic s. 136 Osborne s ta te d y ears ago "we can, fo r th e p resen t, tr e a t a s in d i­ v id u a l p r o te in s only th ose products whose ex ten siv e fr a c tio n a tio n has g iv en no evidence th a t a mixture i s being d e a lt w ith , and we must await new methods o f study b efore anyone o f th ese p ro tein s can be accep ted as tru e chem ical e n t it ie s ." !Ehe Mung bean g lo b u lin s h erein described have shown such constancy o f com position and p ro p erties th at we f e e l j u s t i f i e d in co n sid erin g them as substances o f reasonably d e f in it e ch aracter. 137 LITERATURE CITED Am. 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Gortner, R .A ., O u tlin es o f b io ch em istry ., John Wiley and Sons, I n c ., N .Y ., p. 352 (1 9 4 9 ). 60. Ferry, R.M., Cohn, A .J. and Newman, E .S ., J . Am. Chem. S o c ., 58: 2370 (1 9 3 6 ). 61 . Osborne, T.B*, J . Am. Chem. S o c., 24 : 28 (1902). 62. T is e liu s , A ., Trans. Faraday S o c., 22; 524 (1937)* 63* Abramson, H .A ., Moyer, L .S . and Gorin, M. H ., E lectrop h oresis o f P r o te in , Reinhold Publ. Co., N .Y ., p. 57 (1942). 64. Longsworth, L. G., Chem. R ev., ^0: 323 (1942). 65. Longsworth, L. G-., Ind. Eng. Chem. A n a ly tica l Ed., 18.: 219 (1 9 4 6 ). 140 66. Moore, D.H. and White, J.TJ., Rev. o f S c i. Instrum ents, 12.: 700 (1 9 4 8 ). 67. Sorensen, S .P .L ., Compt. rend. tra v . la h ., CarlsLerg, Vol. 12: (1917)* 68* Longsworth, L.G-. and Maclnnes, D .A ., Chem., R ev., 24: 27 (1939)* 69* D avies, C.W., The co n d u ctiv ity o f s o lu t io n s ., John Wiley and Sons, I n c ., K .Y ., p. 6 (1 9 3 3 ). 70. Karon, M .J., Adams, M.E. and A ltsch u l, A.M., J . Rhys. C o llo id Chem., 54: 56 (1 9 5 0 ). 71. Cohn, E .J ., J . Am. Chem. Soc*, 52.* 696 (1928). 72. Green, A .A ., J . Am. Chem. S o c., 55/ 2331 (1933)• 73* D an ielson , C .E., Acta Chem. Scand., 4: 762 (1950). 74. Ir v in g , Gr.W., Eontaine, T.D. and Warner, R .C ., Arch. Bioehem., I l ^75 ( 1 9 ^ ) . 75* S ta n ley , W.G., Whitnah, C.H. and Andrews, A .C ., Dairy S c i., (1 9 5 1 ). 265 ADDENDA lH2 A HEW APPARATUS FOR FREEZINGwDEHYURATION AID TECHNIQUE FOR HIGH VACUUM MANIPULATION This apparatus i s designed and con stru cted fo r drying p ro tein s and "biological m a te r ia ls. A Figure 1. Freezing-dehydration tank A. Side section view B. Top section view a. freezing chamber, b. center pipe, c. freezing cone, d. 250 ml centrifuge bottle, e. adapter, f. rubber ring Figure 2. a adapter b U clamp c setting screw d wooden block to mechanical pump Figure 3. Assembly of apparatus C. mechanical pum p ~\ lD 1 A. Freezing-dehydration tank D. cold trap E. McLeod gauge B. mercury condensation pump F. dry air inlet G. two way stopcock A NSW APPARATUS 3P0R PREEZING-DEHYDRATION AND TECHNIQUE EOR HIGH VACUUM MANIPULATION By rapid fr e e z in g and dehydration from frozen s ta t e under low pres­ sure b io lo g ic a l m a teria ls can he dried to a very low m oisture content w ithout shrinkage in volume ( l ) . This technique has been u t i l i z e d in th e f ix a t io n o f t is s u e in m orphological stu d ie s and in h isto -ch em istry (2 ,3 )* In a recen t p u b lica tio n (U) th e u se o f freezin g-d eh yd ration method fo r the determ ination o f m oisture content o f dehydrated v eg eta b les was described* I t in volved the a d d itio n o f a la r g e amount o f water to a weighed sample o f dried v eg eta b le, fr e e z in g and drying from the frozen s t a t e and com pletion o f the drying in a vacuum oven or vacuum d esicca to r in the presence o f an e f f i c i e n t water absorbent. I t was observed th at th ere was a marked in crea se in the drying r a te o f samples o f dehydrated v e g e ta b le s when f i r s t satu rated w ith water, then frozen and subsequently d ried in vacuo from the frozen s ta te to a low m oisture con ten t. crease in the drying r a te may be a ttr ib u te d to two fa c to r s . \ The in ­ One i s an in c r e a se in volume o f th e fro zen t is s u e and the other i s the in crease in p o r o s ity . I t a lso was found th at the com pletion o f the drying in a vacuum oven a t s u ita b le temperatures produced the same r e s u lt s a s drying in an evacuated d e sic c a to r a t room temperature. To meet the requirem ents o f t h is technique a new apparatus fo r fr e e z ­ in g-d eh yd ration has been designed and constructed in accordance w ith the p r in c ip le s here d escrib ed . The d esign o f t h is freezin g-d eh yd ration vacuum 1^5 tank resem bles a Dewar f la s k (double-w alled stru ctu re) (Figure 1A) ♦ The open chamber (a) con tain s th e fr e e z in g m ixture, dry ic e suspended in 95 percen t e th y l a lc o h o l, which m aintains a low temperature o f - 72°C. The chamber serv es as a cold bath fo r the quick fr e e z in g o f samples. This chamber w i l l h old s ix 250 ml. cen trifu g e b o t t le s . For the drying procedure th ese b o t t le s are a ttach ed to the tank by s ix adapters which are d is tr ib u te d around th e s id e s o f the outer tank (Figure IB ). When th e a i r i s evacuated, th e tank and the s ix b o t t le s become one u n it under th e same low p ressu re. In the drying op eration water vaporizes from the samples in th ese b o t t le s under the low pressure and i s d iffu se d out o f them and condenses upon th e inner cold w all (Figure lAc) as long as the fr e e z in g mixture i n the chamber remains a t a low temperature. The vap orization continues a s lon g as th ere i s a grea ter concentration o f water vapor in the b o t t le s than in the tank. This r e s u lt s in a d iffe r e n c e in d iffu s io n pressures between th e b o t t le s and the tank. Since the greater con cen tration of water m olecu les i s in the b o t t le s , t h is determines the d ir e c tio n o f d if­ fu sio n ; i . e . from the b o t t le s to the tank (5)» The tank i s so constructed th at when i t i s in op eration i t m aintains the d iffu s io n pressure a t prac­ t i c a l l y zero in th e tank because o f the rapid condensation o f water vapor to s o lid ic e upon th e cone o f the lower part o f the inner tank (Figure lA c ). The volume o f space in the vacuum tank i s four tim es the volume o f the b o t t le s which a ls o co n trib u tes to the movement o f water vapor from the b o t t le s in to th e tank. Hence, the d ir e c tio n o f the d iffu s io n o f water vapor from th e b o t t le s to the tank continues co n sta n tly u n t il the water 146 vapor i s esdiausted in the h o t t le s and the samples are dried* One p r in c ip le o f an e f f i c i e n t freezin g-d eh yd ration apparatus i s th e estab lish m en t o f a steep concentration gradient in the system* How­ ev e r, th e r a te o f d iffu s io n i s a lso in flu en ced by th e d ista n ce through which th e d iffu s in g m olecules must travel* These two fa c to r s are compo­ n en ts o f what may he c a lle d the concentration gradient* This p r in c ip le has guided the con stru ction o f t h is freezing-deh yd ration vacuum tank and has met th e requirement o f e f f e c t iv e ex tra ctio n o f water vapor from the samples in the h o ttle s* CONSTRUCTION OF TANK.— The freezing-dehyd ration vacuum tank i s made from the lower part (IS inches) of a s t e e l gas cylin d er (8 3 /4 in ch es d iam eter). The th ick n ess o f the outer w all i s 3/16 inch w hile the inner tank i s made o f sh eet s t e e l 1 /8 inch th ic k . The upper part o f the inner tank i s r o lle d in to a c y lin d er, 8 inches in h e ig h t, 7 1 /2 in ches in d i­ ameter; th e lower part i s r o lle d in to a cone, 6 inches in h eigh t and th ese are welded to g eth er. through th e t ip o f the cone. (Figure lA ,b ) . A 3 /4 inch pipe 20 inches in len gth i s welded The tank i s evacuated through t h is pipe The two tanks are welded together a cross the top and the u n it i s thus sim ila r to a double-w alled Dewar f la s k . Six openings are eq u a lly d is tr ib u te d around the outer tank 10 in ch es from the top edge. These openings provide fo r the connection o f s ix 250 ml. cen trifu g e bot­ t l e s (F igu re IB ). Adapters fo r th e b o t t le s are welded a t th ese openings. They are made o f 1 1 /2 inch pipe and cut 1 1 /4 inches in len g th . Two h o le s are threaded in to each p ie c e o f pipe on op posite sid e s a t the cen ter fo r f i t t i n g 1 /2 inch lo n g , 1 /4 inch b a ll head b o lts (Figure 2 a ). The lk 7 b o lt s are brazed in to p la ce on the in s id e o f the adapters* U clamps o f s t r ip iro n (F igure 2b) which hook over the two b o lts on the adapter are a d ju sted by a s e t screw a t the other end o f the U clamp to h old th e cen­ t r ifu g e b o t t le s sta tio n a ry (Figure 2c)* A wooden block a c ts a s a cushion between th e bottom o f th e b o t t le and the s e t screw. The adapters are b eveled a t a 90° angle on th e in s id e to accomodate a rubber ring* which f i t s over th e neck o f th e b o t t le (Figure l A , f ) . This provides an a ir ­ t ig h t connection between the b o t tle s and th e tank* B efore w elding, the in sid e and o u tsid e o f the two tanks were p ainted w ith red o xid e primer* Two coats o f red oxide primer were app lied a f te r the u n its were welded to g eth er and th e tank was evacuated w hile the p ain t was wet* When in op eration , the tank i s wrapped w ith an inch th ic k wool blan­ k et fo r in su la tio n * ASSEMBLY OF APPARATUS AND LOW PRESSURE PRODUCTION The e n tir e apparatus fo r freezin g-d eh yd ration (Figure 3) c o n s is ts o f th e fr e e z in g dehydration tank, A, th e mercury condensation pump B, the mechanical pump C, and th e cold trap D which i s connected between the mercury pump a n d the mechanical pump to trap water vapor and mercury vapor. compose the a c t iv e p arts o f the apparatus. These The McLeod guage E and the dry a i r i n l e t F are used in freq u en tly and can be is o la t e d from the more a c t iv e p a rts by the stopcock a t G* The e n tir e assembly occupies a hor­ iz o n ta l space on a bench or on a movable ta b le o f two by four feet* * Cenco F r iz z e ll holder No. 18107 lUg This en ab les th e u n it to he placed on a convenient working le v e l* s im p lic ity o f t h is u n it e lim ina te s op eration al d i f f i c u l t i e s . The I t i s easy to evacuate t h is sim ple system and to keep i t evacuated* The ca p a city o f the mechanical pump should he h igh . Capacity i s measured hy th e tim e required to evacuate a given volume to a d esired pressure* This time i s in proportion to the volume o f the system. A comparison o f th e cap acity o f two mechanical pumps w ill i l l u s t r a t e th ese p o in ts (Table I ) . The data in Table I in d ic a te s that a la rg er volume req u ires more tim e p ro p o rtio n a lly fo r proper evacuation. I t a ls o fur­ n ish e s inform ation th at a proper choice o f the mechanical pump i s impor­ ta n t in o b ta in in g th e b est production o f low pressure. With th e h elp o f a mercury d iffu s io n pump a low pressure o f 10“ -* i s p o s s ib le when a f a s t mechanical pump i s used (Table I I ) . Table II i l l u s t r a t e s th a t the e f f ic ie n c y o f a mercury pump i s dependent upon a '•backing p ressu re” produced by a mechanical pu$p. The data in Table I I in d ic a te th at a mercury d iffu sio n pump w i ll produce a h igh er vacuum when matched w ith an e f f i c i e n t mechanical pump. This i l l u s t r a t e s th a t the u se o f mercury and mechanical pumps togeth er are n ecessa ry to produce th e d esired low pressure and overcome the d e fe c ts o f th e system. Although mercury d iffu s io n pumps backed by f a s t mechanical pumps are p referred fo r h igh vacuum work and have been recommended by many in v e s t ig a to r s (7) * they do have one disadvantage. The vapor pressure due to th e mercury i s so h igh i t tends to d estroy the vacuum in the sys­ tem. I f a pressure o f the order o f 10"^ micron i s to be m aintained in 149 TABLE I COMPARISON OP THE CAPACITY OP TWO VACUUM PUMPS (6) D uo-seal pomps No. 1405 1 /3 h .p . No. 1400 1 /4 h .p . Low p r es, o b ta in a b le 0*05 micron Hg 0 .1 micron Hg Pree a ir ca p a city 33*4 l i t e r s per min. 21 l i t e r s per min Evacuated Volume in L ite r s and Time in Minutes Pressure 5 1. 10 1 . 20 1. 30 1 . 50 1. 5 l. 10 1 . 20 1 mm. Hg min. min. min. min. min. rain. min. min. .65 1 .5 3 .0 4 .3 7 .0 1 .4 2.8 5 .8 101 10° 1 .2 2 .3 4 .6 7 .0 11.5 2.3 4 .5 9 .0 10”1 1 .7 3 .5 7*0 10.8 1 7 .2 3.0 6 .5 1 3 .0 1 0 -2 2 .5 5 .0 9*6 l4 .g 24.0 4.7 9 .5 1 9 .0 10” 5 3 .5 b.8 13-7 20.3 33.5 7 .5 15.3 3 1.0 10"^ 5*1 1 0 .6 21.0 31.5 54.0 20.0 4 0.0 TABLE II PERFORMANCE OP MERCURY PUMP BACKED BY MECHANICAL PUMP P ressu re produced by m echanical pump Low pressure produced by mercury pump (Todd) 0 .1 mm. 0.0003 mm. 1 .0 mm. 0.0003 “ a* 5 .0 mm. 0.0003 mm. 1 0 .0 mm. 0.007 2 0 .0 mm. °*14 3 0 .0 mm. °*3 mm. ““a* 150 a system , mercury, which h a s a vapor pressure o f 0.185 micron a t 0°C. must "be prevented from d iffu s in g in to the system . sure o f mercury a t Since the vapor pres­ i s but 3 x 10“ ^ m icfon, the con cen tration o f mercury may be g r e a tly reduced in the system by the u se o f a co ld trap (S ). Thus, a trap must be used but a tte n tio n should be given to the d esig n o f th e trap s u ita b le for high vacuum work such th at i t o f fe r s o n ly low r e s is ta n c e to the flow o f g a s. The trap used fo r t h is appara­ tu s has an o u tsid e tube measuring J 2 mm. in diameter and an in s id e tube measuring 18 mm. in diam eter. I t has a u n iv ersa l j o in t o f ground g la s s so th a t one o f i t s arms i s a d ju sta b le. Since th e mercury vapor i s ex­ trem ely p oison ou s, i t i s e s p e c ia lly dangerous fo r u se in a poorly v e n ti­ la te d room. Both mercury vapor and water vapor endanger the e f fic ie n c y o f a h ig h vacuum mechanical pump. Thus t h is second condensation trap i s a s a fe t y d e v ic e . The important fa c to r s a f fe c t in g the op eration o f a pumping system are: 1 . leak ages in the system, trap c o n s tr ic t io n s , bends, e t c . o f g a s. 2. The r e s ista n c e of the pumping l i n e , These 'o ffer high r e sista n c e to the flow The ca p a city o f the vacuum lin e s i s determined by: 1. e s t p o s s ib le d ista n ce between the pump and the vacuum apparatus; la r g e s t diam eter p o s s ib le o f the connecting tubes; curves in tubing rather than sharp a n g les, and 4 . the w a lls o f the e n tir e system . 3» the short­ 2. the the u se o f la rg e absorption o f gas on Both g la ss and m etal adsorb an apprec­ ia b le amount o f carbon d io x id e, a ir and m oisture upon th e ir surfaces* Metal u n its are more porous than g la s s , hence they have a higher cap acity fo r adsorbing g a se s than g la s s . In order to lib e r a te the adsorbed gases 151 a t a rap id r a te g la s s mast be heated up to 300°C. w hile pumping* apparatus r e le a s e s gases very slow ly a t room temperature* New However, m etal u n its are not outgased by any procedure other than th a t o f contin­ uous pumping. In a system fo r high vacuum i t i s im perative to remember th a t th e pumps are an in te g r a l part o f the whole system, and a s such, t h e ir fu n c tio n in g i s dependent on the design o f the system . Even fo r a h ig h speed pump the r e s ista n c e o f the pumping li n e s mast be kept to a minimum. Short le n g th s o f sm all diameter tubing must be avoided. If th e r e s is ta n c e o f th e vacuum system i s h igh , the system w i ll be a slow system even i f the pump used has great speed, because the pump speed i s then lim ite d by th e system . OPERATIONAL PROCEDURE*— The reserv o ir or fr e e z in g tank (Figure lA ,a ) i s f i l l e d w ith four l i t e r s o f 95 percent eth an ol. o f dry ic e (2 or 3 ounces) are added. Small p ie c e s Vigorous COg gas e v o lv es. Addi­ t io n o f dry ic e i s continued every 5 to 10 seconds u n t il the vigorous ev o lu tio n o f COg gas d ecreases. ic e . This req u ires about two pounds o f dry Then four p ie c e s o f dry ic e a h a lf pound in s iz e are added to assu re the temperature o f the a lco h o l a t - 72° G. S ix 250 m l. ce n tr ifu g e b o t t le s containing samples are suspended h alf-w ay in th e fr e e z in g tank fo r 20 to 30 m inutes. p la ced in an upright p o s it io n . The b o t t le s are A fter 30 minutes in the fr e e z in g tank, th e b o t t le s are removed and connected to the adapters and fa sten ed w ith th e screws a t the ends o f the U clamps. A fter the sample b o t t le s are removed, s ix pounds o f dry ic e are pat in to the fr e e z in g tank. The temperature o f -7 2 ° C. w ill remain constant 152 fo r a period of 7 to 8 hours* Additional dry ic e must be added i f the operation i s to proceed for a longer period* turned on fo r 3 to 5 minutes* The mechanical pump i s then When most o f the a ir i s evacuated by the mechanical pump, then heat i s applied to the mercury pump. The pressure o f the system i s measured by a McLeod gauge* When th is vacuum-dehydration process i s completed, the apparatus should be turned o f f in the follow ing manner; mercury pump i s f i r s t turned o ff; 2. 1* the heater for the a ir i s slowly allowed to go into the system through the drying tube by turning the two way stopcock a t G- (Figure 3)* 3» *4® mechanical pump i s immediately turned o ff; 4 . re­ move the b o ttle s containing dried samples, these are stored in a desioic c a to r - co n ta in in g P20^ or Mg(ClOlj.^ and subsequently evacuated. EXPMIMOTAL RESULTS,— A* Drying meats and vegetables. Six cen­ tr ifu g e b o ttle s containing s ix d iffe re n t weighed samples were frozen in the fre ez in g chamber of the apparatus fo r 30 minutes then connected to the vacuum tank for 4 hours dehydration. o f mercury a fte r 1 hour. Pressure measured 22 micron Temperature of the freezin g tank was —72®C* .After 4 hours dehydration the samples were reweighed* Sample Wt. Fresh Wt. Dried % Water Loss per Total Wt 1. Lean beef 58.30 gm. 37.15 gm. 36.8 2. Kidney 38.86 gm. 22.20 gm. 4 3 .0 3* Beef liv e r 31* ^5 gm. 18.10 gm. 42.8 4* Apple 54.65 ffn* 30.09 gn. 44.9 5. Carrot 38.20 gm. 24.72 gm. 35.3 6. Cabbage 34.75 gm. 10.04 gn. 71.1 153 O bservations made from r e s u lt s o f t h is experiments 1* When a sample was cut in to la rg e chunks, the cen ter p ortion was not d ried in 4 hours and remained frozen . 2. A ll d ried samples reta in ed th e ir o r ig in a l form and s iz e . 3« Incom pletely dried samples absorbed m oisture from atmospheric a ir . This o b serv a tio n was made from apple t is s u e which became s tic k y a few m inutes a f t e r exposure to a i r . 4. Pour hours dehydration was not s u f f ic ie n t fo r drying th ese t is s u e s . B. Drying meats and v e g e ta b le s. S ix cen trifu g e b o t t le s containing s ix d iff e r e n t weighed samples were frozen in the fr e e z in g chamber o f the apparatus fo r 30 m inutes then connected to the vacuum tank fo r eig h t hours dehydration. Pressure measured 20 microns a f te r 1 hour. A fter 8 hours dehydration th e samples were weighed and stored in a vacuum d esic c a to r w ith PgO^ a s water absorbent. The fo llo w in g r e s u lt s were ob tained. Per­ cen tages o f water l o s s per t o ta l weight were c a lcu la te d a f te r 8 hours o f vacuum dehydration and a f t e r com pletion o f drying in a vacuum d e sicc a to r fo r 7 days. $> Water Loss per T otal Weight # Water Loss a f te r 7 Days in D esiccator Sample Wt. Fresh Sample Wt. Sample a f te r 8 h r. Dehydration 1 . Banana 3 2 .6 5 gnu 8.48 gm. 7 4 .0 74 .6 2 . Green bean 24.60 gm. 3,00 gn. 87 .8 84.3 3* Sweet potato 45.79 20.07 S01* 56.17 6 6 .3 9 4 , Carrot 3 2 .3 3 8.53 g a . 7 3 .6 3 86.48 3 . B eef 46 .1 0 gm. 14.20 gm. 69.19 7 1 .1 4 6 . B eef l i v e r 4 2 .0 0 gm. 16.30 gn. 61.19 67.07 154 O* Dehydration o f f i v e whole rat l i v e r samples. Samples were fro zen fo r 30 m inutes in the fr e e z in g tank which was f i l l e d w ith dry ic e and eth a n o l. The dehydration period employed was 8 hours* A low p ressu re o f 5 micron o f mercury was m aintained during th e l a s t 4 hours* The samples were weighed immediately fo llo w in g dehydration and d a ily th e r e a fte r fo r 6 days w ith continuous storage over Ig®5 d esicca to r* a vacaam No fu rth er lo s s in weight occurred a f te r 5 days storage* The r e s u lt s secured are p resented in Tahle V. TABLE Va EFFICIENCY OF DEHYDRATION BY FREEZING-HIGH VACUUM TECHNIQUE Fresh Weight in Grams A fter 8 h r s. F reezin gDehydration Gas. Water Loss per T otal Wt. $ Water Loss per Total Wt. $ Water Loss A fter 5 Days in Vacuum D esiccator 1. 6*97 2 .6 4 M 3 62.12 62.26 2. 7-59 2 .8 2 4*77 62.84 62*97 3. 7-S9 2.59 5.30 67*17 67*93 4. 6*87 2 .0 2 4.85 70*59 7 0 .1 k 5. 8.87 3*19 5*68 64.03 70.10 U sing th e percentage o f water l o s s a f t e r 5 days in the vacuum des­ ic c a to r (column 5) a s rep resen tin g 100$ water lo s s from a l l samples, the d ata in columns 1 and 2 o f Tahle Vb were c a lcu la te d and show the d if f e r ence in p ercen t water l o s s hy 8 hours freezin g-d eh yd ration and 5 days in a vacuum d e s ic c a to r . Samples 2 and 4 were more com pletely dried hy the freezing-dehydra^ t io n method prohahly because the samples in th ese two cases were so p la ced th a t a con sid erab ly g rea ter surface area was esqposed. 155 TABLE Vb Sample 1. 2. $ o f Total Water Loss by 5 Days in Vacuum D esicca to r (Po0.-) « 5 0*92 $ o f Total Water Loss by Freezing-dehydration 99*08 0.21 99*79 1 .1 2 98.88 0.21 99*79 8.66 91*3^ SUMMARY”. — This apparatus fo r freezin g-d eh yd ration has been construc­ te d as d escrib ed h erein and has y ie ld e d s a tis fa c to r y r e s u lt s in our lab­ oratory* Some o f th e fe a tu r e s o f t h is apparatus are d iscu ssed . The tank i t s e l f i s a device fo r ex tra ctin g and trapping water which has vaporized in cen trifu g e b o t t le s from frozen samples. The apparatus i s capable o f e s ta b lis h in g a steep concentration gradient because o f two c o n trib u tin g fa c to r s : and 2 . 1. th e e f f i c ie n t water vapor condensing mechanism th e short d ista n ce between the openings o f the b o t t le s and the f r e e z in g cone (th e lower part o f the inner tank). The fr e e z in g chamber serv es a ls o as a co ld bath fo r fr e e z in g samples and a s the second co o l­ in g tra p fo r mercury vapor. The double w alled stru ctu re of the tank o f­ f e r s the advantage th at th e temperature o f the fr e e z in g chamber remains low fo r a lon ger period o f tim e. The h o rizo n ta l p o s itio n and th e la rg e opening ( 25mm) o f the c e n tr i­ fu ge b o t t le s f a c i l i t a t e a more rapid d iffu s io n of vaporized water mole­ c u le s . The tank has a la r g e cap acity fo r drying samples: c e n tr ifu g e b o t t le s can be used a t one tim e. s ix 250 ml. The adapters o f the t ank 156 a llo w b o t t l e s to be used interchangeably which i s an improvement over oth er ty p es o f lab oratory vaeuum-dehydration u n its* Further advantages a re o ffe r e d by the u se o f cen trifu g e b o t tle s because substances which must be separated from so lu tio n by cen trifu g a tio n can be dried in the same b o t t le w ithout tra n sfer which prevents p o ssib le lo s s o f sample, l i m i t s contam ination and saves time* The dried samples may be preserved in the same b o ttle s * The s im p lic ity o f d esign o f the e n tir e u n it con trib u tes to ease of co n stru ctio n in an ordinary machine shop* I t has been found th a t 8 hours operation i s s u f f ic ie n t to dry samples to near constant weight* The apparatus i s easy to operate and does not req u ire s p e c ia l a tte n tio n during the period o f operation. 157 LITERATURE CITED X* Gersh, I « , "The Altmann Technique fo r F ix a tio n "by Drying w hile F reezin g 1*, 2. Anatomy Record, 53.: 309, (1932). Hoerr, N.L. and S co tt, G-.H., "Frozen-Dehydration Method fo r H isto­ l o g i c a l F ix a tio n " , Medical P h ysics, the Tear Book P u blisher, I n c ., p . 466 (1 9 4 9 ). 3* Simpson, W.L., "An Experimental A n alysis of the Altmann Technic o f F reezin g -D ry in g ,w Anatomy Record, 80: 173 (1941)* 4. Makower, Benjamin, "Use o f L y o p h iliza tio n in Determination o f Mois­ tu re Content o f Dehydrated V egetables," A n alytical Chem., 20: 856 (1 9 4 8 ). 5. Meyer, B .S ., and Anderson, D .B ., "D iffusion o f Gases", Plant Physi­ o lo g y , Textbook, D. Van Nostrand Co., In c. (1940). 6. Welch Company Duo-Seal Pumps B u lle tin , Welch Co., Chicago (1947)* 7. Sanderson, R .T ., "Vacuum Manipulation o f V o la tile Compounds", p. 30- 42, John Wiley & Sons, I n c ., (1948). 8. Dushman, S ., " S c ie n tific Foundation o f Vacuum Technique", John W iley & Sons, I n c ., (1949)* p. 212, 158 A HIGH TEMPERATURE BATH MADE FROM ALUMINUM SHAVINGS 159 A HIGH TEMPERATURE BATH MADE EROM ALUMINUM SHAVINGS The need fo r a h ig h temperature hath fo r h y d ro ly sis of p ro tein s in aqueous s o lu tio n s le d to th e work presented a s follow s* I t was n ecessary to f in d a m a teria l fo r a hath in which constant h o ilin g temperature o f an aqueous s o lu tio n could he obtained. Since aluminum shavings have a h ig h c o n d u c tiv ity o f h e a t, i t was found that a high temperature hath could he con stru cted from them. An aluminum-shaving hath i s constructed o f a copper hox which con­ ta in s aluminum turnings ahout the s iz e o f r ic e gran u les. The hox i s p la ced on top o f a copper p la te a quarter o f an inch th ick and i s fa s ­ tened hy screw s. The copper p la te i s heated hy three e l e c t r ic elements and th e h ea t i s reg u la ted and c o n tro lle d hy a therm oregulator which i s lo c a te d in th e cen ter o f the hath, (fig u r e l ) . Because o f th e high co n d u ctivity o f copper a uniform temperature hath can he constructed* The copper p la te must he th ic k enough in order th a t s u f f ic ie n t h eat may he d iffu se d to th e e n tir e u n it. The e n tir e hath i s in c lo se d in a hox o f t r a n s it s so that the uniform temperature o f the hath can he p reserved. The space between the copper hox and the t r a n s it e w all measures one inch and i s f i l l e d w ith rock wool a s in su la to r . The hath i s covered with in d iv id u a l tr a n s ite covers w ith notches to ac­ commodate the necks o f The dimensions o f the f la s k s . the in sid e o f the hath are a s fo llo w s: in le n g th , 6 in ch es in width and ^ 1 /2 inches in depth* round bottom f la s k s can he se t in a row. 40 inch es Eight 300 ml. The f la s k s are con ven ien tly 160 p la c e d in m etal cups made from 8 ounce aluminum measuring cups with, hqn— d ie s removed* In case o f “b reakage o f f la s k s , the so lu tio n i s trapped in the cups* (Figure 1) • The f la s k s are cushioned in the cups w ith "Ghor e - g i r l 11 copper ribbon m aterial which r e ta in s i t s form. Aluminum shavings were not s a tis fa c t o r y because they did not r e ta in a d e f in it e form to accommodate the shape o f the fla sk s* However, the bath can be operated w ithout the u se o f metal cups. In packing the bath the 8 m etal cups w ith th e ir contents are placed on th e bottom o f the box* (F igures 1 and 2 ) . Then the aluminum shavings are poured in to a depth o f 0*75 inch, evenly d istr ib u te d but not p ressed or tamped. Two aluminum bars are la id on top o f the aluminum shavings in fro n t and in back o f the metal cups but not touching the cups or the sid e w a lls o f the bath. A fter a l l th ese are in p o s itio n , the bath i s f i l l e d w ith aluminum shavings up to one inch from the top . The two alum­ inum b a rs, 39 in ch es in len g th by 1 /2 inch in diameter, are imbedded in the aluminum shavings 3/^ i^ch above the bottom o f the bath, are used as heat conductors* (F igures 2 and 3)* The bath was te s te d fo r 50 hours o f continuous operation a t 200°0. Recordings were made o f th e temperature v a r ia tio n s at f iv e d iffe r e n t p o s it io n s in the b ath. The flu c tu a tio n o f temperature of a l l the p o si­ t io n s o f the bath was found to be the same, *1*5°C» (See ta b le ) . I t was dem onstrated that the temperature o f the bath was even and constant a t variou s p o s itio n s in the bath when th e thermo regu lator was s e t at 200 C. The therm oregulator was se t a t temperatures ranging from 30°C. to 250°C. f o r 12 hour p er io d s. The bath h eld the given temperature throu^aout 161 th e p eriod tested * D e ta ile d data on s p e c if ic temperatures other 200°C* were not com piled. Temperatures higher than 250°C. could he secured hy adding more h ea tin g elements* The aluminum-shaving hath so constructed has many advantages fo r u se in a chem ical la b o ra to ry . I t i s e s p e c ia lly u se fu l a t or ahove 100°C. fo r h yd rolyzin g or r e flu x in g samples fo r long p erio d s. P a r tia lly hydro­ ly z e d samples can he taken from each f la s k fo r a n a ly sis o f the degree o f h y d r o ly sis w ithout d istu rb in g the main procedure when the two-neckf la s k s are used* c le a n . hath: Aluminum shavings are non-corrosive and always appear Ahove 100°C. th e shaving-bath has several advantages over a liq u id no liq u id escapes hy evaporation; no unpleasant fumes a s w ith a cid or organic liq u id s . The c o st o f the m a teria l, exceptin g the thermoregulator, fo r b u ild ­ in g th e hath i s between $12.00 and $15*00. The thermoregulator i s manu­ factu red hy Fenwal, I n c ., Ashland, M assachusetts. .An aluminum p la te 3/& inch th ic k , *40 in ch es hy 6 in ch es, weighing 10 pounds may he used in stea d o f copper (c o s t $ 3 .0 0 ). shops a t $ .1 0 per pound. Aluminum shavings are obtainable a t machine Figure 1. Front section view ‘<1 300 ml. flask placed in metal cup, cushioned with chore-girl copper ribbon Aluminum shavings Rock wool insulation Copper plate Position o f thermoregulator Heating element 0 0 0 ^ 0 0 0 ? * » » < i . W « ■-a Aluminum bar JfH^ Rock wool insulation Figure 3. End section view • • Aluminum bars i -i ■• • Heating elements Copper plate 163 TEMPERATTffiE VARIATIONS AT PIT/E POSITIONS IN THE BATH Time in ThermoMinutes reg u la to r P ilo t L ight 1 2 3 4 5 °0 °c °0 °C °c 1 2 3 4 5 6 7 s 9 10 o ff o ff Off o ff o ff on on on on on 201 201 201 201 201 201 200 200 199 199 203 203 203 203 202 202 201 201 200 200 201 201 201 201 200 200 199 199 19S 198 202 202.5 203 203 202 202 201 200 200 200 201 201 201 201 200 200 200 200 199 199 11 12 13 14 15 16 17 IS 19 20 on on on on on on on on o ff o ff 198.5 198 198.5 199 199 199-5 200 200 200 200 200 200 200 200.5 200.5 201 201 201 201 201 198 198 198 198 198 198 198.5 199 200 200 200 200 200 200 200 200 200. 200.5 200.5 201 199 199 199 199 200 200 200 200 200 201 21 22 23 24 25 26 27 2S 29 30 o ff o ff o ff o ff o ff o ff o ff o ff o ff o ff 200 200 200 200.5 200.5 200.5 200.5 200.5 200 200 201.5 202 202.5 203 203 203 203 202 202 202 200 200 200.5 200.5 200.5 200.5 201 200.5 200.5 200.5 201 201 201.5 202 202 202 202 202 202 201 201 201 201, 201, 201 £00 200 200 199 199 31 32 33 3*435 36 37 38 39 40 o ff o ff on on on on on on on on 200 200 200 200 199.5 199 198.5 198.5 198 19s . 5 202 202 201.5 201 200.5 200 200 200 200 200 200.5 200 200 199.5 199 198.5 198.5 198.5 198 198 200.5 200.5 200.5 200 200 200 200 200 200 200 199 199 199 199 199 199. 199 199 199 199 164 TABLE Oont. Time in ThermoMinutes reg u la to r P ilo t Light 4l 42 43 44 45 46 1 2 3 4 5 °C °o °c °0 °c ?7 4s 49 50 on on on o ff o ff o ff o ff o ff o ff o ff 19S .5 19^.5 199 199 199 199 199.5 200 200 200 200 200 200.5 201 201 201 201.5 202 202 202 198 198 198.5 199 199 199.5 200 200 200 200 200 200 200 200 200 200 200.5 201 201 201 199 199 199.5 200 200 200 200 200.5 200.5 201 51 52 53 54 55 56 57 58 59 60 o ff o ff o ff o ff o ff o ff o ff on on on 200 200 200 200 200 199.5 199 198.5 198 198 202 202 202 201*5 201*5 201 200.5 200 200 200 200 200 200 200 200 199.5 199 199 198.5 198.5 201 201 201 200.5 200.5 200 200 200 199.5 199.5 201 200.5 200.5 200.5 200 200 200 199.5 199 199 201 198 203 200 201 198 203 200 201 199 3 3 3 3 2 t 1 .5 ± 1 .5 1 1 .5 i 1 .5 H ighest reading Lowest reading Range or v a r ia tio n F lu c tu a tio n t 1