'1’ pl gr 1"“?9' JL ‘ 1r, ,‘111- .1! . ‘ ~. 1.1 . 1. 1 1 i T5131: 1'9 ~ ‘ , ’ 1 . (E ‘ 11! "5,117 111%?!” h ‘11 i-I71lf’,’ 1, ~11 - _ ‘ , {‘1‘ , 3' f 1 “11". 1! If .5 1 “El h: I: 1.11 31,": '. blflv". ” 1 1113'!" 171:1;‘1'1‘1 1! ' ~ {:11 _. " M’fw was: ""1 ,5: F617 ‘f" :11 {“1- '1; '9“ 'l 1; 9:1.1 1 Yr 1 $15? Jfi’: 1:11"; {18 1. 27‘9' ' ’f' .1 . f}??? 155571 {11 (‘3';- 11"}! pug-Jr 1 1.1. My; “nadfi'nhx‘: $1914? a“. fififiu '15}? '1. 1._ gm ”"32",” ' .' 7 {“4 ’1'1‘11‘13 . $9 ,1 [11 . I“ ' I" ' . 3‘1; 41"” '13:» F '1 1 z ' N41, , 135:5”: 1 4 1 1L“ 191111.422]; 91:14.7”? a“ $ ’51:: x .' 1:9, '4 1 ~1 (1". 1., 157%}? 5'1; c'tza “11"“ ~‘L-1 19111;?" "in" 1,-1‘1' 1-;14 . {9311' F11: STUDIES 011 THE PRECIPITATION OF Ens—G 11.31.sz31 TFESIS FOR THE DEGREE OF 3.8. EDWIN G; DOVAHUE 1934 95495 STUDIES ON THE PRECIPITATION OF EGG ALBUMIN A TEESIS SUBMITTED TO THE FACULTY OF MICHIGAN STATE COLLEGE OF ACRICULTURE AND APPLIED SCIENCE IN PARTIAL FULFILLEEITT OF TPE REQUIREIRHTS FOR TPE DEG 7E OF MASTER OF SCIENCE BY EDWIN G. DONAFUE June, 1954 A CI I3 OWL 3 D 43.33 I? My sincere thanks are offered for the help and guidance of Professor 0. D. Bell, who understands and forgives human frailties, but at the same time gives a challenge for hard work and clear, straight scientific thinking. Mam TABLE OF COTTETT‘ A. Introduction. Page. B. 3Definition of Terms................................l 0. Historical. I. Agencies Causing Precipitation of Proteins. 1. Eeat................................5 2. Freezing............................5 5. Bressure............................6 4. Radiation and Vibration.............7 5. Acids and Bases.....................9 6. Salts.............................. 2 7. Oxidizing Agents...................15 80 Organic coripound8000¢ooooooOoOooOOO-L6 (0 Cf" lI. Stabilizing Factors; Protection again- Precipitation..........................19 III. Adsorption and Chemical Combination as Factors in Precipitation and Stabiliz— (>3 ation..................................2 IV. The Use of the Fephelometer in Studying Precipitation and Stabilization .......26 D. Experimental 1. Standardization of Hephelometer.........28. II. "Protective" Action of Alcohols on Nephelometric Turbidity ..............56 Page. III. Heat Precipitation Studied by the Nephelometer and the H Determin- ation Rethods ........................ 49 IV. Conclusions ........................... 55 E. Bibliography .................................... 57 lNTRODUCTIOE Changes in the state of aggregation of the substance making up protoplasm is known to be intimately related to life, activity and death of an organism. In this connection one need only refer to the works of Chambers (1), Addoms (2), darinescolzi, Heilbrunn(4), and Bancroft and Richter (5), as a few of the papers on the subject. These workers draw their conclusions after making direct observation upon the protoplasm of living organisms. Such studies on systems as complex as protoplasm give us highly significant information. However, it is believed that more fundamental knowledge of living processes must come primarily from investigations on simpler systems con- taining pure substances whose concentrations are known and controllable. Protein systems possess many prOperties of protoplasm, especially when we consider opalescence, viscosity, ease of precipitation, etc. And when we realize, (I) that proteins occur as a major constituent of all living protoplasm, (II) that most enzymes are known to be proteins or at least always associated with them, and (III) that the science of immun- 010gy is largely a study of protein reactions, it becomes clear why careful studies on pure proteins are important. Lloyd (6A) states that the proteins of the cytoplasm belong always to the classes of albumins and globulins. It was With the above point of view that the present work on precipitation of crystallized egg albumin was begun. DEIIIITIJM OF TERM‘ The terms, precipitation, agglutination, denaturation, flocculation anzl coagulation are consid er~ a ly eip‘ova1 throughout the literature on the chemist y of pro He1.s, and unfortunately they have often been used rather loosely. For clearness the folloz n~ definitions of these terms will be strictly adhered to in this vzork.‘ Precipitation is a general term that re e‘s to any separation of ma ial from the colloidal state or from true solution. Whenever doubt is held as to whether the aggre~atcd mass is reversible or not, the process of form- ation will be called precipitation. A precipitaJe may vary in properties from a faint turbidity in solution, to a stiff gel. G) Agglutination is de fi as the f imation of larger aggregates after precipitation has taken place, the aggre- gates being plainly visible to the naked eye. Denaturation is a change in preperties of a native soluble protein, brought about by numerous agencies (heat, acids, bases, alcohol, urea, etc.), such that the pro loses its property of being soluble in water or salt sol- ution at the isoelectric point of the modified protein. A. This change does not cause precipitation unless the pH is ‘ adjusted to the isoelectric point, and only water or salts are present. Flocculation is that special kind of precipitation which must always be preceded by denaturation. The floc- culated protein then, is insoluble in water at the isoelec- tric point, but can be dissolved in acids or bases, the protein separating out from the latter solutions by adjust— ing to the isoelectric point again. Coagulation refers to the complete process: denaturatior plus flocculation. to PISTORICS The treatment of the literature reviewed may conveniently be divided into the following subjects: (1). Agencies Causing Precipitation of Proteins. (II).Stabilizing Factors; Protection against Precipitation. tIII).Adsorpticn and Chemical Combination as Factors in Precipitation and Stabilization. \IV). Use of the rephelometer in Studyinh Precipitation and Stabilizati n. for a more unified discussion in this review r=3ercnce ' - w n a - J. , DYOL,1“ ill be LrO‘r “ is on' ‘ n it 1 1'0: 't t b -Ec drtv ' IL vc' xe.ri r en 1‘ i‘. - - .t .- ,, : J. ,. r ( I ) O V— ~4 A ,3 ¢ ‘1 V I; ‘P ._ r L V C) A .L . .1 v - j- l: V . r g 4- ., L . 1 a ‘ . ,w .w - . v . Proeahly as lens as ecc- have been cooked and need he .5. . ..‘ .'-I.-, H . , ° .. -J.- . ,,... V .—. ‘-.,.. 1...“, iweu ;:; COdrhlfithD of egg WhlLd b* that his Lac“ 0. ~;»n7. fl ‘ J—‘\ .‘ "' :J‘ . ' . “‘,.‘ ' ‘v‘o- ‘. -. -. - . '- ’- - v -' a ' -.,-. " " i‘ ‘- 3’ 3 f I} J 1? 1,; —- v 1 T (I Of. LA: 0 L] e 'L ‘1' I; -l.‘ J. 4.; 3‘12); ‘.-'. . 4‘ V '. ‘21 n1 I , . - Ll ‘. 3.13.451 . . -‘ v- 4—3 /~ . O _ : I-~ -—< .. . t- 3" --" 9 -" . " '- ‘I I r. .' 'x ' E \ to nclrlu the DOlllz- 931-- el H.,., .,; ..cA :; en D’s” fr“? ; slink tg'“".rcrt igufd to a white, oparue, nea:ly solid rel. If the albumin solution is more cilrte, the heat will form merely a turbid suspension. The classical work of Chick and Martin (7) in 1910 on crystallized egg albumin, established the fact that the heat precipitated protein is truly coagulated. Also by measuring the rate of denaturation by heat at constant pH, the reaction was shown to be a monomolecular one for both egg albumin and hemoglobin. The rate of the denaturation was a function of the concentration of the protein on y. These workers con- sidered that the usual method of measuring the temperature of coagulation was of value only if the conditions of heating, time, protein concentration, etc., were accurately standard- thloyd (63) quotes bepeschkin, who reported in 19L2 that denatured egg white floccu ates in ten seconds, while two thousand, two hundred seconds are required to coagulate the native product under the same conditions. That the specific optical rotation of egg albumin is increased by heat denaturation, vas shown by barker (8) in 1955. He found that the measuremen was a constant for a given degree of denaturation. it was his belief that the protein denatured by various agencies was very probably a different substance in each case. Later the same worker 19) gave evidence that the refrac— tive index of pure egg albumin is increased slightly as measured by the Zeiss interferometer, which gives the refractivity out to the seventh place. He considered that the change in the re- fractive index supports the contention that a change in chemical structure has taken place. 0i work of others, he listed nine propertic are known to change upon heat denaturatio: ing this (2 U affinity for water decrease; viscosity of optical rotation, refractive index, digest and trypsin, are all increased; nitropr ts ~83 groups absorption not change latter bein IN Cs J. becomes spectra are known to change. \QA ' 4-; \ ”J CB4. ed; a measured ’n urea solution. bancroft and nutzler (10) claimed ed in reversing the denaturation of egg coagulated fhe reverse coaculable as the original eff Freezing. .L! ' O 'Y/‘l ti) an]. v. horetic mobility an, Preperties which molecular weight, t in 0+" the solution, ibility by pepsi Ho 1. positive; and immunoloyical reactions, (LS and previous e3: albumin which the solubility, and Specific V5 b-L reaction for and do (18 931 to have succeed- ting the air white in hfle presence of ether, ann further this to the peptizint action of potassiu thiocyanate. d product was soluble in wet r ofte dialysis was by heat, and gave the same in unological reactions in 1923, neiner (ll) subjected egg white to repeated freezing and thawing and found that the protein began to gather at the bottom of the container. if the process was not repeated too many times the protein could be redissolved when melted, but eventually an irreversible product was ob- tained. D'yachkovskii (12), in 1952 observed that albumin, heroglobin, and vanadium pentoxide sols are precipitated after thawing the sols frozen at —5, -15, and -20 degrees, but that they are completely stable if frozen at -182 degrees. The suggestion was made that the stabilizing effect at the lowest temperature was due to the higher rate of freezing. Pressure. In 1914, Bridgman (15) found that egg white could be precipitated by pressure. Upon subjecting the material to a pressure of 5000 atmospheres for 50 minutes, he observed a noticeable stiffening; 6000 atmospheres gave it an ap_ear- ance like curdled milk; at 7000 atmospheres apparently complete precipitation resulted. The process was accelerated by lower temperatures, but appeared to be independent of the time of application of the pressure. Basset, Macheboeuf and Sandor(14) published the r»- sults of their work in 1955, in which they stated that the serum of horse blood could also be precipitated by high pressures. The minimum pressure for gelification was 15,000 atmospheres. Serum globulin jellied completely at 15,000 atmospheres, while serum albumin remained limpid at all pressures up to the same pressure. Radiation and Vibracion. From the literature one finds that practically all known forms of vibration can play a part in precipitating albumin from solution. This section will treat these from the lower frequencies to the higher, and not chronologically. Hopkins (15) proved in 1900 that coagulation of pure 0 albumin occurred from solution when the system was shaken egg violently. Fe observed that the precipitated product gave a reddish violet eclor with sodium nitroprusside as did that coagulated by heat. Three years later, Rams en 116) brought out that adsorption at a surface or in a film results in denaturation, and that the stiff foam of egg white formed when the material is beaten is an irreversible one, composed of the coagulated albumin adsorbed in the film of the air bubbles. Audible sound was found by Elosdorf and Chambers (17) to bring about precipitation of albumin. lreeuencics of 1000 to 15,000 v.p.s. precipitate albumin from solution almost instantly at 50 degrees. That mechanical agitation and sound waves may be related in their precipitating action is indicated by the work of Wu and Liu (18), in 195 , in which is reported the precipitation of egg albumin from solutions by exposure to supersonic waves. When the solution was saturated with air, hydrOgen or oxygen the albumin was precipitated in fine shreds enclosing gas bubbles. No precipitation resulted when in gas free solution, or when saturated with carbon dioxide or hydrogen sulfide; incidently in the latter cases no gas bubbles were formed. The theory was that gas bubbles caused precipitation at the interface similar to th t occurring when albumin solutions are mechanically shaken. Young (19), in 1922, showed that light rays of the visible Spectrum , denature albumins in many ways similar to heat. Denaturation by ultraviolet light was demonstrated by Dryer and Banssen (20) in 1907. Clark (21) reported the results of her studies on the action of ultraviolet light toward egg albumin, in the year 1922, in which it was found that at pH values below the iso- electric point the light did not precipitate the protein. At the reaction of p? 5.6, 6.2, and 6.8, precipitation did take place, but at pH 7.8 the solution once more remained clear. The conclusion was that the action of ultraviolet light is one of emission of electrons from the charged protein particles, since greater dispersion results when the protein is positive, and greater aggregation when the protein is negative. miss Clark showed that a change has resulted in all the tubes of varying p?, by the action of the light, since half-saturation with (F34)2804 caused precipitation in all of them. Before the exposure, such treatment caused no such chanse. Fernau and Pauli (22) observed in 1915 that radium emanations precipitated albumin. In 1923 , Fernau reported (25) that X—rays precipitate both albumin and eerie hydrox’de sols. Acids and bases. Chick and Kartin (7) in 1910, showed that the H-ion concentration greatly influences the velocity of heat denaturation, there being a minimum velocity at about the neutrality point of water, pH 7, and a very rapid increase of the velocity as the H -ion concentration is increased. however in the acid. part of the denatured protein was Soluble, the total amount precipitating out only when the reaction was adjusted to the. isoelectric point. In 1912, the same workers 324) showed that alkali also increases the velocity of denaturation in a manner analogous to that in nhich acid increases it. Lloyd (6C),after reviewing the work of Chick and Martin, 10 and of Wagner (73) on the effects of acids, poirts out that if the temperature is sufficiently high, denaturation takes place in neutral sclutions; and if the H -ion concentration is suffi- ciently high denaturation can take place at 20 degrees or less. Concentrated acid can also crowd the protein out of solution. Schoorl (25) in 1924 observed that 5 percent erg albumin in strong acid (2.5? H01), bee me opalescent after a half hour in the cold, became very viscous after several hours, and precipitation took place in about five hours. This is similar to the action of salts in hieh concentration, which subject will be discussed later. rhe coaaul ted protein obtained after denaturation By miner l acids is ctllcd by the speci 1 name acid “etaprctein, and th-t obtained after denztrr~tisn by alkili is called alkali metaarotein, according to Pawk and bergeim (26). in a series of qualitative experiments on the behavior of ens albumin towards a large variety of ordinary laboratory reagents, hunter (37) reported in 1993 that the protein was precipitated by concentrated H2804and by the sulfonic acids of the folio inez¢X—na*hthol, dinethyl aniline, salicylic acid, resorcinol, quinol, and fluorescein. Vallery (28) in 1913 presented data indicatine th;t heatine with least dissociated acids gives the greatest amount .1. ‘u . 4".x .9 -, r! v ; “I .1 1m-a -: ‘ -\ 1‘ . ‘ V 'r‘ A 12 lbl e iron serum alou.in solutions or lion P. ‘Q of protein prec albumin in the urine. The reason given for this pas that during coagulation by heat the dissoc'1ted acids indtce ll hydrolysis of the pr rotein giving rise to scluble peptones. Thus acetic acid was found to give more coagula ted protein ne than did trichloracetic acid, F.- H. from serum albtn; in in th u (D (I) (3: at O P. the latter being m re disso . Labes and Janw sen (29 ) in 1950 studied the effect of substitution in acetic acid upon the precipitating power of the acid toward denatured serum albunin. The i soelectric point of denatured serum albumin is shifted toxm rd a nore acid reaction by/9—iodopropionic, dichloracetic, phenylacetic, benzoic, trichloracetic, and tribrouoacetic acids; at the save H- time the zon c of prec pitation is broadened. At the same time, babes , collaborating with Schuster (50), reported an in' creating study of the effect of sub- stitution in aromatic acids upon the optirum prec cipitation zone of nltir 1 serum albumin. The effect of substitution depends upon the position of the groups. Thus a nitro yroup on the para position in beizoic acid causes a much greater f tation of the protein, and a greater shif ;—Io aroun t of preci3 in the optimum zone than a similar group in the ortho or meta position. With hyiroxyl or an inn grOLps the revs Ce relation- shii Lol‘s. The effect of position is explained on the basis of the polar haracter of the benzoic acid molecule. Sulfonic acid groups substituted into aromatic radicals increase the precipitatinn power of the compounds. m l--' d- ’0 O In a review of the subject of precipitation of proteins by inorganic salts, dobertson (51) restates the findings of Hardy (52) that precipitation of proteins end cOlloids in general may he of two kin s: I. One is accompanied by a decom;osition of the precipitating agent, occurring only if the protein pos (Q esses a charge. In this case relatively small quantities of the precipitating agent are required to bring about precipitation.. II. The second kind, whether accom- panied by decomposition of the precipitating agent or not, occurs even at the isoelectric point of the protein and requires relatively large amounts of the protein. The first type of precipitation is brought about only by electrolytes, and it appears to be chemical in nature, but the second type may be actuated by certain non-electrolytes, for example, alcohol. The mechanism of the latter type seems to be a dehydration of the protein by the precipitant, which has greater affinity for the water than has the protein. Robertson, continuing with the subject of salt preci- pitation, again cited Hardy (33 . The latter noted that denatured egg albumin can be made into an anion or cation simply by changing the reaction of the solution. In acid the protein was positive, migrating to the cathode; in alkali it was negative, going to the anode. In both cases the protein was precipitated at the electrode toward which it migrated. hardy (33) observed that when the denatured protein was electrically charged, it was very readily pre- cipitated by traces of electrolytes (1 gm. mole in 80,000 cc), the cation being effective when the protein was negative, and the anion when it was positive. This work of Fardy's shows precipitation of the first type, noted above. Similarly, Bodansky (54) quotes Loeb, showing that gelatin combines with silver cations only when the pH of the solution is above its isoelectric point, the combination being such that it cannot be reversed by washing with water. Also it is only below the isoelectric point that gelatin combines with ferrocyanide anions. Both of these ions were readily detected by color reactions. Pauli (55) in 1899 showed that as a rule the precipi- tating power ( second type of precipitation) of a mixture of salts is the algebraic sum of the separate effects of its components, in other words the effect is additive. Also he noted that the presence of salts lower the temperature of heat coagulation. however in a later paper, 1905, he noted that a number of salts that will not precipitate egg white by themselves will either increase or decrease the precipi- tating power of other salts. This was considered by Pauli to be due to the antagonistic effects of the anion and cation. Citing Hardy, Robertson (51) stated that both types of precipitation can tahe place by the same salt (of alkali metals or of heavy metals). The salt will precipitate the charged protein; higher concentration of salt will redissolve the precipitate, and finally with still more salt tation will take place again. Cervello and Varvaro (36) studied the egg albumin sol- ution made by precipitating with salts of Fe, Cu, Pg, Zn, and Mn, then adding more until the precipitate had just redissolved. The solution gave no characteristic tests for the metals. The temperature of heat coagulation was raised in all cases except the Zn and Pg solutions. In the case of the Fe, heat coagu- lation is inhibited altogether. Before we leave the subject of precipitation by electro- lytes, it should be noted that the isoelectric point (point of maximun precipitation) is specifically influenced by ifferen ions, as one might expect from a knowledge of the Pofmeister series. Lloyd (63) gives a review of the literature, showing that with casein solutions, sulfosalicylate ions greatly shift the isoelectric point toward the acid side; and thiocyanate, iodide and bromide, less so. Acetate ions move this point only slightly to the acid side, nearly coinciding with the .’ 'C O 9 epted value. Cations hove the isoelectric point ton rd (-1. I J. O F.) H J x L; H P.) :3 (D (’l *1 Q) ( J o in 1933, le r111u ( 35) noted that hydrogen perozcide and ozone precipitate albumin and eerie hydroxice sols,and hydro— lyze sucrose, and that X— ~reys, ultraviolet rajS, and‘X—rcys do all of these. The work of Tim ter(27) recorded the fact that eotassiua dichronate and bromine rater will precipitate erg alb1:in ( 9,u Cossu (38) re norted in 1334 that iocine ca,se: preclpi- tation of albumins. of possible in J. of Effront (37), in 1911, that the action 01 sunlight on cro— .- H {:3 Ci- F" teins and amino acids in alkaline so en, gave rise to the formation of ammonia, volatile acids and nitric acid. Starch- iodide paper indicated the :resenc e of hyd103en peroxid_e in solution. To test if h; diogen peroxide was the cause of tee '0 U . O -_ ‘ A . 1 var1ous proieins and O decomposition, alkaline solutions amino acids were distilled in its presence. All the nitrogen was converted into a uonia and a slight amount of nitric ueid and fatty acids were formed. Effront suggested that the per— oxide oxidizes the amino groups to hydroxyl groups. CIA- 16 Organic Compounds. Precipitation of proteins by methyl and ethyl alcohols has long been known. Lepeschkin (59) in 1923 made a study of precipitation of albumin solutions, reporting that alcohol precipitates the albumin reversibly, noting that the action is similar to that produced by salts. Non-soluble substances such as ethyl ether, chloroform, and benzene, which form emulsions in water, pre- cipitate the protein but do not form an insoluble precipitate of albumin on the surface of the dispersed fluid particles. Fowever, rauli and Weiss (40) reported in 1951 that alcohol precipitates gluten reversibly and ovalbumin non- reversibly. dacobson (41) observed that benzyl alcohol precipitates epg albumin and peptone irreversibly even in solutions con- taining 1 p.p.m.,(l925). The precipitation of hemoglobin by K01 was found by Jirgensens (42) to be aided by the presence of methyl and ethyl alcohols in all concentrations, while propyl alcohol sensitizes the precipitation only when present in small concentrations. At higher concentration the latter acts less and less as a sensitizing agent, (1927) nruyt (45) in 1925, pointed out that the alcohol con- centration necessary to precipitate a protein is at a minimum 17 O C) (Q m {D J 4 N at the isoelectric point. Fe considc :ed this to be a nu l) consequence of the lower degree of h; dra ion of the protci at that point. In 1929 Utzino (44) reported that the opale scenes of albumin solutions containing alcohol is increased by raising the temperature or by inereasin: the amornt of alcorol. The first to report that acetone precipitates alb fin from neutral solutions has Weyl (45), in 1910. Lloyd (6?) states that both alcohol and acetone cause denaturation of proteins; also t' at tem . mr ture ubtles plays an important role in the denaturation, Since hardy and r demonstrated in 1910 tha this reaction does not Q m b: :31 H. '5 (D occur at terperatures lone: than 5 degrees. The effect of other non—electrolytes on the precipitating power of ethyl alcohol was studied by ditolo (46) in 95 . He reported that in seneral there was an enhancinr effect. Urea slifhtly increases prec1ipita tion, while glucose greatly O H d 1-“ d' d- increases it. The pre ing powersof various alcohols were in the order of benzyl alcohol and phenol, the strongest, then ethyl alcohol, and methyl alcohol the we Rest. Labes and Jansen (29) studied the effect of substitution in phenol, on its precipi ta inc newer toward de11ature 1 serum albumin. When one group is introduced of methyl, nitro or chloro, a more rapid precipitation occurs over 3 V’der ranre C) of p? the: with phenol alon . When two groups are substi- tuted this actio= is even stronrer. However, the substitution 18 of OH in the ortho position markedly decreases the precipitating action, the para slightly less, and in the meta position (re- soreinol), the precipitating effect is about the same as with phenol. Marie (47), usins ethyl acetate, developed a very sensi- tive tes which is positive for egg white at a dilution of 1: 10,000. The ester is superposed upon the liquid in a test tube, and if albn in is present the white rins forms at the junction of the two surfaces. The first to report that urea denatured albumin was Spiro(57), in 1960. eater it Was studied hy nansden (48) in 1912, who stated tint coagulnble proteins are converted at room tcmaerature into substances havinf firepcrties of acid- or alhali album n, depending on the reaction of the medium at D the time. Strong solutions of are white are chanced into stiff jelly at ordinary temperatures, the jelly rem ininr even after the urea is washed away. Another interestinp point was that NE4OCH and FHQSCN produce many of tte effects Of urea. hopkins (49) in 1950 observed the strikinr fact thit denaturation by urea had a negative temperat re coefficient, if the temper.ture was kept belor that of heat denaturation. Thus at 0 degrees after 1 hr., a rivcn ctperimcnt yielded 86.2% of the albumin in the denatured form; at he degrees, 69.;w; at 37 depress, 61.0p. In the sane paper, HOpkins considered that the red-violet color produced by sodium nitro- e ussidc hid been proved to a specific test for a denaturation. 19 Accordingly he used the test to'observe the denaturing action of various suostances on crvstalliae‘ egg albuvi solutions. Tt :ose ntich were positive were methyl—, ethyl-. and but 31 urea, ns**73 trical dime hyl- and diethyl urea, thiourea, acetamide, formamide and urethane. The following were negative: symnetrical diethyl urea, acet3'- and meth31 acetyl urea, biuret, allantoin, semicarbazide, alanine, phenylalanine, valine, leucine, cysteine, benzamide, creatine, caffeine and asparagine. Fonkins poi1t 1 out that although urea denatures albumin, it also neptives the denature' grotein even at the isoelectric point. Precipitation is onserved onl3 upon (II). Stabilizing Factors; rotection Against It is well known that albumin heated with sufficient acid or base will not flocculat at all. Although these agen- cies increase the velocity of dena uration, they also stabilize (D de na ured protein until the reaction is adjut ed to the isoe lectric point. The work of Fopkins just discussed above shows that urea also de natures , but stabilizes at the ears tine. :4. {2‘ (D Q7 ; 3 $2.! i:A1.narett 0 (5C) obseerd that both sulfur diox ’ormaldeh3de incre.;se t'e viscositv of albumin sols at 25 degrees and prevent their coagtlatien t" heat. Siri arl3, Cabin (51),found in 1929 that the pH range over which flocculation occurs from heat denaturation is sharper in the presence of fors- aldehydeThis was held to be a confirmation of the view that flocculation involves the amino groups. The work of D'yachkovskii (12) has already been mention- ed on freezing of albumin solutions. Precipitation occurred when frozen at temperatures a few degrees below zero, but at —182 degrees the remelted solution was completely clear. Fairbrother (52) in 1929 stated that the viscosity of egg albumin is decreased to a minimum value sometimes as much as 40 percent, by an X-ray dose of definite strength, This decrease is permanent and no appreciable subsequent coagulation occurs. Such albumin ooagulates more slowly than the original when brought to 61 degrees. Mitolo (46) reported that caffeine inhibits alcohol precipitation of protein from concentrated solutions of ovalbumin, but that in dilute albumin solutions it aids precipitation. The effect of temperature.on stability to alcohol and acetone has already been noted (6F). Below 5 degrees, these solvents do not denature albumin at all. Chick and Martin (6G) showed in 1910 that egg albumin crystals pressed dry until they contained only 20 percent water could be heated for five hours at 130 degrees in a current of dry air, and still be soluble in water. Fowever when heated with steam they became completely insoluble in a few minutes. 81 uepeschkin (53) reported (1922) that a high salt concentration slcwel up the rate of denaturation by heat. Jirgensens ( 4) note1 that high salt concentrations stabil— ized eye albumin ass in st precia itation by alcohols. Teorell 55 in 1930 observed'that methyl, ethyl and pr013‘ 1 alcohol C1us ed a lower turbidity in serum albumin which has heated in its presence than when heated in aqueous solution onlv Chis ‘as cons ide 1ed to be a protective action exerted by the elect 01:. Protection "as in th e ordei‘ of decreasing strengths: prepyl, ethyl and methyl alcohol. 70 influen nce was observed on cry albumin. Teorell found that turbidity by heatiné decreased with increasine acetate buffer ‘eustdb :ne t concentration. He suegested that the nephelo oznetrichhid not necessarilv give the amount of albumin precipitated, bu may be partly or wholly a measure of difference in the state of dis pe eIs ion, '3 d- C.) C+ c+ r— \— (D 3.2.» (D 2:5 d" r H L‘ Cf. H. O ’3 Beilinsson (56) showed in 192' v- . V - ‘ V , V! ' ~ Q. w .1 of serum albumin has greatly prevented b3 the presence of N v A . A f q ‘ '- ' sucro e or of r*ldcciol in “"F concentratio C. inc etncd '7»‘ r- " A -" I v A J- . x N ° 4‘ \1 V in was to br in the h ate1 sci : ios b0 p? 5.5 aid t1u1a‘e blt- F v I4. J- r‘- , awn, - O ~vr 'Oc“ t r r ‘ r‘ ~ A . ,A o '7 ' gdtdlmbe’ am onium sullate to a standarl tr1b111t3. The protective act ice with sucrose w,s also checked 0; nitroren detezminations on the centrifused precipitate. Ieuberg was stated by beilirsson in the above paper, to have reported that uany f the hydrotrOQic salts exert a protective action on proteins lwanowshy (58) in 1973 on the formation of alkali al }..J also on relative eloreter. Fe conClnded that formatio> of the altali albu" nrec1pi aeainst tation by in 1932, Duddles (59) reio"ted that solutio tallized ear albumin 'ere stabilized to heat coat glucose, ihnwztose, can? cxz, sh rose 11m11mannitol; glucose protect=d he albumin from coafulation by lifht. his nethaw ”as to run nitroaen determinat filtrgte fret the heat coat lu., after teatime to at 7C ”efrees Pauli 311 Schon (60, applie‘ conductivity 3 in stud3igyf'hindiis of zinc cfiflxr‘bde on serum alb It was observed thtt betfeen s it concentrations ,7 ~nd e x 1c ” 1., the 1031;1t; o; the :2 ~10; 11s W‘s expect d a1d that it WAS wit? a this range th Chlorid‘ W‘””ellj ;rgtects albu if seai st co eul Observ;tions have been n-de on tDe st bi11: suesrs 0‘ col-oi‘al syste s 'tter t5 n t‘e pret=i GIZMleB ii: 11 be Ci'sai"nific;1133 to neftz'tis ols 1) toward h at I,. m. 4.. .0 ,1- stud1od tne e1fect 01 913ccrol buhinate fro: «~e albu in, and albumin fro 1-1, 1"” ZAP. ‘ A-. (‘ fl ". a. c: ‘u. rid by Flycer doe (N _\ v C ,Jo n t s C. G, n soluti n O N fie 1’38 .;— nfler the oes protect O O V». 4- ' r” ‘s Qt. L) 1.0 L» d - GI1*- ya d gal (61) If) L4. -. Joshi “ of A m. 3 : ; ... _ \L MD 1.. u: .1 r nu . . 1o nu Cu 1 L... I; nu no :4 e no a 1..“ In.“ .1. 1 Lb nu . S \I 3 y a o n u ru mm m“ u i1 u ma 0 0d 1 .1 O .1 l .1 n .1. Lb I... «D >1 L o a mu ”a a n O +U u . .l S as .1 11 _.. .. «IQ wnuq 31; w. nu mu m“ .hu C t x. M O ”a W“ 0 fig +u 0.. 9 .. S . a. 8 ma e 11 $1 .a V“ .1 “a .d mp 0U .1; 11 my mu 1 “1 a .l & 1U 4o bu mm as Mu L 11 mu n“ my Av :1 Cu 1); 1 w u .+o e as m h. as a ”1 r6 mu m. Lb C. {\ n flu .a .1 a 3 a . no h“ an ab ma :1 km“ 0 t O. O C .t qu n m” :1 G a e t r w. .0 ”yo 1 Lb 0. “LL 1...“. 1W, L {J r 1 \a nu u e an m no .1 .1 . .u u Cu and nu mu .11 .11 1A ma AU mu +b x. nu ma on my nu o cu Va .11 \11 my mu .1; +9 TL r V Z a I my Lb T11 w“ e t. .1 1| QC 0 .AJ. nu Au :1 av V1 so by m1 nv nu av Ac nu Am r; d S i 0.. to be ,1 1 1 ,. o5: U U V” I;'\‘ ’1 \J .- r ”J ' be a nec- about (3. 5v ) e of r in ) to 7 e1: e 1 ‘r .t um (4 \ e rs takes up T.“ . n l C l a art? L' reatly decrea .) V8 ('3' p C ( lS albumin by hru hat t rela da 'VOl “7:?" J. l U 1n 4“. '7' U A vya V 5351 albh‘ 0-1 laud T‘fi rd“ a oi nu A y in 8‘? Oil l?1 ‘ or x k .1: a 1." It A '.' c.fl V A e or lcohol ovsh e 0' C '1‘ E O .. [I .L 7:1?A ,~ L— .._ C' r‘ 1. u dried era albu .1. 'fi rela t. ( 6 T1 LU 1 L 4H5, uu' voluw or! -ved 1‘ he mini: Water «e Baker )._ (5 1 V. m ('V 1.) 1V8 ercen 25$" LA. and o"1 exaan C'I’W‘ L. (\j #3 essary result of the lower degree of hydration of the protein, corresponding to a lower charge, since the two are interrelated. Weber and Versmold (65) in 1951 reported the results of their work on cryoscopic measurements on egg albumin systems containing non-electrolytes. By calculation of the volume of space occupied by the solvent, protein and solute, it was de- termined that the hydration space was found to tive the value, 1.53 to 1.56 ems. of hydrated matter per gm. of protein. The binding of glucose and glycerOL attains a maximum with rising crystalloid concentration, when about 0.084 moles of slucose and 0.048 moles of plycerol are bound by 1 gm. of the albunin. Under similar conditions urea is bound to a much larger extent, and even at 2 molar concentration its binding does not reach a maximum value. 0n heat coagulation the hydration space di- minishes 0.1 gm. per gm. of protein. The authors thus believe that Serensen's idea of denaturation being incomplete dehydra- tion is substantiated. In 1987, hundén‘fréselported that the sweetness of sugar solutions was decreased by the presence of 0.005% to 3% egg albumin. Strong evidence that glucose is adsorbed by erg albumin, has brought out by de Anciaes and Trincao (67), who established in 1928 that the reducing power of the mixture of the surar and albumin was less than the sum of the reducing powers of each one tested separately. This difference was ascribed to adsorption of the plucose on the albumin. The reducing power was determined by the netho‘ o: Fayedorn. If the albumin was first dissolved in a 5 percent sodium chloride solution, th (D adsorption \as almost conpletely prevented. In the same year Boutaric and Banes (68) sttdied the , casein, and alb— 5.1 U} 0 (-4 bindinn of eosin on mastic resin, goli unin. It was found that wlen the micelles were thrown down by cent Lh‘th”, no eosin was found in the preci Mi 3-0, but when the; were precipitated by aluminum chloride or by Heating, and then separated, the freater part of the sosin was found in the colloidal material. Chick and iiartin (7) reaorted a decrease in P -ion concentration durinn heat coagulation of egg albumin when below the isoelectric point, and a decrease in GE -ions ‘34 when above the isoelectric point. This was attribute to a binding of the acid and base, respectively. T'ne work of Cervello and Varvaro (56) , already dis- cussed, Moi ts to a combination of ions of the heavy metals L DJ with albumin when the salts are adde to the point of re- dis Wi g the initial precipitate. Such solutions did not give the usual tests for the meta s. Ito (69) in 1927, studied the conductivity of potassium '...J-' . .. SOlublOflS upon rJ (D chloride, zinc chloride, and calcium chlorir tie allitiou of er” albumin. In all see es the added protein brought about a lowerinn of conduct i;vity. The author concluded - .'°,.‘ -‘- ,. r, 4.! ..,j., «.J..0 . o ,, J- that this was caused “y the unsciotion o: t'e 8&1b. 26 Galeotti (31) showed that at ttc vo“ent .cn pre die itat ion begins, upon the addition of silver nitrate, the precipitate is of constant coneosition, containing silver and the protein. (IV). The Use of the Fephelonzeter in Study inn Precipitation and Stabilization. Kober (70), in 1913 publisi1ed a nephelometric method of deterzn.in nin nroteins in milk. He used a three nercent solution of sulfosalicylic acid as the grecipitatin; agent. mention has already been niade of the work of Teorell CO (55), on the u e of the nenhelonetc" in studyiné the orotect1ve action of alcohols on serum proteins to heat; also, that of Iuanowsby (58) on the stabilization effect of glvcerol on egg albumin toward ereciTitation by ammonium su fate. these workers re 01 ted any studies on the nature of the pro— tective action: whether the nephelometric meas1rements show differences in actual amounts of protein in th preci:i— 'Lr ‘L re 4,1111 1"““0' Q ’1 t mg 2... f d' “\ 1710' r. f June U3 be , or .1.L;lC_..d C -dhcfleu. 1L1 fie Clvr er O lS.J£44.QlO_. O [1.6 same at tut of orecifiit1te in all cases, or a combination of both factors The value of the ne1he lo eter was discussed in 1957 by Wells (71), in a cri 'tical revicv of the literatu1 e on turbidity measurerents. he stated that turbidity is a measure L7 of other factors in ad dition to conceo ntration. Wowevcr it ’.JI was s rrested tha th3re s coed reason to expect that the other variables would be of interest also. If the limitations of turbidity me asu enents are reCOfnized, he considered that the use of the tur bi dimetric instruments can be invaluable, especially in work that requires quick observations uithout disturbine sensitive adjrs t nts in the s;stem. Then too, minute amounts of mater ial can be e1t11,te1 vvhich cannot be weighed on the most ensitive balance Wells fur’:her considered that certain limitations must be recognized. In the absence of suitable st1n dards of tur- bidity, which be pre pared in any laboratory, it is now impossible for diiferent workers to duplicate turbidity meas- urements. Wave length and intensity of light, shape of instru— ment, size of cups, rate of mixing of solutions, etc., have been found to eifect readinns considerably. Among the "other variables" which Wells mentioned was an observation made by Eechhold and Vebler (72) in 1932, who found that oarticle size has an influence in the tur- bidity as measured in the nephelometer. They prepared barium sulfate sols in 013 oerol ar d ethyl alcohol, the particle €1ze ranging from 3.5;gto 4 a“, ‘fith increasinr size of micelles, th e tur b1r1t increases rapidly up to particles ECCiny(in size, then it falls off rapidly a first, then more slowly. XEBRILEYTib 3y? albumin r s prepared in the pure crystalline form by the method of Sérensen (74), and kept in concen— trated solution under toluene in the refrigerator. The more dilute solutions were prepared by dilution of the CF stock solution, and then checked by duplicate ni royen determinations using the micro-Kjeldahl method, modifi- cation by Allen (75). for nephelometric studies the bausch and bomb nephel- Ometer attachment was used, pith clear—fleas flanged cups, to be placed upon a black movable base in the Dubosque colorimeter, with fixed plungers. (I). Standardization of Lephelometer. 1 A solution of egg albumin was oreoareu h- ‘- , containing 0.1500 mgms. H per ml. The standard suspension was prepared always as iollous: From a 10 ml. micro—burettc, 1 ml. of the above albumin solution was measured into a clean, dry 5C 41. Erlenmeyer flask. then, 9 ml. of water was added to this from another burette, and the contents were mixed well by gentle rotation, care beine taken to prevent the formation of air bubbles which cause denaturation. Then 5 ml. oi a c.2tGC molar solution of sulfosalicylic acid was added from a pipette by allowing it to run down the side of the flask. Again the contents were mixed by rotation. A turbid suspension Iresulted which was stable for about lC minutes, after which time a gradual agglutination developed. The "unknown" suspension was prepared in the same way, except that the volune of albumin solution was varied, and that of water adjusted so that the final volume of the suspension was the sane: 15 ml. To keep constant any gradual changes in turbidity after the addition of the sulfosalicylic acid, the stan- dard YRS made up new with each "unknown" and the acid was added D? 0 means of two 5 ml. pipettes, simultaniously in the standard and the "unknown". The nepheloneter cups were rinsed out once with the suspension, then filled to the flange. After wiping off the outside of the cups with a dry cheesecloth they were placed in the nephelometer and adjusted so that the plun- gers were in the centers of the cups. The standard was placed on the left and set at 20.0 mm.; the unknown, on the rifh.. study it was found that in case .‘ the standard settinw had to be lowered, say, to Z.C mm. in LC order to take a reading of a very dilute "unknown", the reading (within experimental error) corresponding to the standard at 20.0 mm. can be obtained by multiplying the observed reading by 10. The observed reading for each sample tested was taken as the average of five different settings of the "unknown". In Table.I are the results of the standardization. Each recorded nephelometer reading is the average of four separate determinations on entirely new suspensions. The pH was measured in a few of the suspensions, by the quin- hydrone electrode, and it was found to be pH 1.35. It was a constant, regardless of the amount of albumin present. Fig. 1 shows the relationship between the total mgms. N in the suspensions and the nephelometer readings. It is obvious that there is no linear relationship between the two. The logs of both values were then tabulated, Table II, and plotted as shown in Fig. II. A perfectly straight line results. The equation for the curve in Pig. II was obtained in the following manner. A straight line curve is of the general form: 1), a=nb+c If we let, a=logR; b=logW; andnandcbe constants, Table I. Standardization of the Nephelometer. Standard suspension. "Unknown" suspension. 1 m1. alb. soln.,(0.15 mgms. N). x ml. alb. soln. 9 ml. water. 10-}: ml. water. 5 ml. 0.2.M. sulfosalicylic acid. 5 m1. as. acid. Set at 20.0 mm. arias x 10«— x W R 0. Ave. No. [ml. alb. (ml. Ego) (total (neph. Detns. Deviat. soln.) mgms. N) read.) of R. #1 0.15 ml. 9.85 ml. 0.0225 mg. 167.0mm. 4 20.7 mm 2 0.25 9.75 0.0375 98.8 4 $3.3 3 0.50 9.50 0.0750 42.2 4 t0.7 4 0.75 9.25 0.1125 27.5 4 $0.3 5 1.00 9.00 0.1500 19.3 4 1.0.3 6 1.25 8.75 0.1875 14.3 2 $0.4 7 1.50 8.50 0.2250 11.7 4 1:0.4 8 2.00 8.00 0.3000 8.2 3 $0.2 9 3.00 7.00 0.4500 5.5 3 $0.1 10 5.00 5.00 0.7500 3.5 1 -—- Table II. Derivation and Use of the rormula. (Data from Table 1). (>7 “- ‘N t v eries Logic)? LoglCR W weaie’g 79 230. (known mes. (mgs. L by' Error by nitrogen) formula). Formula. ;}1 8. 52-10 2.223 0.0;25 mgs. 0.0‘40 mfs. +5.55% 2 8.574-10 1.915 0.0275 0.0374 -0.33 3 8.875-10 l.C25 0.0750 0.0783 +1.70 4 9.051-10 1.43 0.1125 0.1093 ~2.87 5 9.l76-10 1.286 0.1500 0.1474 -1.75 6 9.273-10 1.155 0.1875 0.1905 +1.60 7 9.352-10 1.068 0.2250 0.2247 -0.16 £3 9.477-10 0.914 0.3000 0.3023 +0.77 E? 9.653-10 0.740 0.45C0 0.4270 -5.86 10 9.875-10 0.544 0.7500 0.6225 -17.0 * By formula, ave. fi dev. $2.4 * This figure was omitted in the calculation for the {percent average deviation. Afglutination was very marked. n0 bu MICHIGAN STATE COLLEGE DEPARTMENT OF MATHEMATIl ji“) 1111‘]! II 1 1 1 I1)11Ii“l!j III.I !‘ ))1llllll "IJIII I“|‘.IOI‘I1IIFIIIJ‘I.I‘IIIVIII{JICIC I'll; .dYIlQIIIIIllifi‘iIJ'.1I.-"111Iv‘n -l‘tl“)j1ll1'. \‘11‘1'11‘ . I. I. I . . , . I .. . o. . E . . . I.. I . I o I . I. . . . I . . . . I . fi.¢ I . o . H I . a . v 0| . . I . ..OL. r..I I c. — _ I. r.... . ,- . o l . I .I I. 06" Ie§ I o I v V. v). . I I . I I I I I I 4 III I I ll . 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E . _ r o- . —- I~OII O _ H p ._r If r . d a ,. i. d d I I . In. .. . ..H _. .. . I. u _ . — . . . . . . .. . I I w . .. I I . . . . _. . .v . .. . . . I. . I. U I I . . I 6 ._ I F — I _ I I . .I I '. _ 1 I . _ . 2 . ....WA I I I . I .1“ II D I. IIIIO 0 tilt I. . . . .. I < .I 0‘ ‘A o I . I .u I. . . a I .. ....I.. ... . .... . I .1 .. . _ . . . I... u. .0 . I. IT I I .h I . v I .. .4. I. ... c U ‘ A . . _ ..IIII . o.-.¢IOI¢A + I . . ‘ . I In; I ’gug . . .... I I.” v o I. ..I..*O A . ~ ¢ I ... . ... 0.... fi LlIrslr. r L. I .I V I — IR Irwrp DEPARTMENT OF MATHEMATICB 35 Where R is the observed nephelometer reading of the unknown in mm,, and W is the known moms. N in the total unknown suspension. Then, substituting, 2)“ logR=niogW+c To salve for c, the curve extrapolated through the point (x) on the graph, where 103 W20, gives 5). o=iog R=O.3OO Point (y) on the graph gives .4). log R:l.840 ; 10g Yi'=8.'700—J.O =-l.500 Substituting 3), and 4) in equation 2), 5). n=-1.185 Equation 2) then becomes 6). log R:-1.i&:5(1og W) + 0.300 This gives for Fig.’ I, 56 +1.185 ’7). RW :: 1.995 A somewhat more convenient modification is the fol- lowing: [0.300 - log R 8). W = antilogk 1.185 which was found to hold to $2.4 7o, over the range of 0.02 to 0.45 mgms total nitrogen in 15 m1. of suspension, as will be observed in glancing at the last three columns in Table II. Using the same instrument and cups, there is no doubt that the formula will hold for determination of alb- umin in pure aqueous solutions of the crystallized product. A standard solution made up from a new batch of crystals and compared in the nephelometer with a standard of the old, gave the same reading within limits of error. (II). "Protective" Action of Alcohols on Nephelometric Turbidity of Albumin Suspensions, when Precipi- tated by Sulfosalicylic Acid. Qualitative experiments indicated that the turbidity of albumin suspensions ( made as in the standardization experiments), was greatly decreased by the presence of methyl- , ethyl-, and prOpyl alcohols, glycerOl, glucose, fructose, mannose, galactose and mannitol. Since it is known that the sugars and mannitol protect erg albumin from heat coagulation (56), (59), that the alcohols decrease the turbidity from heat precipitation of serL 1m albumi (55), and that glycerol decreases the turbidity upon the addition of ammonium sulfate to albumin (58), these qualitative obser- vations were though t to indicate a new angle of ap roach to the same problem of protective action. The author believes that previous work points to he gimportance of the hydroxyl group in the stabilizing action of the added substances. Consequently the simpler alcohols were studied in the present work. 1yl alcohol was st11died first. The H The action of standard suspension was prepared as in the standardization experiments. The "unknown" suspensions all contained th e same amount of albumin as the standard (C.15 m3118. E in 15 ml. of sm1s ension), but varying amounts of alcohol. The mixing was alv.ays c1rried out in the following order: 1 ml of the albumin solution(C.15 1sms. E/ml), was added to the flask; x ml. water was added, mixei by rotation of the flask; (g-X) ml. of 10 1. alcohol added; mixed by rotation; at once 5 ml. of 0.2 m. sulfosalic lic acid was pipetted into the standard and "unknoun" malt a so slr, mixed, and sad. The results for methyl alcohol follow in Table III. 58 Table III. Effect of Methyl Alcohol on Hephelometric Turbidity. (Pptn. by sulfosalicylic acid). Standard suspension. "Unknown"suspension. 1 m1. alb. soln.,(0.l5 mgs. N). 1 ml. alb. soln. 9 ml. 810. x ml. H20. 5 ml. 0.2 M. sulfosalicylic acid. 9-x ml. 10 M. CH3OHo Set at 20.0 mm. Contains 0.15 mgs. N. W is obtained by the formula, calc. W = antilog(0.3001- 10: R) usp. Final 11 w w -W as No. 5F"? No. molarity (neph. calc. calc.(pro- Trials 09308 read.) (mgs. N) (mgs. E tection) "protection") #1 0.17 M. 20.0mm. 0.1455mg. 0.0067mg. 4.47% 5 1.35 2 0.55 19.5 0.1477 0.0025 1.55 2 —-—- 5 0.67 20.1 0.1424 0.0076 5.07 1 ---- 4 0.84 19.0 0.1492 0.0008 0.55 1 --—— 5 1.55 20.9 0.1578 0.0122 8.15 1 ---- 6 1.67 22.5 0.1292 0.0208 15.87 1 1.55 7 2.67 26.6 0.1126 0.0574 24.9 2 ---- 8 4.00 55.6 0.0925 . 0.0577 58.5 1 --—— 9 4.67 56.8 0.0854 0.0646 45.1 1 --—- 10 6.00 56.8 0.0595 0.0907 60.5 2 1.27 11 12.00 (a) 75.6 0.0465 0.1155 75.7 1 _-_— 4.7 {b} 65.6 0.0525 0.0925 65.0 1 1,25 59 Eotes concerning the tab1§,_ probably I""Idolarity" isAthe incorrect term because of expected volume changes. However the assumption is made that the difference would not be over 2 fl, which is within the error of the nephelometric measurement. (a) Made by adding 9 ml. of 20 E. 08308 to the suspns.; slightly turbid before adding the sulfosalicylic acid. (b) Made by adding 9 m1. of the C.P. CUSOE to suSp.s.; very turbid before adding the acid. (c) Measured by the quinhydrone electrode. Assuming that the turbidity as measured in the neph- elometer gives the actual amount of protein in susgension by use of the formula, methyl alcohOl shows marked protective action against the precipitation of pure egg albumin by sulfosalicylic acid, the action increasing with increasing amounts of alcohol up to 12 molar. The change in pH is hardly sufficient to account for the decrease in turbidity. In exactly the same manner, ethyl alcohol, propyl alcohol, ethylene glycol and glycerol were studied. The results are shown in the next four tables. Table IV. "Protective" Action of Ethyl Alcohol. (Pptn. of alb. bv sulfosalicvlic asid). . J . Standard and unknown susaensions, the same as in Table III. Euspension Final Lolarity R * Fercent 1 1 To. 1 CzfifioH ‘(neph. read.)‘"grptection" : : : u 1 -1 - 1 1 t #1 0.17 35-10 1 300': T4911. 1 5 O40 ‘3 1 : 2 : 0.53 20.0 4.75 5 0.67 25.2 15.81 4 2.00 54.0 59.0 5 4.00 56.6 60.4 6 6.00 65.2 64.8 7 9.7 * .1... 74.0 68.4 * Kine m1. of 95 p 025508 was added to the suspns.; slightly turbid before adding the ss. acid. a 1 , 4 . .. . ~ ~ . . Cl lhese results sho“ a slightly greater r1se 1n protect- . \‘ o | . y o o T‘ y 1ve action it the lower concentrations than those Vita 0.509. However, the maximum "protection" is slightly less, with the concentrations use . Table V. "Protective" Action of Propyl Alcohol. (Pptn. of alb. by sulfosalicylic acid). Standard and unknown suspensions the same as in Table III. uspension linal Molarity R Percent Ho. Egoh (neph. read.) TProtection" #1 0.17 M. 20.1 mm. 5.06 m 2 0.55 22.5 15.0 5 . 0.67 51.5 54.5 4 1.55 54.8 59.2 5 2.00 57.0 45.2 6 2.67 29.4 51.1 7 5.55 24.9 20.4 8 5.67 25.0 16.0 9 5.87 56.0 41.9 10 4.00 41.6 48.5 11 4.67 167.0 84.5 12 6.00 400. 91.9 15 8.0 * 1000. 96.4 * Nine mls. of 0.8. propyl alcohol used to give this concentration. one observes that propyl alcohol has a maximum "protective" action at about 1.55 molar alcohol, then a min- imnn.at 5.67 molar, followed by a very steep rise, reaching almost complete "protection" at the highest concentration used. The suspension # 15 was practically water clear. 41 42 Table VI. "Protective" Action of Ethylene Glycol. (Pptn. of alb. by sulfosalicylie acid). Standard and unknown suspensions the same as in Table III. uspensibn Final Helarity R rercent No. 0239§:9§398 (neph. read) "Protection" #1 0.67 E. 21.0 mm. 8.47 % 2 2.00 21.9 11.6 5 4.00 26.8 25.8 4 6.00 45.8 50.7 5 10.7 * 190. 85.7 t Nine mls. 0.P. glycol was used to give this con- centration. Here again, the results show the same phenomenon. In general glycol is weaker in its action than any of the alcohols studied so far, except at the highest concentration. In Table VII,on the next page, are shonn the data for the effect of glycer01 on the albumin suspension. Slight- ly greater "protection" is observed in the case of glycerol than in that of glycol, when equal melarities are com- pared. The effect of glycerol and of methyl alcohol are similar. . All the results in the last five tables(III éVII,inc1usive), are plotted in Fig. III, giving a perspective on all the data. 43 Table VII. "Protective" Action of Glycerol. (Pptn. of alb. by sulfosalicylic acid). Standard and unknown suspensions the same as in Table III. uSpension Final Molarity R 'Percent No. CHgOE-CHOH-CHaOF (neph. read)"Brotection" #1 0.67 m. 20.7 mm. 7.40 % 2 2.00 25.5 21.4 5 4.00 52.5 36.4 4 6.00 59.2 61.8 5 8.2 * 115. 78.2 * Nine mls. of the C.P. glycerol was used to give this concentration. From an inepection of the curves in Fig, III, it is plain that the factor which is being measured,increases with increasing concentration of alcohOl. as a general rule. The peculiar curve shown by propyl alcohol has no explanation that the writer can offer. If the curve is continued from x to y, a smoothe curve results. The experiment with methyl alcohol was repeated, using this time twice the amount of albumin (0.5000 m.ms. N in the total suspension). The results are shown in Table VIII MICHIGAN STATE COLLEGE _ . ..-..-"_,_. - -- _.-- _ W7H~u.. . u Jul . ...- “fl-v-v-fi u—‘p’rW-fi— u—h’ - «flaws—fulfi- - , . h < 0 o u . i ‘ r p - v w I ‘ u 1 . p o e - ! , . - 1 e . 1 v 1 r 1 Q ~ u u. o o e 1 I . . , . . Per (”7 ca pm... I “4‘ I -vb—‘u4§ 1.9a, 1.1 - 31' '1 . . m (I) if , 1 )1?“ '(3 .. . . . ti. 1, . . v . . . : . I ' ' I .fiffif‘. 'TY I 7 .. IT' . T. -i. 21"? ? ‘~ ‘ E ‘ 2:1: , f ~ ~ - 1 «f: v. . , , 1 . _ I _ 1 i ’ i 1 1; 1 : I1 ; j» " ' 1 1; J ; i g _ - - Fig. III. - - g ; 2 Concentration of Alcohols , r" . ,- and ti 1 ~ "Protactive"’ction. 1 From Tables III—VII. 3 .. 1 - . “mo—- W... a..- ..-.....4.h__. ...-MM __—.. - _,_~_-._. -- *_ . l 1 - v I o ‘1 . 1E . . u v ‘ - r . , | ‘7 LGEBHC d, Methy1.1lcoh01. fer—«~10- Ethyl alcohol...“ 'Pr01y1 Alcohol. fllycol .-y. .. . . ... ... . . : .,. ' I’ O A. -- ‘ |. >— -'- ——— 4. ' . t - |v on I .... .. -. . . a . I | .. . .. ......... ‘. . .- . .. . . . 1. , II .-...4’.. .. 7.. 6. . .... -". . 9 .. . ‘5‘ .. l6 ,‘8 . .. .. . .. ....t ...... q-e. .o.¢-t - .. e e ’- .V 1 A . l . ... 1 . . I” .. .4.. 1.; 1 ... - ... - , , _ 4 nnnnnn >1 - u». n on.‘ ‘k. Jr ~0- . I . .l- . ..... t. .. .-.....I i . -' .o . 71...... .-I ..... L» ...—o » ¢~ h.....'-~.o <~~o—.— 1.; 1.. ... l o :_:-!.-:, . ,...... ....J ... . . « 1. - . ~-— ........ 1 ------ thfinflirz? af’.3flk' I -1 . ..... . .. ....... .... . ,_ .I 4 ._ ... ....o . . '1111 b ..... . s. y . I....,. ~ .. o... 1. . .... ..-. .1 ........... ... , ll _, . >.. ,.., -... ... O .1... '.p¢ ooooooo , <<<<<<< e I. ....,.,, ., . ... ..... H ..... o o ....................... 40+LebH 4..-.4.‘ '0-"—- 91 ....... . .. "'§‘~1 .. k. . . . - + Y I: I. .1154» A 4‘5“. r 6741-1‘: ¢¢¢¢¢¢¢¢¢ 6.. ::".Tl" » ~.¢A..~-- :1," 1 ‘- h11111111ri‘1'141 1111111111 111 1111i 1 11111- -111“ 111 1111111 111 1 1 ;1;.' 12111111 11"l;_ 411---; ' " DE PARTM ENT OF MATH EMATICB - e ' . t“___ -- ... :vqr—JL...-_..._._~ 4... -___.. ...—..-. -1 1.4 ._ .-.. _- _ . 45 Table VIII. Effect of Higher Concentration of Albumin on the "Protective" Action of Methyl Alcohol. Standard Suspension. "Unknown Suspension. 1 ml. alb. soln.(0.15 mgs N/ml.); 2 ml. alb. (total, 0.5 mgs E) 9 m1. 820. X ml. H20. 5 ml. 0.2 M. sulfosalioylic acid;8-x ml. of l0 M. CfisuH. 5 ml. 0.2 M. ss. acid. Set at 20.0 mm. ~___‘ uSpension Final Molarity R Percentage No. Cfing (neph. read.) "Protection" #1 0.67 s. 8.8 mm. r 4.60 % 2 2.00 11.0 21.0 3 4.00 16.6 44.2 4 5.55 21.8 55.6 5 10.66 ' 56.8 71.5 6 13.1 * 48.0 75.0 ' Eight mls. of 20 M. alcohol used to make this. * Eight mls. of C.P. alcohol to make this. Upon comparing the above table with Table III, it is seen that the same percentage"pr0tection" occurs, re- gardless of the different amounts of albumin in the two series of experiments. The effect, then, is independent of the albumin concentration for these two concentrations used. The above results are not plotted in iiggL III, because they correSpond closely to the 08508 curve already plotted. 46 To obtain additional information, the attempt was made to check the nephelometric studies by the determinations of total nitrOgen in the filtrates from the suspensions prepared as for the nephelometer, both with and without the alcohols. The method was as follows: A suspension containing 5.065 mgms N in the total 15 ml. was made up in each case. The suspension was allowed to stand five minutes ( the nephelometric readings required an average of about 8 min.), then filtered through qualitative filter paper. The first 5 m1. of the filtrate was discarded, and a 5 ml. aliquot of the rest analyzed for H by the micro-Kjeldahl method. The results of the experiments follow. Bach recorded value for % mgms. E in the filtrate is an average of two separate determinations on entirely new suspensions. Table IX. Comparison of N detns. of the Tiltrate with Eephelometric "Protective"Action in the Bresence of Alcohols. (ppt. by ss. acid) 5.065 mgms. N in total suspension; 5 ml. aliquot for detn. Kjeldahl bephelometer Suspn. N0. Alcohol Molarityfi M in flt.%"protection" 41 none 0.00 m. 1.24 5 0.00 a 2 Methyl 5.55 0.85 50.0 5 Ethyl 5.55 0.28 64.0 4 Bropyl 7.1 8.48 95.0 5 : Glycol 9.5 0.84 81.5 6 I glycero- 7.5 8.16 72.2 7 1 controlw . 100.8 159.0 L—- The "control" in the table consisted of only the aqueous solution of 5.065 mgms.H in the albumin, no alcohols 0r sulfosalicylic acid present. The"% protection by the nephelometer" was obtained by inspection of the Fig. III. From Table IX, it must be concluded that the "pro- tective" action of the alcohols, as measured by the neph— elometer, is only apparent. The albumin is almost quantita- tively precipitated by the sulfosalicylic acid, both alone and in the presence of the alcohols, but the alcohols lower the turbidity considerably. rropyl alcohol and glycerol do show protective action by the I determinatiOns, but the actual protection is only 10 h of that calculated by the nephelometer method. Some doubt may justly arise as to the fairness of. comparison on the two suspensions; the njeldahl determin- ations were made on those containing 5.065 mgms. N per 15 m1., while the neihelometer was used on those having only 0.15 mgms. N perfikml. However, the author atte‘:z;pted a few h determinations on suspensions containing 0.500 mgms. N per 151m1.. taking 20 ml. aliquots, with similar results, though such small amounts of N were difficult to check. Another criticism may also be brought up, in that the time of filtering for suspensions containing the more viscous alcohols was very long, taking upwards of an hour in some instances. In the continuous presence of the sulfo- salicylic acid, it is possible that there is a gradual increase 48 in turbidity, and also in actual amount of albumin in the precipitate after such a long time. Therefore a check was made on the effect of time on the turbidity. A single suspension was prepared, containing 6.00 molar glycol, 0.15 mgms. N in the 15 ml. of suspension, and precipitated by the usual strength of sulfosalicylic 8016. and was read at once in the nephelometer, compared with the standard. The reading was 44.4 mm. After half an hour a new standard was prepared, and the glycol suspension read again. The reading was exactly 44.4 mm. as before. Therefore, the writer believes he is justified in concluding that the nephelometric observations on the effect of the alcohols are measurements almost entirely of factors other than true protective action. Differences in particle size may have something to do with the observed phenomena, as pas suggested by the work of bechhold and Pebler (72). However this seems doubtftl, since the filtration readilv removes the suspended particles in all cases. It appears to be a tenable hypothesis that the degree of swelling or hydration of the suspended particles would explain the facts more cloSely: the more the swelling, the clearer the sus— pension. 49 (III). Peat Erecipitation Studied by one chhelomcter 03 .5 Q..- a .4 p.» d- F) O ”“2 (2‘ L3 {J (1) Cf. CD *5 E} P, B 9.3 C 1‘ H 1) ...) 0 (‘1' (‘S‘ O Q: (1 0 v ,- 4.1- - . - ...,. .....- ., 04.. ... . ~~.« Checking the nephelometrie he sure 1uts 11th nitro an detai- Y.- .- - .1 . , - ..-J. .,_. I... 1. -. .. -1... mlndtlo s on the filt‘ate or prec111tate. It 1 as found that a pH of 5.2 gave a stable suspension, when a solution contain- . o . r .- “v14". -.. . 4“ J‘— 1‘ ”I" f - .‘ ‘4 W - o v 1 ' - — V. r« (N 1 . I‘ :D‘ ‘ ... in" a bulls ub tle coicentiation of C.7050 aims. Y in 20 ml. \ . of total volume (ex;crinents of Puddles (59)). was heated at 70 degrees for 10 minutes. each of twelve Iarse test tubes was placed 5 m1. of (.4 :5 sodium estate acetic aei:3 buffer solution (0.157 moles Yazo. CD and 0.051 moles H‘e. per liter), sufficient al‘L 1:.in sol tion so that there was 0.7050 apps. 3 then water was H. O {11 (1. f O (D «\J U measured into the tubes so that with a g ven volume of the 10 1. alcohol added the total volume was 21 ml. The solutions were prepared in triplicate, A, 3, and 0. After heating all the tubes at the same time in a water bath at 70 degrees for 10 minutes,A_was determined in the nephelometer, compared with the usual SL llfosalicylic acid standard suspelsion; and B and C were fil eered, and the filtrate or precizitate .nalyzod for nitrogen by the micro-Kjeldahl method. J. The results for methyl alcohol are shown in Table X. ...). T 5' (D (D CL; The column "urns. Y in ppt. by nep.. eq." gives values is» 11 calculated by means of the equation, 0 9}. :.1-_-_ 1.555 antiIOg 0.500 - where elometer readin: in 3' y" o 0 ' ‘ , u 18 the ngms. O 1., Y in the pot.; which corrects for the difference in and the unknown. Effect of methyl Alcohol on Heat Precipitation. Table X. R is the observed neph- and the 1.353 is the dilution factor volumes of the standard (Measured by H detns. and by nephelometer). pH 5.2 ; 0 0.7050 3338. E in 20 ml. heated at 70 C. for 10 min. Each value ave. of duplicates. Baht. eerie 11551 R 1ggs. N in ppt. Percent of t6ta No. No. Ecler.(neph. by by by by _ ile...read.) gjeld. Nephel. Kjeld. Nephel. Control . #1 2,0 11.1 mm. 0.5441 0.5155 48.8% 44.55 # 2 m/4 8.4 0.4550 0.5957 54.5 55.2 1 5 m/i 5.7 0.5554 0.5497 94.4 77.9 4 2.5 M. 5.8 0.7052 0.5424 100. 77.0 1 5,0 15.4 0.2950 0.2255 42.1 51.0 2 5/10 15.5 0.2952 0.2255 41.9 52.2 2 5 m/4 10.1 0.5570 0.5587 50.5 48.1 4. 5/1 5.1 0.5559 0.5190 89.8 75.5 1 5,0 11.0 0.5205 0.5155 45.5 44.8 2 m/10 10.5 0.2870 0.5275 40.7 45.5 5 5 m/4 8.8 0.5200 0.5805 45.4 54.0 4 5/1 5.9 0.5525 0.5555 89.7 75.7 In the suspensions containinn l n and 2.5 k. alcohol, agglutination was observed at the end of the heat treatnent. The table shows two new facts: the first is that CF50? accelerates the heat precipitition of egg albumin, the action increasiny(with concentrations above 0.13), with increasing amounts of alcohol; the other is that in the ease of heat precipitation the nephelonetric method agrees fairly well with the Kjeldahl method. The latter is surprising, since the suspensions of albumin are prepared by two entirely different methods. Where disagreement occurs in the table, it is a question whether the nepheloneter or N detn. method is more accurate. In filtering for the N detn. it was often found that it was difficult to get a clear filtrate; further- h more, filtration often caused slight meaanical denaturation, which would introduce considerable error where such small quantities of N are present. The disagreenent between the two methods at the higher amounts of albumin in the preeiaitate 15 to be expected. The figures in Table II show that when the formula is applied to suspensions containing 0.75 mgms of nitronen in 15 ml. value _ the calculatedhis l7 % too low. The same experiments were carried out using glycerol, as a different type of alcohol. The results follow in Table XI. Table XI. Effect of Glycerol on Heat Precipitation. (Measured by N detns. and by nepheloneter). pH 5.2 ; 0.7050 mgms. N in 20 ml. ; 0 Heated at 70 C. for 10 min. Each value the average of duplicates. xpt. eries Final 2 Mgns. T—in ppt. Percent of total N0. N0. Molar.(neph. in ppt. Ale. read.) jeld. Neph. hjeld. Neph. 51 8.0 11.4 mm.0.2999 0.5051 42.5 2 45.4 4 2 m/4 9.5 0.2894 0.5540 41.0 50.1 #1 3 2.5 M. 24.5 0.2580 0.1644 56.6 25.3 4 5. M. 56.8 0.1648 0.0789 25.4 11.2 1 8,0 12.7 0.5570 0.2795 47.7 59.5 2 m/4 14.1 0.2950 0.2521 41.8 57.2 2 5 2.5 M. 25.8 0.2542 0.1645 35.2 25.5 4 5. M. 47.0 0.1599 0.0925 22.6 13.1 These results confirm bailinsson‘s observation (56) that glycerol stabilizes egg albumin against heat coagulation. The nepheloneter gives the same conclusion, however the ab- solute values do not agree by the two methods. In high con- centration of glycerol, the nephelometer gives lower results. This may be the same type of phenomenon as was observed in the experiments with sulfosalicylie acid, since the turbidity increases more slowly than the absolute amount of albumin in the precijitate. E HIGAN STATE COLLEG MIC 1 I _ . 4 . .. .. v,, _ It » ‘. ........ . H p . _ . dahl x L ' .‘Iv ‘ a F 51 1‘K5 .7 ’11- l .‘and I Yeah. Readings W...” l .1955 Tables x and 21} r 1 I x I . c With‘methyl-alcoh01.g With‘glycerolg" ‘be_end.’ o ?x b F M ATH EMATIC' .5 DEPARYMEN 4,; 5m..,.m,. is... .14 2 o 4., 5;.t, .4 3,4. .1. urxcuuyaox gssrwassrwweeec. Biblit'fimt L.I..I.L.. ) in' TM.» -.. Y.A.:Lll}|| .Mt... ‘lxl .lllb’(.l|.1 54 When the nepheloneter readings are plotted against the observed mgms. N by the Ejeldahl method, in the presence of methyl aloohol and of glycerol, Fig. IV is the result. Ber- haps if a larger number of experiments were carried out using other non-electrolytes, such as glucose and acetaldehyde, the curve may be of great value in studying, by the nephelometer, the effects of such non-electrolytes on heat precipitation. The nephelometer method is by far the easier of the two meth- ods. A single experiment was carried out with ethylene glycol on heat precipitation. Table XII contains the results, obtained by use of the nephelometer, only. The assumption is made that glycol follows the same curve as in Fig. IV, the N values being taken directly from the curve. Table XII. Effect of Ethylene Glycol on Feat Pptn. (Detnd. in duplicate by the neph.) pH 5.2 ; 0.7050 mpms. N in total 0 of 20 ml. ; heated at 70 for 10 min. _5ries E0. Final R Hgms. 3 in Percent L Molarity (neph. read.) Precipitate. in Ppt. Glycol. k 1 H20 12.4 mm. 0.297 mgs. 42.1 % 2 M/4 14.1 0.285 59.2 5 M/l 9.8 0.550 45.8 4 2.5 M. 6.5 0.590 85.7 Glycol ircreases the amount of heat precipitation, as judged by the nephelometric rethoi. She accelera effect, however, is less than that for meth"l alcohol. (IV). Conclusions 1. The nephe crater can be used in determining minute amounts of pure, agueous egg albumin, vfi en sulfos alicglic acid is used as the precipitating arent. The formula has been worked out, and found to be accurate to 2.4 percent, over the ranre of al‘umin concentration containin a 0.03 to 0.45 mgms. nitroren in 15 ml. of suspension. 2. Then alcohol: are present in a: cunts at ove 1 molar the formula is valueless for determining actual amounts of albumin, with sulfosalicylic acid as the preciaitant. 5. In the presence of about an 8 molar concentration of methyl-, ethyl—, and propyl alcohols, rlycerol and glycol, .he turbidity shown by sulfosalicylic acid precipitation de- C) x.) C) :3 Ho :5 l O 5 d- T? (D {‘3 H 0 O .J‘ O .J o L; d. creases about 68 to 97 percent, de M5 the same time, 5n drotein is nearly Chantitative y precipitated. with propyl alcohol and rl;cerol, slight prote ctiie action is 9'! observed by nitrOfen determin tions on the £11 rate, but the amount of protection is far below that which would account for the decrease in tc rbidit Ly. The snares tion is made that differ- U ences in particle size may explain the decrease, but more likely (21 O} a difference in the degree of hydration of the suspended particles is the cause. 3. hethyl alcohol and glycol accelerate the heat precipitation of egg albumin at p? 5.2. The effeCt is weaker with glycol than with methyl alcohol. 4. Glycerol protects the albumin from precipitation by heat at pH 5.2 . This confirms the report of beilinsson (56),who obtained his results by a different method. 5. A comparison of the nephelometer method of meas- uring heat precipitation of albumin, and the micro—Kjeldahl method , was made.. The methods agree, in that a high tur— bidity corresponds with a high amount of precipitate, and conversely. However, the absolute values by the two methods do not check as well as one would like. 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Allen, W.F., Accurate and adaptable micro»Kjeldah1 method of nitrogen determination, Ind. Eng. Chem. Anal. 3 ., 3 z 239, (1951). {‘14 ‘1» )_\ Q ‘ '9‘ m3 ’ ‘ 4 .. , . O k ME'r‘J‘ ’ 1512.015 "f“n‘en ' ‘ 95495 4111.99nahuell W7 'Vf*rfif . :slcn-the_precipitation of egg albumin. Donahue' ;1pg “Wm”.- , 7mm- _‘ p ,. z.1 IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII (m4))))))))Il))l)))))))l)l __. . .A. ‘ ,._.—_ -_ -.-..._.~.