III

I

      
   

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IIIIMI‘IWIIII

I

AN INVESTIGATION OF ACTIVE FRAGMENTS
AND’ THE ACIIVE $§TE OF SWEET

POTATO BETA-AMYLASE;

é MINIWWIIl

Thesis Ior Hm chmo OI DH. D.
MICHIGAN STATE UNIVERSITY

Rene Evard
1959

 
   

LIBRARY I“?

Michigan State
University

  

 

 

AN MHCKTION 0F ACTIVE mm AND THE
ACTIVE SITE 0F SHEER POTATO p—mss

Rene Bard

ATHEIS

Submitted to the School of Advanced Graduate Studies of Michim
State University of Agriculture and Applied Science
in partial mlffllmmt of the rewirunmts
for the degree of

DOCTOR OF PHILOSOPHY

Departmmt of Ohmistry

1959

I @5215;

ACKIIO'VEEDCMENT

The author wishes to express his sincere appreciation
to Dr. Jahn C. Speck, Jr., for his interest, gxidnnce and
advice during the course of this hwestigation.

Acknwledgnmt is also due to Mr. John P. Riebm for
his assistance in setting up the chromatography column.

finally, the author wishes to express gx'atimde to

Michim State University and the National Instimtes of
Health for their timely financial support.

W

 

 

All mnamm 0!" AG?!“ mm AID THE
W em 01" m1- mm W

mm

AN ARTE“)!

Wt‘bd to the Sebeol «Advanced mes Studio- or
' mu University of Aux-1mm and Applied Science

DOCTOR 0F PHROSOPHI
Department of cm”

You 1959

Amad_ _ m:

 

 

 

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'mmmmmamnmmwmummmm
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nah mum: m, poplin «mm the «m puma B-ufhn mi-
umuswanmmm. mWorgatma-imm
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nmaeuumanmmm,mmupmmm
ummmmm,umw1mccmuwm
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mwmammmwmmmwm
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3’
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I. ”mm mmwcnwOODIIOQDOOOOUOCIOODIOOIOOIOOOGDOCOCCI

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I. 3181031081: JINWOIION

Very little is known about the intimate structure of. ma, the
name of active fragrant: of miniml size, or the amino acids summon
in the neighborhood of active sites . Inch valuable intormtion about i
the nechanim of mm action can be'cbtained by experiments carried
cut with native protein” however it amen! reasonable to name that
infomtim concerning active sites and the mechanism of may!” actim
could be obtained more readily in an investigate of enzyme {manta
um posmaing some catalytic activity. Using specific .mdopcprhidam
can should be able to split an enzyme under mild , controlled cmditima
filming the formation at impact: which conceivably could retain sane
activity. A crystalline B-awlaae has been chosen for such a study.

Amine: are mm: which catalyze the hydrolysis of starch» and
glycogen”. In nature they act with other carbohydraaea in carrying out
the rapid breakdown of starches to mm! which can be utilised by the
living cell. They occur in higher plan“, molds, yum, bacteria and
are also meant in may secretions and tiamea or the min]. body.
Moreover, they have been and as tools for elucidation of the Mom
of the mbetratea upon which they act. Much of the early Mcmtim
cmceraing the acficn of the We is conmdictcry, minly’ became
o: impairs meantime. However, as these enzymes have been crystallised,
it has been possible to my their properties me): more quantitatively.

The-whee: otnltedherleyendwhuthflehmmnly
Widbynnyotthearlymemtm. Oneofthefiretrepwte
ilthtothymnndhreos (1)1:honotedthetthetohionofminflnim
oft-lied berley greinupm starchieettributable tothopuemo ore
mum Wax-nuns agent which they named dash” (m it! «mud
mpeflyotupentingtheinteriorofthaeurohmmleemmouw
envelop and mooning duh-inn and snare). m inventipuone or
o'mm (2, 3) pro informatics rem; the tor-mum at glucose,
film. and am: vhm molt mote rotated with eta-oh. O'Sallim'a
results and than of Mn (h), and 3mm and 3mm (5) showed that
poalihly mo Mastic ferment! were mi: in Much! of ulted barley:
«10 Main; alto“, and little dextrin, dutroynhle at low mpmmre,
the other producing more dextrin and lens Milton and destroynble at a
high» temperature . Suheemmfly, than we tux-mums were called
mocha-omits or p-enylnee, end dutrinogmio or ammo. The “conto-
me wluee m mama by their ability to liberate mom
rapidly without dimming the rminder of the mm moleonle. thin
mum, in the 9 tom exhibits a; high duh-create” mum, hence
the me fi-wleee (6, 7, 8).

An inventiphim (9) for the wee or when in a miter of
mm indicated that «Immune an abundant in minis, puma,
Eastern, fungi,'hut no $1:th could he found in tissues of mint].
origin. Meme! Joeu (10) Wannabe:- of plants torthemoe
at Rimming“ and “summing“ was... They found awn-e in
eoybem, pen, henna, lmtila, corn, rice, rye, barley, wheat, pontoon,

turnip, and oer-rote, them fiwlue m mly present in soybeans,
we, barley, .poteteee, and cute (11).. At the mint time it 1- mar
nieed that fiwhm predominate in and: (both germinating and

unpainted) . N

The morale in uyhee nativity which occurs curing. mimetic»
m attributed by early investigtore to en eotul anthem“! of the
m . hkesr (12) found m ditremt dieeteue m unmhnted Wiley,
and their notion we Guru-mt before and um sanitation. Emma: (13)
ohm-m that hath ctr-whee m fiwleae were mount. in the early
emcee or development of that kmele, end as mflretion manned
mm. (lineman-ed when” flawless deemed. Upon mum,

the meme uu noted, (Ir-whee developed only, am fi-wlue increased

toenximnleter on. mmmfim (11.) mm thetwluee
were not woteine, but «scouted with the lather, and liberated through
the eetion a: We meme. We]: and mm (15) some that
the We of mix: is went under two differmt tom: one fiwleee
eotive, and another inactive. m latter can be activeted. by the action
a! woteolytio meme, each a; pupil-1 which doubles the ectivity. Hills
and hiley. (16) confirmed the obeemtim that peptin iterated the '
nativity of unmanned barley by 100 percent. This inane” in
B-eayhee . activity we due to Isa-atomic loosen anointed with .e tater
insoluble mm. Chrzeeece end Jenion (17) poemleted the meme
0: en whee inhibitor “site-aqua.” 0n germination of berley, the
protein donation Mote omntmot the effect of f'eito-eMeee,"

and restore the m1 eotivity of the whee. seven]. other 1%th
have been nude (18) .in order to explain this increase in when eotivity
upon mam. Fm the more reliable Momtion, it new! em
this Banshee artiste in ocean and gains and is «tinted by the eotim
erratum, mob inthemeroioweotrypeinmdtrypm.

 

me fi-uyluee tum din-mt am we been mined byw
madam mm or them an eloohol wipitetim moon by Mme
(19) in 1895. and a1» curled m by show and Sehlesinm (20), van
mm; (21) and nylon- (22) . By repented alcohol motimtime of
mu of barley unit, and Manning: with maxim mlfete renewed
by Wu, ohm n 5;. (23) and mm and Doehbelinz (21.) we
able to obtain 1 premium which no momma B-em'lue activity. .
m. no mm m mm. m and Hollmheok (25) mm the
Men-mm ammo: of not mm», and om... that the «mm
«me he precipitated with a 56-68 percent new). cmconmum never-r
them-e, an overlapping ed! «mum merited e elect-out mum of
m w. More new nethe teohnime of Wohmidt'v-heite we
Parr (26) who fwd that the mlt Bwlaee could he eeleotively «mowed
mum ool'emet 13113.8. One ottheeeeieet methodetoeepente
mm mm fiwluee mono-ted by Gh'Lum (27) who found that the
Wee in completely destroyed by memo at an acidified m1;
or unit, a pa 3.3 and o° for mecca minutes. Ohleem also ohm

mtmeamnucmeodowwmmwmm-ormmnu
by heating the mun melt me tor mm mm et 70". 31m,
M end Bree (28) need lees dnetic omditiene for inactivation by herb
end low pH end 1w mpenmre . mole vex-km round that “autumn
o: a melt whee pup-um a 20° end 133 m mum in en alum
ample“ decimation of. the c-emyleee in four hours. Beating the mall
came momma at 60° for one hour destroyed the BWleee activity
0! the mum. 01am, W and Dickem (29) completely meeti- ,
mined the When or an aqueous mt. extract at pH 3.3 end 0°; beefing
the m «treat at 70° for fifteen minutes completely removed the
fi-m‘leee nativity.

Using a amiable]: at these “0mm“, Meyer, Fischer and Piglet
(m) were eble to cryeteuue uelt fi-enyhee. Le e. starting material,
mu woe-km need e emu-m1 nit met, which me am mum at 0°
and pH 3.6 to move the amuse. m: operetim wee followed by
manna eulfete meeipitetim and acetone incantation which finally
ave e. crystalline mean). having e. cmetent ectivi‘by, and poeeeeeing
ell the enumeration of a pure protein. However, prior to Hayes-ta
oryetelliuticn of mlt B-emrleee, Elle, Wen, and Thomson (31, 32)
m: M obtained a cmtelline trapezium o: a P-enyhee from event
potetoee. Thane workere fractionated en extract of sweet potatoes with
mm was. and obtained a. crystalline protein mum; A high
activity.

Since «when appear during geminatim, use of e nmgernimtad
’ seed loaned to be more reasonable for the mmmtion of B-emleeee .

 

.m’ 8m end mom (33) prepared 4: cryetenine Bwleee mu fruhly
gramme flourbyeeembiellythe eene procedure“ thetaeed for
the premtion or nit 9%.

A comprises: of the ' proportion of. the oryetelline Bwheee (M
three different sources ohm interesting relationships (33) (m1. I). .

we I
memcm 6:? 0315mm owes

   

 

What mt Sweet Petite

Percent Kite-om 1h.3 1b.]. 15.1
Sulfbydryl Greece + «e 4
0mm PH 5.3 5.2 14.8
Mobility (pH huge) b.s-9 .2 the-8.0

Inleo’erio Point 6.0 6.0 h.8
Abeorptian m. (m ) 280 280 280
Inactivation by Metal: 4- + +

A - , __A_. 4‘.

 

V f fiv—

Soybeene have else been investigted for the mince of fi-wlaee.
Herein and Eaton (31;) obtained a exude preparation tron this source
which, after precipitation with alcohol, we a powder boring the oldie-
lytic activity or e $‘NWLRIG. Theme group mrther puii’ied bbie
reputation (35), and, recently, obbew workers obteinod e crystalline
fiwhee trm soybean (36)-

 

‘rhe methods for the determination of fi-emrlnee, are based upon
the increase in reducing power of e. solution of asteroh. Widely need is

therebboddevieedbymm (37)endnodifiuibymeuuflmdebede
(38) no Jam by SW (39). ‘mm, thin method involves the
Moflm of tmioyenide by nel‘boee, mum by treatment vibb‘"
Winn iodide end titre-um of the liberated iodine with momma.
Mm many, melting momma (he) um the 13mm warm
by waiting oolorimbrioany the extent or reduotim‘ o: 3,5~dinitrod-' '
ulieylio acid by maltose. Pom nabbed: (hi) and new: re"
deceim {ha} lure eleo hem propoeed, but they do not amour to heve
one into wide nee.

 

Mob of the early work with {3th wee-done to emoidebe the
seine-me of the searches. Crude magnum were used before the
man of men-nine ensynee . Snob erode. new meantime ommnee
veryingtncee at Whmmflmltease. mimeulteeno cementum
rm eerily investiptione of the mechanism of 97min” action are om
to reflection; In the following diamaeim, no attempt will be made to
diaflngeieh bebnem the specific owns», since they an we» in
«mtielly the me rumba-1. The node of action of own» oen be
Wood no follow: Only a Luv-gimmick: linkages are attacked.
Bebe. ultoee is the any low molecular weight product of the action of
banana“ (6, 7, 1:3). Severn lines of evidence indicate me the .
attack by fi-eawl—eeee mm at bbo nmrbdubing end of the aux-ab mlmle.
Hel’oodemtntne mm by Windite (bk, to) or by mercuric and. (b6)

 

cen.be hydrolysed hy'fi-Iuwineea, to give meltoee, end a.dentrinic laid
at 1am complexity. notbynmaa studies (h?) show the pawn» lime
dutrinotmlepoctin emtehnallotthew mthllhmohing
points around in the mam mama» molecule.

Pertinent to this is the work or French and m: (be) who «mice
the action ef’p-enuleee as it.IJmE©BGhDI e 1,6elinkege. Emunehedeligoé
aeoehnrideu of law moleculmr‘Iuight‘uurc prepared and sebaceted tO'the
tetim o: pm». The mane-Mn may or the action or mnu~
tn‘Vtriane aligns:echerideewenllogoue to the‘brnneh.petnte inmenwleyeettn
led to the canolneien thnt the fleenwleee-eotian atepa‘botere the glucose
unit nut to e~1,6 bmeh. These ”mu as Mad in mm. II.

TAKE 11
ABTION 0F fimfl m BRAHCIED OLIWCCHARIDB
====z=~mw1-v>r+éu ' - ' ‘%"'1. ~éwvn====zzza

 

 

 

 

 

 

Nanvfieeietenttirnmnganente

WWW—mi,

0‘0 0-0..
06M 6-0
W 0-0.-
0-0 0-»0-0

0.0. I 0-0-01-
0~D-0~O 0—0
o—e«-o~ 0.0.0...

0
0e Ganeeeo'with ite reducing group 0 - 0 - 0

0~0~ 2 glucose: linked l,h.

. 0‘
u» o 2 glncoue 1mm 1,6.

--——v——n wr—w ,. r fir ‘ arr w

 

m: investigate: continue the outlier convention that ultetrioee in
not ethane by fl-uyleee (1.9, 50). Home, as Mob pointed out,
model cab-tutu or low mm:- mm do not manually mat in
th mmyuthenetnnlly com: Mote «WWW.
Weemtotthem election offiwhuhubunindir
pate. considered for the mloee mm of starch, the Insomnia“ my be ..
or e. nouns-omen" type, where the m’ ettaoke one when when»
one degree“ it cmplotely before attacking mower. On one other mo,
11». any be a Imam-own" typo in which m m wlm moleeulee m
mm in e mndm attest. After careful review or the evidence
“enable, it appenre that in «see when cryetelline B-Inyleeee m 4 ~
need the notice my be MW“ between these two mechanism.
Mob 3 Q. (51) chewed that the template momma of mloheptloue
libmtee two mlmlee of nltoee and one or mltotrioee . No wlo»
put-none found wing tm course of the hydrolysis. Kerr end Cleveland
(52) else obtained date much they consider ae nearing the "einfle-chein"
melanin. Headwineofintu'eediete aineomldbetmndinMroly‘
eate- ot envious the partially Wed mplee had the me degree
a pomeuntim u the m1 aimless. firewood and coworkers (53)
Illa W good widmoo of a ”mac-chain“ notion. Ne mm
film“ ovum be identified in the reaction mixture: on minutiae of
the iodine affinity of the wloeo and its eedimtstion emetent. This
nation in amt-at with the remarkably high min-over number (256,600)
romeo for this mm. (514). However, money and Whom (55) mom-
aided the action pattern of $111311.» Imder e variety of omditima filing

 

 

19

u . anhetntee tue momm- and e mthetie wines at new chin
1mm of to mite. with on the nab-tutu and under «:11:th
the notion pattern we always iota-meek” hm eingle and . miti-
ehein. their result: «some the m We: Multoeeunite
«ring. not mount» with; mbetmte .mo. rm. nether. unmet
either tint them We contain. maul active W at the
may» reaction can cow at a single active neuter. A WW?
Wanna-.30 omnlmg'ohoxhb glanmunitem syntheeieedby
May and Frank (56), and they labelled the nmredueing md with 01".
Brief entice by 9W0 m the synthetic We are memo-
entire um». and their resume mm: that on. m... meme 1...;
mm mite per effective emanate «counter. the possibility
of nltiple entire cum toting smelt-anomaly on a. duel: mime!
Wmelinimtodbymotenginterieflyhbelled 313de
when type. use this me 11; 1mm om the notion poem be
Pwhu- depends upm the relative men of the kinetic amt: which
were the tomtim, dismietimme reaction of the internedietemynv
minute complex. Therefore cam ”mltinmin' or completely
Mingle-em“ notion ere «tram mm of a canon notice: gotten.
mm upset of the notion of 9m“ in the eWWioiw.
noon; on as a tram, it in been possible (5?. 58) to emu-mt. tut
both an and $.an oleem I. e 1,.hngluooeidio bmd of Shiloh or glycogen
m the 01 am. no .019 non-tat or the prudent, mom, consumed
to awn}? 90 meat 91' tm theorioal incorporated: from. the -
mm. In ordar to noomneror the inversion a: configiretion o: the

 

pmm with 9W, mm (59) mm um mun hummus
e direotdnplemtet the mmlumm muuew;w
rename. in 11min momma scum be u rename: the infidel. stop
inherent“ 10 When «them-Mp WM: 2mm am

in which as clawed at m 01 m, «a in uniting mm in“:
mummmm. mummy mumflthtm
mmw- (60).

 

Tm integrity ear pertain mm tor 5101mm ecthity of
Wheaten: Mmuumtwwm. W, It the
mttine, Mam tobammidmewmhmmthet
mmmnwmmmmmmnmnmmumm
mama]. activity. 80 hr, the but tool! to 30¢in protein but
been the gramme enzymes. “ting War we and amtrolled «minim,
mlhnnldboebleto split swig-33y «mum: mm prawn
whale with the” reagent: and be «his to inflate fitment: pemeaing
at least Motmectiutyofthemgml man.

thet active my” manta em he wanted an um claimed by
W Q 51. ($). than worm entnlned a. crystalline W some
tin and unrated the peptides produced by ultnemtrifupflm. M
We, pomumgamolemnrmghtbmmm 30m, remand
the to 1m mt or them-1311111 panoramic activity. m m
mp at worker: as also able to dumtmte that active flaunt: an

 

 

be shunned upon mm digestion at woman. mm (62) repel-ted
the {which or mum active, W10 inflame during the
thm of pepsin. However, Wflliamm (63) in attempting to
upset this work mm: the ective Inmate in the nmdihlyuble mm.
chemikav (61:) also reported finding dialyuble active peptide. true the
mtolyeie of Win, chymetrypainandpepein. Ream, mmwm
(65) tame some manual]: active commute in trypein cutout-ta,
ehnuingtht therein» producodmmllmutitiue mtealytio my»
mm the trypein.
mummdwmcuummsbmm (66).
From this a. precinct of relatively low melamin- waicht we obtained.
the "pawn modified" cytochrome c contains the prosthetic mp of the
was linked to a. peptide Which rem-amiss part of the original protein
m of cytochrome c. The hamopaptide exhibits interesting magnetic
gropertiae. diftm'mt frm those of native cytochrome c . the mm
1: inactive in both the anaemic We and the cytochmme W
Meme. Mt it strmgly catalyzes the caution of ascorbic acid ..
Getflue (67) he: also how digested with pepsin. This tram reunite
in a remake inantintim cf the catalytic activity. of cam:
War, the was activity tour-:1 pyropllol increases . the
'pepeinmodii’ied" humane we isolated and further identified an a pro-
thin of kirk high molecular weight. It. is interesting to note that
even though the specifieity toward hydrogen peroxide is lost thrash
«ligation, eons of the more amen]. (mi-dam“ properties eppeer.

Le e result a: wotoolyeie with mama, mama: (68!) has: hm
ebutoruweapeptidefrmtheH-termmlmd ortheribmuoleeu
molemle. The peptide and the reduced» protein 80me hive 1m tun.
nu meant as the mama activity of the cams. Under «ruin
cmdifims, nomination at the two from“ malts in mu Maw-
tim of ribonuclenee totivity. A distinction is anally ado m V
W compound solely of amino acids and thou containing a nmnino
mid prosthetic group. mt figuration or the ribmnclaWbtiliem '
man into an mm mm and a mm mm meme ”'oomctow"
suggeete tint this distinction my not be so slurp u hippos“. It Should
be 01' interact to mine the possibility that the peptide Inseam at
rihmnoleeee contline the active eite and the minder of the protein
man u an ”spams.“ ,

Although not as striking as name modification by adopoptidem,
me my be modified through the use or mopeptiduee . m. m
11331!th that the integnl protein 1. not any: necessary for bio-w
logical nativity. Cubmpoptiduo (69) 1113th three amino acids
tron the Win molemle withmt eltsring its activity. W,
me dwimt in the work of Smith with 1mm amnopsptidueo (70)
on ppm.- The H-tminsl portion oi maul-innit: in Mink
denuded by leuoine minopoptidue, and, elthoagh about when to tro-
thirde ofthelflDuinowids combo moved, there is noloesin
wotoolgtio activity after motim with cysteine and warm . The
I motivate specificity does not change, and the sum of the amino Acids
moved and those in the residual, domdod enzyme is in ommtiel

amount with the cmpoaiticn of the anti" protein. Eartha aperi-
Imta indicate that m of the mm produced during mtolynis o!
lupin are native (71)-

Tho than «puma indicate the Apoanbmty of degrading W
WWW otthoirmtflityinmmamm.m.
mum and Waterman o: the «tin mm W mid;
m1. 3.31th «3ng the mm or flu we mid: At hi -
mun atom load to the W1"! dmedol «mm. mum ideas
in Mud, mat acute Bank» at dim with em}. Mommas"
«mm-manna Immvuththohopc mtitumhmmd
to “an“ mm mm: still pun-m om wlmm whim.

II. WW

l-énmm

 

, .-., M... The Beam 3 model 31209th and
1m am 0011: m and for mm mans-nut. in a. ruin.
my. Abnorbmoo was in the umvioln m undo filing the
new mu m .1me with l-crn gum: calla.

-..- Thu Thelma meta-optional. appamm (Nodal

138, Pm Elmer Corp.) In: and tar moving human-y aloatrophorouI

studies. For tba conductivity was, an Industrial batman“
09. amhctivity lands. (Medal 30-1 B) wipptd with a cell of 0.14893

ma was used.

For paper WW, tho type In 3276 Pam Bloatrephm-Odl
mm was um, («13le by Ivan Banal]. Ina.). am pm My!
mo Iran Sam-tar and Semen, ho by £510 In.

W.“- A Beam Modal H2, 311:: meme, lino 99mm
pH actor as usual.

,_ x. m _- .-- A11 wotaolyua digestion: of langu- ish-n In
hoax-Imummo carried out nanomoappnma-MWW
mum more “”1113“ by filtration. The apparatus emulated at a
Pym mm mm «mm to a. 35-31 mm- tube which no fitted

withucapflhwtommmuthdnmgotamplosdurm;d1¢omm

 

 

15

16

withmt amhflmtion. The apparatus no sterilised at 15 lbs. me
for 15 Mint-a 1n the mtochve before every run.
Mgr-A Vining Cellophane tubing was and.

 

.- ;._.;_ mu .-- The Ghrontombo maul B (Raoul-oh Eminent
corp.) m and to:- doaamdinz mo. Incoming chrmtomna were
developed in the chrmtompw amt-tun minim-ed by Univmifw
Appnha Go. The ahromtoma were dried in the March Mimi:
corp. ova constructed for thin pan-pone.

am

w«-—- The sweet potato 9%» was a Moe crystallised
mnfion obtained from Worthington Mechanical Goa-p.

' _ , 3:: 11.2.4-.. .- Crystalline clvnotrypoin, (alt-tree) , mum,
twice cmtllliaed (containing 50 percent mane-inn lulfhta) , papilla
(twice any-stunned) um all obtained from nutritional Biochemical Corp.
The crystalline pepsin one-pension, in "mono-cysteine buffer, and the
carbuypeptidau (twice cryatullised in 16 pox-amt lithium chloride) were
obtained from Worthington Biochemical Corp. Subtillam m a. gift nun
m 1.111160.

The potato mopeotin nu pup-ted from potato starch according to
the procedure or Th. Sohooh (69) and Melting 93 3;. (no).

 

 

_ 3‘ .,.... This buffer used in the paper electro—
phoresis Wt: has prepared by dissolving:

17

8.712 g. Vermin].

1.872 g..Sod1um hydroxide

6.1376 g. Sodium 0.0mm

60 1.1.6.1 H Hydrochloric acid A
endthe totelvolhme ndeuptol 1-. ulthmtm'.

_ _ _ ,_ . ,.... Into 500 ml. of ethnol saturated
with We chloride is dissolved 0.5 g. o: Broxuphenol Blue.

I ‘ _ _ _ , _ -M- m.» The mutant need to detect
maltose in the paper electrophoresis marinate no prepared «mm.
to Blah”: end Stage (81).

h Peromt Aniline in 95 Wt ethanol 5 vol.

1. Percent diphenylenme in 95 mount ethe. 5 vol.

85 met phoaphorio mid 1701.
All other reagents used were at the (LP. or reagent grade «polity, unload
We specified.

 

 

 

The fi-uvleee ectlvitym determined bythe method on! Melting
and Bemfeld (1.0, 82), wherein the colored reduction product from the
reaction of maltose end 3,5-ddnitroeeliaylic acid 1e themed spectro-
photmtrieelly at 51.0 nu.

We". Tm we a! 3g5~m1trou11oylio told no moistened
withefewdropeofmter. menace ml. 012me43“:
added and diluted to 500 ml. with water, after complete solution et roan
tapenture, 300 g. or Rochelle alt we added and the volume brought

18

to l 1. with later. This sentlon m M10 in the shame ot-e‘ubon
dim fro the air.-

W." retell WM, unlm otherwise specified, the
em wooedore m ens-led out. in the fellatio: mar: 0.1 :11. ot the
me solution to be seared end 0.9 ml. of ndlstllled new m
M into e 25-.1. glut-stow Vohmtrlo m1 mm: m we
mum in s. mm hath st 20" c. One mama-r at e 1 meat Wie-
peotim eolntionmeedded to this, and themotlonmmoved to
wooed for 180 eeomde. m reaction was thm etopped by additim of
2 .1. a the 3.5-d1n1trosalieylie acid reagent. scum; this, an
mm m pls-oed in. boiling at» both for 5 mm. on. running
solution! were then cooled and diluted to 25 ml. with new. The absorb-
maenereedstShOmepmetthst oteblnnk grenadine sinner
nehim, but in which the" we solution use neplsoed with water.

 

A mltoee stool: solution use punt-ed by dissolving 101.6 3;. o:
mltoeehudntemIOOnl. cluster. 0.1.1.1., 21.1., 3131.,sm5nl.
Wot mmotivelywere diluted to 19 ml. and one m1. of eeoh diluted
solution was used for the emulation of the aunt.

 

The oxyetuline fiwleee use moved by means of a systole, and
0.231; g. placed into I. test tube. Thin was diesolved in. 7.5 ml. of

mango»- utter (pH, 7.0, em a) and placed in s only-mg mm.
We nethmelloued to proceed spinst the mbufterumrueed
toe' dissolving the sample, for 18 hmrs at W. At the end at me than
thsvolmsems Menptolw ml.u1ththeo.01!(phosphstehu££er, end
a S-nl . sample us moved tor determinstim of the electrophorstia
pattern of the fi-swlese Much. To the reminder of t» solution
modded 0.01 g. etemtslllnetrypsm. metwptiedigeetimm
Illuedtowoceedmthesterllsemtoetorm houreatBOOB. At
the no of the digestion peeled, s ample sue moved for em of
nativity end the W see We... on. cold roan eminet'm ml.
or phosphte butter and need he the electrophoretlc enelysie .

 

6 O G__"_t'lt_" £32" rig-‘51} .I,

A 0.212 g». ample of sweet potnto flawless m dissolved end nude
up to S ml. with phosphate buffer (pH 7.0, 0.1 It). To this solutim
meddedo.01mg. otelvmotrypsln. Ammedthlsmtu’ensmwed
lmedhtelyforuelyoffl-wlneeeotivityam thede
solution ass placed in the eterils spmmhas snd incubated at 30°C) to:-
12 hours. Another mph m then removed for may or B-mlue activity,
and the Minda- m milibnted haunt phosphate buffer for the
electrophoretlo “13110.

 

A o .175 a. mp1. of met potnto own“ was dissolved into 6.5
ml. or eeetste miter 0: pH 5.10 (0.05 H) and e. ample we removed for

2O

essay of activity. Atten- edditim of 0.0213 g. of pep-in, the eolntim
was placed in the sterile ammo. Digestionm allowed-to W
tor 1.2 hours at 30°C. um- removal as a mp1. for any, the reminder
of m solution on damned am son :31. out phosphlte mm (pH 7.0,
0.1.x) m use in the .1»th main. . ' ’

hwhmtemoleoportmedetelm onshore mum
active. At pH 3.0, the met potato own» .. inactive end molpi~
um rm the mean, but, upon digestim with pepsin, the minitste
dimlved. No activity could be :10th in this mime, and paper
demotion-tic exp-mu indicated that the protein had, hm broken
dosh considerehly.

 

mummy experimmts with pap-in indicated that «ligation of
met potato 9m” Mdlt take place." indicated by s decrease in
activity. However the“ «primate wen not slurs reprodxoible. A new
sample of m... was obtained, and non moise detemimtim were rude.
rheyaeumtion o: the couples we carried out seem-ding to the meme
o: Kimsl mo ounces).

A town hotter solution m moved by dissolving 2.91: s. or
aodims oitntemteihetewml. oro.1nnc1mtnthe poem
6.0, and than diluting to 100 ml. A. Wane solutim'm also
more up free 0.605 g. at cysteine and 1.1.6 3. of. dis-eodmn ethylene-
dimine Wetste diluting to 1:90 1:21. The digestion We ens

21

moved by mixing 2.5 m1. of the citrate butt», appraadmtoly 5 ms. of
the awn», 1 ml. of the vague-cysteine valuation, 0.3 m1. of. the
lupin companion and 1 m1. of mum: water. A sample of thin air
taro me mad baton tho addition of the ppm, and used as a cmtrol'
in tho any and mm fleotrophoresia apex-1mm: . Tho solution m
an incubator! at 30°C in the sterile app-mm. The results of obese
Wamreportodinkblu Irandx.
Inordortomaythoofloctoftheoddfiimottruhpnpaintoa
sample which m M been digested with mph, the tolloflng upm—
m m can-19d cut at. duoribed above, but after new digestion lad
than plan, In indium by a. decrease in activity, 0.1 m1. of fresh
pup-An mansion m added :ftor 2h hours, and the fiwnao activity
of the mixture diam. The result: 01‘ than «pennants are reported
in M10 m. '

 

Piper olmtrophoruia was utilised in a umber of experiments boom“
it Word. 0. me of ”panting wotein and peptide ompmmta of such
W on a very null scale. It is also possible to determine the
enzymatic uotivity of the compmmta directly on the paper.

A ' = .-— 81x hmdred ml. of mom].

 

buffa- pH 8.6 (o .125 H) was placed into each electrodo nasal and a 11mm
junction «ubluhod batman the maul: by means of o. siphon to @111.an
the liquid Mole. The paper Itrips were wound in the buffer, mm

“mummrwsmm,mmoodmmmmo Nowa-
mmeMamm-mmm‘ummmatw
ummmmutohm 1+. magnum. Mrtowm.
muwmappziwmachunpbymormmappumm
Wuiththoinmm. Umllyamtotfiuporstupns
aypludtorlztoléhmtormqlwmphwn. ~

5.~m¢rm1ofthonr1pu,thoymmcutm
WMMuaMmUWdemmmntlm°O£orWM6m
mm,mm1pnmmmmmmm1bmwmgm-
outwlomunmmwmmm. Theatriplwmtm
mmuozmwtmmmmuhmmmwmmm. m
thmuahinohwd.

° The other nation of tho Mp um};

 

 

m w for «any of the fiwlaao activity. Tho mm strip was fim
allowed to dry tt room tamed-ma. It us than spayed with o. 1 percent
starch solution and 0.119de to dry in the air. Following this, the

Mp w my“ lightly wifih tho diphlmyhninflml‘hm ‘mm. m»
drying in m am for 2 to 3 minutes a, 110°, & brown band, indicating
tho W of. alto“, oppured share native own» in: wt.

 

The Woman“; that ofsum, mmmr (8h).
Ayn-mm: 5 as. of mat potato am” was dissolved in 10 ml.
of acetate buffer (pH, 7.7, 0.05 H), and a ample moved for new of

23

activity. MayormbtmoinmflmnmddodtothoMmmdfihe
IoIntionincuhutodatao°m Wmmmmtomm
Wacoardingtothonmlmodnro. Thromlflmnporhdm
M101. ‘

 

6”!»me in an comm” from {aha menu which catalyzes
the lam-pm: or peptide bonds at tho aux-owl teminns a: mum and
peptides. Itl‘acuou on met potato 9w)...“ m fiddled utilising
the memo or Mahdi—amt £1 51- (85). .

' ' wwonemummxw
crystallin- e‘arbmpoytiduo «amnion ma diuolvod in I. tau :1. of
0.011! MB, and {implants butter (pd, 7.5} 0.1 u) an added until the
volume of the solution on 25 :1. One ml. or the vaulting ”mom con-
tained 0.thon 0.2 mg. of my”.

--_« v"- For the mine laid (imam 1 ml.

 

 

digests vars moved m1 2 m1. of Mahlomeetic acid added. The M“
cipitntod protein in centrifuged from the aim". One ml. of the nip!!-
nltmt solutim was placed in a. 2511. woman flask and 1 ml. of the
nimdrlmhydnndmtm reagent (90) described elsewhere as added to this.
no nuk- were then 1.1.... in a boiling mm but): for 15 mt».

um cooling, Mmdflnted to thowkwithSO parent ethanolamd
the option dmw read at 570 m1 apinat that of a. bunk.

x; .. a . . .- m odfi‘ri‘by as tho outm-
poptidoo'ono tented on main inthotonawingmmor. Into: me
are phood thou «mum:

3 I1. 01 o. 6 moat Mun mum in phoophoio buffer pH 7.1:.

2 n1. phoopho'bo but» pH 7.5. '

1 n1. urn-ponds.” solution.

m on indnbotod of. 30°, and mo :1. oligaoto mo moved otter 3.9,.
60, md 1150 ninntoo. Amino mid (imam we carried out on then
until.” u indicated obovo. The results. mgim in hblo II on W
indicate that, under the“ conditions, the mbmouddoo is mun.

,_ ~ showy... _ 4...». .'-- Prior to when with
“ammo, tho 9min" ample no Mini: dialysod W.
phoophlto mm (pH, 7.5; 0.1 x) w 2°,- m cm: 1:0, mm m of on
medium in: which interferes with the amino acid dotornino‘him . to m
dulnod fl-uwnoo caution in added 1 ml. or the dilutod cox-bow-
poptiduo and tho whim no node up to 10 ml. with pine-pinto Wow.
this mm" on than mono-mo at 30°. Sonploo Iron moved of. road.“
morni- row uooy of outivity ond mac ooid Mum. use), in
mor to chook whothor or not commando» ontolyooo, o cmtrol m
m up in tho following manor: 1 ml. of the oorbourypopudooo ooluticn
”Mouptomfl. «mm phoap‘hoto Mfumdollwod to “and
undo! tho m condition; as tho motion mixture. The rmlto of the
:1th with corbaxypoptiduo are reported in Tabla XII.

25‘

 

rimmed proteins on nanny we mcoptiblo to tho ottték of
wotoolytie cum“ than are native protein: (86). For thin mun,
the effect of um on mot potato Emma «hiring motoolyaiam‘
inn-tinted with the hope that under these conditimo the gawk“
night be in» naiotnnt to protoohrtie attack. The «not or um uln-
tima 11am on this meoma also Mar-tinted. ~

Proliuinory uporinmto inBHum indicated that what potato
fi-uyhu in active under than vanadium. but that it is 6mm in
EWW 6 to 8 hours, depending ‘upm the cmentntim M the enzyme.
rho rfiuoibility of the motion was studied in tho {allowing mom
small: 5 mg. at mot potato fiwluo In diuolvod in a “antler:
which no 8 H in urn ona 0.05 M in acetate. The pH of this Wm:
5.0. The flank» ootivity of this ”Station m :1me Wkly
mum‘sndagam at mm duringa. period, of standing“. 30° ei'
Na. umber of hours. The rmihder of the solution was phood in a Mme
m museum manned apt-net acetate mm (123 5.0,: 0.95 2:) wt 2°
to roam the m. The outside solutionm magic: moral mm
m; tho mu at dialysis. A white precipitate appeared at the
button of the dialysis hug indicating tint the dammed protein is in-
soluble in buffor. The result! of this eminent are reported in
181310 XXIII. I

To 5 m1. of 8 Hum was added

 

9.th 5 mg. of sweet potato fiwlau. One ml. of this solution

26

no moved for central, and 1 mg. pepsin m added to the War.
Both the digestion mixture and the control were incubated at 30°C .
Samples were moved for assay of activity, end 1 ml. elimete were added
to 2 ml. of 7.5 meant hishloronoetic acid. The Supernatant Gelatin.
mm the triehloroeeetic mid precipitating men-u were tam mm...
mutate st 280 m in order to check for proteolytic hetim. The
rmlts of this experiment we reported in Table 1W.

 

. .-- Since experiments in 8 14 um indie-ted
a rapid dmtumtitm of what potato fiwhee, the effect of 6 n w ‘
m studied.

To: 10 m1. solutim of 6Mureamadded appraimtelys mg. of
sweet potato fi-mylue, and the activity determined at different 11115th
to otter the demturotion under these canditione. The remlte are
reported in table m.

 

. 5.-.- As indicated by the mime aperi-
mte, there is a slow demmmtim of :3th 11pm treetmmt with 6 M
urea . Omemmtly, the urea dmtnmtion elmg with peptic dimtien
were studied at this area cmomtretion also. Ta 10 ml. or e 6 M urea
mum m added approximately 5 mg. or met petite fiwh‘oe. A 1; n1.
(newt of the urea-flawless satiation we moved, and to thin ample
m added 2 m1. of e pepsin aolutim calming 6 mg. of poplin. Both
continue were mated at 30°C . am! ample: were removed 1t regular
intervals to determine the flwhee activity. Several lame carried. out
in thin mar an the reunite shown in Table XVII.

2'?

 

amp-cums mumtanahnmmlupemumhnmum
Want. maul. othllmmaddodapmtaySu. arm
mute p-uyhno, 1 :1. um um ant for neutral, and to we minder
m addud 1 mg. or poplin. Both solutions worn haunted w 30%, and
“12100 taken mt at Worms minds of the. Th3 results are 31m in
hblo XIVII. Paper electrophoresis an also carried cut an a umber of
then staples.

art-3111191.] a -1;- .:--.1 w _* 5. £15.-.-“ To 5 m1. at «I sciatica of 6 H
m us men npmtoly 5 mg. at mat petite 9m», and mo
:1. man out for any w control. Ono lg. atymotmain m added to
the ran-under, m both solutims mo insulated a. 30°C. Again, mp1“
are mad tau- fi-wluu any at ram” intervals. mo remit. of
this Wt “a sham in 131910 XVIII.

., _ An apartment oinihrto m can
«scribed far. Win-digestion m pan-roman um; firms-1n ma
at Okinawan rm. ave the results recorded in hble XIX .

.55; = 35-5. -EILIL.L1..1"_--.J! 55-55 Amman Wt m par—
found an ordor teaming the stability of m potato-awn to
hunt, and tbs ”ability of cmbining a but ailment in conjunctiva:
with mmmu.

prmmtoly 5 mg. of met potato Bwhu no dissolved in 5 m1.

 

 

of mats but!» 133 5.0 (0.1 H). m, activiw of this aolntim m
astound. A ”pic of the mixture no then halted in boiling um

28

for 5 m anu- which the Ewan nativity m $0th spin.
MW at mmmmm Mdmamwmthnt 70%
in: 6 man, and this imam m m “and tar- Bwluo activity
aim cooling. To this «mm, mm at 70°, I“ was: 1 :3.
twin. wrmungmmmm a. 3096 m in mm. the
remit- 016:1:th givmin‘hblen.

 

In order tn teamm- tor m at the units chained m a» mum
of manly-bio mu a: mat. pouto awn“. it- hm mm to
M m amps-ulna 0: this me. This in dm {1111'- -mnht1vlly
by mm ehmtomw-

in“. 1 ~22: 9.: «- ‘5: The ample of met pouto 3%
In W06 101' up” mnemm ween-ding to the method at 6mm
m Pardon (87). About 5 as. at m potato Famine m 413801!“ in
0.1 ml or mutant. boning, gnu-distilled max-mimic mid and mm
mama!” (internaldmtorchm.). Mambo“: phm mica-
ntor, mm?“ at the am pup and sealed. . Hydrolysis ml mama
to mend tor .12 hour- a 115° in menu. man- this ire-mint, m
hydrolnlto in: light yfllw with 7617 m1 traces a: bikini: mm.
the What. was then evaporated to dryness in a vacuum desiccator war
aediun mum. 0m n1. otntorm added to tha rum, and thin
mum “1.119331 and in the animator. {the dried ample mu man
up in 1 ml. at 10 pan-amt howoml alcohol, and. stored in tho «mg.-
mtor mm and-d few mm away-ping.

29

. .w m .-- Artu- a. tow uranium“ with varian- 0011mm,
tho Mob-mm and humolaGOuc acid-Inter W m rm to
sin m'mmuintm armannmmdmimuwnrm
chromite”. the Molmm nixhire was named amending to
mag. (38). In on!” to obtain the but remit: it was 110W
00mm mantMcod pram. Onohmdredg. of piano).
am 20 m1. at an mm solution attaining 6.3 mouth «dim; aim“
m 3.? parent sodhmdihydrogm phosphto mo than mind to pram .
tho ammo uoluticn. The munalmwbic solutim. m ground by mixing
manned n-butmo], am. And mom acerbic Item in c Int-5‘31 ratio»,
Miami-ink. The moon: phase at this mixture m and to militants
the W. and than ermine phase and far resolution. Th1: solvent an
prop-med trash before any run.

W0“ mm In. 1 mm mm Ma m wt to fame.
mmlfixlB inches. mummlmchfrmm ofthoodm, 0M
3 1113119: {rm an adjacent edge. The mph in: applied at the Juneau:
dthelinu. Forthondrolynm,um unplan- 0.121311ch
mm]. 90mm, numbing tho spot I. m. in am. Solid We
of 0.5 mg. of each amino acid we diualnd in 10 percent icon-owl
dcohol,andmmamto ehrmtommmtocmflmm
ulna.

Bunny the wont-som- um tint ahead in the phenol walnut
“mammmummgnmrorahwaom. Iii-thin
mm”, the ”but travelled than 25 on. Iran the origin. um this
mm, the pmmmdrw inthodnftonnatromtenpmm

30

form hm.- ‘m, thyme phone in thy other cabinet for dam:-
ing 6111‘”me in mmaaeom said for 19 to 12 mu. m
drying the sin-«150mm, m m cm nth-a 0.2 percent. cannon
«Winfimemmlwflhsmmwnmm. Tho-pots
tormwmowidl ammmmmgtorsmm in the M
mun u. 90%.

In later WM, 1% m feud preamble ta m m WW
minbumolnccflo widtirut mums-union, :11th
IfiMMtommofmnm. mamas, theremlting
mmm m ran in M01 water. no but Mantegna-m
obtained by this 1mm method.

”taps! wm made to an cum- calm Wm, Inch an 001mm.
mm. but rm not found amt-mam been" of poor mohtim and
Mg. Tho ”lulu at tha mm 0113011.”me mm 1::-
mn m. ‘

 

W»; A rectum mum 001103159: m and to comet '
the «Imam: from the column. A mm B Spectremm swim
with an mm: for test tubes In: used for absorbmca «mums.

W.“ The standardisation a!” the test tubes as carried
«at warding 1w the woman“ of fitoin and Moore (89). A ‘30ka of
mmm3uwoammmidms prom-ad to givemoptiml
duality of 0.6 to 0.7, using inter as a, blank. M W 15081". mu,
m 5...... 150116 N. um obtained and checked an that they would not

31

my by m than t 0.005 opbiul duty-m.

. - ._ ,2 _ m.” A Maiden Sammie 00., (ounce, 133..)
mmggum 11:12th to m-m comm.» 50°C. during
that mi. of u m.

Ema-A WaOMIGSm. lummmm
at 0.9 an. m and for the chrmtomw at the natal and scidie
nine new. I'm: tho dmtim at the has amino acids, 3 cm
fithu 1m W o: 0.9 an. and tittndvith sutured automated.
um pckoduwmupto mam mm.

m.- who want. 09-120., mm 2 (zoo mu) m the wane
51,693. 31:13le other um unrelated tram mm Scientific Go...
Welt: wwmmmem, Word, 111. may
as ( .‘ .. no 13111391 mam) mam; taunt In chum m m
an. W 0.. W as mm mm m mmmm mach-aim

 

Gem-p.

rummmmmammgtovmmmm
mum-.00). 10203.01m1nm500m1..01nmat90°m
macs.dmubiummmn.unmnyo°,uum.
OWMWMWtomm for 30 musflthmt. Mar
hating. “firth“,thoaolnuanmamemmmmm~
wwwammammwmmwwmm
mum. WWmZLBJSa.

WWI-inmt.wmrwnomdu Mmimtimuapnmd
WMWEpotninlwdflnmeJ 3.1vdr1ndat1nm75fl. arm].

32

Woke. To Imam was «ma 25 ml. of h M 01.0th mrm- (pH,

5.25). This mgmt was usually frauhly mm each. m.

r... LL 3-..... 1.“ in m to obtain tho but mu.
mlyrmn Midluoi’thonppromto wemuflayod 2...me-
tompw. The madam used for ohuificntim of the min partial”
m that row by mm 11 u. (91) am: a. tow Wimtim. 0m 11:.
a: nun uu awarded an 10 1. of (ii-tilled um, um and .11....
to mm forEtoéhom-i. Thiatmtnmtms mmtodthmtimos.
mm.» 311mm mum movedby siphoning. The resin
mammmuamgammmmmw slowlynth
2 I. of h H Wehloric win. This treatment was ton-wad layman;
fiasco m1. ofdiatflladuw. mm... than mtpModin? 1.
othfisodmmmdomdmwmnmmwformhmr. Fin-.113,
the resin was collected m a. Buehncr funnel and mm with distilled
“ta:- untfl the flaking! worn neutral. For the ”nation or the rain,
I. 2d. septum mane}. m «mm to a domed: which was in 1mm
Joined to .a cit-tilled mt» up. The min a; poured into m mm.
and: mm of gun tubing, titted witha stopper, m placed on top a!
fiamnnel. fiwmmhpm~dlmd tomhthroughthommi'
mmbmw wtthetop, thenwntubm cents-WWW
at the Itop'cock. Th5 alumina of resin in the overflw m collected
on a. mm:— mm. m omditiona for ”mum are tabulated balm.
Eu
Discarded

filled this fraction for the
15-031. COIMO
Used this factim for the
150-0111. comm.

 

.33

Mhmzmmmsmmoumwmmwmmmmnm,
MMQchmnahadm¢WmnmlvithlLo£hfltodmn
We until the nitrite ml 11mm . This optimum m fallen“
bynlmngvichI.otmm. Anmdnfnctimmeatandinthe
:0me unam- 0.23:1:de warm.

Mm.» hohotthebufl'm (pa, 3.25mu.25)wmmmrod
12:181. mitiuammmmhoMmm. mw5.2ebmwm
M01351. Why. mam 35mtm¢agmtmdthiodimcolm
“Mathew“ mix-“ad aunt haters nae. .

_ - _ . _ .u--'.--.-Thocodiumsalt oft-he
alumnammmthmw,mdthmuithmmlmmmml.of

 

0.2 H butter at xii-1.35. Both ealuma were poured with a. mammal
ummmamm‘ mmpnngmlso-cn. colum,90m1. arm
suspension an poured and 2 can. of min £1110de to attic under purity.
um this, 3.11-5qu we applied at the top of the colum (6 1b]./
In. 111.). This an a. «cum of ram RpW‘bfly 30 an. in 1mm.
Th1. woe-dun m then repeated, moving the mpamtant liquid etch
tacky slimming. When the colum had reached 150 cm, 0.2 H swim
We as mama to flaw through we comm under mity amt.
m the batman: pH 3.25 m mhflimtod and allowed to now until the
cam had ruched mum-m. the mm was then ready for use.

The 15%. 001mm Medina Wanner in cm auction.
' Boson use 11: m swam with buffer at pH 5.28.

. 1 In ordor to chock the flow ram and the
mm m‘ of tha calm, a mthatio mixture of. amino acids as

3h

punt-ad ”"1an out one 31111301. Inch 01'.” taunt-n amino acids.
MmdiuolvddwmmBml.orélhydmchlwicwm,mm
mammmhmtelom.uthnw. mmmummkcyh
mfhemugamtw. mmrorm.1m1.0£thostoekaomum
m muted to 100 ml. with mm: (:11, 3.25) ms 1 :1. at the mm
aohtimphoadmthocolnm. marmamuhhmammdmm
thumpmweoxhhecemm kept .0 50°. chewamlmhe ma.
Wcmmlm.otucham0001dmphcadmhhomm
mmadtomm under gravity. mam. 0fbuf£orph3.2§ numb
mm and spin moved to «the man:- meaty, after which the eemm
m mated with than ”Mm mam}. containing the buttar, and air
Woot3pmndnmuminehmappliodtothamm. The
humanism theneenoetedmzm. methane. M. tiratanewnte 0:
17m18n.mhm_mehm,mmhhenmm,emrthe
mudutfladthsnheMflmflmppodtolztolhml.pormimto.
Mammtimmflwntadmnotmtatfieotmmomumaf
.mnomidnbythecolnm.
Anolumtntpflh.25uaintrodncodintimtommumetomge
with‘the nonhuman. Awarding to seam and hem (91) this was in
tohondoatmofmmtvolnma2.15hmmtatwhiohtheumrtio
pone ha anal-god. stem-ed hears use noted that the poaitim to! Wine
nmmtiwmpfi.mmtitummtodetammo
«Whamehwuheermmmmmrumteynmw
hemmwmuwmmmmmm. mmmmkm)

mwsmmgmmmtnahigharpfiummoismtedflth

35

-Mmmaalouupflcyimtwmmnnu. upfimga
tronh.9toh.5uatr1¢dbutfitthm. nmmm'vwa
mmhmtmmmtomuflfimWIrWMBE
twammmmmtnwwmmm mm
«mummcmmowmmmwumawmmswtun
$13.25“! mmnmmatvmms twang-Dakar
«WammtsotoMfl. mw’mdmo,vcmemmm
nmwm mm,amagoedmnumorumanamtmm-
W. WWdMnafiaflwtmmmflmofmm
mom.

mmmumotmlumm, 0.2I-Iod1uniy6rmdo'm
mm “to am through the calm «enlight- mea- gnaw a. room
mm. WWII:- atpflBflSmapin inn-caused. Arum-equi-
Wmmbhwmeelmmnmrwmrue.

‘ Haw—Alcummmmummmmw
mm 0.131 g. a: Lamina 1n "unfilled mm- and nuns up
thavolmotomnl. 0mfl..MthiaMklmdmuolnt1mw-dmm
teamm.mo.5tozn1.ummmumrormnumatm
mm cum. Similarly,- mmm o: 1 to 1,0» dilation-fie: m art-m
solutimmeum tar Mmmomphainthe0£$tom2molom~
mmmabmndoupteém.vmm,md1n1.arm-unwa-
Wm mmtmmtoach. mount uh“ (mmnm
mm)m¢tmmm1nnbonm¢mm bah fox-15 minutes. mm
99011113,.10131. at‘EGpomm‘h'ethnol was added to etch tube, maths
wmconarudatmm am» that ofablmk. For tubal mm;

 

36

mofiimdmfitymdmgmlfi, mmsu. ofSO mount “Mel
mum. mwmam Wumahmjnngn'QS.

n. W o: Mania mum much an or ammm
Wdutmmudmflhdnbwe, «03“th
mum-damn . mmmuummmm 30.9120
Wunmawmmwmmg. mm~
Mammy-m. comm, mummwutmm 03mm.
moment «Almond!!! mtmmleuntodmm alanine
mimmtlvhichvm divided by the eoloryiold @ «awoken! as
ram-a m- m- mgant. (90).

a... m 5- m m potato meo m Wm
mind smut distilled mm for 1:8 hairs, in the rainwater, ohms-
‘mtfin outaido Iolntion moral tines. ‘ Th9 ample m thm dried in the
um daicoator aver phosfioma 13mm, and atom in this manner
until lauded for mohair. The Weani- m carried out with a fix-
Mails 1502033. MW protainaulhnl. domtmtboiling
wax-caucus acid, in an mud, mild tube. The mpnm far
mm m 105°, and the m m either 13 or 72 Win. smut.
“plea was mpn'ad far the basic amino maid dumtimh . it the and
at the WM“, the mount" in” drnd under am over sodium
M. Dunn. atwbormthm‘ddod to this mums, and Wdrying
Wmnwwmmduwmmomplmmmldmm—
magnum. mwmmmupmmm. otdthm:
pH 3.25 or pH: 5.28 buffer, and am used main-m or stand in m

37

mm. Thoreau: of mmnohcm mag-1: ofmctpotete
fiwlm we reported in mm. m.

1h man uh f a“ In W to W the amt of
WMWWMMWWMWWMfim.
“WED“. charm mu mmpru-muth
6.9:)... umwxmsmmsrmorwwwem
We. MMmmthometz-MW
Wt. u m. the mtein mm bleak, huh. m a m ham
115w todimlu. m. collation” mpmtedtedrmeel inn
um anteater, and. than, the sample we Mind the described
above. homelmmteeheckthemteetmtmendulm.
Newmmwdetmmtheowmoadde intheseumplee,
maitaypeu'ed tintmofthemhedbeendeetroyedtoe cmeidemble
demo.

in“... u .4- a a.“ Since Wynn in mm min;
mid hydrous“. belie merely-Hie was carried wt in order to act-em”
whether ornot thieenmoccid in wt in “root potato hawk-e.
swamammmmmammhomlm. $0.381!
When-means added. me tuber” thmeucuetedat mum
pumpam gonad. mammal-med mtinan mun 119°C 2091?
hours. the hmmm m thm names: with dilute mm ham,
and the precipitated mum whee moved by mtrifugina and washed .
mmmumtmmdmmvmdeeicutwmmdaupte
e em on? 1.0 m1. withmw when needed tar the chm“; determinants:
of awesome. rho groom followed In that or Spies and SW (93).

38-

fishtnl.et23.8Bm1m1-1cmid,mdl.0m.atflfimcmid,
emWMngBOLg.a£re- -.-__, . f ‘ . aha-do,m
nixed we cooled to 25%. To this mm mood 1.0 :1. at 51M
ammo! dimmcmmtntimorthemmte.
mmmmnmm, itmkeptmlthemtorme
hm. ennui-tenth ml. eta-.03; mat Minn unite usthmadded to
mm,w1&muwmw¢mg,wmoo1oohnmato
develop far 30 mm at room whenth in 15% dark . The optical
dendtym moot $0mamt that at: blank. Standards chums
0.20 up, 0.10 mg.,mdo.flh mg. «mommminorder to datab-
nlhuehmme. mmwotMmm-hmtemrepmwm
mum.

   

 

The importance of W1 mm in theaetim of $11010.“ has
1mg been meow (77, 73). Accordingly, Wt! were pet-foamed
to may the inhibition of met mate {3th by iedm -- : -~-
mmmmm onto, and Mthwlmlmwe.

shook sciatica: of 1mm, mhlormemrihmoete and W1-
mleimide (1110“ H) were properw. For use in W Wt:
these mlutime were diluted to 1x10“ H. ‘ .

Appradmtely 25 mg. of meet potato fiwhee WWW was dis-
aalvecl in 30 101.0! placaphute Emitter at pH 7.5 (9.1 K). In ewh of three
10-111. volumetric mm was placed 9 m1. o: the 9m» eohtim one
1 mi. 01' 11:10“ h ”which or inhibitor. The solutions We momma
at 30°42. mo ”and from time to who. After a ooh-mu period of who,

 

39

1 ml. a: mod n on! nah inhibitor w W mourn: to each
fleck. >

Another Bwlue eunuch we prepared under the m conditions
an share, and ma how of incubatim with 10" u inhibitw, 1. m1.
of 1:10“ :5 inhibitor eolutim who am added to the metian nun.
when the humus was completely inhibited, 1 ml. of my" x mm.
wedded in omertodetminewhetm ornot thin inhibitim cafldbe
W by thin mhetmce.

'rhe inhibitim by mm was also WW, fella-in:
the relation of the W mp3 of flwhaa with this mbetenoe
epeetmphetmemm, along with the decrease in activity of the
Bmleee pollution. The method used here m that deem-med by W‘
(71.) . Alexander shared that when present in means, Hamleiuide
mote stoichiometflcem with MW]. cmpamde , and the deem-ma in
9.me 3.15300 membeuaedum eenymethod for uni-W mph.

A 0.601% E hthylmleimfide stock eolutim no mannered, and 2 1111.
hi this volatim we added to 1 ml. of a fimlue eolutim. A blank In
wowed, remains the mowmumeo common with distill-ed at»,
and the abearheme read at 360 m. The reaction of We
with the W171 group cf the fiw’hee we allowed to proeeed directly
in the quartz anus and for spectrometometric mmmmte. At rower
intervals, 0.1m. sample: were moved for new of B-awheo activity.
The protein cmentution in the reaction W was dammed on we
blank by the Remtml-Gamditf him-st procedure (102). The remlte or
fine emporium are molded in Table EVIL

he

16. MW” of mg: EEWW

The photocaddation of meat potato 0W1.» he carried on. word-
:mg to the poem 01mm am 30113108 (191). Award, 5 mg. at
mt potato fiwhaa m diamlved in a fan :01. at phoephfle buffer
(pH 7.5, 0.1. n) and 0.5 m1. of a 0.02 pea-0m; solutim a: Mlm'bmo
«find. The emotion mixture was Wind at mom’mremre 30 an.
my man a. 15 watt! Imp. The activity of the awn» m oheokd at
mam ottheupmmmdafieréhws. 1001.0001de ‘
experimt are reported in Table mm.

III. WIRE AND DISCUSSION

 

met pants mee showed no decrease in aotivity as indicated in
mm 111. matthawownusnot 1:061:10!!me indemni-
mm by e. naturism of the electrophoretic pattern before am utter
mwmt,uohmmmaaland2.

W111

mum's OF Tmsn DIGETIOH
W~1A* » T

 

 

 

 

 

Activity
33mph (Absorbmeo at 5140 m .)
0:131:31 aohticn 50" 4‘90

mar trypuin digestion .500 .105
-_._...2L._0m__.,e 3.204 2-5. .-- Rosana similar to than cab-med with
trypein were chained upon trams“. with meta-pun as indicated in
kblo IV and figure 3 , which rapt-um the electrophomic puttem etha-
ammtryptic eotion.

 

 

 

I+2

   

Ji- _.

Ascending Descending

FIGURE 1.-— Electrophoretic Pattern of Sweet Potato Amylase.

( In Phosphate Buffer , m 7.0, 0.1 M, for 7200 sec. at 10 m)

.14..

FIGURE 2.--‘3LmTROPHORETIC PATTERN OF SWEH POTATO -AIVIYLASE
AFTER- DIGFSTION WITH TRYPSIN.

   

————*

(In Phosphate Buffer, pH 7.0. 0.1 M, for 7200 see. at 14 mA)

 

1+3

 

ii- a;

Ascending Descending
FIGURE 3.- EWROPHORETIC PATTERN OF SW POEATO MIASE
AMER DIGESTIOH WITH SEYMOTBJIPSIN.

(in Phosphate Buffer, pH 7.0, 0.1 M, for 7200 sec. at 14 mi)

   

.——-————_
Ascending Descending

FIGURE u..-ELECTROPHORET10 PATTERN or SWEET POTATO qAMYLASE
AITER DIGESTION WITH PEPSIN.
(In Phosphate Buffer, pH 7.0, 0.1 M, for 7200 see. at 10 mA)

 

an: IV
mus OF enema EWEOI

   

 

 

. Activity
m. (”3ch 0.0 5109 m .)
Origin-.1 minim .501 .502
After diwticn .510 .510
0. .r «:0 0 you.-. .w Papoin also in inectivo on sweet potato

B'thpBSu mwm~madu reporecdinhblev. A: in
the cue at moan-m and 121-7001115110 change: either or electrcphoretic
pat—m own be detected upon 0mm 0: thin mdopeptidaae. (Figure 1;.)

TAM V
W 02‘ FEED! DIM-STICK

«nan-db 9W“...

 

 

 

Activity

 

3W“ (Abeoflaance at. 51.0 m.)
0mm]. soiuoion .950 .950

After digesticn .950 .955

“ _A___4._

i V W - —wv‘ :— w—v—

hmtemealeo performed at: lower pflwhare pepsin could
conceivably be more active. At pH 3.0, the mo potatc fi-mhac pm-
cipitn'hcd tom the 001mm, and. upon digestion with poplin, the min--
0.00 dissolved. No Emilee activity could be detected in this mm,

and pupa: electrophdwouia axpurtnlutu indicated that the yrdduhn.hed
boun.digasted, aided nc‘bmnd upmnnrcd'upcn trocinnnt*Iithflbrandphandl

 
 

' .” RIM-B 1| WW3: me am the

_ union m m damned in crystalline
.tdnn.€98). Prudindnuxy-axpenmmduta indidntad that thflwa‘ifl a.d00rlnaal
in.ectdlidy at the flfiumvdnin‘upan truntncat‘with.pnsninn Firthcr'axpuri-
taunts indicaxed.|.decraulchin.actirihy‘np>tots.cnwtuin.rmdmdinflstddhdd
WWtonop. mlamemummmod
in iihlbu VI and VII.

 

   

  
 

 
 

  

 

MB VI
mm or mm on sum mm human
. , Digestion Mod Activity *
3“” u I (W! _. . __ WWG' g" 5103-) __
’ (Ran do. 1)
Cmtrol o 4.80 .085
Digested 17 .350 .365
Dispatad Zn .350 .Bho
Gunirdi 0 .235 .237
mm 6 .150 J55

Bdgpcted ha .128 .122

‘__._.

1&6

 

 

 

 

 

 

m VII.
mam mum on sum Mpums: ‘
W ' ,. ;. 1-2 :MMW
_._ -. squire). _ W . hunted
Mod Activfl-i; Pix-ice “ Activity
(Hours) (Anathema at She an.) (Rom) (Lhaorhemo at 5m m.)
.210 .210 0 .210 .210
13 .200 .mo 13 .m .11.).
21; .200 .mo 2h gum .137

.d. . . h- ..

g .4. ‘VA_ ._ ___‘.‘

mm «a mu um mm m: to hm; ebonh Wis of neat
pate!» Pawnee, and that most at the which the: place in a. chart.-
mid affine. m,m6hwuafd1mtim,.thmilnom
decrease in activity. An uplmtim for the” data in either this the
B-wiesomnodflmmmehemyee to mummwdrmm

under the 1211111ch or pepsin, or that pawn mm as inactivated.
mewmw5mamMRsemndeWemeb
m e tux-thaw loss in activity occurred as indicated in Tables VIII and II.

 

 

W VIII
woman or W TOANALRmm mmm W
(m Re. 1) ‘ -
Period Activity

5839? m _ mm) (Ahaewhenco at 5140 ma 3
6mm). ' 0 “5 ”8°
Digested 5 .380 .370
ingested 3 "320 '330
91W 5 .230 .239 -

 

40.1 ml. trash pepsin ended hora.

1:?

 

 

m n
mom or mmmnmnzmmmx
. Fm “Ce 2)
a a Peri, 0d Aetivity
# ‘ -._ (lime) _ Ahecetehce at 5110 neg.) .

Control 0 .580 .580
Digested S .570 575
mm 6 .510 «530
Digested Bit- .th .hhe
Digested 3 .1319 .m
Mane-ted . 8

e329¥ '0st

 

ab.1323}... mehppinlddedhefl

In mum mm» by pew elmtwoworem, chive hm
Wing to modified awhile appeared. no evidence toteotive
mm“ m actuated. In than WWW digested. unple-
a: met pateto awheome mnmgnthe emtrol. lie We.
Wm” could be detected. Cmtrnl and digested temples me also
mmthemetrip,emlm1ymehmdeppeered.

. shaman is a. hector”). preteen mm
mm. Iva-www.mw iteectimie ofthe
momma- w (99). the ammo: went-1n on m potato
awn» 1e «pelted in Male 1.

- ~ age Since, with the «caption of rerun, the ado-
heme m to have little or no effect on sweet poteto 3W,
m of the aopeptideee mmmu wee tried. Althmgh the
”the «Mmemeemmet muemm itmto
aetflyeeuwwdromuw mohahlybenotuueemleathetot'an

 

 

1:8

   

 

m I
mmormmmoummmmpmsn
Moa{ Of Digestion Activity ‘
..M A M - “(Absorb-nee“ 51:0 an.)
0 .“e .670
2.5 .665 .560
1b .660 .669
2‘4 £85 .629
35 .600 .510

 

endopeptideee in sexual term of mixing this woblm, it should effect
certain modifications in the integel structure of flawless, and give
infurution ebmt its compositiun.

Thetthu WWWH them experimte, undm'theea
umditiune m native wee dmunetreted by its action on new, (share
in table II). ‘

 

 

 

 

 

m! XI
LOTION 03‘ “WW OH mam .
mlw ge}. ,:;ri‘p.;.r fih“'#-eh.mm :. ni- fir . rim
Digestion Pezded Ken Protein Nitrogm
(minutes) ’ . (Aheurbancu at 570 m. )
0 .330
30 Jim
59 .860
1159 1.10

 

 

 

Digestion Period . Activity Hm Protein Nitrogm
_._ (Mtge) -- . W atLShD y...) W at 579 m.)
(M NO. 1)
0 .580 .580 .297 .305
30 .615 .620 .310 - .315
60 .620 .620 .315 .315
1-20 ‘a‘D 06119 0315 e310
(mu No. 2)
0 ~310 .315 «035
30 .338 .316 .035
60 675 .365 .0145
120 .370 .355 «Otto

IMO

“

 

These mlte indicate a striking increase in Bwleee activity “pm
The absence of a aimiflcent 1mm in
nmwotein nitrogm, ee W by this method, am he «mm
is mention at mm mm at m below the emitivity or the
mm. Thin W by uarhmptideu same: surmi- immi-

oarhourypeptideee troeMt .

ption.

The romance of sweet potato fiwhee to proteulyeie with try-pain,
Wand herein, um thmghe little harm-ins, mindeedbo
me emthmm unwanted at first. Steinendriupm (78) report
thetembu u: when WWW emeem also mietmt to

W,

Wumebywmmmmm- WWW“
their physiological amm... for the moot M in the mum at b
m of protease: and can to be motectod from their action through
binding uith motel ions. This m denonetretod by ooourrmoe of proteolqais
b: themhmwhentheym habitat! with either tripe!!! or anym-
min in the menus or completion; agents such on ethylmudiendne
tetreeoetie acid. W. under these uirmnetencee could be stopped
hytheedditiono: ualcinmion uranium” of othordivelont ions. Ina
Wait piblioetim the“ authors report that all We“ inverti-
ated amteinet lent l 3. atom oxfimlybonnd oeloimn 1m particle.
Stanfield Fischer propose the following echem to explain the resist-mo
of m1.» to protoolyeiee

metal ion binding agent

 

 

Duties when i. a "motel poor" when
(active (active)
‘ r :—
metel 1m . protease

bruit down products. (motive)

A similar observation we reported by Ami and Foamy (79) in the one
or omelbtmin and trenetorrin. These iron binding proteins form very
einfler stable umplexee with 2 atoms 01' iron. Tbeyvm famd to he.
more resistant to hydrolysis: by obymobrypsin or trypein than the m1-
froe proteins.

Althmgz no motel ion has hem reported to he unscented with
$-owlnu,(Belle (32) found traces of lead in the ash of crystalline
flawless), its etehility tonrd protoolyeio could be explained in e

51

Nainflarm. Tmmmtmummhuuupum,mduym
to tho solution of thymus.

2 . in»... M? ,

 

3.... potato fiwluo 1. «one in a H um, but 1: very rapidly

mm in this median. out this in an irreversible mom, is
indicated in Table XIII.

ME XIII
mmnmmmm OFSWPOTATO fi-AHIMSEBIB Hm

   

Sample Mod Activity

 

”A M Quin) (Abloibmoo at she y...)
0m D .085 .090
mm 5 .000 .300
After (1min am 18 .000 ,...

' Wm“ butter

——.-.— W v.— . a—fi

The motive new potato fiwhn 1- nolnblo in 8 14 um, but
charm; the cm of dialysis, it precipitates out as the urea concur-‘-
mtim «om...

Atwmnmsxmwmmmtmmwmooof
pop-sin, and the mu (reported in Male IV)1nd1cate that in 8 M urea
lat-990013318 took place. Halter, dmtnm‘bim m too rapid to obtain
«moon-y tum-emu. ‘

52

   

 

[um 11v
honor or mm m 8 x m
m. Bimtion Period Activity . Hon Protein Nitrogen
# _ {@0532 A. (..th at 01.0 m.) (AW gt; 380 m.)
Digested 30 .012 .015 .1193
mm 1.2 .000 .000 .635
Digest“ 69 .000 .000 .630
Control 60 .082 .082 .000

 

mommumméfimionotmflyumpwuinenum
no woman: shown in Table“ indicate.

   

 

ME XV
mmmum 03‘ m IOTA?!) fiWE I73 6 H um
mum: Period Activity
# (W) . 4 ‘ (£19an a 5an m.)

0 316 .535

13 .1130 5W

23 .255 .265

25 .200 £10
m . .000 .000

 

A: indium above, there in a slow damnation in 6 M m, and
this could ind” tho protein more amount; to motoclyua.
swath, tho um (lanthanum along with peptic dimtion has been

53
“tidied. Themlta ofthiatypoofmperinmtammportedihhble
XVI.

mm:
muonmmuwormmmo ammonium

   

 

 

 

.H” ”a; 7 v: . :7“ W. :3; w‘ mg _
Bigaa‘bion Period Activity Period Activity

(Himtea) (Ab-moo hf. SM) 111:.) (HomXAbaorbmce 0.35140 m.)

o .500 .531: ‘ o .6oo .601

15 .325 «350 h 625 .5150

1.5 .200 .200 10 51.0 .5m

60 .185 .190 19 .501 .510

75 .220 .110 25 .300 .395

120 .095 .105 28 .220 .230

two .055 .050 ' 33 .080 .082

550 .033 .025 an .072 .975

-; _._ .4 A. . .- ' , A _AL-A‘u

 

w v—vpy . .., fix— ...7 . _vw—r r———. ‘ ——w~ ‘ —,____

Sines the homo]. um o‘ohcmtmtion 1n the previous experiments m
h 11, other the addition of the pawn solution, determination of the
ponies whim on mot potato fiwflan ma carried out starting with a.
h H um ”much. I , N

In this mm“, the interesting chumflm was made that trott-
mammumemaeméhmmmmmmu
differm’s war, (was though the tow}. omomtntim at the time of
Moms in the mo. Papa-r electrophoruis of the digested samples
wdidnot mummimh cunmmthowotm Moms. Tho
activity remind with the min component which matted within experi-
mttl error, 1.1; the some volooiw as did the control.

mm
Aormnormmonmmnmmmanmhhum

 

Digested Sample Actiflty emu-01 Activity

 

(M) (We at she ha.) (Emu) (Ahmed at 513.0 m.)
0 $1.28 . 9125 0 .123 .125
3.5 .031. .085 3.5 .329 .125
21; w .070 .070 21; 433.5 .110

a ._ _ A r LA , m 4 A . .A.

W *— T—y wv W ... r ‘. .——-—— , ~ru—r w ——.-w _ .— wr—v

mama ohmhaduihhnypaanamwmm, Win
hhlel m and In an he mama mm change in activity bcymd
h... which can be: ..th ta ammuah mdcr than munch...
Wmmmmnméxmuthnmam mom-1n.
MWfldMImt-omtupmmm‘ho 9W.mm
mums-um.

WIVIII
M10! 0? cmrmon: H 6 Km

 

 

 

W!» I“ WMWMWJ

Mod( of Mgeotim Activity
My” - (Abnowhmoo at 51.0 m .)

mutt-o). 0 .365 .360

mm 1.6 .212 .209
Digested 2o .218 .223.

.w ‘7. w.‘ ,v‘ .- WW 1w 1 F‘Y'rv'

55

 

 

 

w XIX
ACTH)! 0? mm m 6mm
Period of Digestion Activity
5““) “f A -. (W) . . -.<:b-W°:~t_519° "ea-L
can‘t-o]. 0 .110 .100
mm 3 ‘5 .1110 ‘ .110
Digested 18 .065 .065
amoral 18 .065 .065

 

 

smoomtaotamohintho ”mum (80), mum-amt.
an: carried out to Wipho the mum of more potato 0-minute
hmmtmnh- pomhmtyorpa-omlymunnymnma chm
hut mm.

mm H
W! 0" EH? m SHEET P013110 WHEN-33

 

 

 

 

 

Activity
MPH _ , (Ahoorhmoo do 51:0 3.1.).
Original .160 .170
Am 5 animate. ht 100° 4390 .000
Am 6 minutes at 70° 195 '115

 

56

A mp1. or hha hwnsa which hsd been trotted at 70° for 6
minutes, was inmhnted with mm M tho activity checked after 1
and 18 ham or digomm. no decrease in activity was round.

It. AAA-AA A» oh... .oh-A. 9.1" AA A-.- ".
a... .o A 12. _v - 1 A. A.--

Two dimeioml paper ohrmtompw of sweat potato Bwhle
hydrolyuta revealed the wee of 16 amino acids. 30 hthmpt in.
ads to «pout: and distinguish bottom leuoino end 1001mm. Thou
WI indicated on: the pro-moo of histidine tn doubtful.
Baum, nub-want ammo: chewed that 11'. id promt 111 mu
m... The mm o: the paper chrmtomphy uperlmtn are re-
porhod in Table III. In this Table are also given at “In“ of amino
acid: chained with h synthetic mixture of the amino acids believed to
he mt in tho hydrolmte. The R: values reported in two different
ammo: from the 11mm for these amino acids in the «solvents
employod in than experiment! are also included. It. ahmld he noted that,
the literature .morda wide diam-once: in the romlta of paper charm:-
tomphv of amino 0.01310, so that one mot ma known human for 1W1-
os‘bim wrpoaes instead of relying on reported Rf when.

 

The malts o: the amino holds analysis are reported in Table;
XIII and XXIII and Figures 5 and 6 remnant a. typical ohromtompMc
Moumflm of the amino hold. of sweet potato fiwhao momma.

57

mm
nmmmamormummowmmnmwmsn

“manta-90...... -

   

 

 

 

0.80

Lancing Purple 0.80 0.79 0.8).
PM W 0.80 0.80 0.78 0.85
m. my 0.75 0.75 0.61. 0.78
Praline know 0 .89 0 .92 0.85 0 .88
We Bram 0 .51 0 .h9 0 .52 0 .51
Alanine m. 0.50 0.51 0.60 0.55
methionine Purple 0.79 0.76 0.73 0.81
fizz-0mm nod'dish 0.1.3 0.38 0.39 0.50
Armin. nix-pic 0.1.1 0.38 0.1.1 0.09
moms Roddinh 0.32 0 .29 0.30 0.1.1
am Purple 0 .21; 0.22 0.21; 0.36
Lysine 201-010 0.21. 0.20 0.39 0.81
0min. W 0.13 0.09 0.08 -

Amrtm Blue 0.11 0.09 0.07 0.19
Glutuic Purple 0.214 0 .25 0.16 0.31
mama. ? 0.55 0.69

 

.50

.§§ uflafl ogufion ggoguzggt

. [it

3.4”

[E

Vrl. . . III...

 

 

0.3 .1... 1.... «0.0 £3 «04 mm...” RA 5.854
00.0 mm... 03 «3 30. c 00.4 and 00.0 «0.0 0.5.0 05.000
d...” 0.3” d..." «$.93 wmé Mn...” «urn mm.” 0.30. .010...” 3.03.003
0:0 .00 0.0 0.0.0 00.0 «0.0 «10 00.0 00.0 0.0 05.3
00.0 $0.3 30.3 ««.0 2.0.3 9.0 2.0 00.0 £0 $0.9 00.0 05333.0
«0.0 40.0 30.3 8... 5.0 00.0 00.0 «0.0 «km 0.0 «0.0 8095.
om.» 000 «m0 000 20.3 3.0 «3. 0.00 3.0 0.0.; 3.0 03080
«00 00.0 «00 mg 00.: Go. 3 3.0 00.... 5... 5.0 0.0 030000
«.0. m 00.0 _ 0... 0 «MA «0.0 300 00. m an... «7.. «0.0 3.: 0.08200:
.34 0.0.4 0.2.." .03 H3 1... 8.0V 40....“ an.” «3 1..., E0
5.0 om.m «0.0 00.0 m«.m 00.0 «wm 40.: 00.... «0.0 1... 05.3
0.0 m»... 2.15 00.0 00.0 3.0 00.0 00.0 00.0 «0.0 00.0 00302
00.4 «0.0 2. n 40.: n3 «0.0 .00.: «0.0 00.: an...“ 00.0 05008
00.0 . 00.0 ««.m 0.0 5.0 8.0 3.0 «0... «3 «0.0. 3.0 800E
00.0." 35 and 8:9 «0.3 00.3 3.0 and 00:: 0.3.” 3.005 0.0330
RA «00. 30.3 3.« «fin 0m.« 0.1m .36 Sun 3% 3% 05000
. . . 3.0 3.0 «0.... ..«A 000 «4.0 00.0 00.0 wo..§...8.§
. 00.9 8.9.. $0.05 R8: 85 00.2 0%.: 1... 30003

«5 [~00 «g «30 080 03 «80 4.5 1

111.410.010.00. 10 0.0.1! 11111311.... .. 300 003

 

E08010 £00000 hamsfi 303g 050...? .3003800. 00013.. 003200000 08019.

.1..».3 04m. -41?“ . 001.11. .1 ...1..1...M;...w

 

 

 

 

«0.3010 808 53% ENE; gnu-0400.5.
a3

 

I I I a I I I I II I I I I
I I I II I I I I II I I
I I I II. I l I I I I II I I .
.l .I II II II II II II
I I I I
\ I
I I I I I
I.
. I I I I I
I.
I I I I
I I I I
.I I I I I I
I I I I
I I I I I I I
IIIIII
I I I
.l I IIIIII
I I I I I
I I I I I
I
I I I I I I I
I I I I I
I I I I
I
I
I I I I I I I I
I I I I I I I
I I I I I I
I I I I I I I
I. I I I I

 

59

 

 

   

 

m XXIII
mormewmmmrmmm ormmommm
mm Acid Pu- 100 g. mm m . Molamm- Hum 01'
. _M . w m _ M “9552'“? (5")
I‘M 1m ’ 185
We 3.6 5h
swine 3.1; 60
music 11.0 130
Praline . 5.3 83
Glycine 1..1 . 109
W S .1 109
Mine 55 35
w-cntme 1.1414 21
Kathie-am 3.8 an
”alum h.2 57
Lamina 7.5 101
Tyrosine 6.3 62
We 6.2 . 62
Lynn. 6.2 7D
Histidino 1.? 19
Armin h.8 h?

 

 

. I V I A A . , § . I v v ’ r O .
. . , t “ . . . . I O . . . .
: .|. . . v A t. V . .
. . . s..- - ..L 1 ‘ ,4 . .. , :u . J
‘ l« q, . I .. A V n l . it (A, . d
. . . .
. Mu _ MI‘

 

O
6

.oamaau .ma Hm.H a you .numom ONHIQNH no gasaoo .ao omaxm.o a do mqoc mm: aoaamnamom one

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

fill-.0

.mmmahadr. eunuch aoozm no cadahHOHdhmnd 309% ada04.od«a4_ouddod.dqd Hmnpnoz one no“ ophso pqmaéflmfi In.“ mafiMah
7:: 28:3 .8 was?»
own can a? no: com fin om +1 j
* d 1 fi 1 d1
. . . A
z j j i j
nu
.d o“ .d
. m. m. m
m w n
I m
m. u A k s
m m. m. ( m
I s m. m.
I 3 U;
o "N v. x
9 n? O
n U. u
0 I. I. 9 .L.
I. o u T. u
u u a .A a
a u w
a u
9

 

autoneq

swam-3

 

 

orgiedsv

 

 

' m 045 49 aauaqzosqv

 

 

61

mamaam .ma 5:.H a you .nwmom omHIQmH mo menace .ao maxm.o a no meow mm: qupmummom may

.omwaha4r ondpom umozm mo camshaondhm d aoum added onfiad owmwm 0A» mo mppso anodammm Ic.w mmame
A.Hav pqmdammm mo oasaob

 

 

 

 

 

 

 

 

on Om OW 0d ON ON OH o
d u d a & q u
1 ‘0
f .N.
v m
a s
on a,
I. I.
u n.
1 m. m. . .
a e 3
1 .50.
m
m m
1 9
m. .w.
u
8

 

 

 

m 04g 49 eoueqxosqv

 

6.2

th was indicated that urine, throw. and
mm undergo the moat «mum under than acidic own”.
More, mmmmmdthoaommame WWW
exhumation amazing to one method om. fig. (95). As W
mum, on» m moor in the oomm ohrm'bomphy out two '
Woo: m in «mum-o: amino and “31”.?‘arying’tho pa o!
the W butter how be and MS 61:! not new to mflnmeo m . i
mum of than into amino town. However, upon holding the pH 3.25.
batter put tho recommended volume for changing, nun. ammo «notion
whim the buffer of pH 14.25 an applied. Although “lino did not 31'“ a
m pom, the «mum m «taxonomy. That the mar of clamp
on mod under those auditions, was proved by employing wiomio
«momma. Pim?iltypd.oulofthemtimobfl1md
m o. mimic mm oxidized ample. Only cystoio acid and um. m
mm in this one, «since other amino mid: .moh 0.: Maxim.
some and tyrosine showed comidmblo destructim under this mount.

Ememdonfinomthon-unoacida Mmtmthelmfll
W. Assuming a molecular weight of 152,000 (51:) tor mot potato
Bamboo, than would be 19 to 20 histidino residuu- andvzl halt Wino
mm» per molecule . In an determinations , ominlont wants of
Wandalmimmfamd, ”well”; 2 tolutio of lmoina to
inolmodne . Aaportic and ghtamic’ aoida mam“ mks up :1me
25 percent of the nut potato fiwlooo.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

' m 044; are eouaqaosqv

[ I I v ‘4‘]
Gunner; t:
Zofiioootosx
euttoA -¥$
Strum-[v /
2’-
euron‘o c g
q
c:
, “KP
euuoza g
91“?“10 _
_:] C)
c>
“n:
eaves (
auwooxqm “c:
*:::3 o
snag-[us 91111101114814 X ‘9
“—
Ora-redsv . a
OPTIQIUIS eutuomaen
+§
-R
ptov oxeaaxo "*1
'H: “i: via} ;3? ‘6? 1 O 0

Volume of Effluent (1111.)
Figure 7.-- Effluent Curve of a Performic Acid-oxidized. Sample (1.61 mg.) of Sweet Potato . quylase Hydrolyzate.

O\
U

 

 

In sacral, mm W foe memo acids of mat mute
Whoa am with than or 3311331“. (32), on W ”MW
for mm amino acids by specific mourns. Balla' rah-u m as
follow“

Arms 6 mt

Wino 7.0 pol-mt

8m 0.76 promo

W 11.32 mam.

Theabomoo ottryptoplunmindioaudbytheramlm of them
mu and oolmmo (Imam. rim is reported in M10
HIV.

WM
WIRE 0? mm 'W ”le

 

mm: as. awn mmhMa mm memmmwfliflmenH ' I

 

33-31910
0020 .900 .919
0010 ‘M oil-25
0.0», .150 .115
Basic wax-ammo .015 .012

Mo wwm mg. mph. added .095 .090

A _._.._

IWW . v‘vv .. wy . . r 1' z - v

This also aback: with tho mm: or Bounce) who did not rm: my
Mom in mot potato 51min“.

65

 

simwflrntpmohtothu mmwmmmmu
hoped for, it was decided to mm. m mm of inhibitors on hummus.
W amp- hun boa: new as «mental for we oouuty of. ,
53-min. (72). Singer a“. (73) Mona the: action at a lumber of I
admin-Lama mommioauiohm mum «Jim W
you}! orthh mm. Inthomtamoh, mattmptmndoto
and: mum masonic! zommodmwmh the mandwhmh um-
oould be. and u a "tag“ for the active 3100. The inhibition propane!
at HWhimido, roflormomibmzmto and iodoaochnmo were
mind. The renal“ obtained we ”ported in M10 m.

w m
-5
AGTIM 0? mm (At 1119 H) OH sum mm W

’ a
ISAW-w.‘.-Q-_qm-‘.gu~‘-.e~.-.——.’—,n.rou~—. - .. . - .-.,--_ «. - —.a-- 6. Fr... -.—- v----‘—1---o _. .. _‘bOIr'u-fivl-"n. wmn-fl-m .-a—w....__ -—~-.—-r—.- .-er——l'vl-~.I -«- .uo“~._~n~.— .—. u..n—W.wnfigl_-",n. ..u. _.

   

 

 

 

 

We .
control
n—mmmoo .5140 .530 ' .530 .5110
Iodoaomnido .600 .610 .600 .590
p-chlorommribmouto .325 .305 .300 .305

.4“ w _n. 'A a , ‘ ‘;.1 ._._

 

.1... —— Wy r‘rw ‘ _w— w— var—.— _

The attach or o high” concmmtim (0.01 M) or the” inhibitors
mthomomplumlhmminhblam.

mum
37100001100310! ormmnonmrmm z -.

.L.‘

   
 

 

 

 

 

- _ H M _ 1300mm--- . 90mm
WM .190 .200 .065 .063
Imam .500 .530 .00 .505
wmmmh .000 .000 «- m

 

m inhibitim Wfl m repainted with Immune
and 1000000000100. a. W as mm mm £1. mommoim
o0 1:15.10"8 K or each Miter mpootiwly, m1, arm» 2 hours, 1 ml. of
mo" '0 mimoodoo. 10:. mu no mom in ma.» mm.
couple-to inhibition of 0m «may with WWO ‘ took
1510091100390. period 015m,mrmm2hhm oiinoubatim
with iodonocumido, 8.me 30 pea-om of tho 0mm activity
00111 mined. ’

7110 number of mlfmrdm mp0 necessary for 0%“ activiw
on W wording to the mom of 11mm” (71;). m 011»
Imammmbmmowmdwm ammo-1:
mm by mo mohr «motion ooamomo (620) of 0m. mama, ma
tho M10133 mutant 1: ml to WWW emu”. at
tho III-mp3... M dam“! by tho bum “001m; the ample 0mm
amaludmt poutoflwhu. Mumummortaommu
mm.

67

   

 
 
 

 

 

 

 

 

mac! mm .. mm mmommm
5 5 I Activity (Absorbmco at 51.0 m. ) '
ample tum-2536135 "Lita-511m 'm;%
M ‘ ~'1n10‘:.‘ll - 1110.01! Rm
mm. .610 .600 .010 .010 .000 , .000
law .639 0625 «km w 0215 .220
smattefimmWot wand .670

 

 

MadeiInoubatim fang; I 5 w 001mm
with x-uhymlmmo boo rbanoe {Atom-hm ’ ' 03010.! SE

 

(Hams) . at 51.0 m.) at 300 an.) Aboorbamo Groups .
“0..-: m--- A w--- 1*.“
0 .560 .570 .900
5 .015 .020 .305 .135 2.18
17 .000 .cm .678 .302 h .818

 

M a vault 0: thus «pox-1mm, nwmm 00m to be a
m Win: inhibitor :00 mm potato meo. ”10“.,“0 m1
mum-ox mummy. mmhapro‘heinmhmlo omnooboootmu
by 3000010011031 m: reagent, moo Roberts and mm (103) team out
Kama mated with am; 60 moat on may almond poop:
in 0mm albumin as did p-ohlmrihmm‘oo. Beverbholou, with

mat pohto fiwluo, tho motion with the «11000271 group 00000013.].
for activity in preterm, 000 it. my 1:011 be possible to label. as:
0.001" 031.0 in this am». ‘

 

Photomidntion with methyl” 0330- m and by W011). and 80110100
(101) to fluctuate the importance of mum. :03 the W10 activity
at fibumolm. foul inhibition no 003003700 wt. an uptake of 3 010198. ~
ngmpornoh ofthiamzm, atuhiohpomttho mlyaimitiomt
ohmgoinminowid mummmmm 0:30:33.» 0:
Midis” out of a total 01' 1.. That 0110101100 in not 110000001? for
aofivity o: m pouto own» was momma by comm mum of
obstetivityotthiomflueaflaréhm immuthomoo
at W and mothylom blue.

mun

mmormmwmmuwomwnm
WWEOFMEMMBIMEANDOXYGEN

 

 

 

 

 

4:3” " 1 r4“? ““4w5i21: 14. r ~ firs—2m “fig-:0; r}:
Paid Lotiflty
(Hours) (Aboarbmoo at 5140 nu.)
.200 .275

6 .280 .285

thmm

‘ The behaviour of met white 9% Ward. protaolytie m:
mflhufimmw autumn: of theme mid camper
amm. Twin catalyzes the Win or puptidu bond: involving
”311131 and W1 groups, W W with a few exceptions,
am the W at bonds involving tyrouyl md W1
my. 3mm than amino acids were!“ to be mi. in M mute
5m”. mmmtowmmwthmmmu mm
5mm me by the canton-whim a: the mums.

.31qu pepsin is . relatively nonspecific Wag trawling
ppm in this roa'm‘b (80)}, it, Mme, Mm «mm.- o:
W of bond- involvinz swim and ”m1 mp8. mum
mm at'MMnoaddsinmpoutafi-‘mmmuphm
mmia at m: min by mm Imdcr new; cmum, M won as
the mum of pepsin 1n urea ”mums. .

The inure-so 1n lawns: activity upon taunt with aubur-
popuduouybomudbymwal ofmlminoacid mime makingwe
active site.

we active Womtim of the met pohto 9mm» my be W
tam-mgr: Wm bending involving mostly animus, “Mic acid: and
We. The traversal: mmum 1n urea. ponibly ocean thaw
rupture at these Inch-03m beads, and than my net be sufficient Wide
hwylmmmWMmdmmmeflwmo:
tha um.1+. in aimifleant mt mm (97) 1a reversibly motivated

m8nmaolutim. Aelmguthndimelinhguattrypfin
min intact. the unfolded 30ml. m: the annuity to mama to
the not!“ emfmtim on 1m; the m emmtntim. Mptura of
maimlfidolmhpbytmopriete Manama“, ladeteemem
«ban-m meal-am at the We which men because so diatom
that We «canary structure comma]. to:- eetivity can no longer he
”wanted by male dilution.

finwrkoffims-r (73) Wmmtwmmam 91‘
Mel srmp- mm 1:: met potato awe». and that upon mew-
mlemler mum of the” mp3 to We linkages, the activity
of. the flamingo m loet. Rowe andWeill (96) chewed that the inhibitim
oat EW with ueerbie acid in umvempetitive. Wee authors suggest
that neat-hie geld inhibitim -md its mm by cysteine chm «um '
”cattle acid is eating by some melanin ether then a direct motim
with the 0.30th W131 grams of the memo. From these mastic“
atima itapwatht nemflaexozfdimlfldelinhgeammemt
lag-ugh"; 2) threaten.” mwflwdwlmupa, m3) metim
of me «13ml group meet minimum is entricimt to completely
inhibit fi-myheo eetivity.

Sineethe origimlemhtethio Weidnot mathemats
hoped for, the nature of the active site of fiwlue could be investi-‘i
weed thong: the use ai‘iehi‘eitore. Thin type or." emu): m M
been mcmumy «wind at tor a. murmur out new (phosphoclncamuae
(75). elm-Wm (76). We: (77) and meta (109)}.

N.W

1. Undcr ordinary cmditieqs m moteolylia, mot potto muse
Waneuuncetohydrolyeiehytrypain, Winpopainand
ubtllinin, u indium by tom mention of activity an no mm
in m electrophoratic pm before and utter incubttim with than
W.

2. Pepin dog-ado: fi-Wlase rapidly to mill. tumult: which do not
rue-in any mlolytic aetivity.

3. The actim of arbmrpeptidue on sweet potato fiwme lame
the activity of the latter .

1.. 3mm petite amuse 1: my dam in a 24 m.

S. In Bummdinéum meow" meet petite flwme
as indium by a. rapid loss in activity, but no tit-amt: poemlmg aw
flawless activity could be esteem by paper electrophoresis “patients.

6. Ho protealynie by twin or ehynotrypein could be detected in
mm solution. .

7. A complete moo mid we; of met putato 91mm has been
performed. Seventeen mine acids are mt. The remlta indicate a.
high amount of 51ch and “Mia acid, equmom mmnte of glycine-
amd amino, and 0.me a two to me ratio or mono to 1301m-
eam. Rotmtophaniamt. mmmdllcyetinamidueumd
20 median mid“. we wait per molecule 0: met pot-to B-mlue.

 

a . The inhibition or met petite Ewan. with gmmum
inducetanide and p-chlormmibumta has hm studied. A couplets
inhibition or met potato iii-whee is chained with E-ethylmlainide \
in 10" H emmtidze, in 5 heart: at which time enly one sulm'dryl

mp upper: to hen reacted.

73

ME

(1) mm and 1...... m cm. Hora 53.. 73. (1833).
(2) c. atmuo, J. aim. eoo. &, 125. (1876).
(3) 3' mm“: w" 35.: 770: (1879)-

(h) mm mm. vmh-eeat. 69, (1879), quoted by H. 1'. Bram
md Jo m, J. cm. 806. . 599,13” 0 .

(5) n. e. m, J. Baron, mm. a... 35, 596, (1879).

(6) a. em, Liobioge m. ma, 1, (1925).

(7) e. Ohlum, 2. Mel. cm. my, 17, (1930).

(8) e. 9. Emma, a. H. Hofidna, 31mm. J. 3.9. 151. (1936).

(9) Mary L. cum, ma... Adan, Amoco. 1n Gtrbohydnte chem.
5: 329 (1950)-

(m) H. a. Gore. 5. Joan. Ind. and m. cm. g, 102, (1932).
(11) .1. 1.. m, J. am. 306. m, 11.77, (1992).

(12) J. L. m, a. r. Exttm, J. Chem. soc. 1655, (1,929).
(13) P. 3. among 310cm. 2. m 71., (1935).

(3.1.) J. a. mine, a. u. Wt and E. mom. eml cm. 15, 629, (1938)..
(15) x. Welt, 2. (cream, Bloom. z. m 107, (.1937).

('16) c. H. 11131., c. a. May, cm Chem. 15, 27,3, 351, (1.933).

(17) it. om... J." Micki, Bloom. 2. ms, 250, (1933).

(1.8) 2;. Keen, cm em. a, 30k, (19%).

(19) 1'. a. Osbourne, J. Am. 01m. Soc. 31, 587, (1895).

(20) H. c. Shem, a. n. Schlesinger, J. m. cm. soc. 11, 61.3,. (1915).
(21) o. A. m mums, z. Phyaiel. Chem. 392, 253, (1932).

7h

(22) 39:3)hflw' n. smear, 15.301110, J. Am. am. soo. 111.. 3037,

(23) 1-1.0. Sheena hymn S. F. Doebbouna, J. Biol. Chem.
591. (1931.) ’

(2h) WW1. s. 3. mm: J 3101.0“. 119. 739. (1935).
(25) a. m, 0.11. Wok, om cm. m, 399 (191.3).

(26) z. Mom-1911.1. A. mz-r, Hum-nun. 29,. 251., (1932).
(21) E. 9m, Bowls. and. m. m WM, 1, (1926).

(28) J. mm, A. 3.1, 3.31:... z. W. cm. 252. 1011. (1937).
(29118ng 91cm, 3. 11. Wt, A. n. Diekaan, Com-l cm. 3.9, 126,

(3c) 89$)m' a. a. new, A. 91...... 3.1.. am... we, 3)., 316,

(31) 89:3}3015, R. R. temp-m, M. K. m, J. 3161. cm. 3.61, 571,

(32) 11. 1.- mn, 1. 1.11m, 11. K. mu, 1pm., m, 9, (191.3).
(33) 1({19 ghm’ r. r. em, a. a. 3mm, 801?. 61.1... m... .16. 1921.

(3).) v. 9. mm, J. Harem, emu cm. 15, 1.56, (1933).

(35) 9. am, a. M. 111.2011, 1. m. Haylor, m” 39,, 23, (191.3).
(36) J. Mambo, 1. 131mm, 361mm end 1.... (1......) 39, 232, (1951.).
(3?) a. 11. Wt, 9m cm. 19. 189. (1933).

(38) 3. Ken... 1. n. Mame, m" u, 237. (191.1).

(39') 5. W. We. 3)... 167. (391.7)-

(m) 9.. mm, 9. 3mm, my. 011... Am 3;, 286 (191.8).
(1.1) n. c. Gore, J. Auoo. omom up. then. m, 1.03, (1933).
(1.2) o. mm, m1. 360. am... 35, 1935, (1906).

75

(113) 6:. Go M R. A. Hem. m. J. 39, 1.51. (1936).
(1.1.) x. Wok, a. W... Moons... z. m, 231.. (191.2).
(1.5) €19fi31m' ,9. mm, J. hen, 11.11. 911.... Let: a. 11.65,

(1.6) H. T. m, J. 11. mar, J. Ohm. 366.15, 996, (1899).
um '1. a. mm... mm... 1. 1.95. (1952). .

(1.6) n. m, n. we, J. 9161. cm. 323, 1.69, (1956).

(1.9) K. Wk, 1‘. 1...... 31mm. 2. 33.1, 213, (191.2).

(59) 9. v1.1“, J. 9111.9, 9. More... J. em. soo.1293, (1953).

($1) 1). Franc 1!. I... Lavina, J. hm, 3.801‘bcr3, J. in. cm. 80c.
12. 171.6 1.950).

(51) 1. Km, 0mm. 11111., 11. 21.21, (1951).

(53) J. 11.1). Goalie, I. D. Ma: 0. T. W, 13. W, J. 91m.
3090: 697: (1958).

(51.) 8. mm, 1. m, J. 8191. Shem, m, 213,, (1950‘).
(55) J. u. 311:9, w. J. mm. mom. J. 51, 51.9 (1957).
(56) J. 1.. may, 9. m... J. Biol. 91...... m 1, (1957).
(57) 18.92;)W’ J. Leibmita.m11. no... emu ma. ,6, 1, 131,

(58) r. 0. m. J. Lamar, Bioehim. Biophye. late, 22, 1165, (1958).

(59) B. 2: mm J26, in 506mm 0: m Aam’ We D. W,
B. fleas, 608, (1951)).

(m) Ho 1!. m, E. N. M, Jo Am. cm. 5000 12’ 3M, (1955):

(61) 3.3 .mm ..v Glimwfi. ninetel, nakwmd.
Hunk- 85316. 567. (195h).0 . LE. 13755. (19511).

(62) Gertrude Pea-lune. Nature :35, 1.06, (1951.).
(63) u. 3. Wm, no. Prue. 15, 135, (1956).

76

(61.) 11. 2. War, 3% 11.. 2‘95 (1956.). 9.1. m. 191.69 (1956).
(65) 6. P. am, 11:16 Human, J. 1.... Ohm. Soc. 3, 501 (1958).

(66) 6. 1.. M, mocha. J. m, 367. (1.951).

(671 r. I. Am. J. meow. (191m). 115. 227.. (19581.

(68) r. x. mm... nu. 1.1.1. 5.1. u. s. 11., 162, (1953).

(69) 6. WJ. Knuth, J. n16. Chen. mg, 911. (1951.).

(7n) n. 1.. 1111. 2. 1.. m... J. 21.1. cm. £311.. 117. (1958).

(71) n. 1'. m, 2.11.. Smith, J. me. one. 139, 669 (1958).

(72) c. x. 11.111, 191.991.. 9.1.1.611, J. A... one... so... 11, 212, (191.5).
(73) 3. mm, a. Seat, 1. singer, J. 3161. cm. 111, 217 (1951).
(71.) n. a. 11mm, 1n. em. 3, 1292 (1956).

(75) D. E. 1mm, Mary Jane m, J. Am. Chem. Soc. H: 2657 (1957).

(76) E. K. sewer, R. We, 1.61m“. R. Drinko, 3. Horseman,
Mention Prom 15, 31.17 (1955). ' .

(77) J. A. W, 1. we, J. Am. am. fine. m, 1263 (1958).
(79) n. 2. 1m, 11. 3. m, J. 3161. cm. 131, 293 (1958).

(no) gigamm, a. 1. mm, Advance. in mm Chan. 11, no

(81) J. 1.. mm, B. 1. m, W11. 1191 (1952).

(62) 2. mm, um in meow Anemia mm. 1166., How rm,
1955, vol. II, p. 11.9.

(83) J. R. Kimal, n. L. smith, J. 13161. 811911. m 515 (1951.).

(811) I... 6. 81m, 0. K. Bola-m, W. W. Brunet, J. Am.- Chem. Soc. 12, 2805
(1957)- .

(85) H. Framkd-Gmnt, J. I. Innis... A. L. Levy, “Mods of Mechanical

Antigua" Wanna Publishers Inc... New York, 1955, Vol. III,
Po 7.

77

(86) K. WW5, Cold Spring number was. “118.3161. 11,
11:1 (1919)

(87) R. 11. 68113111 and H. B. Moo, "Win in Biennium W11"

(88) Helen K. W17, H. E. Sutton, Louise Gun- and J. S. Ben-7,611.11.
1m 1661 ... 25 (1951)

(89) 8. 116m, 1m. m, J. 8161. am. 116,, 367 (191.8).

(98') n. Moon, 1:. H. 88m, J. 3161. Chan. m, 908 (1951.).

(91.) 8. mm, 12. a. 81mm... 8. n. Stein, 1661.61»... 19, 1185 (1958).

(92) 8. 11661-6, w. 8.. 8m, J. Biol. cm. 111, 663 (1951).

(93) J. n. amna,‘nm 0. 0mm, Anal. Chem. 19, 3.0 (191.8).

(91.) n. J. 81661:, g; L. 1mm, G. 21mg; "1 m1 o: 1.661- Chrxom-
1; fl: 8133 par mactrophm-ema, Acudsnic Prue Inc., our Kerk,

(9S) 8. 8. 111m, 11. 11. 61m, 8. 86m, J. 8161. 01.61.43, 91.1 (19-51.).

(96) A. 11. Rm, 0. 1. 116111, cm cm. 35, 289 (1958).

(97) J. 2. 11m, J. 8161. 8m. 15, 1061 (1957).

(98) A. K. Bills, 11. Wm, J. 8161. 81m. m, 669 (191.8).

(99) A. V. 011mm 1!. mm, Oomph. rand. 81117.13)». 6111118138“,
326.011.16.21. 36. (1951.)

(100) E. L. Smith, Fodmtim 1:66.11, 8115 (1957).

(101) 1.8.1111, 1. 8.3lb108, Arch. 81666.... m: 816mg. 1., 868 (1955).
(102) H. 1.. mm. H. 1. 88661.11, Olin. 8m. 1, 391. (1956).

(103) 1. new, 6. 11m, 16.1.0116... m, 1291 (1958).

  
 

CHEMISI‘RY HERA“

 

 

Hill!

56 2668

030

1293

3

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