Preparation of egg albumin solutions and observations on their optical activity during heat denaturation
by Norman D Cascaden
A THESIS Submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree
of Master of Science in Chemistry at Montana State College
Montana State University
© Copyright by Norman D Cascaden (1950)
Abstract:
S.P.L. Sorensen's method for preparing crystalline egg albumin, or solutions thereof, has been modified
to permit a faster and more economical production of albumin solutions‘for a study of optical activity
changes during heat denaturation gap 9%
ea$ TpmgIB (8WW&- A G i & w r m B i m m & p
Sr ..
m m #
33+ w g a s #
. v
& ga#]&'
Submitted to the tiradnate B1
Seuitj
iii■' partial fulfillment of the requirements,
for the degree of
latter OfrSeienoe in BhemistrJ
#
loutant Stete Bollege
A^projed;
'#a^er
'
n, Graduate BiXleibu
Bozeman^. Wentana
1^0 -
Y
^
-2-
TABLE QF CONTENTS
Page
I.
ABSTRACT......................................
3
IL
INTRODUCTION....................................
k
III.
Historical and Literature Survey....... .........
I*
Statement of the Problem Involved.............
9
METHODS.......................................
10
Discussion....... ..........
17.
10
EXPERIMENTAL RESULTS AND PROCEDURES................. 17
A - Preliminary Studies.... .................... 17
B - Determination of 8/eights of Albumin in
Albumin Solutions........
22
C - Polariscopic Determinations.... .......
27
D - Table I
Series A............ ................. .
2U
Series B...................
2$
Table 2
Series A...........................
28
Series B...................
29
V.
DISCUSSION OF RESULTS.................
31
VI.
SUMMARi.............................
3k
VII. ACKNOWLEDGEMENTS......................
3$
VIII.
36
LITERATURE CITED........................
83220
Aigstraci
■Sf Pk-
g method: for preparing e%y#&!i$me egg ;
@O.WAl% or .-Solaiiew thereof^ has heett
petislS a faster-
and more eeowmieal'proittetioh' of alhttSiB'soltttlotts'for‘ "Stttdy of.Optiesl activity elWges daring heat -henstttraiiOB®
"'. '
Preparation of
Albumin Solutions sftd Observations on Their
Optical Activity During Seat Treatment
Introduction <mi Dloterlcai
Tslh&n. an egg is eoolcbd it# internal persistency changes and
what once was fluid is .after cooking quite solid#
the egg M s been'
coagulated*. 1'W.s phenomenon has been Imw n for thousands of
but it was only more recently that heat 'coagulation was recognised
as being one of .many possible ways' that a.protein eould. be changed '
chemically,
denaturation.
The term applied now to. thi.s. change in proteins is
.The. phenomenon of heat coagulation of proteins*
particularly the albumins of the egg and blood* has been the object
Of maty studies in the last seventy-five y e a r s . Denaturation is
now being studied intensively in all its aspects and especially so
with respect to the effect that denaturation has on the original
properties of the protein*
However*
in spite of .all attempts' to
learn what takes place when the white of an egg* which consists of
albumins and globulins* changes from the liquid, state to- insoluble •
Ooaguluis^ it still can be Said, that little is definitely known about
the actual chemical and physical mechanisms involved*
tk$. f#' well, known facts pertaining to beat coagulation are
given as follows%
I " The liquid pari of the egg or the white of the egg on
heating turns t© a solid state,
fhis" is the. primal fact of coagh^-
laiion.' % ''»■Sulfhydryl fvSi;)’groups*, which .are- @how;with tha .■
'
edtem
albumixn
appear !■$$ # A heat # W W W
iWg^.
T k # e groups &P& Aat erigi-pa^l^ p#s6at in, the natural,
-white of eggs*
'3 " If a solution ©f egg aroumin in a s.alt*fr#a'
medium is shifted f $ m its IsoeleeWid poiht %
the addition of; U d M
of- base and heated^, it -misias elear»,^: fhe@e are same of the -faets
that are htiowu about heat denaturatlou*
ISsi^r Ideas, have been
.■■
offered to. explain idiat o#urs during d@naturation> M t -they usually
are refuted hgr doffidohd- Wo- couid not
they w r h preSloatfdy
the results on w M & h '.
4h #**&#!» of this is. the ease of f/u and ifu^"
who suggested that tyrosine is eliminated, -iihes alWmin is coagulated
"by fiedt* hut lastin'•and Sees^ -c-euld not .confirm the finding.of Su
M d la*
.
'■. Wewrtheletdd some constructive work has been done in determjdi.ing wMhf # d # s then s$hwih is dd#twsd. %
Meatv though
disd#^e#ht is to he Aw&ad id. the wdrh: of #ildk
and .feurafh.^ M d 'Mao#erso#
@3^
two types, of linkages are ,affeoted %
It does support the ided. thath#at Mring -demturutidh$.
v
firsts the :p8 SehSitlws.* # M e o n M the $E iwensitiw linkages*
they state that th# pS sensitive linkages are Very possibly salt
linkages and result from, the &a#b%yl and-. Mine- groupings in- the
protein molecule,:
The ph insensitive linkages are declared to be '•
hydrogen bonds betwoM tdo adjacent peptide cliaiiis.. The MStam1
O tiM
of #aa$ boWa Igp t&& a^li&atioa of 'ha#
diepla&ai&eBt of t&a
peptide ohmlns in relation to one Mothorv ISdoper and WeuWth^' also
the
wty #a***
UwA
$»&#&#&. ef tb&
.
«Aa t W b * # L $ gf tke&rwad&f B&a#a5&
dbtW
#9- W W » &
'4km*6iWNA$9m i» aagr.
medit'iea&on ®£ Hjatk VXfitqptb
p#o&g±a*
®£ & W x W
%» W W L t e Qh*#g#8 la dbaalgal, #%&&&#&&& #&'.
Mo^gitial;
&$- W
W w ptot©to that w # w 4.
.litahg
Uwmg# #:',
W
iR##a#0& reactivity W
W w
euwMWh*'
the Ctwtittxent gpoBg##
<B@sget in meieonier # e p % tod •a Aqgg# in the hate &£
-
&yaz$$g#l8**
The tern ^ewpwteblyiinjir .as- # #
Senrath' ®$k
in. the- ieilniiien
# $ estimation *E the m S M & to& limit#
33% B W A m g # & h f # p # & t# # » # w y dW##se ip
#Mk*xxA»
aad
pab%w#& that %ay %e tb* # w # W 4# d e w W W A W k
-entirely
that- nn incipient
.
-. - '%
#»
fa t r w ,pwt#*-
'••
lytlo)' may W the nrigih of &enatnhati@&* #n@tnFatl@n in it* .
bwndeat &#& w#t.$BA@w. # # # %##&» @ w ##*@0! ± & * pwtd&a
ganping; #
t# h##*
BBtny&l, etate»
M s i&it#
n#3&W U w B B i A W k it b&A i& W
$!W9#'#b.o8a * w m * W&at
-
a
drbae&dbf %&# t##& W # 4 W i # # 4 # *n&y W
%g%gQkL<N%
to pa%WkB* oaae they have b e m iwl# # , .
% * h&gg*8t - B W A l W hlAek i# &»#$%% idbast #81## i& % &
'Me#
*otnel3y In
pmneea ie w
IgmwwB*.» 4 # #
% # tAWqF
* p#t#4
%M^«AA'B%yWd%6 %RaB
'
greatly ad^atieed !by the Wdrk Qf Bmll FlschQif W
M s students^- f0r
.it was their work with synthesis of polypeptides, that gate M a e to
their idee of peBddaW Ijboaseyaeis # Ishe Badaa linkages la ^fqteWv
^ieebeyfa # # l d @ QhMos fire,, stated to he omtpeeed of enaple aitW
aolde W W
;f
t&Wtkgh their WfbA^L * M gfW#a*lh&o gyw#b,- # & #
-###& a@@@» ao# to a%a# that %& addition to gept&de iiakagee oth#
W - M w a w w do g w A M y # W - la Ithe t W i b e W ^taia#* Se&otl# grMpe- a w thought to Aa#t ta proteim
M w b e l group IyfGBB),,. pheholie gro#
W W
sii#batl&-
# 9dp (*#)*.
shlfhydryi. (wSH).awd M s iAfMe &mup& { v W k ) m $ : F W M h B y otheys-«;
FMiewihg the adreaati&a: of pelypegtida o h & w as a
^rpteia linkage* other idea* fallowed, M t a# yet aaoe of them h #
keen Quite' able tm displace Fischer*# concept,,, ©ne veiy serious
defect id his. theory is that it. dees wot. attempt to explain why
IxrdftsqLewa ha#6 a.
soiuhility^ aspeoMlly when the sa%# aaoyat#
of the SOsieiPmino acids aye pyssent ip the protein molecule*
Buripg;'
the last 3'S years i#§y h # protein structures bat# been proposed*-'
AWeAMdsa
<&*,&*% mg^sts that proteips . W W l t of oyc&lG
<*U&%9*kltAyt4W&)iaj&dah9;&Lp*%g*B93b8af;pij;gMa W & t^ethee by W a A VS&4RO&
IM&r%%#dhfH0ad*%k;&8wa bm&g&t - W si
p#tg#» that.#
compos# of ti PdrMs of d M # u n a # t r W W M m g w *
^ s W % w . a w aAwagad.aa that f # -emy
!Bkwwatt ^oldl'
tMss
i
M a a s W aym%# # m e & t 6d # it*. W h thawiee hast W #
however, only with reservations=
'
'& regteie
ties
%%&
structure
&A# i?roieteie.
acMs* ■# $ #
s%o& acids Iiave be%
■$&.pretepMWio' {WfceaSv
e
Mt* •eatiy
proper*'
amM*
imm either proteMs
$#era%, f#WL& # -Sn tiptia
Seii- .
coc^
arid ishen two amine acids: 1
SSte. a dipeptMe. is formed
‘-/v-y-
<, gash dipeptide contains # #
and $ % groups
through WhiOh the peptide m # he IsagtheBed Mdefihiteiye
the' Imil SMpher ■peptide oohoept,.
this 4s
(#) -Wfher proteins- giv%- bn
alpha amino acids and hos^profsSh
for
Ohromoprots'i#- whioh pissws a -owceoplaore or. dolor ,group- af##.##
on the
moiooprot.'Sino. whidh. *#&#$& ',wreieio. *0-14 attadhdd
to a -protein,ji; M d phospho,protein f/feioh -yield. d^phOSphOM-e' aaM. M- • ■
the "BOB'/protsin gro#,.
of proWns MOludo ths -'
artificially systhosisod: ones' as well as- those which are deeompo^
sitlM products, of the 'first two Binds m W & s W f
It- is In this third
group that. sets, proteins^, mentioned- in this papery are fa >e .fbttisd*^ '
Egg albumin, the material used in this study^ may W
regarded
as -U peptide chain protein^ whole: constituent parts may form layers
in close physical prorifftify o % if no't;
^ UMtpd- ’
%• secoWary yaldhcd
forces'.or Bydrogen hri.sges»" #erefore^ t w #r more alhumM .moleoulei.
may he, held in place hy the Ubore- forces do that one may considerah- aihmih system.- as a giant meleehlo,#. IB-M-s- would proWhly he true
of albumin^ eepeoialiy in- OOneentrated Solufionsa,
-In the course of this Worlsi5 it was observed that 'if an,
alMmin Soltttion is- held at pH
to 6.H # lftsaluble protein
preeipltatigs feoa the sointioh^ 'this protein goes Ihtd Sdltttith
Ih .Sl slight, exoes's of either «tiid or Ms»* 3j W h W * Whh h
dialysed albumin ablution is neutrslieed almost to the point where■
•it fails to precipitate h h # heated^ a precipitate separatee .thich •
also-.'dissolves readily in -either aoid or- base# ■$% thie W-ork> this,
precipitate is referred to as albumin acid seta protein because
the properties of this precipitate are- like those of the- sold seta
proteinSo"®^
Statement of the Problem Involved
ihO: purpose of this wprk- was to- secure d suitable method
for
the preparation of -egg.albumin
solutions:
and at the same time;,
to prepare -a solution that was comparable in purity, with respect
tO:albumin and water, to ones obtained by the established methods
of past investlgat©rs>.
It was
hoped that any
egg albumin s o l # ! #
SO Obtained might bo suitable for optical activity studies.
Discussion of Methods
% e isolation of a specific protein is Of interest because ''
Of the unusual nature of proteins#
difficult substances to
A protein is one Of the more
and crystallise=
In the first p l # %
a protein fiiay not be heated without some change and thus cannot he
'hrystaHihei' .from a hot concentrated solution like some Pthefrorganic
cwpoundap 'Second!#^ e#u. though crystal's of proteins can, be dbW^ned^
they are invariably .hydrated .and # i n absolutely thy p a # ,into thi
&aoiph©ud State6
The first definite reforesces consulted by the author were '
by Harnacl^ and Osborae^ who mentioned that Hofmeister prepared '
g&g ediumu&iaaayatalline
SdbjLtgfiWbwS '#l#Od W
iatha
free egg.
a froth* W t Otmd for Bit hours with an eepa&'-
volume of saturated ammonium sulfate- and filtered to separate the •' '-'
globnlM^
The filtrate, whs allowed to sfaporute for seyeral days
-stt room temperature*
The- substance was recrystallioed s.eyeral time#- ■
from half-saturated'ammonium sulfate solution by evaporation^ but hot
to dryness* as before* until clear -crystals umixed with spheroids '.
Were obtained.
Harnack3'^ however, stated in several of his papers
published in Berichte Ber Deutschen OhemiSdhOh Oesellsdhaft that
Hofmeister never was able to obtain crystalline egg albumin from, the
dialyzed egg albumin solution#. HarnackA obtained, crystalline
.
- •''
> .
'
albumin by the following methods egg white was. treated, with e slight
eaneee of d&lt#e sol&trlea of acetic W d until all the (gloWiW
•
precipiMSeA*
fh© 8 Q # e # lo K o f
# s £iM&z®&* MS Site
f% 3.W te was saiS,m'Mi5©d # , # aa. ,weesS o f ct>M srbyrated sodiW
MaA^wwtw MoZtttioHit 0 # # $ # % # & $ & copper atiffat© S8&&fc4en:
suMed' and> i f the oo^per aS)ti®i’h $M SiS Hssfc" p rec ip itate oat at once
i a f s ia t gr&@B fZohS j Waww sodim c#A.#a$e Mo-ItttioH wad added.
It SWl
%«, pr&aipltatw w#6 B w % w # W Ba water SM,- dil##
'potaaeitiiB
w # a# $ s # t i % ail tiie pswipitate diesolw^
fhe- a % W » W M 'fepweSfitateiS. hy p M W w hehtraflaatloB, #Lth a#tie
W#U.
TNBWR ;#^atw. # A $ 1 1 % 8
■etiiaiheii*
M#&tgr W&P "
'
ttsed aeetCi M i S %&
.,SdpKitiM-
satwated aeitifidh of # w w #
■# % ;W y ^
W A t s aoltitioa 'Cihihg
•
BetfeodJ m & tlaiaieii t# h a w ^ieatly fWi3jL#&# tte
_ %r t M a seMs.# W m T stated they W M
alfewiB
'fWS ttysWlS ffSotit # # # s t W asd W
wMM M
Of1
a W Se Saewe-
$ W A U # Sttlfats crystals
pw&e&t*
■--■
aad:
ami
'fsusi> that, if tfe# wed
a c M instead of asetfe acid^ the s e # r a # m of tfee
, eiystal$. e o e w e d mere w W 3 y ami iaore- sewpleteiy*
dialysed to- Hemew
%
w^tratioh^
Soltttlese. W w ' .
befom the -erystade were allowed td f t #
•
•
.
,.' ■ the following method is that &t s/rensen as given- by Iiorht^v
'
■
.ft is one.of the .most comaoh methods, used for the preparation of pure-,
egg afbm-ltt solutions m € was the One used at the beginning of thl#-
work,
^Thoroughly
volums 0
the nhitos of
fresh oggs: with -W 'tighal
saturated sure, anmonium sulfate eolutieh# a M fliter off' -
the preol#t#%. of owglohulls W
eyeasu. Io the ,clear recMfSt-^eSoW;
tatarafed mmsx d m '
flitratej,..CWtWhed dto- a precipitating
Wlfate -eolatiou W t f l the slight
W i e h is iadichted-hy1
.a milky tiaW44ity>, ceases to dissolve a W heoom# peraaheut^
fhen
<td6 #s.S' S- sulfario $ 4 # W t i i the mixture has # hyirogea ion ooncemff&hien of |K
ihS $ @ M e # for oaystslliwtioh,
.Iurini tW- .
addition of the enifnric aoM' thh' pfeotpltafe is first .dissolve#
the color of the. solution changes from re#10h^y$llow to yellow^
and finally there is. formed an amorphous 'poeolpltstef- ahieh is -at
first-'easy> h # .Iator- more- difficult, to dissolve hy stirring,
#0%$ p#
Jb$ '
it should ho .g#t p0##K&# tO' diOsOly© thi-S •predipitstBw
-,itir the- rooulting' opaiosowt solution Tigorowlyi orysiallloation
■will W W X i y W g i n in an hour,
& S 1 W the
-Ih- stand at r o #
twgeratnro tw$® $ t o - s t l r r l % fraquwilyv
% e u fiitor-ovef1.
night on throe large gravity fwneisi -she,## -«% mother lifuon iwWke
. '
. "
' "-Vabove the ypreolpitato m # day# ##**# it -with a pipette-#, -Wash each
precipitate twice # t h .an-SMiohiua Shlfato solution of such 'cohOeli* •
t#8ti#R that the gweolg&te&ie#. gf $%e
-
&%#&#&&* $&%,
' ■
&tl#lag, ugh the
HWbi)# #%%&&&*
&wWi&gr
t W 'mt&ep
and. tbi&
-&& #**- '
Iermihe- the concentration of th®"'Wshihl solution, prepare.' a s # # s •'
IQdT twMKt thhGS* B&8&
'66^
#&#&%#&
WLW#
solution #d. a
TOlnme of distilled '^atefi to e # h add a
few oiilaio oent|$$tet@^s of the clear filtrate. ■ When, for
all
the thbes- up to end indlndins that containing -6»5 ee# of wwter show
Inrhidity9 while all those M o b t W * * ?-#«, dr more-of watef aye d l # %
it is evident that the eenoentratlon #
the sashing solution should
he iii the ratio of 10 ec-t of armonim Sulfafeisolution to f. #8* .#f
distilled watohy
.
•’
. . .
,
.
the second washing solution has filtered, oye# si^b%
remo# ndth a pipette any which, has net run through*
Sdrape the-' •'
prooipifata from the filter' ptpew into -% large, evaporating diah^
Uding a porcelain spoon.
Dissolve the precipitate remaining on i
So
%*p#r* with d3.stilled water and M d to the evaporating: dishi, then add
With.thorough' Stirring, just- enough Sd-re distilled water to dissolvethe precipitate completely* .If necessary# filter1the solution and •'.
pour it into the precipitating jar; again aid saturated ammonium
sulfate solution tq permanent turbidity, and adjust to a hydrogen-ion
concentration of pH U»5S by the addition of 6.2 ® sulfuric acid.
Stir the solution vigorously and allow to stand j or k days*.
Filter
the CzystaliL^ed egg albumin#- wash with an em&onii,# -sulfate solution
of the- proper concentration, and then dissolve the precipitate in',
distilled water.
Dialyse in a. large hardened collodion 'Mg until
-free from sulfates.
Early in the dialysis, .observe whether the-
•■ .''.collodion bag .has been sufficiently hardened,.
.
■
testing the # #
aiysate for the predenee of -albumin aoob'rding to the method of
'The p m d f i M
.alburaiii fjefebios xvlll gtfesnytm its "
pmpdPkMti fat- Siohlihs if m&mS With a Mvg®- ghaytW -
hf toMend.j,
md -kapt at- shout #%/& ■
®te®$ a#th0d# h&Y4 W w suggestei sitoee the'Wthod- of
S^reasoti #6» published W b the. principle h&& usually heeu the s'W&$
# h e # W w .eugga#tei *#4#g ebdlesi- .sulfate la place of # m w # w 'sulfate i-ba? the -ffceGipithMyg a@ex%.
Ia W s S ^ iutesl the Chtothlfhge •merely to shorten the time i% ••
sopa-i?atitog the pfyetallime egg & # # # # *
She glohulito;s
##-
ceutrifaged off instead pf w m o # % it by filtebitog?. Ihie SiBp' alone
h-sye'd hiit.-titnnh time and offWii.i ■
'
fhe method'- used in ‘this #%%#$ ezcept f w sli#i hliemtle#^
is the Shms ns that used '% d^besseh and
After a reilew of the methods suggested for sinking egg.
.albumin solutions ? a modification was decided on ns being the most '
Oonvenietot for the purposes- of this
% b w # # W @ method Whe tiiid
6 HijSTibgi4 of times Ptod--SuhpetoslOtoh of Crystalline albumin'WOrd Obthiileda.
fhe crystal
e s m M W d under •#
- # w long needles -#%%
yieible-$ ulsfo rosette# made up of toeedle^llke o^rsfals^
the p%^'8@&- of t w #
%r W p $ a W $
hb#ye%:#$ '
# l y eoWia&s- Of ogg PZMpwa&a* wareW%
'@!^o<kpo*W ehWLto w W l W
&t W O &@3&a&
Of
###&
■' "
■
'
. V-
referred to #- solutlew- of pure albumi# i» W W s • The- wdifled"
m#wd gafe- results
OOBipurabie to these obthitoed mitb .i^renniats
•■
V
■i-'
. 'Sb
&t W m g
w W W W: #»
rapre econ.OEieali-- •■
•Stft * W # W
60
#3 ■ :
,
is #
f
C
- ' •■■
f
#
M%?$& #tth SA 0.%%#,#!%$% 4^.sisswiit^d .#wWLm%
# W W S ' is
SbiSiEmgW'
W p # W A ta# gWWitt f^ote t M SA&W#*
&A
# W w » is the# # # e & #
th-migfe lSfgfe gravity ,funnels- using ,# coarse- filter paper*
tration t#fees
4 td % 3w W
stissl pdssiialy logger depeMing-'-0&
the-s^eunt uf w W b W m and precipitate .pr#se%t&
tt$W ysllw fSt'fSte
ughiu#
Wvterial
Fil­
the # k W # '&$ ,#
uttisss W p t SS % c#&d # 0%
sfaeuM: W
WkWs hy the- use of toluene*. She .
alhmlu id--'precipitated out of selutiou Wth an excess, solid
sulfate '(-%.$*% -grade is deslruhlej which.ia udiec! -to the uihwis
sdlUbidu mtil s-hesv white: precipitate- appears that is .p@*%#a*d& '#vlgereus- sti-frihg-e; ,IlldiffW-Wttie out Wer.night- in a W M g s N * * * '
'Sodiiug"dsttt to-0^0 ,appears td ehodura# crretailiuatidn # #
U greater percentage uf aihuaia is ShWttS* #
the -first
IlgatldU- under euch -couda'-tidhs# Ihe filtrate is treated -WitIr a,o-r#;- .;
solid ' s # W w sulfate to saturate# md.- allohed- to- settle-out upier--' • ■
the & m & douditidtts- am he##-*- A % @ # UlK- the Ulhumih^-hr$i:stall-lh#
put •the .second- tlise and; the filtrate,is- generally clear w M & teste#
-d8*h *
# % # a W a p W W t t * -% e
t&9@8' -Wttg ft l%ge'
SB f&lteW: TBfTC
-
W A W & P t t W&*8- ttWk'k 4.4B""
hdhre dep$adiug on the umonht of @dl#leA present*
lhe./hlhu#h,:
'i I h U i
'
,,
■
-
&b e, faint pink colezv
is, t#$ smaiiest
precipitate
of 4isti’
Xie<3:
i&iefc. fom # a ^#X%#r'
a # W W * - - S # then te. feprecipitated WitlS solid, aipioniwa Pnlfat# '
' %#, -
PjftHfSpHgk1GS %Ws#
t&w* %@!% th# #o%- • .•
■■ • ' W W r 'ths third preoipitatlos- the slhtmim, is #dlssol#!i . '•
.is tiis ifMst smosst ■of w a t w ■-* 6:0 eggs of ###**** -sise o»#iL%r
-Spteut',i'SS
'
'
liters of 4 high% #0!6ae;tttraisd, solntion at c e M room •
feiuens is seed as a preservative $ # t M solatisk-,
is': placed is dialysis tubes for dialysing^
$ M ottSlde 4ia$ysSig
Yiatei1'(:distilled vstdr) is changed every half-^hotir for the first
•five h&v3f0j S M then ev#ty Ii«•6 hours for & total of &L hears=* %#''
seiutiott is filtered^ neutwlisei with dilute sodium hydroside, (#oht'
.0,5 S) and again
dialyzed*
Xhe'weter used for- dinlyting ■%&&■ eeeen# and third time is'hw
.changed only -as often as indicated Dy using Saturated barium VhMrlde
solution es- an indiseter of' the.amount ef the sulfste going intw the
YfSters- ■fdually only four- or five ehn-nges ere needed, if & # liter"
'oomteiner is used for dialysing purposes*
% & solution IS diulysei''
at least three times before it is free enough of sulfete for the ■
purpose of this investigation*
—If*
Experimental Procedures and Eesnlts
Aw
• ■
Studies
After AA egg albumin snlwblen bad been prepared #id dialyaed '
until appmentiy- f r # f # $ '#%enii# sulfate and. suifnrie '##(% it
#*»- beaied in
W f t - W it would W
WlW#WA*
'WaePIwrsen #
%& WQf
-
suitable f w bpMbdl
*sl& W W K M m A
#&# W # - # # *
.
al, ^ bad .produced m c h clear M % W W # -aelutiens- »f-;..
eg#-AliwiR- VKbjboti W d pB'Wjwp*'of iS.# dad
c(%^a&e ,vhea.
a # * & W i fdiied 'td-'-
%&» d W # W a s W l e a n w . W W # # &$&#&'
and sitrntiW' paper and tke pB was noted in be between
and
Ibe solution was heated slowly to boiling, -and -kept at that ..temperature.
^for twenty M m t e s to enmre pmp.lwtk .eddgW^tibA^ =Lf
oobw*
#*@#& ,,-
-the ooagulnm wkinh formed was filtered o f f fhe filtrat#
4' fbis thesis ewolwei out of an observation made by Bv Ii dbhneo%
MbBtdm State OollQge3.while W Was trying to duplicate the heat
denatnfition properties of egg .albumin, as teoribed by ©r» %
Qorther^^
1»-: •
fhe. particular property involved, was the reported, heat.
sensitiaatiOB of ad Pgg albumin Bplntloa. whd.ch bad been bo ,tiiorpdghly
dialysed, that, all eieetfoayieg had W e A removed* Such a, eoiutioh # ''
thib lb reported to e#lbit heat, seneititation {the- first step -Mt
heat
.eoagulatldh) but, not the oufditig' or the IloeifLatiob * W H &
IbWevers- it is Stated #
the. same reference, that when the heat
sensitised egg .albumin, soibhich is- treated with an OlOotrolyte3 I of
eMained.qxi tSearbm^ni
$ f w dsbps of suHuipie a e M (approximately.
B) a.n<i he$t gatre rise Se w
farther edaguiationi,
# #
that- tte eoagalatloti'in the first treatment had been complete. ' '.Msitienal ptntioBS' of. the above
.eolations wene then
neirbraliae'd vdth sikllvjn hydnoxl#.; the pH being renghly folieWW with
both iltmne and nltraaine papers*- fh Oome instances * the Beckman pH
.WWf- A * **#&-%»
# # #!&#&. g# at tdsjkdb the
#%# lhifeh to .appear -on
tkwe
',
■‘
'•
# # W w # Af # W W k -
shlfurie- m M , h e M hy the
;
the pli* This swplelWn##'" -
j&km the feat that efW" speudk^ysidhBdstidMaiRdkblk
W.
'digitsie> ooagulatlm eeennred a loser pH i
Valueh which tended tp
.e^ajspie# sodium OhlQrMe9- the floculation reaction is initiated®. ■
' .
Wtile- lohneen see .#&%%- interested Ih determinihg whether heat
ssitsitiphtioh-dihsed h PhaJige in 'optical eptivit^. (the #as' # # e # of this change should, he quitp possihie M t h a, dlhar Sensitised
h i b W M selutionl he. made. # e obhewation that it Was Mpohsih-ie $&■. •
dialyse, an, egg alhmih tolntioh to- s w h & point that it sonld fail
to 'giw. the ordinary eoaghiat-ioh reaotiofts...Because these tes-hltS
■seemed to eohtraciiot- the above whtiomed contehtiOhS in © o r t W h M
%(#i t W
.$e## *#» W A W d i W
t* h# W W t e ^ y
aoilL -ft # # then found that tope^^d & W # a i & # d hot i##'- th#. '
m
|ios£?ibIe ,Iistiii1
Bg v@&%e%
W # more Mmey-
WX d W y # * * * #@ im $& ilts $S . #
ihe-; ie i t s ie ii v #
$*%*#*, W W l d jin#
#0##*,
followed hf $ i W W
# $ # aWsge &a #%# - ' '
papers,
,AiMiittiiy is W&f& ie Mierfeyy- %&Wk Mai
AAotay # #
Ibwae*;
#
W a A * #a%ya%* 4 A W » W W * *f egg %&&#&# & # # *%*$4&aiHdbQ*&Bdaa<&* '^bwwey Mmw &gf#3x>3gr63b&# BSt M
# # # * ‘ ike i&eQLi A -Gtwa
iBBsgfipdMFGB** # A # c W B $ w A d&#o
1
.QitBiiSM ere =MdeiMabie; Ses
.
a# »& & W 1 w A # # A W W ' '
ty #^A 4 W t yiii-.#3& tWab
might be # # $ # $ # as the- cause of
w y MatngM ih optical aeM#&y that mifbi e e e W 4 W M S M'fti-
Ii #a# obseryed tObadk* # # a a #%&%#&. egg. a%&#da
a o M i ^ %g%pqm8<dLQ&Kgy'* IKtws a M M i a M M i l M W & # 8% ae#M$iged: 'ib'.
^ # m w #&&& *##a# kgrdu&BOB&da W t
M^rMghIy agaim
TW
%#0k ci1 s peMtMe' i e # i W W A # W i 4 M 1# the dialyslW # * # # # '
t # m 6e proof % M i the d W y # $ # # w cempitete* 'Ihe .wlu-tMa #.s:
difQyped* M d iiten. %##%#*$ W i th e %#e& w a g W M tm ■
% b # M w e #Axl&$ Ai M y # # Q d ask Af a* 3&tt&a W # e
•. ,
'
# % W # d Aay 8itK%y&#g the .aptMal MAWNQF Af *%# d@#WMad egg
d#u6l%
%uii# t y SfBldMby M b m # # h W * d $, BeMraiieed-Seluiim
W -egg .alb w itt # # iheu^ht the- s e iu ilo a 'iWas- M-sted i # W # # # %-
odagBl&a. a#####*. X&odj&Bdb&osgp&xp
*##* #%& #B W #
Wl
after repeated dislysis failed to- coagulate #% Iteatiags A arngiilw/i'.
WQliaiebKByeTbe eMalaed cm a # i % a fear dree* a# said (api^rWjmately .
*# "Nb** &<%&&
SKbadf e#g9&&w
iaa 86*8 #f t&e earn aa&4
rdetilte
t h # #* # 3 W A # W # m W a ie produced <b*&% # #
'ha#
V_
ekaa*** # & t W - W#.dW^B%b*dw! #4# twsitwr ##.'"
pW: # # # e
.
in t&a 41W & s@&#am. %&*a#.. -.
d%t#apily # # Sn
. -a% %#& # w
'to#
SStoli iaetepPdtelme* .
t o w t w a#;, &iw*i& t o w n # ito t o t o W i t o w w -
aW&$y # a & M W toWSo#
' d W % .toe
]
!GixbqfLozi t o # &5WL1 ^ w t o S * hmto to)wAtoW&r
.##. such solution Tfas found to coagulate ai PM 9oB> tMs m # Saird
t o # w , i & & # # # # & & at# 69-#mo#.#. euM&te and w d & m 9#Sste:
..{both suspected to he present It small amounts) Srlaich might tet. #
'
h e # eoagutotid# W f W S Q g a$eHte* $her#ihre* fit amaa SltoSy to#-.alkali Ridtapratoia formation might dce.w # « V # too# the is#
electPih ppiat of alhmia W
lt' . . .,, .. ■ . , ..|.-‘..: r - - . |-.- , ’■•■
that sway.W i u # ta ohagu&dW # # & W
:^._.._L.^_^.._,.:i.:.,.L
^ k*,',,','I,,,, ,.,
the mmeridal values* ttough #@11* ware- mesurable and .si#ificaat..
t®$U:
A t o w W * fladiagS disagreed with the statemeats. in
Inasmuch aa. s o # w
#»# added to neutrality^ an electrolyte'
'sras' present 'nevertheless'w
c<sguiatldh tdok places Se also f W #
that Aeld-toto&tW; # # # # # toiaW*%toA to toa&lag*
'
r ■ ■
.
W a
to to
.
opposition to HeuraWirS= m x & ,recent statement, that alight departures .
fr# the tototodtoto
# #g'#tototo itoWto wegoto##*-
^•21“*
'dy.e to this cause' alonet
"
Since It w.a§ # ought that the duration o£ heating sight be %
factor involved In the- failure of #gg aibnmia solutions to coagulate^
a properly prepared, albumin solution was- hosted for one and One half
hours at the temperature' -of boiling water (i?£dG at i$S'0O feet die*
vation) Without a15/ perceptible coagulation being, noticed*
Bolari-
scopic tests said op. this solution were no different numerically than
those obtsdned after'- a heatihg period of twenty Binute s*
■ ■
It wan !Botedg-. tdo^ that highly concentrated egg: albumin
8'<gLnt&a# sfBagaukffwi
##t&&5p thi^ #@'4** #
to heat #agul#ie- than
m*W# #
being
WL-
therefore to he coagulated# or to the.1absence pf certain coagulating'
Soils# #
per SortBor (m ® footnote 1# wan not investigated*:
" 4 final, .observation Was-, that though albrnlh eoXutiens were
adequately prdtected from bacterial action by the use of toluene and
kept in the-' chid,# a Siight precipitate formed gradually which w&s
GbnSr
M erei to be probably a- Betaprotein because of its-.-SoXnbility in acids and alkalis#.identity* ■
% work was- done to ascertain its ewact
S *
of
■A AS##
ss%iw#ti% t M SQBbSAtMtisn ȣ egg albrni# '.
present i& A specific volnnte pt
# 4eiswte6.tbe # # # of a&bmtin M
w s M xiseds
^
1% albumin solntidns
viA des*@4 .&&###$%
tm abpwaeties
the weight' 4f tietidtiired Aibnsiln- tree dete^ained
and seoondg this same Weight was estimated by the
.
Method#' 9S&8 £&#s& 8#t&@d # 1 1 be- sailed the d%y
raethdd m $ the SecoM3 the Kjeldahl weight method.
for the d # Wkgkk d W * W L # i % # # # ial* s'attples .of s #
SCibdBtln soistid&s
wM»
'Beostxse hwst dddgtxl^tios # thss#
nhtwdiei nMilxitei, Sblubieas $**# ii'ss to ada^Siltirubis flees,^ .
WMeh. W d M W #
Sentriftisutioa#.
%##}
,ixxsupsbl© of %@iB$#@d&8*atd& W # W « 3y Isr
%%. #£.$ ^ W # g W & sullue #. gp, of Ms# pe#-'
TBBGMB W l W -&B A.j3A0MlWk*i % W $». 68#. '0# #31*
.-
of slbtiftin. solution h#fbr# beutiug,'*.'M-Strutiaa sf the floopuim wsb fdtuad impassible but a#u#l4
-#.&#& **p4*b. £6* 4bOk%aia@a&wa%&;y8xB#j&GK% a](#@s&
(MGdBbw -
.meututie.% -Wd h e n # this- frooddurs wss follow# in getting the 8%$
weight of albumin..
$U Mmdve #d. sodl# ShloiWs^- the s#d###@d fldos "Wits
'Suspended ;in distilled mbep uftiu the rdmowsl of the suphiwbunfe
halubienv #wy were -seSasntdd a s o # # time #d#r thu #%#
'■■■,- - =
ditlons*
'
QnA huhdnad fifty m%. df distilled water W r e ns0^ in
making the above suspension.,
About IQ- si* of sndimentud UlbWain=
-
*0*
■sini water Were left
eatih tiae when remwtzig the ehpe^hataz#
W & q t & w * ' M Q&$®3M%QSi of the eohhehtr#i9h ef N # 1 rewaSei '
that # e amount ef remaining: Iladi %a& negligible &
seilsieniei fleas were, t^mferye# lusntitetiwlj ta -■
weighed
liehee nni geWltted te -4 % ; first. #
tempe^twe^- # i then"# wsWant. weight a& 33#^P*
'Were then ehttl## W
difference*
$ Iw
% e i%' weights.
■
f W the SgeliWl Method ef
tW' weight W alhWlhi
# the egg, ##**&# eeiniitn.wre % # & % # W e.#p!e .wee''
.
aaW&'
Id
0&ai& ef
(*# g # ( i W W w & 5wa3d%Begb& # « w * 4
,
Sn|S6|y, I- gra®- -of e # # r W £ i % -nni the eenteate ef W e h
eWile were ilgeetei f w ninety mtohtee er rsitii the digest Wsatnd
d--'transparent green w S w #
1Iaen # e nto:# digest. W L eealeif 3 # m U
of iistilled water and 1 # ml* of aatsrated^ aarbenati^ffsk Sodirb hydroKd# solution # # e aided ewtiotiely to the flask # & plated -#
the SjIeMahi app&rhtns* Wi M h -pretiwslff had, been readied for iistito
litM n v
tte flash' was shaken rl#rpwiy to start the W W t l o h and.
i
then heated until the WsfrM- amount M d distilled over, fh® acid ■
id the W d d M M g f l w k was. then ha#*tltr#e# 'to determine -the.,
m # o # a present- in the #&#!$*.- from the * # W a dldtlltod '**#%% t M
■pro-teln wad natonM#d^ by smltikying the.- nitrogen W i n e by #*-'$&
the ^Wpilation @ W m in f # M 1>;Serie®. i W d B' gires the weights
of albumin, in 65 -m3,* of # -alWain oolnti# ns Wbsined %
iwthodd*'
'
both- .■
TABIjE &•«*•Series Jk
t>y Biylng aaS
Sample El* HlhmiB
Solution
Weight SI.: HlbBsih
BacIc 6eiciiSats<i. Weight,
Hlhmin
^Solution.. Titration .. H l h m itt. . ....
- Gkl^l
i.
HJeMahl IfelhoM
3?^
;s" ’‘
9t3279
.3'
'
&
'
.3
Hvg..Seisaality of aeifM0.29f5
HeiSiality of Base-S0Ilihf
SMhfc Titrations Aye.
Iitrogen-PifOtein f'acter-6.38
lndleator$•Methyl ©range'
37,%
^ @ 3
lost
iW96
.. ...„
Table. I -
B
Albmaa by Bryiag and by KjeMabl 'Metbods'
Sample
..
* Albumin El0.Back .Calculated !eight
St?_ Albmia -Bry Weight Ml*
Solution , Titration . ...Albmin
., Albmia
Solution
.1
2$
■2-
'S'
>
. gg
25
.
0.33^5
k
.
lost
5'
4hrg*’
V
9#6T
Normality of .base.~0»099?
'. Bitrggea^rBOteia factor-6.38
• Iaaioator-. %e%%l orange.
25
•
m e
-
m #
“
m ?
-
m.#
-
10,8
-
map
w i #
.
-
IieldSil # # # ware mada #
(T, +• Tz.| S $r
gAmgr #% p W # m
T, ■»■ ml* of BaQti iia back titTaMag M W k test
Tz # mli cf BaQBt' 4& back titrating
M »• normality ©f IaQi
O.Oli # grama M nitrbgea. per ml.-, of I B soXation of BEL
6*38 "' .nibrogen^-proteiEt noversion factor
The differetted to, the results ussng heth methods j W the
©| t w
of albumin i$i definite volumes of
SdlotiOOO oppeayed to he due to experimental difficulties,
dayM##i^ht ,deteymioatiep* 8ev@#a& pBd&idm#' eppe&ped*
la the .
%
#6 speed was fouad! that sedimented all the ' # p # u m d ditmi#
%e .
supernatant liquid, as a result, In spite Of all preoautione.;,. earriei
off pome of the deahtured protein^
Secauso the olhmlh #a# deimfeured.
is. a different container f r # the one in which it Ifhs centrifuged,
looses occurred unavoidably in the trdhsfoy 'ddSpito groht care*
In
the ease of the KSoMthl method*-the use Of methyl orange ia-ii not
lend itself to M g h precision in- hack^titrating,. However, the yoS'ulW
p h # o # M iti IahM tp Berios A A M #
Obthihdd by uae -of t M
IndicMor .(hyoHi^eresol green -and Bettgrl yoi) irhlph afforded
high precision in obtaining .pnd points,. M l aspects -considered# the
Kjeldahl method Was probably the more accurate of the twb*.
#
'/-
PolariScopic Sewrrninations.
Solutions of egg albumin were dialyzed at least throe times '
-for a period of tmntyvfour hourn -#d .also hdutralitod betwodn # # '
''
dialypis before they were used for- polari.sdbpio d#e##.#tld%
These egg alburrdn solutions were tested, with litmus and nitrazihepapers to determine their -approximate acidity asel also amples of ■
them- were heated in boiling water to test their ability to coagulate*
;
.
%#nty*Mve milliliter- portions were, transferred to- 0$ ml, volmotrid
flasks and- filled to the mark, with distilled water*
Other portions ■ .
Optical. Isiivity Qhangig- During Heat Denataratioh '
-;
'
Eotatlo# Observed in. Samples Rotation -Oosaphted in Relation to
of the Same Solution
.. One Oram Rased on Weights #tained
Sample !CL; Base Hsed Gale, Wte Run Reid I'leu. Men. Sol*
Acid ■' Men.= leu,, Sol.
...No,: .in..Bnekijtr*.: Sample
Ho* Sol*
0,1223
I -~0d^
i
t
.
'
• 2
.-
'
■! ' ' -
"
.5 .
Sol, and. Heat -
0;%32
6
&.3ge%
Sol.
Sol,
1..Q
I
he-OQ -LoB 1
-OoB -
-IoO
■z
.OB -ItdOS
-1*0
3
-lidOB
%
.-Ii.,08- ■-LoS
.-SdlS
,B- —t-o.oB —LOB
-8,11
S
formality of addr-OiOpSP
.
.Monaaliby of base*0* Q993
litregda^irdteiii faetoa^BilS
ihdieator-#ixed (hrom-cresol green and methyl red) - t o
scale divisions '
.
—8.16
-8.18
.Blank — BOi.2
'--Rotation
and Heat
'
•3 w0*B —o»B
$
Io.-
'
^ Beriae B
Optical
Sesl Sssata^aM®a
■■&otatie##a@reed is SsBpleS SolaMan. ©cwiputed'
Selatiea ■%$•
' of the Sim®- Solation- _,,, , # m Gram Based e# Weigllts Obtainsd
Sample Ei*.dase- Ssad Oald«. Wtie
Acid Sea, rnn. S a %
,Acid -' SeUtt. ' lieu. .Sol,
^nl« Sol, and Heat.
.■ in Back Yiir
SSls.
Bk
I
^,@9
%
39*18 .
$
39.^6 .
&0#8
k
39^8 .
% # #
$
39*#
■6
.
.
OL##
39.^ .
IfmwlitJ-of aPid’-0»Q9l9:
formality ef Mse^O^S^J
Bitragen^-ProteiB. factor-6,38
s-Botsbien is scale m:,?islPns
’I
-Oe 9
—0*9 •
^1.8
%,
.S
-Oe 9
”0»5 •
—1»0
S
•3
-0.9 — 0.3
-IeS
3 ' -3tt..o6 —9.o6.
&.
■S
■- 10.12
-10.16
—9o.08 — 3o:08 .' ^10,16
-10.11
—9» 08 -3o08.. -IOttli
-30*.
spirit'lon wtire treated with, sodiusi. h y d r o # # (about 0>5
Qt
until points w r e .^eaehed at Wld h -they Swt- failed to coagulate ■
heating.
Samples, ware, then measured oat ant neutralised .in
the saftie manner as ab.6ve>
Half of these samples W re placed in
Watery heated to Wilihg and ^spt at this, temperature for SO to 30
minutes*
-Shis
was- .found by-tests to W
coagulate % W Sgg albumin completely.
cooled
to room
temperature,
euffieieht
to
All heated solutions were .
the various Saznplesj, i.e, the imneutral^
Ised diluted solutions, the solution Which dust fail to coagulate oh
heating, and the heated neutralized diluted solution were -placed" ina
cm* polariscepe- tube and the readings taken on -a Soleil^fentsW
saccharimettiri, A- series of readings was always taken on each sample
but little or no varlation#i#,ed between readings,thig paper are g l m m In
All readings,
scale divisions.
in
T?31Dis cvission Cf the Hesuifes
-Sithesgh the
$@#e'
ef. heat, on
* * be 4«®e W # # e *
ei^s* W t a IWbjBb #g#ww&
is
lisa been.
u h #r###im g W iii
*#
Tdaazt &
* Those i n v e s t » r h o
g^AWW b #0tW si# af
W # a W » sW% that # &
W the. $ **W A ih t# SLagmewM* # f
d##b**blW*.
to. #% ■ ■.,
W *& WwgwA:. *#@# xwf
W # y * q A g*w#Wi- a # < W # g . #.
btw#
(Sbea- Iby tb&a WWA)4% # * # # %&%& # a e#*kw
vw w #»
% * W ^ # # $ W R W $ ^ bfea# 3(t # i # proWhl# th&t
a W W p i f $ W # W # ^ 1 a w g W W A y jW**4 # W g W # - t W g # *
#
some $ s # M e
ofeh# l W ^ # a is -#*a
oonvsapw'iy thy (otmafeiBS of as# i W A % e s mey 3 # # to # h # h r # 4 m * . -.
B' was not the- intention in this, wo# few- Study the che^istlf,.
.W RasWtAhatawbA^hf W k.W *##11##. t h # Aybkw^ a c tiv ity A# . modliRW
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now '# speeninte # to what m # ebaogsy MgWk al@e#» Seew e a i l . '
AAWr A b W 0 # » g%WW # * OjgtiChliy A e tW * p%#@W < * * W *
a^mnsteie AWwm '###-»,
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-exy $ ^ w W # % # % % # » w W M * A*. W
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W W *#
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I a sib alite soitSioh a p # # le n iiy to- thb he-atedenatusst ion, f f w W
.
Sueh changes could pospibly be allocated to a lk a li metaprotein
#om atid%
# i th e .a c id side' o f pH f i t io -ssposoiW' to h e a t* - ■ ■
■don.atiy?ste .a M .obtain a .system which is s u ffic ie n tly eiiea*1 to studypoSArisodpiaallyf 'Bat at pH ?j or a&ightly above* it Se posolhlo;
to f W L -&.paW-.-aW# # »
and
'IbswpeBgr AAl#'to
a& t W * ^ w W -
eafe to o t t w # to dmoaat### a w # ;
I n optical: a c tiv ity -caused % o t accompwihg Heat^denaturatibn-*
#
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thm fofmtion # w * p h p W m l W % g $ 0 & o m -noting W n o m d
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f
-
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#w W 3y
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p o lW o b o # n . p ( # ^ 'l» .id # o # W W e t W
racW h a tio n
an <f%ual
at- the p w W
and love
n it h ipmspo# to-
e partiou l& r m##o W # , 3* doe$ net W $ 8 » * t W . the' c h a n ^ -1#'
Saeeooariiy duo to .this <#*»**. She f i n a l e ffe c t* t n the- ess# o f
r W W e & t W ^ may W- * # * W o or mow W *iw % W t
' '
thewe A i n
be & ch#go #RQe# in-on# th e o re tic a iiy peag&ble W tance^.
W tW
chance that- As# groups # f both Iev o and desfcrct character o f atich.'- .
w w & W
o p tic a l
*w W # b W : W W
Wwg
&e p m o t W ! # W l*'
to
fo r W
' ' . .'
ia the # @ W W i
roferpbd to db#&* -but- such a eW.ee bf changes ewid* Of oouh#*- '-.
JfitOt
detected
optical *ethodd».' '.My etaiig© actually HOted could,
tleyaisre Pa-due- to racemiaation, one Y.iiich would, he truly of a .•
heatM©uat\u?atioS' &iud- and not q# -due-to alkali, denaturailon- |l*dv.v.'‘
Barker’s raeOffiiaation). !'he optical change' could equally well, of
course,- fee-due to hydration or dehydrating^ or to-an actual IntewChdnge-'of groupfe.. Whether it- could fee-due to any unraveling- of the •
globular protein^ or to assaoiation. fey virtue of oeootidury valence ■
forced Id highly problematical*
The success of any attempt to -isolate and identi# #y heat
facemissed amino acids .if rdoemitaiion is -the cause- of the result
noted. Is indeed remote, unless one were to find a protein in-#lCh
Such changes are fdr more numerous than are now -suspected, to Occur
in -dehatuWbioti. {& small effect, it may fee holed,- lb not necessarily
U measure of the -extent of the reaction), fhs isolation Cf the
enantiomorph of thfe opposite sign to the one originally1present Mthe-u-ndenaturateA protein would fee more profeafelo-if hoat^demturatio-n
brought ufeout # iuterohange- of #y ievo ,groups.about a& asymmetrlo ,
carbon atom,
'
Ip- conclusion 0# fee drawn at this stage of the study Ub to
what cheatica.1 m pfeysiOUl change caused the change in optical
activity during heat-tienaturution,-
•
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.#a%#
; '■' ’'
W hi# j#AWA%
<t£ pg&jgWhig Htese M W W W # : ■ .
■
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•
and'.weighing of dried, sampies s £ '# d e ifie ' Voimes o f i t e aihimiW:
soluhion®
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•
."
.
ip hhsm' that' peapippptiop *y test. W
’ ■ ' ■.
■.••’
.■'
Acteov&edgemettts
.. the author wiahea to take this opportunity to express M a
sincere appreciatlen. am* thanks tO' Srf Mrg e r -t* Johnson ten? h # .
'pegmazl gni4nne© ahi inspiration haring- this work* also to the •
,,,Other members of the Chemistry Department ■&£ Montana. SthtS College;
for their very helpful suggestions tencnrning. this work?. Be also
wishes to. os#rsss his apprsolation to MiM- Boris I.,- Wilson of tte •
Hhraty-'■staff' for h,sr yaJrFhla-- -as-sistunoo^
I*
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