The enzymic activity of egg-white. Its bearing on the problem of watery-whites

Onderstepoort Journal of Veterinary Science and Animal Industry, Fol1tme 5, Nu·mber 1, htly, 193f';.

The Enzymic Activity of Egg-White.

Its Bearing on the Problem of W atery-Whites

By

'E. VAN MANEK, B.Sc. Agric. (S.A.),

~I.Sc.

Agric. (Comell), Poultry Uesearch Officer, Onderstepoort, and

CLAUDE HIMIKGTON, :M.A., Ph.D., B.Sc., A.I.C., Resea rch Fellow under the Empire Marketing Board.

ONE o£ the hypotheses advanced as an

explanation of "

,,·atery- w·hiteness " in eggs is that there is present in the allnunen of

such

eggs enzymes of proteolytic nature, by the action of which the protein 1-ecome,; disintegrated, thereby decreasing in viscosity.

]~xperiments

designed to test this hypothesis were commenced as far back as the spring of 193:3, but the results then obtained were considered insuffi(·ient to suhshmtiate the view

,

and the

expe

riments were discontinued.

A puhlication appeared recently howeYer by A. K. Halls and T. J,. S"·enoon of the Bureau of Chemistry and Soils, \V.ashington D.C. (19:34), in which the claim was made that a proteolytic enzy me is present in egg-white, parlicularly in thick albumen, and that

" the disappearance of thick wbite from eggs in

storage is due to

a slow proteolysis catalysed by t ry pi.ic proteinase " .

In v

iew of the fact that, the

methods employ ed by these a uthors appeared in our opinion to be open to crit

i

cism

,

it ,yas d

ec-ided to

investigate the problem using a suitable technique.

In the first place, a repetition of the

\Villst

iitter titration m

ethod

employed by Balls and Swenson convinced u

s

that the aeeuracy is insufficient for the detection of such small

changes as wer

e

to

h

e

expected. In no instan

ce could increases

0£ the order ohi;;cr

ved

by the American workers he recordecl; the end-points were neyer particularly sharp in such large Yolumes

.

Attempts were made to increase the accuraey by titrating to a definite pH st anda rd in a comparator instead of " to a faint but true blue which required a little practice to recognise " but without much suceess.

The choice of the above method for the purpose of the investig

a

- tion would

seem

moreover to have been singularly unfortunate.

Recognising, as it does, any increase i

n

carboxyl groups, it would fail

t

o distinguish between lipolytic and proteolytic activity . The presence of an

a

ctive lipase in both egg

y

olk and egg white has been demonstrated by Koga (1923) anwng others.

329

ENZYMIC ACTIVITY Ol' EGG-Wlll'IE.

Any attempt to

detect the

presence of small Cjluniities of

proteo-

lytic enzymes (proteases, ereptases, etc.)

,

in egg albumen demands the employment of a sensitive micro-method

wh~rh

is specific foi· the determination of p eptide cleavag·e. Of

the

various methods available, the

well-lmowu

formol t.itration technique appeareu to us to he the most. suitable. Using

de

cinormal carbonate-free sodium

h

ydroxi de, contained in au automatic micro-1mrette graduate(l in twentieth s of a cubic centimeter, and titrating

to

a standard

pH value

(pH

8 ·4)

with the

help

of a Cole-Onslow

comparator,

no difficulty was experienced in attaining an accuracy of ±

0 · 02 c.c.

This is c:leady

eY

ident from the

protoeo1s re

('orded.

·

ExrEH.IMEXTAL.

Having

in our

previou

s experiments (not recorded) employed only the one substrate geln.t.in, -which is hequenily used

in

the

determination

of tryptic activity, and obtained negative results, it was decided to exte

nd the scope

of the investigation by including sueh substrates as easein and peptone and by working over a wide pH range, any erepsin-like fermen

t.

would l1e expeded to attack casein and peptone but not gelatin.

K o

search

was

made for lipolytic

Ol'

other types of en;r,yme.

:1lbumen solntions.

Various

numbers

of eggs

were

used as recorded, either new lai<l (1 hour old), fresh or old

from

white Leg·horn pullets, intensively housed and all

fed

on a graiu and mash ration. 'l'he albumen "

·as

separated into the two portions, thick and thin by the sieve methofi of Holst and Almquist (1931). 3 c.e. portions of eith er fmdion were diluted to

1() C.e.

With Water ann aJJmYed i.O Si.and for

:3()

minuteS

:d

35°.

In the majority of these experiments, the precipitated mucin "

·as

centrifuged oft

after

shaking

the

diluted solution with glass heads, thereby aYoicling the difficulty of removing a repreile11tative samplt>

of the digestion mixture by the pipette, and exeluding the possibility of precipitated particles adsorbing the indicator.

It is noteworthy that the proportion of thick to

t

hiu albumen did not appear to he

markedly

cliffe1en( iu normal ancl so-called water, Y

-

white eggs, except those which were very stale,

leading

us to su spect that

physical

variations in organisation of the

profein

phase may he intimatel.v bound

up

with the phenomenon of

\Yatery-"-

hitenPSI'.

Su hstrate.~.

Two and 5 per c-ent. casein solutions were prepm·ed in

t

he u

sual

way b,Y grimling the protein with the requisite amount of alk ali , ancl

diluting to volume. The pH

was adjusted

if necessa.r,

v to 8·4.

Two pPr cent. gelatin

wac; prepared

l1y dissoh-ing

comn1 "l'cial

gelat.in in

warm

water

(U0°) and

adjuRtinf;

the

pH.

2

}Jer cent ..

pepi.oue ·was similarly prepare(l

hom Parke, DaYi;;

& Co., " Tin.rterologie

PerJtone ".

A :few flrops of toluol were added and

P~1ch

stoppered solution

kept in the

ice

box when not. in

use

Huffers.

A rang-e. of pho~pbatp :llld

ur

horatp buffer~ 1\"Pl'P prepan·d h OJll

('lark and L11b::; tables; ihe ::;olutions ,,·ere not. ho11·en•r, diluted a,; a high buftPrtttg JIOII·er "'<lc' olhin"d. J11 the <·mnp;tr:l!or n honile hui-fpr of ]dl N·4 was Plltploy .. <l.

l'i·.IJJisin .-.\11 :l<"ti n• il·_q1:.;i 11 -;ulntion "·;IS pr<>p:irHl hotn ptp;,;

ji<IIHTP;i,; <I("<"<JJ"<fing to ('o]t• (l!l:_!(j). Jj~ <l<"tiYif.\· ^\\·a,; i<•-;tt•d ll]IOII t'<l't'ill ht>t' <-out rol t'"\jiPritllt'IIJ :'\o. --1) .

. .\ gly<'<'l•d t'"\lr;~<·t ol p:i!'<TP:~:; \\.,., ,tJ,o prt"']l<•red :111d 1 Jti, n_qbinogt•n 11tili'<'<l io dtPck i lw ::<"iiYii~ of tlte eni<•J·okina:.;e prep:lr[l- tiott ical<-ifit-d 1nilk nwthodl.

l·:ll!emkitJa:.;p 11·a.-; pn•pan·d t'oli<JIIing JLtll::; <llt<l SIH'll,-;un (I!J:;-±,1 .

• \n Erep,;in ~olutiou 11·a,; lll'Ppan•d h.1· griuding pig',; inte,;ti11al llliH:o,;:t "·itb li<llld and 11<1ieJ, ,;(r;~iuing and filtPring·. It,; :1di1·it.1 i-; dt>nwn.-;tr.ttt'd in <·mlirol ""]ll'rim<·nt' ~o'. (i <ond 7.

Formol ,')olulion.---TlJi,; 11'<1" marl<' up a<·<·oHlitlg to(',,],. (lL!:!G). Une thop of .1 JHH <·ent. alcoh"li<" jlhPnolphtltalein ,;oluhon !wing nd<led JlPl' ,._,._ anrl :;Jlffi<·iPnt ,;odi1llll h~·droxi<h· ~ohHinn io ln·iug· tu p}l ·'-'·-!. ThP r<'<~dioJt ^II :to" e:~n•lnll·: ad.iu-i<·d t'<t< h .!a,\·.

l'tlnrliun l'l'l'hllliJIII'.- 'l'l:e di:..:·e-tion lllixtl'n•.-; \\·pre lllY<~ri:ilJl.\·

lll:HlP up Ly 111i:,ing-, in ,lll;d] ,;iop]H'n·cl fb,b, 10 1'.<·. of ilw dilnte<l

<'gg· ;dlnlllll'll, 10 <'.l'. of ,;uL,;ir;JI<' .-;o]utioll :111<1 ]:-, r·.<·. of lll!llPl' :111d

;,dding 4 drop,; of ioluoi. :111 :<ll((:l!llt ]ll'l'l·iou,:l\· d<•tPrlltillPd "'' ,.;ulli<·iptl( to it1l>ihit iJ:Il'IPri:~l :l<"ti,·ity.

In

l':l:ie,; ll'ill•rt• kina,.;t· 11·:1,; 11"1'<1. <·.l'. of tlti,; ,;olution rt'pb <·e<l

<·.r·. of hul'i'PJ'. Till' hnfr<·ritq.!· pol'<'r ,,.,1, "tllit'it'tlil\· ."tl'llllg· to i'll<lhlt•

ilii,: to ht• .lont• 11·ithout :ll'i'Pl'ling· the fit::tl pll ol tlH· !llixtPn'.'· 'l'he ,:oint ioJL-; 1\'l'l'P lpf( in a l'Oll:;iall( tPllljlPI'<Itlii'P \Yilt<'l' hath :1( ±:J'i⁰

An aliqnol of 10 t'.l'. \1':1,: 11·ith<lra\\n for iitr:tlioll ai ZPI'Il time. Tn ihj,: 11·:1~ ~1dd<•d ]() dmp,; of l JHT t'l'llt. nL·ohnlir- plieJJolphtlwlein

~olntion and det•inonnal sn<liutl! ]Jn]nJ.\i<lt• run i11 until the .. olonr a~ ~t'<'ll .in tltH t·onlp:~rator ll!:tt<·l!t·tl t'ht• .sta11tlnnl of

pH

i'\·-J.. l·'i,·e <'.<·.

of forJilOl soh1t ion 11·a.-; Jl<m· :l<td•·d «llll ¹ ht- :11 ixl ure :1g:1in tit r·:ded to pi[ 1'\·4, (i]p <(ll:lll(it.\· rPqniretJ llPill!.l' :1 :lin•!'1 11'<'"'~\ll'P of ill<' :IIIH>I!JJI of <lllli!lo nitrog"t'll jll'l''PIIt. 'l'}lt' t!P}if':t<'_Y of tliP titration ^1\'H:i ,.;ur-h thai a diffPrf'liC'P of Oil<' dmp, eqn:tl 1o 0·!12 <·.<·., <"onld he POI,;il_,.

di:;tittgniod!Fd.

_\ftpr :1 iiliH' itdt·n·al \\hi .. h ,·ari<'d in <iillt·t·<·nt <'"\]!Pritn<'llb fro111 I to :.!4 hour~. a ~<'<'OJl<i ,;alltpk 11·;!,.; n•lli<>l"t•d :111d ~intil:trlv iitr.:li<'<l. :1 fitt:ll :i:lllljllt' la•ing i:~h·11 a1 th .. t'ltht' ol tltt• <'.\jiPI'iiiH'Iil. TIH· titr:dion dillt'l'<'lwf',: l:Ptll-<'<'11 ill<' "Pro ;tw! hr~l :<nd c;<·•·ottd tinw i11it·n·;!l,; ;lJ·p

!'Pt·nr<lP<l in th<' pmiot·o],;.

('nul ro/ H.tp1'1·illlt"Jil-<.

Tit<' <'(J!trro] e:qwri1nenb ol TaLIP

T

,:pn-ed to dPtnon,:tt·ate thf' Idi;!lrilit:v of tltl' nwillocl a,.; f:tr a:; l'f'lH'Odndi' it_.,- of re~nlt;; was t·ont·erned and thP 8hSPJlf'P of ktdel·inl inh•rfPren<·e. En~n ill the

EN?.YMIC .-I.CTIVITY Of' EGG-WHITB.

presenee of ente1·okina:;e no proteolysis ocemTed in the various substrates after 24 to 46 hours, which ju:;tified the eonc.lusion that the increases in amino nitrogen observed when egg albumen m· enzyme

soluhous

"·e1·e present could be attributed to the action of the latteT.

En~psin

was acti'e upon casein and upon peptoue buL had no eftect upon gelatin.

The use of casein as a substrate by Balls and

~"·euson

cau be crticised on the grounds

that it

fails to di

stinguish

between erepsin- like. enzymes and true protea.ses (e

.g

. trypsin). The eondusion arrived at by these authors that a " tryptic protei nase '' i

s

presen

t

in eggs is not justified by their data, if one discounts the reported auxiliary effeet of entero-kinase. In some

caser;

no increase occuned on the addition of kinaRe, in others,

slightly higher

titration figures -were reeorcled, but considering the limitations of the met hod these

figure~>

do not, to our mind, carry much eouviet.ion.

Autol.11sis l!J,l,perimcnts.

Were a protease responsible for the break doml. of egg

a

lbumen, leading to the eondition of watery-white, it would he expeded that measurements of amino nitrogen in su<h albumen

solutions, suitably

bufferecl, would reveal the presenee of sm·h

^<Ill

enzyme es pecially wheu incubated at a temperature of 37° for periods rang

·iug from G

t.o 24 hours.

lt ;;ee me'l natund to look

, in

the fir,;t. place, for

snch a

ut.otlig·es- tiou of egg album en alone. Au to lytic aeti vi ty of tissues u

sually

proceeds most rapidly in a slightly aeiclmedinm. Test,; were however perfmmed with thick albumen solution

s

buffered at

reac.tions

ranging from pH 5·5-6·0; 7·5-8·0.

No action was

obsened at any pH although these eggs were Rhown

to

contain an active erepsin as

sub-

t>equently reported (see Table II, trial 12).

Autolyti

c

proteinases seem,

t

h

e1·efore, to

b

e

d

ennitel)·

ahRent from the eggs under consirlernti0n in om·

experiuteutr;.

In onler to meet the possibility that. a proteina

se

'"at> present hut that egg albumen "

·as not a

suitable substrate to

reve~d

its activity, experiments were earried out in which egg albumen was added to gelatin

solutions buffered at pH

7 · S

01'

S · 4 and with, or without, the ad<lition of kinase. K o increase of amino nitrogen could he demonstrated (see trials 1, 9 and 13 ). The egg coneerned iu trial

1

was a " watery-write

"

a<' understoocl

h~r

the tra(le

.

'l'hat

the majority of eggs do

contain

an

erepsin-lib'

enz_yme.

part.ieularly associated with the thick .a lbumen was clearly proved by the u

se

of easein

·

or peptnur Rolntion;; a,;

su!Jstrate~.

The activity is not great and requi ref:' a micro-method for its flemonf'tra tion. 'l'he enr.yme wouhl appear to he associated in normal

eg·g-s

with

the

thick albumen frac-tion; e\'en in a five-mouths-

old

egg· a feeble ae.tion \\'as found in the thick white hut none in

t

he thin. In

egg-,;

having·

Hoa.ting air

cells,

this enzyme waR J

emonstra.ble

in bot·h

fradions

(see trials

6

and

8).

In the ease nf trial G, t

-he allnm1enR

from two

eggs were HliXefl aiHl a

g

reate r

activit.~'

fouml in the

t

hin

wh

ite than

tlu· tl1 ic-k It lllll~t lw 11:'11\Ul'i'ed. ;l<J\\'1:'\'t'l, that i,, '-'"l' :d ; l1e'(' ··~!.!-"

u11l\ ;; oltlall 11\!<\Utih· of thi1·k ,,·hit1.• L~ih·d T11 pn~s lht> ~j,.,.,_ ll 1' l'U~.-iltl,. lhnt. ,; rli.fru~ioH nt 11w .nzyJnt· bnd rll';'llrn·d l'nHtl tl11,.], tD dt! II.

lt l~ ,.:]~'lll.fi('<lJll t\l;t\ jJl'Oit•())_\''il' ^\\'it' oiJC.pl'\'l:'t\ lljHII\ [tt'jl[illlt' :11111 , " ", • 1 " " , ~ ¹ ;·I 1 ~ 1 r·, 1 1 ., , ~~-il h , 11 1 1 1. ,. ad .1 i t i " 1 1 "

r

1. , 11 , . 10 \, i 11" "-• .

. \~ l't•g'<trd~ tilt' pJI "l'lillllllll, lhP pn·illllilt:IJ\. trial>.\~,,,_ '.!-l.l :lj>jll':lr·••d \1) illtlh·illt• :1 Vl'<'<lit'l :1di1·1t,\ .:ll pll 7

S

t\Wll <tl· ,..;·-L It

11'<1.' dt·•idt·d. tlt<ll'f,,ll', \(' ttl.-'1'1'\(' til•' :t•liYit\· ol i~tdilid11:1l

;tl\:tilll('lt .;,dnlillll'i. poolt•d l'rl>lll 1 '''' ··.~·g·,, (>li-'1

",,,t[[,,.:,. :

1!_,. 11id;• p!l

r~l il g·l~.

Tit,.,,. l'f''itdh an• t·••<·nnll'rl i11 tri:~l..c l-t-11; iilld Fig·'·!. :2,:11111:\: l-':g-. -J. ,-.,prc>~('Lri.iu;.: a ··oJII[lO'iill:' 11f I lit-> ~n<>l ;-l,:>llg·p.; oi ,,-n,·d .11 :h,·

1 :. d i 1· i d '-L' I '·' 1 11 • r i n 11 • 1, I "_

]j :'Pt'l\l:' I \<'Iii' 1\L:ll t\\IJ rq_::ii!l" 11J lil;t:O.:illilll\J ;:i'lll·it\· ,.,j,j; ¹irP

••llt i':i1rl.1 ,.:l!,t·tds· ltH•:ilisPd ;;I 1dJ ^_-,.,-1. tlu· ^ntl:t>r 'l"''':tditlg r>~t-r 1h1·

i:ll:p·p pfl 'j'·()_,'-; (), ,\(' liJtt>J'lll<"di:t(l l't'lltii>.J'I' ll t·t->rtHill llllttl\IH1 l)f ill'll\'il,l l'lolll:l 'til[[,,_. d!•lo·!'lo'ti.

)1 :--t'•.•JJJ~ to 11:--; llHIJ'v iii"·l"\. 11Jd1 t\.\"11 ~~·Jt~ll'~de· t~::;.-:_\.ll'lt'."' ila\-lli}.!'

di!lt'l'elll' optitll<l :~n· l'llli•·<'l'I!P<\. ;,11d I

It>

'11.'2'!2>'~1 in11 i-; Lnnlf' o11! !1_\ I h(•

'2'1'1';ti_;']' )'(•\;Ill\'<' l!l'ill'll\ it• \)It' 'o]f ~ (}_,"1·(1 li'!'.i1111 i11 !Itt• l'<tc'l' II')'I'J'-

~I'lllt'd ^h_1 [<'ig-. I, 1111 ~\;t·h ,Jj]lt•l't:lll t' 1 l'l'\1"1';11).2'.i!l I lit• 111\,t•)' \\\'(1 ('11~1':'.

]( i, \\l'll k1111\\'1J tlt:d :1 nli>.:(Pn• ,,j· l11n •'liZI'llll'' l!:l\'ill('.' dii'fl'l'<'i\1

"[>1111111 ,,·ill :dl'11n\ :\11 <·>.:lt'iid,-d Jlli-;H·Iilil,\ ·,·111'\t' ,.,jl;l1iting 111·"

hiilllJ"- _\-;;til 1'>.::\111!'1''· IIi<• pll-•ll·lilil,l ,_.111'1.1' ut )''-'P~ill ;wiing· llJI""

<'~!..! :Jihutll<'ll (.\ol·lltrop l~Jl!l1 l1<1:' lwt•Jl -;t·h·t·l>.·d <111d lt'}l'"dnt·<•d lll'i1·•·

:tpttH tllP ~~ll111' gT<tph PCIJ"t'l' -""'~~ I]J;~t ~1H' ( \\"11 1''11'\"[\> lit• _..,_, illllH·li

;,-;,1]_,

;tl•lllll iht• po:ld ,,j· thc>ir iiiiPI''"'''io;t_ TJ,j, gi'"'' 11 11,\jllditt·lil·;l\ ' ' ' ' "

i·t•!l!]':llltiiiP 1•-ii\1 lilf' t·•IIH]ilioll> oloL1i11itl~ in

•.·g·:.::

11l1il1'. It i~ '''I'll

rr""'

Fig . . , il~<tt t!J,. '"·,·rhJ>Jiil!g· i'''ldl-; n " totnpn.;it~ ,.,,~.,-,_._

f"Xl1ihi1i11!..! tin• ntaxilll'> a11cl <tiL l",it'lldt•d inlf'l'li'l'ill!.<' t:iJIQ'<' nf lt·-~Pl' llt (:\it 1· .. \\';• t·tllt~irl<·r. lilt'lt'Tnlt', 111:~1 il1•' •·ut H''. ot t>i!..!·. -+ ltl~\.1

P8t.·li 't;.- l'i>:'<dl-1'11 into (1\'1\ ji<LJI iall,l il\i'IJ[\jiJ':II}.t l'Oit'llJOIIc'llh,

n'jli'<'C't'Jdirl!!' IIYO <'llZ_I'lHC~ ll'hn~c nplllilil [i,. <II <t]tJtl'fiXlllt:liPLy pff .-,.;, and 7·()-N tl.

j) I< 1'1. ,_; --.1 l I \ .

:-->1'.\'t•l':d l'l'l'IIT'd~ :ll't' 1"

IJ,.

ftll\1111 i11 lltt· litc·r;l\111\' ol itil.l',(l_~·;J11111\.­

Il)1<>1: 1111' (-'\IZ_\'lllil' ill'til·it\' II) t')-2!.:''· )Jill ilt 1\lilll\' • ' i l ' ' ' ' 1\11' !'l''i!\t~

\\.t•!'l' ~H¹1 n~J;tlnh1p·u~1l!~.

\\'•dli!2t'lltll1h rl 1tt1."1) J'<']l•JI·I••,J ti;,. [lit''•'ll<·•· or " pr"J,.,,J_,·t;,- l'liZYHl(~ Jn egg-·-\·nJk, hut \\".;J;-.; llll:.::hl'.\ tO (lP1110I!-.:lJUj<¹ ill!\' ~ll('fl Cll·jjyj1\·

;n ;,~f!- alLmiiPl.L T\iJg':l I ]!J:!:i) dt•n:,,, 1 i1P pn·.;ew P 11t'. <i tryp.;in-1 ik~

i'llZ_\'IIIt' i11 yoJk. <lt•IP:·Iill~-. (Ill tJw nllh'l' li:IJJd. !I !l<oi-illl'lll!,i,lpr:.Jl]p l')'f'l'ti(' ilt'ti.\·jj~·- J•:g·~· \\•hit~•. \!(• i'lllhitl(•b, ('II]Ji<\iJt~ ^<I Tilll'il\l)hf.il' PliZ\'llte iu 'lllall amm1111 . . \tltli1imt<d n•ff'l'f;ll,.,., arl' qnolc·<ll)) B:ilb

:11111 "''""' 11:'011.

HNZYMIC ACTIVITY O:F EGG-WHITE.

It is evident from the present work that at least two peptide- splitting enzymes were prese

n

t in the albumens of the eggs studied,

whether norma

l

or " watery-white ". The em;yme with a pH ()ptimum about 7 · 6-8 · 2 is probably similar to tissue erepsin.

Alth ou gh our data do not pretend to ofter any explanation

of

the cause of " watery-white " it is of interest. to discuss our findings in the light of some of the

h

ypotheses which have been put fonYard to explain this condition.

In the first place the conclusions of Balls anrl Swensou a1·e clearly untenable. It is difficult to understand, however, how an ereptic enzyme could in any way modify the c-ondition of tlw native protein. Our a

utolysis

trials indicate that no breakdown occurs under these cond itions at pH valu es ranging from 5 to 8.

It is a

lso

signific-ant that

a

number of eggs examined within 12 hours of

laying have been found hy us to

exhibit the condition known commercially as " watery-whiteness ·'. Storage is not essential to the development of t.hi::; condition and iu our opinion the ooudition induced by shaking, as reported hy

variout~ a

uthors (e.g.

Canham 19M, Ha

lnan

19:33, Dryden

1934),

is not identical with that which appears in certain eggf'. a few hours after having been laid.

Our observations suggested some association between a tendency towards natural watery-whiteness and imperfect shell formation. In support of this, it may be recorded that imperfectly shelled eggs picked at random from a day's collection pr.oved subsequently, although not in every case, to develop watery-whitenef'.s within a short time after being laid (12 hours)

,

and our experience would iiJ.dicate t

hat experiments

taking cogi,i&auce of this fnet, might help to throw ligh t upon the problem.

Few, if any, eggs with perfectly

d

eveloped shells of fine texture

and

good ealcifica tion, result ultimately in watery-whites.

The

·

white of

an

egg is an elaborately orgaui,;ed struc ture as may be verified by observing its behaviour when poured upon a sieve.

The thick albumen formil a kind of p.ouch supported no d.ouht by interl acing fibrill:oe of mucin. Within this pouch the bulk of the thin white is contained and is liberated when the structure gives way upon the sieve surface. Eveu so, both thick

aud

thin fractions contain mucin which becomes visih

le

upon diluting the materials with water.

As stated

p

reviou sly, a low proportion of thick to thin albumen is by

n

o means an invariable accompaniment to the \Ya.tery-white condition,

in fact our experiments

have sho,vn that this is rarely the case. The

lowered

visco ity of the white seems to be due laTgely to a loss of structural organization. It would be of interest to determine wh etLer the total amount of mucin in watery-white eggs is less than in normal eggs and also what dterations, if any, t.hi,;

protein has u

ndergone.

Artificial disintegration of the albumen, as for exampl e by the injection of t

rypsin

or erepsin into normal eggs, may be expec-ted to result in a

lowered viscosity of

the white, but this in no 'vay proves that the naturally oecurring watery white is produced in the same way.

334

]<~. VAN ::\IANEN AND CLA UDN ltUlll\GTON.

A hypothesis

whi<·h makes

,;ome appeal to u:;

i::~

that the cause may

be sought.

in

nn

uvset of the L0und- to the free-water ratio.

Proteins are

known

to

<:ombine with a certain proportion of the water in whieh they are

<lis::~olved

in su<:h a manner that this quantity of the sohent is bound or eliminated in suGh a "·ay that it uu longer participates in osmoti<· phenomena.

A.ny

release of thi.H bound water cau:ses a diminution in the Yi,;co:sity of the system. The point

i~

H

usceptible

of experime11tal irlYestigation mul ,.:houl<l lw considered in any attempt to explain the oceurrence -of " watery-whites ". St.

John (1981)

has

found

thf'

hound water in thick Pgg white to be

llO le~R than

2(5 per cent.

Heg·anlrng the whole problem from a <letadred

~tandpoiut,

it

;q>pears to

^UH

that ·nany ta(·it a:-;sumptions ha,·e heen made whieh rf'lluire

experiment~tl

proof or <lisproof. Spe<·ulatiou base<l on false premi:;e,; is obviously useless.

l'hysico-ehemical pos,;ibilitie~

have not received the attention ·

they would

appear to merit. in s1H'h

a

problem.

lt seems to

us

that

the fin;t

point to bf' eluei<late<l i

n

an approaeh to the

problem

it> the actual nature of the differem:e between the

<'olloi<laJ

~y,;tems

of protein in '\Yater comprising the total white ill normal and \vutery-white

eg~J:~-

Thf' function of the muc·in apl•t'Hl'H to be to preselTf' a structural m·g-anizution in the whitf'

ib

hehYeen ihe allmmen aml the otber memhets of the colloidal complex, hut how this

function

is di;;eharged is obsr:ure. Somf'

disorganization

,;eem,; to take plaee in

'' watery-wl1iteness ".

One cannot help recalling· the somewhat similar

problem

of gluten

dispersions

in water. The protein from a

"

shong " flour forms a highly viscous colloidal solution. that from a " weak " flour a less viscous one.

The

reason for this behaYiour is not known. Even a highly vi::;euus solution undergoes a more or less rapi

rl

spo11 ta neou,;

rleerease in Yiscosity \Vithout, as far as can he ascertained,

an,v

markerl or obvious change in the protei

It;

the action of enli,Vlllf'S has been definitely exclurl ed. *

Our

knowledge of protein systems with their possibilities of

hydration,

dissociation and polymerisation is still "o meag-re that.

much work

will have to he done before such pe<"uliar changes as that above mentioned can he understood. It is along-

these lineH, howe\'er, that we feel the solution of the

"·ater~·-wl1ite

problem will

ultimatel~·

be

,,ttained.

SrM~fAHY.

1. The

enllymic acl.ivit.y of solutions of thick awl thin alhumen

from norn1al and. \Vatery-white eggs has been studie<l, using a Jnjcro-

titration method

d

etermining peptide cleavage in tf'rms of tlw increase in amino nitrogen.

2. Thi<·k

0r

thin albumen

~·lone

at pH value" ranging from

5 · 5

to 8 · 5 s11ffer

no autolysis

at 37°,

neither

can any protein-splitting enzyme

be detected

at. any pH by the addition of g-elatin as substrate.

* Since thi~ was written, an article has appeared h~r Blagovf>schemki and Yurgenson (] 935) in which it i~ claimed that there is present in wheat flour an enzyme which exercises n definite solvent action on these protPins by virtue of n disaggregation rather than an hydrol~'si~. It is claimed that there is no increase in amino-nitrogen during the process. It would sPem highly desirable that egg white be studied in a similar manner.

335

EN~YMIC ACTIVITY OF EGG-"WHITE.

3. The

v•ork

of

Hall;;

and

Swenson is

criticised

011

ac·eount of

insufficient sensitivity

of the

method

employed

and the fad that

it

is

such

as •vould

fail to

tli~:~tinguish

betwPen

an inl'rease

in acid groups

dne to lipolytic.

and due to proteolytic actiYity. .Moreover,

the use of casein as a

substrate is ineapable of

differentiating protease

from erPptase activity.

4. The presenee

in egg white of at least hvo erepsin-like enzymes

having

tlifferent pH optima (approximately 5·5

and

7·0-8·0) has

been

de1rwnstrated.

'VJH>ther

or

not these euz;nnp,; play ~wy

part in

the

tlevelopmeut

of '' "·atery-white"

i;; nneeJtain hut

for rPason~ <li:_;r·nsse<l

it woulrl

seem 1m I i kely

8omP

ohRernttion:=:; upon

"

"·ater.\ -wh

ite " i 11

eggs

arP

recorderl nnd

th:.~

problem subjected to a ,;l,ort di,;cu:-;,;ion.

BALLS, .-\. K., AXD :-iWE".'\SOX T. L. (193+). Proil>olysis in Rtored Egg,, Ind. En.o. Ohem., Vol. ~W. pp. i5i'0-2.

BLAGOYJ•:SCHEXSKI . .-\. V., AXD YrRGEKSOK, :\L P. (19:30). On tlw ehanges of wlwat proteins under the action of flour and yeast enzymes.

J/iod1r'111 . . J., VoL 29, pp. RO,)-RlO.

CAXHAM, A. S. (HJ::l:3). \YatPry \Yhites of Eggs. Heported. Preliminary Jnyestigations. Ondr•rsteponrt .J. Fet. Sci. nnd A:nimn.l Ind .. VoL 1.

X o. 2, pp. 02!1-;366.

COJ,E, S. \V. (1920). l'ractieal Ph.\·siologir·al ('ltemistr~-. HeffPr &. Sons, Cambridge.

DRYDE:\, \V. H. (UJ8-!). The Effec·t of Hiffennt :\f.,thods of Packing on Quality of Eggs. Hm·per .-1 dn ms Utillt?l Po11ltry .] ournu/, Vol. 19, No. 5.

HALKAX, E. T. (19:3;3). The Cause of " \Yater~· \Vhitt>s" and Otht>r Defef'ts in J•:gg'. Poultry Ilnndb., 192::3, p. ^B.

HOLST, W. F., ^AND AL:\1Ql:IST, H. ,J. (J 9:ll ). ?lleasnremeut of Dt>terioration in ^tlH; Rtored Hen'R l~gg. Tli/.oordia, Vol. (i, Xo. :J, Augnst, 1!131.

KOUA. T. (192:l). t'ber diP Fermente im Hiihnerei. nior'lielll. Zeit., Vol. 141, pp. 430-4<1-(i.

XOHTHJtOP, .J. H. (1919). The Effec·t

or

\'arions Acids on the Digestion of Pmtein by Pepsin . .J. G!'il, Physiol .. Vol. l, pp. 607-M2.

ST . .JOHK, .J. L. nn:n). T<>mperatUl'P at 1\'hic·h unbound water is completely frozen in a biocolloid. J. A.mcr. Ohnn. Soc., Vol. .53, pp. 4014-9.

WOHLGEMUTH, J. ll90i)\. 7-r•it. J. l'hysiol. Chnn., Vol. 44, p. 040.

336

TABLE

I.

Corres-lnere~st' 8ubHtratP. Enzyme. ponding pH. Kin~se. Timl'. c.c. N/10 RemarkH. Trial No. ~aOH. Hour.-.:.. Gelatin 2% .... -1 I 7·H 18 Nil

I

::-fo action. 42 Nil Casien iio/o ... J i i H 2 ·;) :\'il

I

Xo action. -I 24 Nil T'eptom• 2% ..

· 1

-:l I i·H

I

I

1 Nil I ~ (

I

46 ::-fil Xo action indut·cu by added kinase. ~ I 1 Nil

~ _,

r 411 +0·02 ~ Casein 5o/o ... Trypsin 8·4 r 1 11-Ui 0 · 2 c.c. extract of fresh pancreas. !..-'

.

I .... 46 I 0·72 :.- '7. Ert'psin I 8·4 () r; I Nil ~ ~o dotting of calcified mill; in 18 hourR.

z

18 I Nil

I

1 c.c. extract in intest. mucosa.

~

I ... CasE'in 2% ... I l•~repHin

I

7·H

I

u ;j +O·JJ Definite action on casein at 7 · S.

~ 18 0·90 (") -< ;... P<'plonc 2o/o .... Erepsin I R·4 I I 0 li 0·20 DefinitP aetion on peptone at pH H · 4. d !:;) IH l ·O;i ~ G<'latin 2o/o .... Erepsin I S·.J I O·il I Nil Xo action. ,...

""'

18 +0·05

""'

,...

z

0 ....;

z

t"<j 'l'AHLE 11. '7. ;:-.:! >< ~ H

I

Number 0' Increase 0 Trial

l

Kind.

/0 :.-No. Substrate. of Age. thick pH. Kinase. Time. r.r. N/10 Remarks. () f'ggs. white.

I

NaOH. ..., H

< -

I dum.,.,. ---H

I

I

I

..., 1 -0 35 This egg had a floating air-cf'll ~ 5

I

^-

0·35 0 I ":1 . 1 . Gelatin 2% I 2 days Thi~k .J'/·7 8·4 1 -0·02 Albumen not centrifuged. t"<j 5 -0·03 Negative results, probably due to bad samp--;;) 1 ;-.;il ling. 1 c.c. kinaee used. :! ' 5 Nil

I

:::: Thin I J +O 03

I

~o action. :.; 5 t-0·02 H ...., t<: -0·03 Thi<"k I ;) Nil

I

1\>Iembranf's centrifuged out. 1 c.c. kinase I l I

I

^-0·02

used. ;) -0·02 D:> 2 Casein :l

I

I hour 44·() H·.J. 1 -0·02 No action. D:> I 00 :) -0·02 Thin I l +0·02 [)

I

⁺

0·02

I

- I

--~---

-

Same 2 +O ·10 Pf>rformed day following Experiment No. 2.

I

three

I

Thick

I

:H·5 +0·30 Albumen solution kept in refrigemtor. eggs 2 +0·30

I

2 c.c. kinase used. An f'reptase probably

a

I Peptone 2% as in I

I

21·5 j-0·43 present; action increaser! by kinase. ex peri-2 +0·02 Greater action with thick white. mcnt 21·5 +0·17 :\o. 2 Thin 2 +0·05 21 ·ii +0·19

-

--- I I +0·13

I

An ereptase probably present. 1 c.c. kinase Thick I 21 +0·25 had no auxiliary effect .. I 1 +0·12 4 I Peptone 2% :~ I l day .J.il·O 7 8 :H +0·22 l Nil No action with thin white. 21 -0·02

I

^Thin

1 l\ il 21 ~o

o-t

~---

c.: ~ cc

- Trial Substrate. No. ,'j Casein 5%

--

~---- 6 Casein 5% ---- Casein 5% 7 Peptone 2% ---

---- ---

Casein 5%

s

Peptone 2% ~--- Uelatin 2% () Cas~in 2~·0 Peptone 2% --

----

Number of eggs.

I

3 --- 2

---

I ---- l ---- :l

I I

'_I1Alll.E

I!.

(continued). I I

I I

Kind. %

I

Age. thick pH. KinasP. Time.

I I

white.

I I I

hours. 1·5

I

Thick ! 18 1·5 5 months 41 !J 8·4 18 1·5 18 Thin 1·5 18 ----------~----------- 18 Thick 66 -j 18 4 dayR 41·!l 7 8 66 18 66 Thin

+

18 66 ----------· --- 7·8 2 !) 1 hour Thil'k 43·4

I

24 8·4

I

2·5 24 ---

----

---- 7·8

+

2·!3 1 day Thick 4;) ·2 24 8 ·4 2-5 ____ I __

I

24 ----- 7-8

I

2 22 8·4 :l

I

j2 1 hour Thick 53·S 7·8

I

2 22 8·4

I I

2 22 7·8 2 I 22 I --

.

-

-- Increase I c.c. N/10 I Remarks. NaOH.

I

Feeble action with tkick white only. -f-0·10 u ·15 +0·05 Nil -1 c.c. of kinase used which had no auxiliar y -0·15 effect - -0·25 --- 1 0-10 1 These eggs had floating air-cells. 1n ow -f-0·25 pnctically no thick white left. 1 c.• -f-0 -10

I

kinase used. Action greater in the thi +0-15 white than in the thick. n +O 30 +0·40 +0·18 I Kinase had no auxiliary effect. -f-0 36 +0-25 No action on plptone at pH 8·4. l~efini1 +0·20 action on casein at pH 7 ·8. 2 c.c. kinaf .e -f-0·07 used. -0·10 --- +O 30 This egg had a floating air-cell. Condu~ior +0 25 of former trial confirmed. -fO ·05 0·10 2 c.c. kinase used. - +O 02 1

No action on gelatin or· c·asein at either pfc -0·04 -0 ()!) Definite action on peptone at pH 7 ·8. I Nil -0·15 2 c.e. of kinase used. I Nill -0·07 0-05 0 03 11·2.~ ---

---

t:j

< ""

7, ~ >

z

t:j

z ~

tJ (') ::-< ;.. d

~

::::: ~ 1-<

z

Q H c !7,

I I I - ':;umber Trial

I

!\o. Su bstrato. !I of egf!~.

· - · - --- , -

C·tsein 2% I 10 2 PPjJione

2 '!{ , I ·-· --- 1 ~ - -- - - _! __ _ _

II

I

^CaHB

in 1'-;;) PoptnrH' 2%

·-- - j--·

I I 12 1

I

Nil

I I I

~

1 - --

1 j Same eg?;s n~ 1u experi- ment ~(l. 11

T.un.h IT. (l'ontllllll:'r1). ·-- 11

%

A)(l'. Kind. thiC'k I pH.

, - I

ll day ...

I 1-- - -

1 l hour I I

Thick

'ddt<>.

!

7 ·8 ,ti)·O H

~ I

7·:-l

I I I

K •I

' . . - -- - 1 ~ - --· -1 I , s

'.L'hi<·k 1 ;;z. 7 I u ·ii I I ' I

- -[- - 1 I

'l'hi<k I

;) ·0 ,) ·;) H·U t) .;") 7·0 ~ -i ;l

J.~! nr:..o.;c. J~im.C'. i -I _I_ I

I I

^Hour

s. 2 22 2 22 2 22 :! 22 1 ·,) ;W I :; 20 1 . ii :.'0 (j :!~ (j 2-t. (i 2~ ti :!4- (j 2-l

__ _ _ !_ ~ - · - -~ - ~,--1- -- - ·- ·

Uelatltl 2 /o 1 : I --, --I I'--·-

~-0 -·----1' .. 7·S

(j ~~ 24 13 Peptorw 2% :! 1 "'""' 'J'hi<·k .)() 0 G·O !J8 20 4+

bwr·ea~e ('.C. X/10

.\', , oH.

1\il

- : o ·

:n Nil i-0 (l;j f 0·15 1-0 :~3 I ~ 0 · 11

Remarks. Definite action on both fubstratcs but mar(' marked at the lower pH. Similar J-esuits on pepton<' ut f.> II 7 · 8 when mucin nof, reJnnvf>d. :! t·.f·. h::ina . ..,~ UI"Pd.

-~ o ::_[ I

0 li'i l:efinite ;u;tion on both substrak>' in cac:h -0 4o.· I l'asc: aet.i vity appears to be. l<>s" at the +0 ·]:) loWPI' pH vahlPH. 2 C.C. kitHLHC URCCl. -f 0·3!) Nil ·I 0 03 --0 OJ -0·02 () l)J () (I] +O 02 ~ 0 02 ,-O·o:l .I O·O:l I ,-0 ·03 !\ il .,-0·04 )iii ;.;'il \' j) ,-0 ·OS ·• o ·II

\'o action at any pH although th<>se <'gg,; kz1own to <·(nlt.uin a.n active enzvnll': 1-lf'e previou~ (''>/'<'rintent. " 'fh<·n· is no (•videnee of an <tutolytic proteas<' tH:ting on the nlbutncn as suL~tr.:1tf>. l\'o actio11 on gt'latiJ'. J)pfinitc 'ldion nn peptone. \ ... u kina:--;~ ll~f'd.

t'l

z

N ~

~

0 :... n ~ ....

< .... '"

><I 0 r.j -~ ~

- ~

~ ~

~

,...

1-'

Trial No. 14

-- I

!

!

I

Substrate. Peptone 2% If\

!

Peptone 2% I '

- ---- 1 ---

1

- 16 I Peptone

2% ! i i

Number of egt-.rs. 2 2

A.ge. I hour J hour -·--·-·· -··· 2 1 hour

Kinrl. Thick Tl:id< 'fhiek

i I I I

I

'LIHI.E II. (umtinuerl).

(xl

thick white. ·+;; ·H ,;;l k 60·0

pf-1. lGtu1sP. ;) () ;}-;) (i·O (i·;) 7 0 7-[) ~-0 R·f) ( Bor"'t0) -···-·· ;) ·0 ;) ·fi fi 0 (i .;, II 7 ,) H·O

Tiln<·. Houn;.

[rH:rca'L' i C,(•.:N/LO :\aOH. 20 Sil 4H "lil 20 -i 0·17 4S +O 2!1 20 -1 0 07 4H +0 2:~ :!0 ·1 0 .II 4S j 0 4:!

i

:20 + () 07 I 48 I

o

47 20 tO l:l I 48 : 0 ·67 ' 20 -l-0 ·ll I 48 : 0-58 I

:w

-~o 1 :r>

l{enla..r.kf-'. Definite action on peptone ; apparently two optimfL at pH 5 ·5 anrl within the range pH 7 0-8 ·0. (See Fig. J ). *For pH H ·;) l>orate bnffer u:;erl anr.l the figures <:orrecLerl to tlw Hamf' ha.RiR >cR phospha-t.<' bnffE'r. No kina:-;e usPd. I 4s -! o 2:~ 1

___ , ____ ---·----~----·--·--·--·---···----- __ ·)4 : I 0 · OU 1

lkfinite aetion for first 24 homs, I ittlc pro-. I 48 I tO ·ll grcss r.ft.cr· this time. 'l'wo optima. at , 24 1 ! 0 17 1 pH ii -.~ tcnrl range pH 7 ·O-S ·0. (Sec ' 48 +0. 17 I Fig·. 2.)

i

I

24 ! () ]() 48 1 0 ·10 24 I 0 J I 4il tO II 24 0 ]() 48 -,--0 2:l 24 0. ];) 48 --0·10 24 41:\ . 0·21) .C.(). :22

A11 ren.gentK ~terilizcrl by auto('b.ving nnd thl' r.mount of toluol pr·c'seni increRsecl. .\'o kin~s<· '"ed. I --w ·-··· ---! 24 ;j ·0 4S -4 ()-()2-h-iefinite fwtion on peptone. Two optima ;).;") 24 48 ti·O 24 48 {j. ;) 24 48 7 0 24 48 7 .) :/4. !S S II 24 4S

f-0·0:\ I 11,(: pH ;'\ .. ti ancl mnge pH 7·0-8·0. -1 0 2:l -t-0·31 (:-it'(' Fig. :L) -! 0 JH I 0·27 -'--0 ·13 -;-0. 26 -1 0 28 I 0 23 f-0 10 +0·20 . 0 12 i 0 2H

Ko kim1se used.

~ <j

.... z "" "" ....

^{~ t,;}

z ,,. z

t:l (') ::-< .,... d t:l t:j P:l H ;,:: H

z

C':l ...; 0 7:

E!'ZYliiiC ACTIVITY OF EGG··WHITE.

· ••

. ..

. ..

, .

'"

"'

..

rll.

E. VAN MANEN AND CT.AUDE ltlliUNGTON.

70

·60

Fig. :!.

70

c.o ... poel!t..rflt._l,2:&ndl

..

pH. 7 0

• . .

Fig. 4.

34~J

ENZYMIC ACTIVITY OF EG(; \1"1-JJTE.

ll)"'-'l~~u,.,tcun•· oll~•tr~oln~ '"~ ('.ornl>lnf'd .\cU<W yf '""

Jlfl~"'t"' •·n,1rnc• u>•r .,. Hltnhd r~nu of ptl

Frg . . ).

;)4-1