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T H E E F F E C T O F D E N A T U R A T I O N O N T H E V IS C O SI TYO F P R O T E I N S Y S T EM S

BY M. L. ANSON A~D A. E. MIRSKY(From tke Laboratories of The Rockefeller Institute for Medical Research, Princeton,N. Y., and the ttospital of The Rockefeller Institute for MedicalResearck, New York)

(Accepte d for publication, D ecembe r 2, 1931)Conc en tra te d so lu t ions o f p ro te ins in ac id o r a lka l i may becom e very

v iscous on dena tu ra t ion o f the p ro te in and un der su i tab le cond i t ions amod era te ly v iscous so lu t ion can be conver ted by he a t ing in to a c learge l. These s t r ik ing changes in v iscosity , a l though they have beenknow n fo r a long t ime, have no t been s tud ied in de ta i l in recen t yearsand in genera l the s tudy o f dena tu ra t io n has been neg lec ted . Themai n reason fo r th is neg lec t has been the be l ief tha t dena tu ra t ion i s ani r revers ib le reac t ion , p robab ly an i r revers ib le sp l i t t ing up o f the p ro-te in , and tha t therefore dena tu ra t io n i s o f l i tt le physio log ica l in te res t .The exper iments descr ibed in p rev ious papers ( rev iewed in Anson andMirsky , 1931a) led us to the conclus ion tha t the den a tu ra t io n andcoagula t ion o f p ro te ins , co n t ra ry to th e usua l no t ion , a re revers ib le ;and t ha t therefore dena tu ra t ion i s a bio log ica lly possib le p rocess. Weaccord ing ly became in te res ted in resum ing the o lder invest iga t ions o fthe g ross change in v iscosi ty accompanying denatu ra t ion . The p res-en t exper iments mak e more p rec ise than was possib le a genera t ion agothe cond i t ions fo r ob ta in ing very v iscous so lu tions and ge ls , and the yshow tha t one can con ver t no t on ly a concen t ra te d p ro te in so lu t ionbu t even a so lu t ion con ta in ing on ly one per cen t o f p ro te in in to a c leargel . Althou gh, as will be seen, ther e is an increase in viscos ity due todenatu ra t ion i t se l f apar t f rom any aggrega t ion o f the molecu les , in thevery v iscous so lu t ions con ta in ing l i t t le p ro te in the dena tu red p ro te inseems to be in the fo rm of aggrega tes , inv is ib le p rec ip i ta tes , whichocc lude water .

34 1

The Journal of General Physiology

Published January 20, 1932


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M. L. AN SON AND A. E. MIRSKY 343with an equ al volume of water, shaken with a f if th the to tal volume of toluol andallowed to stand in the cold. The next morning the tuluol and swollen strom atawere removed b y centrifugation. 1

Relative viscosity of centrifuged solutions was measured with an Ostwaldviscosimeter. A few measurem ents made with the du N otiy coaxial cylinderviscosimeter gave essentially the same results.Ef fec t o f A dd . - - In t h e f i r s t s e r i es of e x p e r i m e n t s ( s e e T a b l e I ) s o l u -

t i o n s of 4 p e r c e n t e g g a l b u m i n c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s o fH C 1 a r e h e a t e d f o r 4 m i n u t e s i n l a r g e t e s t t u b e s k e p t i n b o i l i n g w a t e ra n d t h e n c o o l e d t o 2 5 C . I f n o t e n o u g h a c i d i s a d d e d t h e p r o t e i n i sp r e c i p i t a t e d . I f j u s t e n o u g h ac i d i s a d d e d t o p r e v e n t p r e c i p i t a t i o n t h e

TABLE IEffect of pH on Viscosity of Heated 4 Per Cent Albumin

HCl~l i t c r

0.0040 . ~ 50 .0080 . 0 100 .0120 .0400 .065

pH

4.293.96

3.70

Appearance

Preeipitate-gdSlightly opalescent--almost a gdClearClearClearClearSlightly opalescent

Relative viscosity

8 .083.922.091.52About 40

s o l u t i o n is v e r y v i s c o u s . T h e m o r e a c i d t h e s o l u t i o n s t h e l e s s v i s c o u st h e y a r e a f t e r b e i n g h e a t e d a n d t h e n c o o l e d, u n ti l f i na l l y f u r t h e r a d d i -t i o n o f a c i d m a k e s t h e m m o r e v i s c o u s a g a i n a n d s l i g h t l y o p a l e s c e n t .A s in o t h e r p r o t e i n p h e n o m e n a , a f t e r a c e r t a i n p o i n t t h e a d d i t i o n o fa c i d h a s t h e s a m e e ff e c t a s t h e a d d i t i o n o f s a l t. I t m a y b e s e e n i n t h e sea s in o t h e r e x p e r i m e n t s , t h a t s m a l l d i ff e re n c e s i n h y d r o g e n i o n c o n c e n -t r a t i o n m a y b e a c c o m p a n i e d b y g r e a t d i f fe r e n ce s in v i s c o s it y , e s p e c i a ll y

1 All the exp eriments described in th e presen t pape r, ~xcept the o ne on th ehydration of hemoglobin in urea solution, were done in the winter Of 1927-28.Since then we have found it desirable to use alumina cream to facilitate the re-moval of toluol and s tromata. The remark is made to avoid giving the impres-sion tha t the use of alumina cream described in other pape rs has been abandoned.It ma y b e t hat the commercially available Filter-cel which is very useful in thefiltration of some protein solutions can be substitu ted for alumin a cream.



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34 4 DENATURATION ON VISCOSITY OF PROTEI N SYSTEMS

in the range near the precip i t a t ion zone where the so lu t ions are veryv i s c o u s .Effect of Salt Added before Hea ting.--In the prev ious sect ion therewas descr ibed a he ated so lu tion of 4 per cen t a lbumin in 0 .012 N HC1which had a re la t ive v i scos i ty o f 2 .09 . I f NaC1 i s added before theheat ing so tha t i t s concent ra t ion i s 0 .005 M then af ter the heat ing theso lu t ion i s more than th ree t imes as v i scous as wi thout the sa l t ( seeTable I I ) . The sam e ef fect i s p roduc ed by 0 .0002 M K,SO4 and b yeven smal ler concent ra t ions of IQFe(CN)0 . Simi lar exper iments canbe done in g reat var ie ty wi th the genera l resu l t tha t whenever a mod-era te increase in v i scos i ty can be caused by heat ing in the absence ofsal ts and the presence of a small concentrat ion of sal t resul ts in vis ible

TABLE IIEffect of Salt on Viscosity of Heated 4 Per Cent Albu min

Salt

NaCI

K2SO~

Concentration

0.005 ~t

0.002

Time af ter heating

024

22024

Relative viscosity

7.311.813.417.47.3101311.5

precip i t a t ion of the pro te in , then a s t i ll smal ler concent ra t ion of sa l tcan be fou nd which can cause a g re at increase in v i scos i ty on heat ing .I t i s character i s t ic in these exper iments tha t the v i scos i ty of a heate d

sel f -conta in ing so lu tion gradual ly increases wi th t ime. Thu s the so lu-t ion con ta in ing 0 .005 M NaC1 in 22 hours reaches a v i scos i ty a lmos tn ine t imes as g reat as th a t ob ta ined i f no sa l t i s added .

Effect of Salt Added after Heat ing.--If sal t is add ed to a su i table solu-t ion of a lbumin which has been heated and then cooled the v i scos i tyrises and co nt inues to rise for a long t ime. The gra dual r ise in viscos-i t y can b e s p eed ed u p an d m ad e m o re ex t r em e b y r a is in g t h e t em p era -ture. Fo r instance, a solut ion whose relat ive viscosi ty is onl y 1.55 isob ta ine d by adding an equal vo lume of water to a 3 per cen t so lu t ion of



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M. L. ANSON AND A. E. M IRSKY 345egg a lbumin in 0 .006 N HC1 which has been hea ted 4 minutes andcooled to 25C. W hen a n equal volu me of 0.002 ~ K~SO4 is add edins tead of the wate r the viscos i ty i s 2. Af te r 22 minutes i t i s 2 .76 andaf te r 2 days the solut ion has become a s l ight ly opa lescent hard ge l .The h ard ge l m ay be obta ine d in 30 seconds by plac ing a tes t tub e ofthe solution c onta ining 0.001 ~ Na~SO4 in boil ing water .

A s imi la r exper im ent i l lus t ra tes the fac ts (1) th a t one can obta invery viscous solut ions whose prote in conten t i s only 1 per ce nt andwhose sa l t concent ra t ion i s only 0 .0008 M, and (2) th a t whe ther or nota lbumin i s prec ipi ta ted when i t s solut ion is hea ted d epends not o nly onthe composi t ion of the solut ion but a l so on i t s h is tory . An equa l vol -um e of 0 .0016 M K2SO4 is added to a h ea ted 2 per cent solut ion ofalbu min in 0.004 N HC1. Th e viscosity is 1.28 and increases grad-ua l ly . Wa rmin g to 37C. causes a gross increase in viscos ity andhea t ing in boi l ing wate r for 3 minutes m akes th e solut ion s l ight ly opa l-escen t an d alm ost a gel . I f an eq ual volum e of 0.0016 M K2SO4 isadde d to an unh ea ted 2 per cen t solut ion of a lbumin in 0 .004 N HC1,then on be ing hea t ed th e prote in i s def ini te ly prec ipi ta ted.

Effect of Alkali Added after Heating in Acid.--If a solution of al-bum in in ac id i s hea ted and then neu t ra l ized the prote in i s prec ipi ta ted.I f , however , the amount of a lka l i added i s jus t not enough to causeprec ipi ta t ion then the resul t ing solut ion in t ime becomes very viscouseven if the concen t ra t ion of prote in i s low. For example , to a hea ted3 per cent solut ion of a lbum in in 0 .006 N HC1 is adde d an equa l volum eof 0.00268 N Na OH . Th e viscos ity at f irst is 1.67. Afte r 19 hou rsit is 6.82.

Effect of Addition of Native Protein.--Th e addi t ion of na t ive , sa l t -f ree egg a lbumin to a lbumin hea te d in ac id has the sam e sor t of e f fec ta s t he a dd i t i on o f N a O H . T he so lu ti ons be c ome mor e v isc ous a nd t heviscos i ty cont inues to r ise wi th t ime . Very charac te r i s t ic in thesesolut ions i s the grea t increase in viscos i ty produced by warming thesolut ions only a few degrees . The na t ive a lbu min probably ac ts notonly by combining wi th the ac id to produce a less ac id solut ion inwhich the dena tured a lbumin i s less soluble , but a l so by combiningwi th den a tured a lbumin i t se l f, s ince, a t the pH produced , den a ture degg a lbumin would prec ipi ta te in the absence of na t ive prote in . Ingenera l , in m an y so-ca l led mixtures of soluble and insoluble prote ins of



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346 D E N A T U R A T I O N O N V I S C O SI T Y O ~ P R O T E I N S Y S T E MS

al l sor t s when the condi t ions are such that the inso lub le p ro te in does no tprecip i t a te , a l though i t would precip i t a te in the absence of the so lub lepro te in , then the so lu t ion of the two pro te ins may have a var ie ty o fphys ica l p roper t i es no t possessed by so lu t ions of e i ther o f the two pro-te ins t aken a lone.

The exper iment cons i s t s in add ing to a 3 per cen t so lu t ion of a lbuminhea ted in 0.006 N HC1 a 10.3 per cent solut ion of dialyzed nat iv e al-bumin . Wh en an equal vo lume of nat ive a lbumin i s added , the v is -cosi ty is at f i rs t 1.73 and the next mor ning 7.50. W hen a third morenat ive a lbumin i s added the v i scos i ty i s a t f i r s t 1 .75 and the ne x t morn-ing too great to be measured .E~ect of Water Added before and after Heating.--If an acid solu-t ion of a lbumin i s d i lu ted wi th water before i t i s heated , the wa terhas a much greater ef fec t in lower ing the v i scos i ty than i f i t i s addedaf ter the heat ing of the more concent ra ted so lu t ion . For ins tance , i f a4 per cen t so lu t ion of a lbumin in 0 .008 HC1 i s d i lu ted wi th an equalvo lume of water before heating , then af ter heat ing the v i scos i ty a t 25C.is 1.48. If the di lut ion is done after the hea t ing th e viscosi ty is 3.85.Even more s t r ik ing resu l t s can be ob ta ined by us ing somewhat morev iscous so lu tions . I t i s easy to ob ta in tw o so lu t ions of the samecomp osi t ion whose viscosi t ies differ mo re tha n five t imes.

Acid l ikewise has a g reater ef fec t on the v i scos i ty when i t i s addedbefore ra ther than af ter the heat ing . As has a l ready been shown, nearthe precip i t a t ion zone the addi t ion of a l i t t l e more ac id before theheat ing makes the v i scos i ty on heat ing much lower. The addi t ion ofthe same am ount o f ac id af ter heat ing has t i tt l e more ef fec t than theaddi t ion of the same amo unt o f water .

Wi thout mul t ip ly ing examples i t may be sa id that in genera l i t i seas ier to p revent the format ion of a v i scous so lu t ion by adding wa ter ,ac id or a lkal i , than to decrease gross ly the v i scos i ty of an a l ready v i s -cous solut ion, just as i t is often easier to prevent the precipi tat ion of asubs tance than to d i s so lve i t once i t i s p recip i t a ted . He at coagula tedand precip i t a ted a lbumin does no t d i s so lve read i ly in a concent ra t ionof acid which is sufficient to prev ent the precip i tat ion on heat ing.Effect of Urea.--The addi t ion to sa tura t ion of so l id u rea to a veryv iscous so lu t ion of denatured egg a lbumin a lways resu l t s in a g rosslowering of viscosi ty. For instance, 4 gin. of neutr al urea were added



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M. L. ANSON AND A. E. MIRSKY 34 7to 5 cc . o f a so lu t ion whose v i scos i ty thereupon droppe d f rom 8 .18 to2 .14 . Fur the rmore in ma ny cases the v i scos i ty i s abo ut the samewhethe r the urea i s added before o r af t er the heat ing . I t i s imposs ibleto get a very v i scous so lu t ion of denatured a lbumin in sa tura ted ureaso lu t ion wi th the concent ra t ions of p ro te ins and e lec t ro ly tes used inthe exper iments a l rea dy descr ibed .

The exper iments wi th egg a lbumin can be var ied in many ways ,a lways wi th the resu l t tha t i f on ly one fac tor is var ied a t a t ime then arange can be found in which the v i scos i ty i s ver y sens i t ive to th a t fac-tor. I t is in this range, which is alwa ys close to the precipi ta t ionzone, tha t the h ighes t v i scos i ti es are ob ta ined .

Exper iments s imi lar to those wi th egg a lbumin and g iv ing s imi larresu l t s can be done wi th o ther de natured pro te ins . A few exper i-

TABLE IIIEffect of pH on Viscosity of Heated 5 Per C ent Hemoglobin

HC1 Relative viscosityr,~ls ~ llt~

0.0021 7.600.0024 3.180.0063 2.37

m en t s w i t h h em o g l ob i n a r e s um m ar i zed in T ab l e I I I . T h e h em o -glob in concent ra t ion i s 5 per cen t , the t ime of heat ing 4 minutes .The so lu t ions are f i l t e red before the v i scos i ty measurements and areall clear.

DISCUSSIONViscosity Change Due to Aggregation.--AU the theor ies o f v i scos i ty

agree that the v i scos i ty o f a so lu t ion depends in some w ay on the f rac-t ion of the to ta l vo lume occupied by the so lu te . Kun i tz (1926) hasshown empi r ica l ly tha t when par t i a l v o lume i s p lo t t ed agains t v i scos i tythe same curve resu l ts fo r a number of d i fferen t co llo ids . By th e useof th i s curve i t i s possib le to ca lcu la te in a new case the par t i a l vo lumeof the so lu te f rom the v i scos i ty o f the so lu t ion . Wh en the value soobta ined i s h igher than th at ca lcu la ted f rom the concent ra t ion and



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34 8 DENATURATION ON VISCOSITY OF PR OTEIN SYSTEMSdens i ty o f the so lu te , t hen one mus t as sume tha t t he so lu te con ta inswater . Wh en the am ount o f water is g rea te r than can reasonab ly bea t t r i b u t e d t o o r d i n a r y h y d r a t i o n t h e n o n e m u s t f u r t h e r a s s u m e t h a tthe so lu te i s no t d i spersed in to molecu les o f the o rd inary s ize bu t tha ti t has a s t ruc tu re which cons is t s o f aggrega tes occ lud ing water . Sucha priori cons idera tions l ead to the v iew th a t i n the mor e v i scous so lu -t ions o f dena tu red p ro te in the p ro te in i s i n par t aggrega ted . Th i sv iew i s in harmon y wi th a l l t he exper imen ta l fac t s .

1 . In general the condi t ions for a high viscosi ty are close to thosefo r v i s ib l e p rec ip i t a t ion . I f an ac id so lu t ion o f hea ted , de na tu r edprotein is brought jus t a l i t t le closer to the isoelect r ic point or a l i t t lesa l t i s added , the so lu t ion becomes much more v i scous o r the p ro te inac tua l ly p rec ip i t a tes . I f t he so lut ion befo re be ing hea ted i s s l igh tlyfurther away from the isoelect r ic point or contains s l ight ly less sal tthen the v i scos i ty o f the hea ted so lu t ion i s very much reduced . I t i scharacteris t ic of col loidal aggregates that in a narrow cri t ical rangesl ight changes in ionic concentrat ions should cause ei ther further ag-gregat ion or dispers ion.

2 . The grea t increase in viscos i ty which fol lows the hea t ing of a sui t -ab le p ro te in so lu tion can read i ly be p reven ted b y the add i t ion o f wateror acid before t he hea t ing . The add i t ion o f water o r ac id after t h ehea t ing has mu ch l es s e f fec t on the v i scos i ty . I t i s known tha t i t i seas i er to in f luence the fo rmat ion o f aggrega tes and p rec ip i t a t es tha n i ti s t o d i s so lve them once they a re fo rmed .

3 . Urea which can d i s so lve dena tu red p ro te ins a lways lowers thev i scos i ty o f a very v i scous so lu t ion o f dena tu red p ro te in . Were thereno solubi l i ty effect , the addi t ion of sol id urea ought to increase thev i scos i ty .

4 . The increase o f v i scos i ty wi th t ime in many o f the so lu t ions canbe accoun ted fo r by g radua l aggrega t ion .

Viscosity Change Not Due to Aggregation.--Although the gross in-c rease in v i scos i ty , wh ich may under su i t ab le cond i t ions accompanythe de na tu ra t ion o f a d i lu t e so lu t ion o f p ro te in , i s p robab ly due toaggrega t ion o f the p ro te in , t here i s ev idence tha t dena tu r a t ion o f i t se l fcauses an increase o f v i scos i ty apa r t f rom any aggrega t ion . Concen-t ra t ed so lu t ions o f u rea no t on ly d i s so lve coagu la t ed p ro te ins (Sp i ro ,1900 ; Ramsden , 1902) bu t a l so dena tu re na t ive p ro te in (Ramsden ,



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M. L. ANS ON AND A. E. MIR SKY 34 91902, 1913; Anso n and Mirs ky, 1929; Hopk ins, 1931). De na tur ed pro-te in prec ipi ta tes when the urea i s r emoved . The mo re the prote in isde na tu r e d a s de t e r mine d by t he so lub i li t y t e s t t he m or e v i sc ous t hesolut ion becomes. Wh en the prote in has become comp le te ly insolu-ble a t i t s i soe lect r ic point in th e absence of urea , the v iscos i ty ceasesto change (Anson and Mirsky, 1929). Th e f ina l r e la t ive viscos i ty ofan 8 .5 per cent solut ion of hemoglob in m ay be 6 .3 . S ince these exper i-men ts were descr ibed, Burk and Greenberg (1930) and Hu ang and W u(1930) ha ve show n by osmot i c p re s su re me a sur e m e n t s t ha t de na tu r e dprote ins a re not aggrega ted in conce nt ra ted urea solut ion. The molec-ula r weight of den a ture d hemoglobin in urea solut ion i s n ot grea te rt ha n t he mole cu l a r w e igh t o f na t i ve hemoglob in i n w a te r . Bur k a ndGreenberg, however , in order to make the osmot ic pressures of solu-t ions of d i f fe rent prote in co ncent ra t ions propor t iona l to the p rote inc onc e n t r a ti ons ha d t o a s sume tha t he moglob in is hydr a t e d t o t he e x -ten t of 2.8 gin. of wa ter per gin. of protein . Ku nitz (1927) had pre-vious ly ca lcula ted the hyd ra t ions of ge la t in a t d i f fe rent concen t ra t ionsneeded to m ake th e osmot ic pressure of ge la tin solut ions propor t iona lt o t he p r o t e in c onc e n t ra t i on , a nd he ha d show n tha t t he s a me va lue s f o rthe hy dra t i on could be ca lcula ted independ ent ly f rom the viscos it ies ofge la t in solut ions by the use of h is curve which re la tes v iscos i ty to solutevo lume . We ha ve now l ike w ise show n tha t t he hyd r a t i on o f de -na tured hemoglobin in urea solut ion ca lcula ted f rom the viscos i ty ofthe so lu t ion is a bou t t he s a me a s t ha t c a l c u la t e d by Bur k a nd G r e e n-be r g f r om the i r o smot ic p r e s su re m e a sur e me n t s w h ic h show e d de na -tured hemoglobin in urea solution not to be aggrega ted. The increasein the viscos i ty of a concent ra ted urea solut ion of hemoglobin as thepr o t e in de na tu r e s is t hus p r oba b ly due t o o r d ina r y hydr a t i o n a nd no tto aggrega t ion. This increase in viscos i ty mu st not be confused wi thth e decrease in v iscos i ty which is obta ined when sol id urea i s added tothe very viscous solut ions of egg a lbumin. In such cases the urea i sdissolving aggrega tes of a l ready de na tu red prote in .

A solut ion conta ining 0 .385 gin . dena tured dia lyzed horse hemo-globin, 5.5 gm. u rea an d 10 ml. wa ter has a t 30C. a viscosity of 1.6 rela-t ive to the viscos i ty of a solut ion conta ining 5 .5 gin . urea p er 10 ml .w a te r . F r om K u ni t z ' s c u r ve one ca n r ea d of f t ha t t he so lvat e d he mo-globin occupies a volu me of 10 per cen t of the to ta l volum e or 1 .44 ml .



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350 DENATURATIONON VISCOSITY OF PROT EIN SYSTEMSSince 0.385 gm . no n-solvated hem oglobin occupies 0.29 ml. , the solva-

1.44 - 0.29t ion per gm. of hemog lobin is 0.385 or 3.0 ml. This calculat ioninvolves the assumption that so far as using Kunitz's curve is con-cerned, a solut ion of hemoglobin in urea solut ion can be treated thesame as a solution of hemoglobin in water. One can obtain about thesame value for the solvation of the hemoglobin without using Kunitz'scurve by f inding what volume of sugar has to be subst ituted for thesolid hemoglo bin in the urea solut ion to get the same visco sity.

CONCLUSIONSThe v iscosi ty o f a p ro te in so lu t ion i s increased by the dena tu ra t ion

of the p ro te in . Th is i s t rue bo th when there is the fo rmat ion of p ro-te in aggrega tes which occ lude water an d w hen there i s no aggrega t ion.Under certain condit ions, as a result of the aggregation following de-na tu ra t ion , a so lu t ion con ta in ing on ly one per cen t o f p ro te in ma y beconver ted in to a c lear ge l. The cond i t ions fo r ob ta in ing very v iscousso lu t ions con ta in ing l i t t le dena tu red p ro te in a re a lways c lose to thecondi t ions fo r ac tua l p rec ip i ta t ion and under these cond i t ious thev iscosi ty is very sensi tive to s l igh t changes in the concen t ra t ion o f sa l tsand hydroge n ions .

R E F E R E N C E SAnson, M. L., and M irsky, A. E., 1929, J. Gen. Physiol., 13, 121.Anson, M. L., and Mirsky, A. E., 1930, J. Gen. Physiol., 13,469.Anson, M. L., and M irsky, A. E., 1931a,J. Phys. Chem., 35,185.Anson, M. L ., and M irsky, A. E., 1931b, Y. Gen. Physiol., 14, 605.Anson, M. L., a nd M irsky, A. E., 1931c, ]. Gen. P hysiol., 14, 725.Burk, N. F., and Greenberg, D. M., 1930, J. Biol. Chem., 87, 197.Hopkins, F. G., 1931, Nature, 126, 328, 383.Huang, T., and W u, H., 1950, Chinese J. Physiol., 4, 221.Kunitz, M., 1926, J. Gen. Physiol., 9, 715.Kunitz, M ., 1927, J. Gen. Physiol., 10,811.Kun itz, M., an d Simms, H. S., 1928,J. Gen. Physiol., 11,641.Mirsky , A. E., and Anson, M. L., 1929, J. Gen. Physiol., 13,133.Mirsky, A. E., and Anson, M. L., 1930, ]. Gen. Physiol., 13,477.Ramsden, W., 1902, J. Physiol., 28, xxiii.Ramsden, W., 1913, Tr. Faraday Soc., 9, 93.Spiro, K., 1900, Z. physiol. Chem., 30, 182.


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