t h e i s o e l e c t r i c p o i n t of a s Tandard g e l a t i n

8/2/2019 t h e i s o e l e c t r i c p o i n t of a s Tandard g e l a t i n

1/15

T H E I S O E L E C T R I C P O I N T O F A S T A N D A R D G E L A T I NP R E P A R A T I O N '

BY DAVID I . HITCHCOC K 2(From the Department of Pkysiology, Yale University, New Haven)

(Accepted for publicat ion , Apri l 21, 1931)

INTRODUCTIONThe isoelectric point of an amphoteric substance ma y be defined asthe hydrogen ion activity of a solution or suspension in which the

amp holyte shows no migration in an electric field. This definit ion,which is quite generally accepted, is in accord with the original use ofthe term by Ha rdy (1899-1900), as well as with the definit ions givenby M ichaelis (1922) and S~rensen, Linderstr~m-Lang and Lun d (1925-28). Micha elis defines the isoelectric point, for the mos t general casewhich he considers, as the value of [H +] at which equivalent amou ntsof posit ive and negative ampholyte ions are present. S~rensen andhis coworkers define i t as the hy drogen ion activity at which the m eanvalency of the am pholyte is zero. Obviously these theoretical defini-t ions must correspond to the above experimental definit ion in terms ofzero velocity of cataphoresis.

The possibil i ty that the value of the isoelectric point m ay v ary w iththe presence of ions other than H + and O H- has been considered byS~rensen and his coworkers (1925-28). If some othe r negative ion,for example, were combined with or adsorbed b y the ampholyte, givingit a negative charge, i t might well be true that this charge could beneutralized, and the ampholyte rendered isoelectric, only at a more

1 Presented before the Amer ican Socie ty of Biologica l Chemis t s , Mont rea l ,Ap ril 10, 1931.2 Most of the exper imenta l work was done by M iss Ruth C. Belden. A few ofthe ear l ie r measurements were car r i ed out by Miss Es ther R . Mason an d by D r .Ruber t S . Anderson. 685

The Journal of General Physiology


2/15

68 6 ISOELECTRIC POINT OF GELAT INac id pH than tha t cor responding to the i soelect r ic point in the absenceof such foreign ions. In ord er for the isoelectr ic reaction to be com-pletely def ined, i t may be necessary to specify the concentrat ions ofall substances in the system, or the activit ies of al l ions, as well astha t of H +. Owing to the more m ark ed effect of H + and O H- on thecharge of amph olytes, i t seems reasonable to keep the def init ion ofthe isoelectr ic point in te rms of [H +] or pH, recognizing tha t i t m aynot be a cons tant quant i ty for any par t icula r ampholyte , but tha t i tma y v ary wi th the presence of o ther ions.

In the case of gelat in the isoelectr ic point was f irst determined byMichaelis a nd Grineff (1912) , who located i t as betwe en [H +] = 1.6 X10 -5 and 3.5 10 -~, averag e, 2.5 10 -5. In te rms of pH these figurescor respond to 4.80, 4 .46, and 4 .60. Mo st sub sequent de te rmina t ion sof this quanti ty for gelat in have been more or less indirect , and in thefew cases where ca taphores is measurements have been made the i so-e lect r ic point has not been very exac t ly loca ted. Thu s Loeb (1922)inte rpre ted his var ied observa t ions on physica l and chemica l proper -t ies of gelat in as indicating an isoelectr ic point at pH 4.7, but Kraemerand Dexter (1927) showed that very few if any of the ear l ier observa-t ions were inconsistent with an isoelectr ic point at pH 5.0, whichthey loca ted qui te exac t ly as the pH of the maximum l ight sca t te r ing(Tynd all effect) for calfskin gelat ins. Th ey showed, however , thatthe f igures obta ined depended considerably on the source and meth odof prepara t ion of the ge la t in. The wr i te r (1928-29) found tha t age la t in sample f rom the same source as tha t used by Loeb, pur i f iedaccording to Nor throp and Kuni tz (1927-28) , had minimum osmot icpressure and max imu m opac i ty a t p H 5.05 q- 0 .05, which was a lso thepH of solutions of this gelat in in water .

Some of the discrepancies in previous results are probably due todifferences in the gelat ins used, and others to the more or less indirectna ture of the methods . The present paper repor ts a s tudy of theisoelectr ic point of samples prepared according to def inite specif ica-t ions of a committee (Davis, Sheppard, and Briefer , 1929; Hudsonand Sheppard, 1929) of the Leather and Gelatin Division of theAmerican Chemical Society; hence i t should be possible for workersin othe r laborator ies to obtai n identical mater ial . Th e isoelectr icpoint of such ge la t in has a l ready been repor ted by Sheppard and


3/15

DAVID I. HITCHCOCK 687Ho uck (1930) to be at p H 4.9 ~- 0.10 by electric migrat ion, and a t p H4.9 q- 0.05 both by l ight scat tering and by alcohol precipi tat ion. Theresul ts of the presen t work confirm those of these workers within theirl imits, bu t since somew hat different meth ods were used, and since theresul ts perm it of a m ore precise locat ion of the isoelectric point , i t isbel ieved that a detai led report of the work may be of interest .

The gelat in used was prepared, according to the specificat ionsment ioned, in the laborator ies of the Eas tman Kodak Compan y.8

IIIsoelectrlc Point from pH Values of Pure Gelatin Solutions

It was shown by SCrensen (1912) th at the p H of a pure solut ion of as imple ampholyte in water must l i e be tween tha t of pure water andthat of the isoelectric point of the ampholyte, approaching the lat terfigure as the concentrat ion is increased. Th us he calculated tha t for amolar solut ion of glycine the pH should differ from th at of the isoelec-tric poin t by only 0.001 pH. Similar considerat ions oug ht to hold for acol loidal ampholyte l ike gelat in or any other protein, even though i tmay not be possible to calculate the pH, as S~rensen did, from thevalues of the ionizat ion constants. Hence i t seemed reasonable tomeasure the p H of a series of gelat in solut ions of different concentra-t ions , wi th the expec ta t ion tha t the va lues would approach a cons tantfigure, that of the isoelectric point , as th e concentrat ion was increased.

The p H measurements were made wi th hydrogen e lec t rodes a t30C., using a KCl-agar junct ion. The pH v alues are based on thefigure 1.075 for 0.1000 m olal HC1, l iquid junct ion potent ials beingassumed cons tant .

The f i rs t measurem ent of a 1 per cent solution (Eas tman StandardGelatine, Lot 1) yielded the surprisingly low value of pH 4.61. Thiswas found to be due to an acid, presumably acet ic , which was quitecomple tely removed from the ge la tin by fur ther washing of a 5 gm .sample with 4 changes, about 1 l i ter each, of cold dist i l led water.After such washing, the gelat in gave solut ions of higher pH values,which were, for con centra tions of 1, 2, 5, an d 12 gm. per 100 cc., 4.90,

The writer is indebted to Dr. S. E. Sheppard for two samples of "EastmanStandard Gelatine."


4/15

688 ISOELECTRIC POINT 0]~ GELATIN4.88, 4.87, and 4.86 respectively. The se values are show n graph icallyin Fig. 1, in which the pH values are plot ted agains t the reciprocals ofthe gelat in concentrat ions in gm. per 100 cc. The value obtainedfrom this figure by extrapolat ion to infini te concentrat ion of gelat inis pH 4.86 -4- 0.01, and it is this value which is inferred to be the iso-electric point of the gelat in.

This method for determining the isoelectric point , as well as thatused in the fol lowing sect ion, implies that the ampholyte used~mustbe pure, or at least free from appreciable amounts of ions capable of

i

i

4.89

4~8

pH 4.87 ~ ' ~

4115

0

f ~ JJ

o~ a z ~3 0.4 ~5 0.6 o~7 o~ o.9 I.oP~ciprocaf of ~e l~n concer,~afion in ~m .M r ,ob c.FIG. 1. pH va lues o f so lu t ions o f s tandar d ge la t in in d is t i l led wa te r a s a func t ion

of the rec ip roca l o f the ge la t in concen tra t ion . By ex trapo la t ion to in f in i te con-cen tra t ion , the p H of the isoe lec tr ic po in t i s e s t ima ted as 4 .86 ~ 0 .01 .combining with i t or al tering i ts charge. Loeb (1922) has poin tedout tha t a prote in i s most readily obta ined in such a s ta te of pur i tyby washing or dialysis at the pH of i ts isoelectric point . The m etho dused in p urifying the gelat in used in these experiments involves washingat about pH 4.7, which is not the value obtained for the isoelectricpoin t ei ther of this gelat in or of that studied in the wri ter 's form er work(1928-29). Theoret ical ly i t should be possible to remov e the lastt races of electrolytes by prolonged washing with w ater, since proteins


5/15

DAVID I. HITCHCOCK 689are weak electrolytes and their sal ts should therefore be hydrolyzable.That the washing of the gelat in here studied actual ly had this effectis indicated b y th e low ash co ntent of the m aterial , which was 0.04 to0.05 pe r cen t, as well as its low specific con duc tivity , which was 3.8 X10 5 reciprocal ohms for a 5.6 p er cent solut ion at 30C. and 5.0 X10 5 for a 9.4 per cent solut ion. Mo reover the me thod s of SectionsII and I I I of thi s paper , which depend on the ini t i a l pur i ty of thegelat in, gave resul ts in agreement with those of Sect ions IV and V,which do not requi re tha t the ge la t in be absolute ly pure a t the s ta r t .

I I I

Isoionic Point by pH Values of Wea kly Bu~ered Gelatin SolutionsS~rensen (1912) pointe d out th at the am oun t of acid or base requiredto bring a solut ion of an ampholyte to i ts isoelectric point should be

independent of the ampholyte concent ra t ion and ident ica l wi th theamo unt requi red to br ing an equa l volume of pure water to the samepH. He la te r (1917) applied thi s metho d to the de te rmina t ion of theisoelectric poin t of carefully purified egg albumin. In a st i l l la terpaper S~rensen, Linderstr~m-Lang, an d Lun d (1925-28) showed thatthis method would not necessari ly give the isoelectric point i f theampholyte were capable of combining wi th ions other th an hydrogenor hydroxyl . Accordingly they def ined a new q uant i ty , th e i soionicreact ion, as the hydrogen ion ac t ivi ty* a t wh ich the qu ant i ty of a ddor base comb ined with the amph olyte is zero. It is the isoionic ratherthan the isoelectric reaction which is given by this met hod of S~rensen.The me tho d was sl ight ly modified by Michael is (1912), w ho deter-min ed th e isoelectric points of the soluble am pholy tes phen yl alanineand glycocoll by m easuring the pH of di lute acetate buffers with andwitho ut the ampholyte. On the acid side of i ts isoelectric point thepH of the buffer was raised by the ampholyte, on the alkal ine side i twas lowered. Michael is was thu s able to locate the isoelectric poin twithin a few tenths of a pH unit .

I t was sugges ted by Sheppard (1929) tha t thi s buffe r meth od might4 Sg~rensen'sdistinction between pH and p al l is here disregarded, as the stan dard

of pH used in this paper is based on an activity coefficient for HCI and not onconductivity measurements, as was S~rensen's original definition of pH .


6/15

690 ISOELECTI~IC POINT OF GELATINwel l be appl ied to ge la t in . Accordingly measu rements were made ofpH in ace ta te buf fe r s of var ied concent ra t ion, made up in each casewi th and wi tho ut ge lat in . The ge la t in used in these exper iments(Eas tman Standard Gela t ine , Lot 48) had been more thoroughlywashed a t the t ime of prepara t ion, and no apprec iable am ounts of ac idcould be removed from it by fur ther washing. The pH measu rementsin th is case were made a t 30C. wi th the h ydrogen e lec trode appara tusof S imms (1923) , in which contac t i s made wi th sa tura ted KC1 in anope n stop-cock. The s tand ard used was again pH 1.075 for 0.1000molal HCI. Th e results are given in Table I .

To f ind the isoionic point of the gelat in, curves were obtained byp lo t t i ng t he c ha nge s i n pH p r oduc e d by t he ge la t in a ga ins t t he pH o fbuffer solutions of the same concen trat ion withou t gelat in. Fig. 2,which shows the da t a of Exper im ents 3 and 7 of Table I , i s i l lus t ra tiveof the na ture of al l the curves, the others be ing equal ly smooth. Foreach experiment the isoionic point is given by the intersection of thecurve with the l ine of zero change in pH. The f igures so obta ined aregiven in the las t column of Table I . The i r mean va lue i s 4 .85, wi than average deviatio n of 0.01. I t is to be not ed tha t this value isindependent of the ge la t in concent ra t ion wi thin the l imi ts s tudied(1 to 4 per cent) and of the sal t concentrat ion up to an ionic strengthof 0 .105 (Exper iment 7). Hence i t ma y be conc luded tha t wi thinthese l imits the isoionic poin t of this gelat in correspon ds to pH 4.85 -4-0.01.

IV

Isoelectric Point by Maximum Turbidity of Gelatin GelsWhile the def ini t ion of the i soe lec t r ic point says nothing about

turbidi ty , i t i s genera l ly accepted tha t an ac idi ty cor responding to theisoelectr ic poin t produces a ma xim um of l ight scatter ing in gelat ingels. In a previo us stu dy (1928-29) of anot her gelat in preparatio n,i t was found tha t the pI - [ of maximum opac i ty was ident ica l wi ththa t of minim um osmot ic pressure , and the la t te r va lue has been shownby Loeb (1922) to be theoretically identical with the isoelectr ic point .Accordingly i t seemed wor th whi le to inves t iga te the behav ior of thes tanda rd ge la t in wi th respec t to turbidi ty . The method adopted was


7/15

DAVID I. HITCI-ICOCK 691T A B L E I

Isoionic Reaction of Standard Gelatin by pH of AcetateGelatin Buffers with and without

E xp. no . Nor m a l i ty o f Naacetate in buffer

0. 001

0.001

0.001

0.005

0, 005

0. 005

Gela t in pe r100 cc.gm .1 .0

2 . 0

4 . 0

1 .0

2 .0

4 . 0

b pH ofuf fer with-ou t ge la t in

4 .474 ,674 .855.085 .244 .474 .674 .855.085 .244 .474 .674 .855 .085 .274 .034 .264 .474 .654 .865 .055 .275 .525 .744 .474 .654 .865 .055 .274 .444 .634 .834 .995 .25

pH o tbuffer wgelatb

4 .704 .794 .864 .904 .954.754.814 .854 .884 . 9 04 .804 .834 . 8 64 .874 ,874 .194 . 3 94 .564 .714 . 8 64 .985 .095 .185 ,244 .664 .774 .864 . 9 44 .994 . 6 64 .754 .834 .894 .94

Change in pHth pH due to of isoionlcgelatin reaction

+ 0 . 2 3 4 . 8 4+ 0 . 1 2+ 0 . 0 1- 0 . 1 8- 0 . 2 9+ 0 . 2 8 4 . 8 5+ 0 . 1 4

0 .00- 0 . 2 0- - 0 . 3 4

+ 0 . 3 3 4 . 8 6+ 0 . 1 6+ 0 . 0 1- 0 . 2 1- - 0 . 4 0+ 0 . 1 6 4 . 8 6+ 0 , 1 3+ 0 . 0 9+ 0 . 0 6

0 .00- O . 0 7- - 0 . 1 8- - 0 . 3 4- - 0 , 5 0+ 0 . 1 9 4 . 8 8+ 0 . 1 2

0 .00- -0 .11- - 0 . 2 8+ 0 . 2 2 4 . 8 4+ 0 . 1 2

0 .00- 0 . 1 0- -0 .31


8/15

692 ISOELECTRIC POINT OF GELAT INTABLE I--Concluded

E xp. no . Nor m a l i ty o f Naacetate in buffer

0.005, in 0.1 NKCI

Gclatin pcr100 cc .

1. 0

pH ofbuffer with-out ge la t in

4 . 3 74.564.774.975.21

pH of Change inbuf f e r wi th pH due toge la t in ge la t in

4.53 +0.164.67 +0.114.80 +0.034.91 --0.065.01 --0.20

p Hof isolonicreaction

4.84

Ave ra ge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 .8 5

* 0 . 3

+0.2C

00

~--ase. -0.2~0C

U

\ \ \

r \ \ \4.6 4.7 4.8 ~.9 s.O S.I4 .5 pH o~ buffer wihou~ ~lcLatin

FIG. 2. Change in pH of acetate buffers, produced by gelat in in solut ion, as afunct ion of pH of buf fer s of the same concentra t ion wi thout ge la t in . These curvesshow the da ta for Exper iments 3 and 7 (Table I ) . The i so ionic poin t i s g iven bythe intersect ion s with the l ine of zero change, and is at p H 4.85 4- 0.01.the s imple one descr ibed in the previous paper , wh ich consists s implyin the inspec t ion in o rdinary dayl ight of samples of ge la t in solutions ofequal volume and var ied pH which have been a l lowed to se t in uni formtes t tubes in a re f r igera tor . The pH va lues were de te rm ined a t 30C.af te r mel t ing the ge ls in warm water .


9/15

DAVID I. ttITCHCOCK 693In the first experiment Lot 1 of the standard gelatin was used, after

the excess acid had been removed by washing, as already described.The concentration of the gelatin was 1 gm. per 100 cc., and the p H wasvaried by the addition of very dilute HCI. The gel showing maximumopaci ty was in 0.0001 N HC1 and had a pH value of 4.85, while thoseadjacent in the series were in 0.00005 and 0.00015 N HC1, with pHvalues of 4.86 and 4.82 respectively. Before this gelatin was washedfree from acid, the maximum turbidity of 0.2 per cent and 1.0 per centsolutions was found to be at pH 4.85 and 4.87 (0.05) respectively,the tubes in this case requiring the addition of dilute NaOH to producethese pH values.

In the second experiment the gelatin used was from Lot 48. ThepH was varied by acetic acid in 0.001 N sodium acetate, the solutionsbeing those of Experiments 2 and 3 in Table I. In each case theturbidity was at a maximum in the middle solution of the series, sothat from these observations the pH of maximum turbidity may beplaced at 4.85 0.03.

V

Isoelectric Point by Electrical MigrationBy definition the most direct way to determine the isoelectric point

is by determining the pI-I corresponding to zero migration in an electricfield. Previous determinations in the case of gelatin have been madeby the macroscopic U-tube method of cataphoresis, and the results, asalready mentioned, have not defined the isoelectric point ve ry exactly.Since it had been shown by Loeb (1922-23b) that collodion particlessuspended in a gelatin solution behaved in migration experiments asif they were particles of gelatin, it seemed likely tha t such suspensionsmight be used for an exact determination of the isoelectric point.Similar determinations have been made in the case of egg albumin byAbramson (1928), who used quartz particles which behaved as ifcoated with the protein.The migration was conducted in a cell of the type described byNorthrop and Kuni tz (1924-25, see also Mudd, 1928), the source ofpotential being a radio "B" b att ery of nominally 135 volts. Observa-tions were made with a microscope equipped with an 8 mm. objective


10/15

694 ISOELECTRIC POINT OF GELA TIN(20 X) and a 10 X ocular. At f i rst direct i l luminat ion was used andver y few par t i c les were v i sib le . Late r the v i s ib il i ty was great lyincreased by the use of a Lei tz Model E dark field condenser, whichhas ample working dis tance for a cel l of this thickness. I l lumina t ionwas obta ined from a Zeiss microscope lamp containing a I00 wa tt ,110 vol t project ion bulb.

The col lodion suspension 5 was prepared as descr ibed by L oeb (1922-23a). Tw o drops of this suspension were adde d to 100 cc. of 0.1 percen t gela t in made up in d i lute ace ta te buffers o f var ied concent ra tion .The gela t in was Lot 48 of the s tandard prepara t ion . The pH deter -minat ions were made a t 30C. in the Simms hydrogen e lec t rode vessel s ,whi le the migra t ion exper iments were made a t room temperature , 20-22C. Read ings were taken at s ix different levels in the cel l, spacedat I , 3 , 5 , 7 , 9 , and 11 twelfths of the total thickness, veloci t ies beingdetermined wi th a s top-watch and an ocu lar micrometer scale . Whe nthe veloci t ies for a given suspension were plot ted against the depthsin the cel l , the poin ts fel l fairly close to parabolas , as dema nded by th etheo ry of Smoluchow ski (1914). The devia t ion of individual pointsf rom a smooth curve was such that i t seemed safer to t ake the t rueveloci ty as the average of al l those observed , ra ther than to t ake read-ings at only that depth (0.211 of the total thickness from ei ther topor bo t tom) which should theore t i ca l ly g ive observed veloci t i es equalto the average , e I t ma y be no ted tha t the average of velocit i es a t s ixlevels gives a f igure which is larger by 1.35 per cent than the t rueaverage heigh t o f a parabola . No reversa l o f the mot ion near thewal l s o f the ce l l was observed , such as tha t no t iced by Svedberg andAndersson (1919) for certain inorganic sols. The para bol ic curvesthrough the observed po in t s a lways ex t rapola ted to zero veloci ty a tthe walls of the cel l . This is in accordance with th e conclusion ofAbramson (1929-30) and means s imply that the pro te in was adsorbedon th e walls of the vessel as well as on the part icles .

The veloci t ies are expressed as obser ved in # per second. Th ey haveThe writer is indebted to Dr. M. Kuni tz for the collodion suspension used in

these measurements.6 This theoretical prediction is due to Smoluchowski (1914). It may be addedthat the same theory predicts that the average velocity should be equal to 2/3 o fthe velocity observed at the middle level in the cell


11/15

DAVID I. HITCHCOCK 695

not been reca lcula ted to uni t potent ia l g radient because the reproduc i -bi l i ty of the observed ve loc i t ies was much higher th an th a t obta inedin a t tem pts to ca l ibra te the ce l l by the meth ods descr ibed by previousworkers (Nor throp and Kuni tz , 1924-25; Abramson, 1928-29). Thepotent ia l drop in the cel l, a s obta ined by the var ious methods of ca li -bra t ion, appeared to vary be tween 7 and 10 vol t s per cm. I t i s be-l ieved tha t these la t te r var ia t ions a re not r ea l , but a re due to the in-adequ acy of the ca l ibra t ion. An approximate reduc t ion of the ob-served ve loc i t ies to uni t potent ia l grad ient may be obta ined by dividingthem by 8 .5 .

Each exper imen t was run in dupl ica te wi th separa te mixtures . Theresul ts a re given in Table I I .

The table shows tha t the agreement of pH va lues in the dupl ica teexper iments was a lmost per fec t , a s might be expec ted for buf fe redsolutions. The a gree me nt of catap hore tic velocities, while lesssa t i s fac tory, i s such tha t the dup l ica te de te rmina t ions m ay sa fe ly beaveraged. The average ve loci t ies were plot ted on a la rge sca le aga ins tthe average pH va lues for each concent ra t ion of sodium ace ta te , anda sm ooth curve was d rawn b y me ans of a f lexible spline he ld by weightson a ll of the points . I t w as a t f i r st thoug ht tha t the points of eachexper iment could bes t be represented by a s t r a ight l ine , but a f te r a l lthe da ta were plot ted a cons is tent devia t ion f rom l inear i ty was ob-served in every exper iment . In order to ge t a curve to pass througha l l the points , i t was necessary to draw it somewh at S-shaped. Thecurve of Fig. 3, which shows the results in a buffer 0.001 with respectto sodium ace ta te , i s typica l. The points indica ted by c rosses wereobta ined f rom a separa te exper iment , not g iven in Table I I , whichwas done a f te r the curve was drawn. The da ta show no indica t ion ofa f la t tening of the curves a t the i soe lec tr ic point , such as would beexpected if the isoelectr ic region were not sharply def ined.

From each exper iment a t a g iven sa l t concent ra t ion, the pI- I of theisoe lec t r ic point was obta ined by the in te r sec t ion of the curve wi ththe l ine of ze ro ve loc i ty . The va lues found in th is way a re given inthe las t column of Table II , the average be ing pH 4.80. The da taare probably n ot ce r ta in enoug h to war ra nt th e infe rence of a t r end inthe va lues wi th sa l t concent ra t ion, a l though the va lues a t the lowerconcen t ra t ions a re s l ight ly higher. I t can only be conc luded tha t the


12/15

696 ISOELECTRIC POINT OF GELATINcatapho ret ic isoelectric point of g elat in-coated col lodion part icles is atpH 4.80 4- 0.01, a value which differs by 0.05 from th at obtained in

T A B L E I IIsoelectric Reaction o f Standard Gda tin by Cataphoresis of Collodion Particles in 0.1

Per Cent Gelatin in Acetate BuffersTo tal E.~.F. = 133 to 137 volts .Pot en t ia l g rad i en t in ce l l = 8 .5 vo l t s per cm. (approx imate on ly) .T e m p e r a t u r e , 2 0 - 2 2 C .p H v a l u e s a t 3 0 b y h y d r o g e n e l e c t r od e .Algebra ic s ign of ve loc i ty i s tha t o f charge on par t ic les .

Conc. N~acetate

xt

0.001

0.003

0.005

0.010

0.020

O. 030

0.040

R at io o f ace t ic acid to N a ace ta te

1.85pH # per pH~ec.

4.52 +4.08] 4.694.53 +3.381 4.704.684 .47 +4 .00 ! 4 .654.46 +4.321 4.644 .44 +3 . 38 4 .624 .44 +3 .23 4 .624 .41 +3 . 18 4 .594 . 4 0 +2 . 9 4 4 . 5 9 [4 .3 9 + 2 . 3 4 4 . 5 8 !4 . 4 0 1 + 2 . 8 9 4 .5 8 I

I4.45 I +3 .4 7 4 .644 .47 [ +3 .70 4 .644 .44 I +3 .78 4 .624 .46 [ +3 .29 4 .64

1.20u per pH5ec.

+1.77 5 .01+1.78 ! 5 .01+2.07 5 .00+2.2 4 5 .01 I+2 . 0 0 5 . 0 0 I+1 . 7 4 5 . 0 0+1 . 8 4 5 . 0 0 I+1 . 7 5 4 . 9 7+ 1 . 6 2 4 . 9 8 1+ 1 . 5 8 4 . 9 7+1 . 4 8 4 . 9 7+1 . 7 5 5 . 0 0+1 , 8 5 5 . 0 0+1 . 6 3 4 . 9 9+1 . 8 5 5 . 0 1

0.48 O.per sec. pH

--2.97 5.15--2.96 I 5.15- -2 .97- -2 .24 5 .18--2.42 5.17- -1 ,68 5 .18--2,05 ] 5.18- -1 .44 5 .16- -1 .51 5 ,18- -1 .45 5 .17- -1 .26 5 .17- -2 .35 5 .17- -2 .20 5 .17- -2 .17 5 .16- -2 .02 5 .19

A ve ra ge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .* I n t h e s e c o n d e x p e r i m e n t w i t h 0 .0 1 0 ~ a c e t a t e t h e g e l a t i n c o n c e n t r a t i o n w a s

only 0 .01 per cen t ins tead of 0 .1 per cen t .the previous sect ions for th e isoelectric or isoionic poin t of gelat in insolut ion.


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D A V I D I . H I T C H C O C K 697The cau se of th i s d i f fe rence i s not g iven by the present da ta . One

might specula t e tha t t here i s an e f fec t of s a lt on the i soe lec t r ic point ,t ending to lower i t s pH , w hich does not van i sh even a t 0 .001 ~ , or th a tposs ibly the col lodion se lec t ive ly adsorbs more of the he a t coagulable

\~+IO.,i t \

\4.6 4.7 4.8 4 9 5.0 ~!pH

FIG. 3. Velocity of cataphoresis of collodion particles in 0.1 p er cen t gelatin inacetate buffers as a function of pH . The isoelectric point is given by th e inter-section of the curve with the line of zero velocity. This curve sho ws the resultswith a buffer containing 0.001 ~r sodium acetate, th e intersection being at pH 4.81in this case (Table II).prote in~in the ge la t in , which has been shown by Sheppard, Hudson,and H ou ck (1931) to hav e an i soe lec t ri c point i n the v ic ini ty of pH 4.0 .

I t m a y be no t e d t ha t none o f the a bov e m e t hods g i ve s a ny i nd ic a ti onof the exi s t ence of two i soe lec t r ic point s a t w ide ly separa ted pH va lues .The infe rence of Johl in (1930) tha t ge la tin ap paren t ly h as two i so -


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69 8 ISOELECTRIC POINT OF GELATINe lec t ric points a t pH 4.68 an d 5 .26 was m ade f rom the in te r sec t ions ofcer ta in viscosi ty curves . The m ore di rec t meth ods of the presentwork lend no suppor t to such an assumpt ion.

VISUMMARY AND CONCLUSIONS

Two samples of a s tandard ge la t in were s tudied, both preparedaccording to published specif ications and washed free f rom diffusiblee lec t rolytes . The i soe lec t ric point of th is mate r ia l was de te rm ined inf our w a ys .

1 . The pH va lues of solut ions of ge la t in in wate r approached thelimit 4.86 i 0.01 as the co ncen trat ion of gelat in was increased.

2 . The p H va lues of ace ta te buf fe r s were unchanged by the addi t ionof gelat in only at pH 4.85 + 0.01. This gives the isoionic poin t ofS~rensen, which i s the i soe lec tr ic point wi th respec t only to hydro gena nd hydr oxy l i ons.

3. Gels of this gelat in ma de up in dilute HC1 or NaO H, or in diluteacet ate buffers, exhib ited maxi mu m turb idity a t pH 4.85 -4- 0.03.

4. Very dilute suspensions of collodion par t icles in 0.1 per centge la t in solut ions made up in ace ta te buf fe r s showed zero ve loc i ty incatapho resis expe rime nts only at pH 4.80 + 0.01.

No evidence was found for the assumpt ion th a t ge la t in has twoisoe lec t ric points a t wide ly separa ted pH va lues .

I t i s conc luded tha t the i soe lec t ric point of th is s tan dard ge la t in i snot f a r f rom pH 4.85.

BIBLIOGRAPHYAbramson, H. A., 1928, J. Am. Chem. Soc., 50, 390.Abramson, H. A., 1928--29, J. Gen. Physiol., 12, 469.Abramson, H. A~, 1929-30, J. Gen. Physiol., 13, 657.Davis, C. E., Sheppard, S. E., and B riefer, M., 1929,Ind. and Eng. Chem., Analyti-cal Edition, 11 56.Hard y, W. B., 1899-1900,Proc. Roy. Soc. London, 661 110.I-Iitchcock, D. I., 1928-29, J. Gen. Physiol., 12, 495.Hudson, J. H ., and Sh eppard, S. E., 1929,Ind. and Eng. Chem., 21,263.Johlin, J. M., 1930, J. Biol. Chem., 861 231.Kraemer, E. O., and D exter, S. T., 1927,J. Physic. Chem., 31,764.


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DAVID I. HITCHCOCK 69 9Loeb, J., 1922, Proteins an d th e th eory of colloidal behavior, New York an d Lon-

don, McGraw-HiU Book C ompany, Inc., (also 2nd ed., 1924).Loeb, J., 1922-23 (a) J. Gen. Physiol., 5, 111.Loeb, J., 1922-23 (b), J. Gen. Physiol., 5, 395.Michaelis, L., 1912, Biochem. Z., 47~ 250.Michaelis, L., 1922, Die Wasserstoffionenkonzentration, Berlin, J. Springer, 2nd ed.,124.Michaelis, L., and Grineff, W., 1912, Biochem. Z., 41,373.Mudd, S., Luck6, B., McCutcheon, M., and Strumia, M., in Weiser's Colloidsymposium monograph, New York, The Chemical Catalog Comp any, Inc.,1928, 6~ 131.Northrop, J. H., and Kunitz, M., 1924-25, J. Gen. Physiol., 7, 729.Northrop, J. H., and Kunitz, M., 1927-28, J. Gen. Physiol., 11,477.Sheppard, S. E ., 1929, in discussion at a meeting of the Standa rd GelatinCommittee.Sheppard, S. E., an d Houck, R. C ., 1930,J. Physic. Chem., 34, 2187.Sheppard, S. E., Hudson, J. H., and Houck, R. C., 1931, J. Am. Chem. Soc., 53,760.Simms, H. S., 1923, J. Am. Chem. Soc., 45, 2503.Smoluchowski, M. yon , 1914, in Graetz, L., H andbuc h der Elektrizitiit und desMagnetismus, Leipzig, J. A. Barth, 2~ 382.S~rensen, S. P. L., 1912, Ergelm. Physiol., 12, 495.S~rensen, S. P. L ., 1917, Compt. rend. tray. Lab. Carlsberg, 12~ 68; also in Z. physiol.

Chem., 1918, 103~ 104.S~irensen, S. P. L., Linderstr~m-Lang, K., a nd Lun d, E., 1925-28, J. Gen. Physiol.,8, 543; also in Compt. rend. tray. Lab. Carlsberg, 1925-27, 16, No. 5.Svedberg, T., and Andersson, H., 1919, Kolloid Z., 24~ 157.

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t h e i s o e l e c t r i c p o i n t of a s Tandard g e l a t i n