88
GEORGE SCATCHSRD AND ELIZABETH S. BLACK
(34) TAYLOR, H. S.: J. P l i y ~ C’liein. . 30, 145 (192G). (35) VESELOI’SKII, \.. S.:Issledovuiiiya Fiziko-Kliini. tclili. duspensii 1933, S:J; Clieiii. Abstracts 30, 7964 (193G). (36) J \ I C I S E R ,I f . 13.: Irtoi,ga/kic Colloid Ciiciikistty, Vol. 2, p . 323. .John \Vile). ailti Soils, Inc., Sc\\. Ywlc (lW5). ( R i ) \ \ ’ I J ~ N E I ~D, . : Tixiis. Fai,ad:ty doc. 21, 3SI (1025-26). (38) \f’HITEYErJL, IT. -I., . A S D F R A Z EJ. Rc. , J . Alll. Chelll. SOC. 46, 2841 (1928i.
w.:
THE EFFECT OF SALTL‘Sos THE ISOIOXIC ASD ISOELECTRIC! POINTS O F PROTEINS’ t2
GEORGE SCATCHARD
AND
ELIZABETH SACICMANX BLACK8
Depnrliirent of Cheitkistry, ~llassacliztsellsInstitute of Technology, Cambridge, Jlasscrch nsetls
Rcceic’cd .I
rcglisl 19, 1948
The term “isoelectric point” has been used with many different meanings, and the term “isoionic point” was gil-en two definitions in the paper in which it \ u s first introduced (11). By isoelectric point we shall mean the pH a t which there is no net motion in an electric field. Obviously, each component of a mixture has its own isoelectric poirit and t,he ni xture has none. Alberty (1) has recently discussed mixtures with isoelectric points very close together. To define isoionic point we shall first define an isoionic ninterial as one which gives no non-colloidal ions other than hydrogen and hydrosyl. This limits us t o aqueous solutions, but it seems a little clearer than a more general definition. Operationally \ y e niay define the isoionic material as the limit approached by successful electrodialysis. Obviously il mixture may be isoionic. The definition of isoionic point) stressed by Sgrensen and his coworkers is the pH which is independent of the amount of isoionic material added. They also define it, lionwer, as the pH of n solution of the isoionic material in water, or in a solution of mother solute which does not, produce hydrogen or hydroxyl ions when dissolved in waiier alone. It is this second definition which we shall use, but let, us first consider t’he three points for some siniple cases. C‘onsitler a one-component protein for which the constant of each of its dissoI l’reseiitccl a t tlic Tn.eiitj--second National Colloid Symposium, which was licld under. t h e :iuspices of tlic Division of Colloid Chemistry of the American Chemical Society a t Canihridge, Massachusetts, June 23-25, 194s. 2 RIuch of this pnpvr is from the R1.S. Thesis of Elizabctli ;If. Sacliniann ( M r a . Donald Black), 1Iassuchusetts Iiistitu‘cc of Tecliiiology, 19-i:. The products of plasma fractionation rinployctl in this 1vorIc wel’edeveloped froin hlood collected by the American Red Cross by t,he Depnrtrnent of Physical Chcniist,ry, Harvard hfedicnl Scliool, Boston, hiassachusettu, undcr a contract recomniended by the Committee on Medical Research betwvecii the Office of Scient,ific Research and Development and Harvard University. 3 l’rescnt uddress: Depurtinent o f Medicine, Hnrvnrd Medical School, Boston, l[assaclinsc. t t s .
ISCIIOSIC' . W D IhOELECTRIC' I'OISTS
OF PROTEINS
S9
ciatioils is independent. of its cuiiceiit ration. Then t8heisoelectric poilit, and the first isoionic point will be identical. The second isoionic point will vary bet\vnen 7 for infinit,ely (Mite solutions t o the isoelectric point for infinitely concentmtetl ones. At) concentrations at \vliicll \\.e usually mslw ineasiirements, the differciice betneen the tn.o isoionic points is much smaller than t'he deviations of real protein solutions from our ideal picture. Figure 1 shoivs the change of hydrogen-ion concent ration per unit cuiicentr&tion of acid as a function of pH for ideal solut'es of varying acid strength. For strong acids it is one-half at pI1 i ,owing t u t'he louffering action of the mater, b u t increnees rapidly to iinit,y as the pI-1 decreases. For weal; acids it is one-third
at the p l i of t'he acid, approaches zero qiiit,e rapidly a8tlower values of the pH, and approaches the ciir1.e for sti,ong acids quite rapidly at, larger values. So ti protein \vith isoionic point less t'han 7 must, have some groups which dissociate at, a pH less than the isoionic point, antl only these groups will contribute much to tmhechange from neiil i-nlit,y. Consiclei, t,he, idealized ovalbumin of Cannan, Ijrclosely the same effect on the iwiunic p H , h i i t tlic effect is m u c h less than for sodium cliloikle at the same ionic* s t rengtli, thougli the diloi~icle-ion conceritiztion is only one-third less. 'l'hc cliffei.cnce l)et\\,een c*dcium rhloidu and sodium cliloride is eTwi greLitrr nt pH 7.4. 'I'hese results indic;ite t h a t the difference is not due t o combination of tlie ullinline w.rtli cation \\,it11 dhiiniin. I t might arise from the higher t'einis i i i tlir elect iust8at'icdtheoiy. Table 1 also includes the results of u f'e\v ~neusiii'enieiit~ in& oil n 1 i ) u i n i i i \\,liicli had been extractled \vitli nietliaiiol to reduce the lipid coiiceiiti,~~t,ioii.This treatment,, \vhich renio\.es about 1 mole of fuLtt,y acid per mole of all)umiii, shifts tlie isoionic point about 0.1 pH unit, in \vat,er or itt any conaentiyitioii of the salts mensui.etl, sodium chloride, sodiiim cyanide, sutliuin t i~ic,lilui.oncetnt.e, mcl calciuni chloride. For t.he sake of completeness, we are including some measurements with 2 = 44.7, p1-I 3.32. The difference between zinc chloride oil the one liantl and magnesium chloride, calcium chlwide, antl strontium chloride on the other indicates u specificit,y probably caused by binding the zinc ion. We liuve not been able to liiintlle these results a t Ion. pH theoretically. This may be explained by inaclequacy of t'he theory or by insufficient accuracy a t the higher acid concentr''1 t '1011 :mtl on the yery steep part of t'lie titr at'ion curve. The measurement's at higher p H give a typicsl Hofmeister series nith very large apparent binding for uiions of tlie acids used as protein precipitants. The great difference betn.een a large organic cation and a large organic anion is shown by the belia\.ior of ti~imetliyl~~lienylaiiimonium iodide aiid sodiuni 2,-toluenesulfonahe. The 1:alues of one hundred or more anions combined \\.it11 one albumin molecule should not lie taken t'oo seriously \vitliout further \ v o l k . There is e\-itlenve, lio\\.evei,,mucli of \\.hich is still iinpul)lished, that fur many such ions one eyui1.nlent \\dl react \\,itti each :mino group of' the proteiii (2,7 ) , so thnt, tlicrc should be appi.osiniateljr one liuncli~etluiiions foi*each alhiimin niolecule. Elmi if tjtic method sliuiild n u t j pi*o\*ecapable of ixetinement t o give ia qliniititnti\.e iiiensui'r of the extent of conilination in concentrated soliit,ions, it siiu{ilil pro1.e 17ei.y useful for a quicli qiinlitntii-e s u n ~ e yof the t'endency for i*eaction 1)et'n.eeiiproteins and ions. If the parameters n antl 6 are not kno\vn for another protein RS they are for serum ull)uniin, the method can still gi1.e a ineasiii'e of the re1ati.i.e tendencies t'o combine \\.it11 different anions. i.4
.IPPENDIX
(3)
T o calculate the purely electrostatic cont'ribut'ion to the change of tlie isoionic
pH with ionic strength, it is convenient to take the isoioriic protein Po,t,o \\-rite the general reaction
+
Po hHf = PHif (pH;+); (Po) = K,,(H+)h ('7) and t o let' k haire negative values as well as positive, ranging from the maximuni I)ase-liintling capacit'y through zero to t'lie maximum acid-binding capacity. The
08
GEORGE RC.\TCH.\RD
-4ND ELIZABETH S. BLACK
summation sign, 2 , will represent the sum over all this range. (This is quite different, from the summation in equation 2.) Then ZK/,(H’)’’h’ 2Ki,(H‘)’’h h2 = Zliii(Ht)”hhL /,R11. = (8) SZ\‘j,(H+)” ’ ZZi/,(H+)IL’ Z K j L( H+)T We shall msiiine that’ t,hc constants are functions of the ionic xtrengt’h,1 / 2 (and tempernt lire). Then 1
By the definition of w, tl_ In _K , _- h2 _ dw~ dl dl
By t,he rules of partial differenti at’ion
At the iwionic point 7;
=
0, and
ISOIONIC AND IdOE;LE;CTHI(' POINTS O F P R O T K I N S
It is convenient to take By equation 2
RS
99
vayiable the square root of the ionic strength, \/I/g.
In 0.15 a l l sodiiuu chloride \ y e found:
F.
=
0.066
+ 13.39(5.37 - PH)+ 5.440(5.31 - pH)'
(19)
= 3.73, E = -2.06, and (dpH/ddF/%)E = 0.1. Experimentally we found about 0.15 a t 0.15 M . The large discrepancy is in the more dilute solutions.
A t the isoionic point li?
REFEREXCKS (1) ALBERTY, R. A . : J. Alii. Cheiii. SOC.70, 1675 (1948). (2) ARMSTROKG, S.H., JR.,A N D MCMICHAEL, R. IC.: Unpublished results. (3) BROW?;, A. : P1i.D. Thesis, hlassachusetts Institute of Technolog,\., 1943 (see appcndix) ( 4 ) CANSAX, R. I
