Jan., 1961
MEASUREMENT OF DONNAN RATIOBY RADIOACTIVE TRACERS
141
MEASUREMENT OF DONNAN RATIO BY RADIOACTIVE TRACERS AKD ITS APPLICATION TO PROTEIN-ION BINDING’ BY ABRAHAM SAIFERAND JOSEPHSTEIGMAN Department of Chemistry, Polytechnic Institute of Brooklyn, Brooklyn, N . Y . Received J u l y 80, 1080
Binding of iodide ion from solutions of sodium or potassium iodide to deionized bovine serum albumin and to bovine serum mercaptalbumin has been studied bv equilibrium dialysis using radioactive P a l as a tracer. The Donnan effect was evaluated by introducing another radioactive ion, e.g., Na24,into the system which does not bind to these proteins. In acid solutions of BSA unbound hydrogen ions measured by pH showed the same distribution across the membrane as did the Nag4 ions. I n addition a t pH 12, where iodide ion should not bind, the 1”’ ratio across the membrane was equal to the reciprocal of the Na24 ratio. Finally a t isoionic H the binding constants for iodide ion to BSM determined by this method were shown to be in close agreement with those oitained by other workers by means of e.m.f. measurements.
The different experimental techniques employed in the study of ion binding to proteins have been summarized in B review article by Klotz.2 Of these, the equilibrium dialysis procedurea and those employing e.m.f. measurements with electrodes which measure the activity of the ion under investigation4J have been among the most widely used. The equilibrium (dialysistechnique is generally performed in strong salt or buffer solutions so as to reduce the Donnan effect to negligible proportions. This experimenta,l approach disregards the effect of the competitive binding to the protein molecule of salt or buffer ions present in high concentration. On this account this method of measuring ion binding has been criticized by Scatchard.6 In addition Scatchard and Black7 have shown that a large change occurs in the concentration of bound hydrogen ion in the presence of neutral salts. E.m.f. measurements of protein systems by means of indicator or ion-exchange electrodes are not subject to this criticism since they can be performed in the absence of buffer and at low salt concentrations. These measurements, which are otherwise quite satisfactory, suffer from a minor drawback, Le., satisfactory measurements cannot usually be made a t ion concentrations less than low4111. These regions of higher dilution are important in the evaluation of intrinsic binding constants and certain other thermodynamic data,8and these must be determined by extrapolation rather than by direct measurement. This paper describes the binding of iodide ions to bovine serum aibumiii (BSA) and bovine serum mercaptnlbumin (BSM) using trace amounts of (1) From a rhesis submitted by Abraham Saifer to the Graduate School of the Polytechnic Institute of Brooklyn in partial fulfillment of the requirements for the degree of Doctor of Philosophy. (2) 1. M. Klotz in H. Neurath and K. Bailey, eds., “The Proteins. Chemistry, Biological Activity, and .Methods,” Vol. I, Part B, Academic Press, New York N. Y., 1953,p. 727. (3) T.R. Hughes and I. M. IClots in D.Glick, ed., “Methods of Biochemical Analysis,” Initerscience Publishers, Inc., New York, N. Y., 1956. Vol. 3. p. 265. (4) fa) C. W. Cam, Arch. Biochem. Btophys., 40, 286 (1952): (b) 43, 147 (1953); (0) 46, 417,424 (1953); (d) C.W. Carr and L. Topol, THISJOURNAL, 54, 176 (1950). ( 5 ) G. Scatchard. 1. 1%. Scheinberg and S. H. Armstrong, Jr., J. Am. Chem. Soc., 73, 535,540 (1950). (6) G. Scatchard, W. L. Hughes, Jr., F. R. N. Gurd and P. E. Wilcox in F. R. N. Gurd, ed., “Chemical Specificity in Biological Interactions,” Acsdemio Press, New York, N. Y., 1954,p. 193. (7) G. Scatchard and E. 8. Black, THIS JOURNAL, 53, 88 (1949). ( 8 ) F. R. N. Gurd and P. E. Wilcox in M. L. Anson, K. Bailey and J. T. Edsall, eds., “Advances in Protein Chemistry,” Academic Press, Inc., New York, N. Y., 1956, Vol. 11, p. 311.
Na24of high specific activity as the Donnan ion, Le., as the unbound ion whose distribution across a semi-permeable membrane yields the Donnan ratio for the solution. Previous investigators, using a variety of techniques including potentiometric measurements, have concluded that sodium ion is not bound to serum albumin.2.8 In addition, diffusion study has shown that sodium ion at the trace level is not bound to a polyelectrolyte which has a much higher charge density than protein^.^ It has, therefore, been assumed in this work that Na24is not bound to the serum albumins. In the study of the binding of an anion A of charge -n to a protein in the presence of an unbound cation M of charge, + m the equations apply
where At Ab A0 yo, yi
= total concn. of anion found by analysis inside
= concn. of bound anion
= total concn. of anion found by analysis outside
= activity coefficients of the ion in the outside and inside soln.
r = (Aoyo/Airi)”“
(MiYi/MoYo)”“
(2)
where Ai, A0 = total molal concn. of any unbound n-valent anion inside and outside the membrane, resp. Mi,Mo = total molal concn. of any unbound m-valent cation inside and outside the membrane, resp.
It is generally assumed that the activity coeficients of the ions being measured are unaffected by the presence of the protein and that in sufficiently dilute solutions concentrations may be used instead of activities.1° Appropriate activity coefficient corrections were applied whenever they were found to be necessary. This equilibrium dialysis-tracer technique was employed to show that in acid solutions of BSA,unbound hydrogen and sodium ions had the same distribution across the membrane, ie., Na2‘ was not bound. In addition the binding of iodide ions to BSA was investigated over a wide range of pH and the iodide binding constants of B S M in the isoionic pH region were determined using both Na24and IIB1as the tracer ions. (9) J. P.Dux and J. Steigman. THIS JOURNAL, 63, 269 (1959). (10) (a) J. H. Northrup and M. Kunitr, J. Om. Physiol.. 7, 25 (1924-1925); (b) 9,351 (1925-1926); ( 0 ) 11, 481 (1927-1928).
142
ABRAHAM SAIFERAND JOSEPHSTEIGMAN Experimental
I. Deionization of Proteins.-Two to 10% protein solutions were deionized by passage through the multi-bed ionexchange resin column proposed by Dintzis." The appearance of the protein solution in the column effluent was determined by means of the color produced with biuret reagent. Deionized, redistilled water was used for the preparation of the protein and salt solutions. Paasa'ge through the column of protein solutions containing M (plus tracer) iodide ion added as an impurity showed that a single pass reduced its concentration to less than 10-8 M. Deionized preparations of BSA and BSM gave isoionic H values of 5.05 and 5.15, respectively, in the absence of sags The latter value for BSM is identical with that reported by Scatchard, et a1.,'2 whereas, the isoionic pH of the BSA sample (5.05) is somewhat lower than that reported by 0thers13,~~ in the presence of salts. 11. Equilibrium Dialysis Procedure.-The technique used for the preparation of the dialysis bags (Visking casing, 20/32 inches) is essentially that described by Hughes and Klotz.3 Whenever the casings were to be used for binding measurements i i the presence of a supporting electrolyte, they were soaked in such solutions for a t least 12 hours prior to their use as dialysis membranes. A 5.00-ml. aliquot of the deionized protein solution was transferred to the dialysis bag which had been sealed off at one end with a double knot. The other end of the bag was twisted so as to leave as little air spa.ce as possible and was then also sealed o f f with a double knot. A length of fiberglass or suture thread was attached to one end of the bag and any excess tubing was cut away. The bag was held suspended by the thread and the outside surface rinsed free of any protein by washing with distilled water and then blotted dry with cotton gauze. The bags were then lowered into polyethylene tubes (30 X 200 mm.) containing 50.00 ml. of freshly prepared dilute potassium iodide solutions of known concentration and trace amounts of Il3].(about 50,000 cts./min./ml.) and of NaZ4 (about 100,000 cts./min./ml.). The tubes were tightly stoppered with corks wrapped in Saran Wrap (polyethylene) and the ends of the threads were auowed to pass outside the stoppers to facilitate removal of the bags. Finger cots rinsed in distilled water were worn while manipulating the bags and the radioactive solutions so as to avoid contaminating the samples with sweat. The tubes were shaken with a wrist-action type of shaker for about 12-19 hours a t room temperature (22 3') in order to establish equilibrium between the internal and external solutions. The bags were then lifted out of tubes by the fiberglass thread, their outside surfaces quickly rinsed with distilled water and the bags dried with a gauze sponge. Each bag was then placed in a clean, dry test-tube and punctured with an applicator stick to transfer its contents to the tube. The pH of the internal protein solution was measured with a Beckman Model G pH meter using a glasscalomel electrode system. Spectrophotometric measurements of the protein solutions were made with a Beckman D.U. spectrophotometer a t 279 mb after diluting 1.OO-ml. aliquots to 50.00 ml. with 0.9% NaCl. The protein concentrations were calculated by means of the relationship, = 6.67.13 Sodium-24 of high specific activity was obtained from Brookhaven National Laboratories. Iodide-131 was a c:wrier-free material from Oak Ridge. One-ml. aliquots of the solutions of these isotopes were counted with a scintillation counter using FI well-type sodium iodide crystal. A minimum of 100,0100 counts were recorded for each sample run in duplicate. Iodide-ion binding was determined by counting the combined activities of and Na2' allowing the sodium to decay (Ti/,= 14.5 hours) for one .cveek or longer and then counting the iodide activity alone. The iodide decay ( Tilp = 8.1 days) waa corrected by means of measurement against a, reference solution. Frequent background counts were made. The pH of the external solutions was determined by a (11) H. 31. Dintsis, Ph.D., Thesis, Harvard University, 1952. (12) G. Soatchard, J. S. Coleman and A. L. Shen, J. Am. Chem. Soc.. 79, 12 (1957). (13) J. F. Faster and AI. D. Sternian, ibid., 78, 3666 (1966). (14) C. Tanfanl, S. A. Swanson and W. S. Shore, i b d , 7 7 , 6414
(1955).
Vol. 65
glass electrode in alkaline pH and by either a glass or a quinhydrone electrode in acid p H . Preliminary experiments with BSA solution in the internal phase and iodide solutions plus 1131 in the external phase showed that equilibrium in dialysis was established at room temperature after four hours of shaking. In order to obtain results comparable with those of other investigators, a molecular weight of 69,000 was assigned both to BSA (Pentex, 1X recrystallized) and BSM (Pentex, 10X recrystallized).
Results and Discussion I. Comparison of Sodium Ion and Hydrogen Ion Distribution in Acid BSA Solutions.--Small quantities of Na24 (about 100,OOO cts./ml./min.) were added to the external solutions whose acidity had been adjusted with standard HC1 to pH values which ranged from 1.0 to 5.0. The concentration of BSA in the inner solution was maintained at 5%. After the dialysis had reached equilibrium, the p H and the radioactivity of each solution were measured. Figure 1 shows the change in the negative logarithim of the Donnan ratio as a function of the pH of the inner solution. The ratios at various pH's were calculated from the activities of the free H+ ions as well as from the activities of the sodium ions. Since the radioactivity measurements give relative concentrations, it was assumed that the relative concentrations of the Na+ ions were the same as their activities at pH values greater than 3.4. In all other cases activity coefficients as given by MacInnesl6 were used to calculate the Na+ ion activities. From the curve illustrated in Fig. 1it can be seen that the ratio of Na+ ion activities inside and outside the membrane are virtually identical with the ratio of their corresponding free hydrogen ion activities. This constitutes additional evidence for the lack of appreciable binding of Na+ ions to BSA on the acid side of the isoionic point. Similar results were obtained for BSM in the same pH range. Accordingly it was concluded that NaZ4distribution measurements would provide direct and accurate Donnan ratio values and thereby permit quantitative evaluation of the binding of various ions to the serum albumins. The validity of this conclusion for the alkaline side of the isoionic point will be discussed in a later section. 11. Effect of pH on Iodide Ion Binding to BSA.Deionized 5% BSA solutions were subjected to equilibrium dialysis against external solutions which contained 10-5 11.1 sodium iodide plus I1Z1, a trace of Na24 and different concentrations of HC1 or SaOH. After equilibration the pH of each internal solution was measured and the distribution of the radioactive cations and anions in each phase determined by means of the previously described scintillation counting techniques. Figure 2 shows the variation with pH of the negative logarithim of (NaZ4)i/(Naz4)~ and (1131)J1131)i in the pH range from 1.5 to 12.0. No attempt was made to maintain constant ionic strength in these experiments. The differences between the two curves a t various pH's i.e., between the ratios of sodium ions and those of iodide ions, are related to the extent of iodide ion binding to the protein. The maximum iodide ion binding appears to be at about pH 4.5, (15) D. A. MacInnes, "The Principles of Eleotrocliemistry." Reinhold Puhl. Corp., New Tork, N. P., 1939.
MEASUREMENT OF DONNAN RATIOBY RADIOACTIVE TRACERS
Jan., 1961
143
which is to be expected because of the increased positive charge on the protein. At pH’s lower than 4.5 there is a reduction in the binding of iodide ion 0.75which is in part (dueto the competition for the same binding sites of the increasing chloride ion concentratioii5 and also results from a decrease in charge ‘2; a, 0 due to the expansion of the albumin moleculejat 0.50acid pH.la l7 The binding of the iodide iori on the 0 alkaline side of the isoionic pH of BSA is of greater G interest. Firstty, there is the indication of extenE sive binding of .iodide ion in the pH range from 7 to 9 although the net charge on the protein ie negative. This means that serum albumin can function as a anion transport agent a t physiological pH. Secondly, at pH 12 the ratio of the iodide ions is almost identical with the reciprocal ratio of the sodium ions In this strongly alkaline solution the binding of iodide ions would be negligib1eI;because of the high negative charge on the protein. There are two alternative explanations for the identity of the tracer ion ratios a t this pH. The first one is that although some Na+ ions may be 0 i 2 3 4 5 6 bound, the extent of the iodide ion binding is exactly pH o f PROTEIN SOLUTlON~nsi~) parallel at the same pH. The second possibility is Fig. 1.-Variation of Donnan ratio-(pH outside - pH that Naf ion is not bound a t this pH and hence and - log (Na24)i/(Na24)0)with pH of BSS (5%) neither is iodide The general chemistry of the sys- inside) after attainment of equilibrium by dialysis. tem makes this simpler hypothesis the more likely one. While it would appear possible to calculate 1.5 ; / a as a function of 2, for nearly zero V for BSA a t LEGEND pH’s > 5 from the data given in Fig. 2, the measurements of pH and protein concentration were not made with sufficient accuracy to warrant such calculations. I n addition the results at acid pH would have been more useful if H I had been used instead of HC1. Experimental difficulties in keeping H I free of Iz for the prolonged time periods necessary to establish equilibrium conditions prevented us from successful1.y carrying out such experiments. 111. Iodide Ion Binding to BSM at Isoionic pH.-Equilibrium dialysis measurements were performed on 5% deionized BSM in the presence of iodide salts whose concentrations ranged from the trace level I:> 10-5 M ) to 5 X 10-3 M, using as the determinant Xaz4as the Donnan ion and of the iodide ion distribution. The ratio of NaZ4outside to ?;az4 inside was the same in solutions of 5 X ill NaI and 5 X M KI containing tracer Xai4, pointing to the negligible binding of Naf ions to BSM even at the higher Naf ion concentrations as was found by Scatchard, et uZ.,’~for human serum albumin. From equation 1 the bound iodide ion concentra-”? tion, (I)B,can he calculated (I)B =
(1)” -
(I)F
(3)
but (4)
and hence (16) J. F. Foster and J. T. Yang, J . Bm. Chem. Soc., 76, 1015 (1954). (17) C. Tanford, J. Buzzell, D. Rands and 8. Swanson, ibid., 77, 6421 (1955). (18) G. Sc:ttchard. A. C. Batchelder and A. Brown, ibid., 68, 2320 (1946).
0
1
2
3 4
5
6
7
8
910111213
pH o f PROTEIN SOLUTION (inside) Fig. 2.-Variation of Donnan ratio-log ( Naz4)i/(Na24)o and -log (1131)i/(1131)i with p H of BSA (5%) after attamment of equilibrium by dialysis as an index of iodide ion binding.
From these data (Table I) a plot was made of = (I)B/(P) and of ;/a where a = ( 1 ) (e-2.303 ~ ~ ’ 2 ~as2is ~ illustrated ) in Fig. 3. The symbols T and CY correspond t o those employed by Scatchard and his co-workers12 who investigated the same system with ion-exchange electrodes. ZA is the
144
ABRAHAMSEIFERAND JOSEPH STEIGMAN
Vol. 65
TABLE I IODIDE IONBINDING TO ISOIONIC BOVINB SERUM MERCAPTALBUMIN (1-)p,
(I-)T,
molality
r (Donnan ratio)
(I-)?, molality
molality
$
(I)B/(P)
?/a
an
1.46 X 3.64 X IOd 1.09 X 1 0 - 6 0.414 4.27 x lo-* 11,850 3.603 X 2.49 X LO+ 6.51 X l o 4 1.83 X .290 7.62 X lo-* 11,758 6.481 X lo4 6.79 X 1.86 X 4.94 X .422 2.09 X 10-1 11,747 1.779 X 10-6 3.86 X 8.05 X 3.05 X .234 9.00 X 10-s 11,174b 8.054 X 3.06 x 10-4 1.02 x 10-4 2 . 0 4 x 10-4 .463 8.55 X 10-1 7,948 . 1.076 X lo-* 1.59 X 4.58 X 1 . 1 4 X 10-8 .527 2.09 6,789 3.079 X 1.24 x :io-3 6 . 3 9 x 10-4 5.99 x 10-4 .727 6,012 4.025 X 10-4 2.42 1.20 x :io6.41 x 10-4 5.59 x 10-4 ,729 5,529 4.232 X lo-' 2.34 1.05 x 10-3 6.49 x 10-4 4.06 x 10-4 .733 2.30 5,390 4.267 X lo-' 2.70 X 1.81 X lo-' 8.86 X .798 3.71 3,893 9.529 X lo-' 5.11 X .LO-3 4.06 X 1.06 X 10-8 .889 4.86 2,593 1.874 X lo-* These quantities are defined in the text of the paper. This point was not included in plotting the data shown in Fig. 3 because of the greater possibility of the oxidation of tracer iodide ion in a carrier-free system.
*
(I
lODIDE(Tracw) = IODIDE(E M F)
0 =
A
i
5
io
s=z _u ,a\
15
2c
P-
\
Fig. 3.-Iodide
/
ion binding to isoionic BSM (5%).
zp
charge on the anion and is the average charge on the probin arid W I = 2~/2.303where w is a constant c&ula{;ed from the Debye equation16 for a spherical protein molecule. The present data for the BSM-xaI system, obtained with the equilibrium dialysis-tracer techIlique, are superimposed in Fig. 3 on Scatchard's (Table I, last column)12 for the published that the results Same system. This figure shoJ%-s obtained with the two methods are in good agreement. The fact that the shape of the curve is concave upwaxds shows that the iodide ion is binding to more than one class of sites. At VI = 0, the intercept is V I / ~ I= ZifiiKOiI and the asymptotic slope is -ZiniK~l?/ZifiiKOi~. At i i ~ / a=~ 0, the intercept is i ; ' ~ = Zini and the asymptotic slope is -Zini/(BiYbi/KOiI). The limits a t V I / ~ I= 0
are usually indeterminate but those a t TI Z 0 (< M I-) are now directly determinable experimentally with the equilibrium dialysis-tracer method. Scatchard, et a1.,12 found that the data for the binding of chloride or iodide ions to BSM in dilute solution could be interpreted as binding to three classes of sites with the number, nil of groups in successive classes being nl = 1, n2 = 7 f 1 and n3 = 18 4. They reported the following values for the various binding constants for iodide ion: K1 = 9200 = 24K2 = 720K3. Other anions, like chloride, fluoride and thiocyanate, were bound to these same sites but with different binding constants. The present data best fit the lower limits of the number of binding sites proposed by them, Le., nl = 1, n2 = 6 and na = 14 and ZqKi = 11,680 (calculated) compared to 11,788 (experimental). The present investigation was designed with a view toward an intensive study of a few well-characterized, mater soluble proteins, ie., bovine serum albumin and mercaptalbumin, with a single radioactive ion ( 1 9 in order to firmly establish the validity of this new experimental approach to the quantitative determination of the binding of ions to proteins in buffer-free systems. The principle involved should be applicable to other water or saltsoluble proteins and to many other radioactive ions which are available as relatively carrier-free salts. Acknowledgments.-We wish to thank Dr. Bruno W. Volk, Director of Laboratories of the Isaac Albert Research Institute, Brooklyn, N. Y . , for permitting this work to be done in its laboratories. We also wish to acknowledge the assistance of Mr. Arthur Hyman with some of the measurements.
