BUFFER EFFECTS I N COPPER-ALBUMIN COMPLEXES
101
(7) ASTRUP,T., AND DARLING, S.: Acta Physiol. Scand. 6, 13 (1943). (8) BARNES,B. A.: Personal communication. (9) BRINKHOUS, K. M.: Proc. SOC.Exptl. Biol. Med. 88, 117 (1947). (10) BUCKWALTER, J. A,, BLYTHE,W. B., AND BRINKHOUS, K. M.: Am. J. Physiol. 169,316 (1949). (11) CHRISTENSEN, L. R., AND MACLEOD, C. M.: J. Gen. Physiol. 28, 559 (1945). (12) COHN,E. J., AND ASSOCIATES: Science 112,450 (1950). (13) COHN,E. J., GURD,F. R. N., SURGENOR, D. M., BARNES,B. A., BROWN,R. K., DEROUAUX, G., GILLESPIE,J. M., KAHNT,F. W., LEVER,W. F., LIU, C. H., MITTELMAN, D., MOUTON, R. F., SCHMID, K., A N D UROMA, E.: J. Am. Chem. SOC.72, 465 (1950). (14) EDSALL, J. T., A N D DANDLIKER, W. B.: Unpublished results. (15) FERRY, J. D., AND MORRISON, P. R . : J. Am. Chem. SOC.69, 388 (1947). (16) GIBSON,J. G., 11, AND BUCKLEY, E . L., JR.:Personal communication. (17) KOLLER,F.: Unpublished results. (18) PATEK,A. J., AND TAYLOR, F. H. L.: J. Clin. Invest. 16,113 (1937). (19) QUICK,A. J., SHANBERGE, J. N., AND STEFANINI, M.: Am. J. Med. Sci. 217, 198 (1949). (20) QUICK,A. J., AND STEFANINI, M.: J. Lab. Clin. Med. 93,819 (1948). (21) MILSTONE, J. H.:J. Gen. Physiol. 31, 301 (1948). (22) SEEGERS, W. H.,LOOMIS, E . C., AND VANDENBELT, J. M.: Proc. SOC.Exptl. Biol. Med. 66,70 (1944). (23) SEEDERS,W. H.,MCCLAUGHRY, R. I., AND FAHEY, J. L.: Blood 6,421 (1950). (24) TOCANTINS, L. : Personal communication. (25) WALTER,C. W.: In preparation. (26) WARE,A. G., AND SEEGERS, W. H.: J. Biol. Chem. 172,699 (1948). (27) WARE,A. G.,AND SEEGERS, W. H.: Am. J. Clin. Path. 19,471 (1949). W. H.: Federation Proc. 7, 131 (1948). (28) WARE,A. G., AND SEEGERS, (29) VALLEE,B. L.: Personal communication.
THE THERMODYNAMICS OF METALLOPROTEIN COMBINATIONS BUFFEREFFECTS IN COPPER-ALBUMIN COMPLEXES~J IRVING M. KLOTZ AND HAROLD A. FIESS Department of Chemistry, Northwestern University, Evanaton, Illinois Received August 10, 1060 INTRODUCTION
Quantitative studies have been made recently of the extent of binding of cupric ions by crystallized bovine albumin (3) at pH's below 5 as well as of the optical properties of copper-albumin complexes (4). These investigations have indicated that in the pH region near 5, copper is bound primarily by the free I Presented a t the Twenty-Fourth National Colloid Symposium, which waa held under the auspices of the Division of Colloid Chemistry of the American Chemical Society at St. Louis, Missouri, June 15-17, 1950. 2 This investigation was supported by grants from the Carnation Company and from the Office of Naval Research (Project No. NR121-054).
102
IRVING M. g L o T Z AND HAROLD A. FIESB
carboxyl residues of this protein. Corresponding studies with amino acids and peptides have demonstrated that the nature of their complexes with cupric ion may change with pH and that the contribution of amine group interaction to the stabilization of the metal complex becomes increasingly important with increasing pH. For a fuller understanding of the nature of copper proteins a t pH's of physiological importance, it has seemed appropriate to extend earlier studies to higher pH's. Detailed results on the effect of buffer and of temperature a t a pH of 6.5 are described in this report. EXPERIMENTAL
The extent of binding of cupric ions by proteins was measured by the equilibriumdialysis technique described previously (8). The semimicro T-tube aa designed by Weber (7, 16) permitted the use of small volumes of solutions. A 2-ml. portion of a solution containing protein, buffer, and cupric ions was placed in a cellophane bag prepared from commercial sausage casing. This was immersed in 2 ml. of the buffer and shaken mechanically for 18 hr. for the attainment of equilibrium. Aliquot portions of the solutions within and without the membrane were analyaed for the total copper present. The copper found outside the membrane may be related to the free copper in equilibrium with the copper bound by the protein within the membrane. Experiments were carried out in an ice bath or water bath maintained within f0.05' of 0°C. or 25"C., respectively. Analyses for cupric-ion concentrations were made by the cuprethol method of Woelfel (18). Absorption spectra were obtained with a Beckman spectrophotometer using a cell of 1 cm. depth, d . Molecular extinction coefficients, e, were calculated from the usual expression, loglo I o / I = scd, where c is expressed in moles per liter and d in centimeters. The bovine serum albumin was a crystallized sample from Armour and Company. Binding measurements were carried out with a 0.5 per cent solution of this protein. The methylated albumin was a 66 per cent methylated product prepared by Dr. W. W. Weber of this laboratory. Reagent grade CuC12.2H20 was used as a source of the cupric ion. All other chemicals were of reagent grade or equivalent quality. RESULTS AND DISCUSSION
Effect of p H ehunge with common buffer Since previous quantitative experiments a t pH 4.8 were carried out in acetate buffers, we shall present first some of the results of new studies in acetate bufTers a t pH 6.5. Experiments a t the former pH were carried out in 0.06 M acetate. In order to keep cupric ion in solution a t pH 6.5, however, it was necessary to use higher concentrations of acetate. Detailed data on the binding of cupric ion by bovine serum albumin in a 0.2 M acetate buffer a t pH 6.5 and 0°C. are presented in figure 1, together with some of the corresponding results from preVi-
BUFFER EFFECTS I N COPPER-ALBUMIN
103
COMPLEXES
mdypublished work at pH 4.8. It is apparent that the extent of binding of copper by the protein is much greater at the higher pH. Inasmuch as the experiments at pH 6.5 were carried out in 0.2 M acetate, whereas those at pH 4.8 were in 0.06 M buffer, it seemed desirable to obtain some indication of the effect of buffer concentration. A few experiments were carried out, therefore, at very low copper concentrations, in a 0.01 M acetate b d e r at pH 6.5. Slightly greater binding was observed in the buffer of lower concentration, as is apparent in figure 1. IS
pH 4.8 0.W U
10
r
5-
- 90
LOG
-4.0
COPPERI IF RE^
-3.0
FIG.1. Binding of cupric ions by bovine serum albumin a t 0°C. and pH 4.8 and
6.5
Although the precision of the experiments in 0.01 M acetate is poor, because the free copper concentration is so very small, it is of interest to note that a twentyfold reduction of acetate concentration is accompanied by only minor changes in the quantity of copper bound by the protein. This behavior is an indication that Cu(CzH302)f, as well as Cu++, is bound by bovine albumin. From Pedersen’s (11) studies on copper complexes with acetate ion, it can be calculated that the free cupric-ion concentration increases by a factor of approximately 8 as the acetate concentration is reduced from 0.2 M to 0.01 M . One would expect, therefore, that the extent of binding of cupric ion by serum albumin should increase’ by about a factor of 3. If both Cu++ and Cu(CzH8O2)+are bound almost equally, however, the increase in the sum of their concentrations as that of acetate is lowered from 0.2 M to 0.01 M is only a factor of 1.9. An increase in unbound copper of 1.9 would increase the number of bound cupric ions by tt factor of only 1.3. This predicted increase in binding of only 30 per cent is reasonably close to the experimental points illustrated in figure 1.
* This emtimate is made by moving a diatance of 0.9 (Le., log,, 8 ) to the right along the abscima of figure 1.
104
IRVING M. KLOTZ AND HAROLD A. FIESS
Since a twentyfold change in acetate concentration produces only minor changes in copper binding a t pH 6.5, it is apparent that the threefold difference in buffer concentration cannot account for the major increases in binding at p H 6.5 as compared to pH 4.8. It seems appropriate, therefore, to consider some of the changes in the protein which may be responsible for the increased affinity for copper. As the pH is increased the negative charge on the protein increases. One would expect, therefore, an increase in binding of cupric cations due to electrostatic effects alone. A reasonable estimate of the change in free energy of binding due to these electrostatic effects may be made from the following considerations. Let us represent the protein at the lower pH by P1and at the higher pH by P’*,where zI and z2 represent the charges on the respective molecules. We are interested in the relative affinities of P’ and P”for cupric ion and hence in the free-energy change of the reaction:
P’CU*
+ P’= P”’Cu++ + P’
(1)
The electrical portion of the partial molal free energy of each species in equation 1may be computed (9, 12) from the expression
where N is Avogadro’s number, z the number of charges on the molecule, e the electronic charge, D the dielectric constant of the medium, b the radius of the protein molecule, a the “distance of closest approach” of a charged ion to the protein, and K is given by
where kB is Boltzmann’s constant, T is the absolute temperature, and r is twice the ionic strength of the medium. Thus the electrical portion of the free-energy change in equation 1may be shown to be:
Under the conditions of the present experiments, with a buffer of 0.2 ionic strength, K is 1.45 X lo7 at 0°C. and 1.47 X IO7 a t 25°C. If we take 30 A. for the radius of the serum albumin molecule and 3 A. as that of the aqueous cupric ion (3, 8), AFelffireduces to the following numerical values: At 0°C.: AFelrc = 2(z2 - z1)(31.3) cal.
(5)
At 25OC.: AFelffi= 2(z2 - z1)(34.7)cal.
(6)
The magnitude of A F e l , thus depends on the difference in charge on the protein at the two pH’s. From the titration data of Scatchard, Batchelder, and Brown (13) and of Tanford (15) one can estimate that approximately seventeen
105
BUFFER EFFECTS IN COPPER-ALBUMIN COMPLEXES
hydrogen ions are lost by serum albumin from pH 4.8 to 6.5. If the difference in charge is therefore assumed to be -17, AF,I, becomes -1060 cal./mole at 0°C. and -1180 cal./mole at 25OC. The observed changes in AFf, the binding energy for the first copper ion, are of the same order of magnitude (table 1) as those calculated from electrostatic considerations with an assumed Az of 17. I t seems likely, therefore, that the greater affinity of the protein for copper at pH 6.5 is due primarily to an increase in the negative charge on the macromolecule. The AF,l, calculated with a Az of 17 (and listed in table 1) is probably an upper limit however, since the binding of anions by serum albumin is greater a t pH’s below the isoelectric point than above (6) and hence would tend to reduce z1 more than 22. Part of the increased affinity of albumin for copper at pH 6.5 is therefore probably due to an increase in the number of available sites. Acidity constants (2) also lead one to conclude that an increase in pH from 4.8 to 6.5 should be accompanied by a further ionization of carboxyl groups (essentially TABLE 1 Effect of pH on free-energy changes CBANOP I N AI
PH 4.8
(BXPEPIYENIAL) ~rpH65
A’);
(OBSEEVED
ur
CBANOE IN (KLECIROSIATIC)
to completion), by an appreciable degree of proton release by imidazolium residues, and by a slight amount of dissociation of a-ammonium groups. Absorption spectra (figure 2) of copper-protein complexes a t pH 6.5 indicate that cupric ions are being bound by C u . . . N linkages as well as by copper carboxyl interactions. The absorption peak of the complex at pH 6.5 is definitely shifted toward shorter wave lengths, as compared to the maximum at pH 4.8. Furthermore, in contrast to the observation a t pH 4.8 that esterification of carboxyl groups reduces the absorption of the copper-albumin complex (4),it has now been observed at pH 6.5 that the complex with the partially esterified protein shows no loss in optical density (figure 3). On the contrary, the (66 per cent) methoxylated albumin forms copper complexes whose absorption is shifted even further toward shorter wave lengths. This behavior can be explFined qualitatively again by consideration of the electrostatic effects produced. The covering of anionic carboxyl groups on the protein by methoxyl groups produces a pronounced decrease in negatively charged groups and hence results in a protein with a substantial positive charge. This positive charge tends to increase appreciably the extent of ionization of cationic ammonium and imidazolium groups a t a pH of 6.5. Thus, although the number of anionic carboxyls available to cupric ion has been decreased substantially by the partial esterification, this loss has been compensated for by the increased availability of amine and his-
106
IRVING Y. KLOTZ AND HAROLD A. FIE88
tidine sites on the albumin molecule. Consequently, there is no loas in extent of complex formation between copper and protein, but merely a change in type of binding. The optical changes reflect this modification in the type of copper protein linkage. Comparison of buffers In addition to acetate solutions of pH 6.5, citrate and phosphate buffers were also examined. Binding data for the three solutions are compared in figure 4. The extent of binding of copper by serum albumin in citrate solutions is decidedly less than that in acetate. desDite the much lower concentration of the former the
FIG.2 FIG.3 FIG.2. Spectra of cupric ion complexes with bovine serum albumin at pH 4.5, 6.5, and 9.6. At pH 4.5 [Cu*] was 0.01 M in 2 per cent protein. At pH 6.5 [Cu++]waB 0.002 M in 0.2 M acetate and 1 per cent protein. At pH 9.6 [Cu++]waa 0.005 M in 2 per cent protein. FIG.3. Spectra of cupric ion complexes with bovine serum albumin and (66 per cent) methoxylated bovine serum albumin in 0.2 M acetate at pH 6.5. [Cu*] waa 0.002 M in 1 per cent protein.
copper-citrate complex is known to be much stronger than the copper-acetate one. The formation constant for the citrate, aa determined by Meitea (lo), is 1.6 X lo", whereas that for the first acetate complex is only 1.46 X lo2according to Pedersen (11). Rough calculations show that the free cupric-ion concentraof those found in 0.2 M tions in 0.01.M citrate buffer are of the order of acetate buffers. The substantial drop in binding in citrate buffer thus is not surprising. In contrast to citrate, phosphate buffers (figure4) permit even greater binding of copper by albumin than is found in acetate. It is difficult to place appreciable quantities of copper in phosphate buffers, because of the insolubility problems encountered; hence extensiye measurements could not be made. It is apparent, nevertheless, that the concentration of free cupric ion is greatest in the phosphate buffer.
3UFFER EFFECT8
IN C O P P E R - A L B m
COMPLEXES
107
Detailed studies have been made also of the extent of copper binding by albumin in citrate buffers of various concentrations. The results of one of these studies are summarized in table 2. In contrast to acetate buffers, there is a marked
FIQ.4 FIQ.5 Fro. 4. Binding of cupric ions by bovine serum albumin at pH 6.5 and 0°C.in various buffers: curve 1,O.Ol M phosphate; curve 2,0.2 M acetate; curve 3,O.OlM citrate; point 4, 0.2 M acetate 0.001 M glycine; point 5,0.2 M acetate 0.001 M glutamic acid. FIQ.5. Binding of cupric ions by bovine serum albumin in 0.2 M acetate buffer at pH
+
+
6.5.
TABLE 2 Effect of citrate concentration on binding of cupric ion by bovine serum albumin pH, 6.5; 0°C.; 0.5 per cent bovine serum albumin
M
0.001 (with 0.02M NaC1) 0.01 0.03 0.05
M 5 x lo-' 5 x lo-' 5 x lo-' 5 x 10-1
'
1.21 0.48 0.13 0.16
dependence of copper-albumin complex formation on the concentration of citrate. From the equilibrium constant (10) (copper citrate-) (Cu*)(citrate---)
=
1.6 X 10"
(7)
one would expect the concentration of free cupric ion to vary inversely with the total citrate ion present, since (copper citrate-) < < (citrate---) and since practically all of the copper is in the form of the citrate complex. The drop in copper bound with increase in citrate concentration agrees roughly with the change one would expect from the data in table 2 for the corresponding drop in concentration of free cupric ion in these citrate buffers. Consequently, it seems reasonable to conclude that it is free cupric ion and not CuC8H50,- which is bound by albumin in citrate buffers.
108
IRVING M. KLOTZ AND HAROLD A. FIESS
Effect of competing substances In many biological fluids, small molecules capable of binding metals are present simultaneously with proteins. These small molecules may compete with the protein for the metal. On the other hand, some experiments indicate that in the presence of certain metallic cations and proteins, amino acids are capable of forming protein-metal-amino acid complexes (1, 14). The latter behavior might be expected to increase the extent of protein-bound metal. In the present experiments with copper and albumin, the addition of either 0.001 M glycine or 0.001 M glutamic acid to the 0.2 M acetate buffer produced a pronounced drop in the amount of protein-bound copper (figure 4). It is apparent, therefore, that these amino acids compete effectively with the protein for the metallic cation. On the other hand creatine and urea, components of blood serum and of milk, did not affect the binding of copper by albumin. Similarly, the presence of 0.002 M calcium ion did not reduce the extent of complex formation with cupric ion. TABLE 3 Thermodynamics of binding of cupric ion by bovine serum albumin P cu++ = PCU
+
Effect of temperature Binding data at pH 6.5 have been obtained a t both 0°C. and 25°C. and are compared in figure 5. It is of great interest to note that there is only a slight temperature dependence in the formation of copper-albumin complexes. Furthermore, as the temperature is increased, the extent of combination shows a small increase rather than the decrease one might expect. Consequently, the combination process must be accompanied by an absorption of heat, Le., AH must be positive. Thus the behavior of copper-albumin at pH 6.5, insofar as thermodynamic constants are involved, parallels that observed at pH 4.8 (3). From the binding data, values have been calculated for AF? (the free energy of formation of the first copper-albumin complex, PCul, from its component protein and cation) by methods analogous to those used previously for anionalbumin complexes (5). Experimental values of AF? at 0°C. and 25°C. have been assembled in table 3, for pH 4.8as well &s 6.5. From the general thermodynamic equations
(x),
= -AS
BUFFER EFFECTS IN COPPER-ALBUMIN COMPLEXES
109
and
AF = AH
- TAS
(9)
the entropies and enthalpies of formation of PCul have been calculated and these too are tabulated in table 3. It is of interest to consider the relations we might expect to find between the various thermodynamic changes associated with the binding process if we make the extreme assumption that the free energy of formation of the PCul complex is due entirely to electrostatic factors. For this purpose we may use an approach analogous to that applied by Westheimer and Kirkwood (17) to acidity constants. If the free energy of formation of the copper-albumin bond were entirely of an electrostatic origin, then AFe1= would be a function of certain atomic radii, T , the electrostatic charges, z , on the species involved, and the dielectric constant, D, of the medium. Associating all of the parameters except D in a single function, ~p,we may write (10)
If we make the additional reasonable assumption that of the temperature, it follows that:
T
and z are independent
This equation may be rearranged to read
If we use the dielectric constant of pure water for D,the temperature coefficient may be obtained from the equation of Wyman and Ingalls (19) loglo D = 1.9446
- 0.001981
(13) where t is the temperature on the Centigrade scale. Equation 12 thus becomes: -aT
-
+ 0.00456AF,lffi
Recognizing that the temperature coefficient of the free energy is the negative of the entropy, we obtain: Aselm = -0.00456AF.1,
Finally, from the general thermodynamic expression AF = AH
we find the following equation for AHe,=:
-
TAS
(15)
110
IRMNG) 116. KLOTZ AND HAROLD A. FIE88
Equations 15 and 16 have been used to calculate and Lw,lCcat pH 4.8 and 6.5, and the results are listed in table 3. For the calculation of ASalso the average value of A f i between 0°C. and 25°C. was used. Similarly for .PHel,, 811 average temperature (12.5OC.)was used in equation 16. An overall comparison of the experimental entropy and enthalpy changes with those calculated from electrostatic changes shows some very striking coincidences. The observed entropy changes are very close to those calculated theoretically; the enthalpy changes predicted theoretically are of the correct sign and order of magnitude. Once again we find that electrostatic factors play a predominant role in determining the energetics of these copper-protein interactions. In comparing the results at pH 6.5 with those a t 4.8,we may discern a trend in the differences between experimental and theoretical values that tends to confirm interpretations described previously in this paper. ks the pH is increased, electrostatic theory predicts an increase in A S and in AH.The observed values, however, show a small but distinct drop in both of these thermodynamic quantities. These small discrepancies could be due to the increased importance of Cu...N linkages as the pH is increased. Thus the differences in optical behavior a t the two pH’s, described earlier, parallel the differences in the energetics of the copper-protein complexes. CONCLUSIONS
From the results described in this paper it is apparent that copper-protein interactions are strongly influenced by electrostatic effects. The intrinsic affinity of albumin for copper nevertheless is attributable to its possession of side chains containing -COO- or =N: end groups. These groups form complexes with cupric ion in much the same manner as they do when they exist as components of small molecules, such as amino acids. In the protein, however, the electrostatic influences of companion groups may affect the intrinsic affinity substantially. SUMMARY
The extent, of binding of cupric ions by bovine serum albumin at a pH of 6.5 has been determined in a variety of buffers and at two temperatures. The extent of formation of copper-albumin complexes is greatest in phosphate, slightly lesa in acetate, and least in citrate buffers. The presence of glycine or glutamic acid also decreases copper-albumin binding. In acetate buffer, binding increases from pH 4.8 to 6.5 largely because of electrostatic factors. Optical properties of copper-albumin complexes at pH 6.5 indicate that some copper-nitrogen bonds are formed in addition to copper-carboxyl linkages. Free energies, enthalpies, and entropies of binding have been evaluated. The affinity of serum albumin for copper increases slightly as the temperature is raised from 0°C. to 25°C. This rise is shown to be consistent with expectations from electrostatic theory.
MOBILITY DISTRIBUTIONS OF PROTEINS
111
REFERENCES (1) BORSOOK, H.,DEASY,C. L., HAAGEN-SDQT, A. J., KEXQHLEY, G., AND LOWY,P. H.: Federation Proc. 8, 589 (1949). (2) COHN,E. J., AND EDSALL,J. T.: Proteins, Amino Acids and Peptides, p. 445.Reinhold Publishing Corporation, New York (1943). (3) KLOTZ,I. M., AND CURME,H. G.: J. Am. Chem. SOC.70,939 (1948). (4) KLOTZ,I. M., FALLER, I. L., AND URQUHART, J. M.: J. Phys. & Colloid Chem. 64. 18 (1950). (5) KLOTZ,I. M.,AND URQUHART, J. M.: J. Am. Chem. SOC.71, 847 (1949). (6) KLOTZ,I. M., AND URQUHART, J. M.: J. Am. Chem. SOC.71, 1597 (1949). (7) KLOTZ,I . M., URQUHART, J. M., AND WEBER,W. W.: Arch. Biochem. 26,420 (1950). (8) KLOTZ,I. M., WALKER, F. M., AND PIVAN,R. B.: J. Am. Chem. SOC.68,1486 (1946). (9) MACINNES, D . A.: PrincipZes of Electrochemistry, p. 146. Reinhold Publishing Corporation, New York (1939). (10) MEITES,L.:J . Am. Chem. SOC.72, 180 (1950). (11) PEDERSEN, K.J.: Kgl. Danske Videnskab. Selskab, Mat.-fys. Medd. a,No. 12 (1945). (12) &ATCHARD, G.:In reference 2, pp. 57, 473-5. (13) SCATCHARD, G.,BATCHELDER, A. C., AND BROWN,A.: J. Am. Chem. SOC.68,2320(1946). (14) SMITH,E. L.: Federation Proc. 8. 581 (1949). (15) TANFORD, C.: J . Am. Chem. SOC.72,441 (1950). (16) WEBER,W. W.: Ph.D. Dissertation, Northwestern University, 1950. (17) WESTHEIMER, F. H., AND KIRKWOOD, J. G.: Trans. Faraday SOC.48,77 (1947). (18) WOBLFEL,W. C.: Anal. Chem. PO, 722 (1948). (19) WYMAN, J., JR., AND INGALLS, E. N.: J . Am. Chem. 600.80, 1182 (1938).
HOMOGENEITY AND THE ELECTROPHORETIC BEHAVIOR OF SOME PROTEINS. I11
A GENERAL METHODFOR
THE
DETERMINATION OF MOBILITY DISTRIBUTIONS* 8
ROBERT L. BALDWIN, PAUL M. LAUGHTON, AND ROBERT A. ALBERTY Department of Chemistry, Uniuereity of Wisconsin, Madison, Wisconsin Received August I O , 1960 INTRODUmION
In the interpretation of any studies of protein preparations, it is desirable to establish the degree of homogeneity of the preparation with respect to the property being measured. When the protein is soluble and stable at its average isoelectric point, electrophoresis offers a quantitative test for homogeneity with respect to mobility (1,5)which is sensitive to very small changes in the protein molecule. A change in bovine serum albumin resulting in the gain or loss of one 1 Presented a t the Twenty-Fourth National Colloid Symposium, which was held under the auspices of the Division of Colloid Chemistry of the American Chemical Society at St. Louis, Missouri, June 1617, 1950. This investigation was supported in part by grants from the National Institutes of Health, Public Health Service, and from the Research Committee of the Graduate School from funds supplied by the Wisconsin Alumni Research Foundation. f
