thiocyanate binding with modified bovine plasma albumins

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 136, 260-267 (1970)

Thiocyanate Binding with Modified Bovine Plasma Albumins’

CHANDRA S. PANDE2 AND RAPIER H. McMENAMY

Departments of Biochemistry and Surgery of the State University of New York at Buffalo, and the Edward J. Meyer Memorial Hospital, Bu$alo, New York l&i6

Received September 2,1969; accepted September 7, 1969

SCN- binding with guanidinated, acetamidinated, acetylated, carboxymethylated, &dimethylaminopropionamidinated and cyanoethylated bovine plasma albumin has been studied. The binding at seven-primary sites is not much affected in the modified proteins. Since the guanidinium groups are the only anionic binding groups unaffected by the modification reagents, thii supports earlier evidence that the primary SCN- binding sites are located at arginyl residues. With respect to association at the secondary sites (presumably consisting predominantly of lysyl and imidazole groups) replacement of a hydrogen on the positively charged groups on the albumin by a bulk- ier radical is found to increase the binding constant of the site. Adducts which remove the positive charge on the binding site, on the other hand, appear to destroy the binding site.

The binding of SCN- with unmodified bovine albumin has been reported elsewhere (1). Binding occurred at 7 f 1 primary sites and approximately 90 secondary sites. The number of primary sites was in- dependent of pH change-7 existing in the pH range of 4.5hlO.7. The effect of pH on the secondary sites, however, could not, be clearly determined from these studies. In the acid pH region approximately 90 secondary sites were present. In the basic pH region, low binding due to unfavorable electrostatic effects prevented an estimate of the number of secondary binding sites in that region.

In order to further investigate the number of sites of anion binding, the effect of pH on binding, and the nature of the groups responsible for binding, studies with various modified albumins have been conducted. Reagents have been used to modify the imidazole, tyrosyl-OH and e-amino groups,

‘This investigation was supported by grants from the National Institute of General Medical Sciences, U. S. Public Health Service (GM 08361) and the National Science Foundation (GB 7224).

2 Present address: University of Allahabad, India.

and SCN- binding with the altered proteins measured.

EXPERIMENTAL

Materials BPA3 (Armour A21505) was dialyzed against

large volumes of 1 mM EDTAS by a thin layer technique, deionized by a mixed-bed resin column, lyophilized, and stored at -20”. This albumin contained -2% cu-globulin and less than 5% dimer.

0-methylisourea sulfate was obtained from Aldrich Chemical Company and acetylimidazole from K and K Laboratories. Bromoacetic acid, acetic anhydride, acrylonitrile, fl-dimethylamino- propionitrile, EDTA and HCEDZ were obtained from Eastman Organic Chemicals. HCED was further purified by passage through a mixed-bed deionizing column. Methanol, ether, dinitrofluoro- benzene, and KSCN were reagent grade.

Methods Guanidinated albumin was prepared by the

method of Hughes et al. (2). 0-Methylisourea

3 The abbreviations used are as follows: BPA for bovine plasma albumin, Fraction V; EDTA for disodium ethylenediaminetetraacetate; HCED for N-carboxymethyl-N’-2-hydroxyethyl-N,N’- ethylenediglycine; 2~ for the charge on albumin in absence of anion binding.

260

SCN- BINDING WITH MODIFIED ALBUMINS 261

sulfate (10.9 g) was dissolved in 10 ml water and neutralized with NaOH. To this was added 10 ml of a solution containing 3.6 g BPA. The mixture was adjusted to pH 9 and kept at 4” for five days. It was then dialyzed to remove most of the salts and lyophilized. The protein was collected and a second reaction step conducted with the same amount of 0-methylisourea previously used. After the second reaction the guanidinated protein precipitated during dialysis against water. The precipitate was washed with water and lyophilized. Chromatography of acid hydrolysates re- vealed that 80% of the t-amino groups were guanidinated. Dimer and aggregate content of the preparation was approximately 50%.”

Acetamidinated albumin was prepared by the method of Wofsy and Singer (3). Methylacetimi- date.HCI (3.75 g), prepared by the method of Hunter and Ludwig (4), was placed in 5 ml 0.1 M

EDTA and the pH quickly adjusted to 9.7. This solution was immediately added to 180 ml 0.1 M

EDTA solution, pH 9.7, containing 3 g BPA. After mixing, the pH of the solution was main- tained at -9 by addition of 1 N NaOH. Three additional aliquots of neutralized methylacetimi- date.HCl were then added at 4O-min intervals with care taken to adjust the pH after each addition. The protein solution was dialyzed against water, deionized, and lyophilized. Chromatog- raphy of acid hydrolysates indicated 90% of the e-amino groups were covered. Dimer content was

15%. Acetylation by acetic anhydride was carried out

by a modified method of Fraenkel-Conrat et al. (5). BPA (4 g) was dissolved in 30 ml of 0.5 M

EDTA at 4” and the pH adjusted to 9.0. To this was added 2 ml acetic anhydride in two installments at an interval of 3 hr. The pH was main- tained at 8.5 by addition of 1.0 N NaOH. Three hours after the last addition the pH of the solution essentially no longer changed and the solution was allowed to stand several days at 4”. The solution was then dialyzed, deionized, and lyophilized. All the e-amino groups were found to be acetylated. Approximately 60% of the product was dimer or higher aggregate.

Acetylation was also carried out by acetylimidazole using the method of Simpson et al. (6). BPA (2 g) was dissolved in 250 ml 0.05 M barbiturate and 2 M NaCl (pH 7.6). N-acetylimidazole (0.7 g) was added and the solution stirred 3 hr at room temperature. The protein was then dialyzed against water, deionized, and lyophilized. It was

4 The error i.n estimation of the percent of the groups modified and the percent of albumin in dimer or aggregate form is probably of the order of f5%.

estimat,ed that there were 4-7 tyrosyl groups O- acetylated. It was observed that the 0-acetylated groups were not stable. A preparation standing in solution for 48 hr at pH 7.6 (4”) lost approximately one 0-acetyl group per mole of albumin. Undoubtedly this instability lead to the variable coverage obtained with these groups. The c-amino groups were 25 to 50% covered in these preparations.

Carboxymethylation of albumin was carried out by the method of Banaszak et al. (7). BPA (2 g) was dissolved in 106 ml 0.1 M phosphate buffer pH 7.5. Bromoacetic acid (8.4 g) was dissolved in 30 ml water, and the pH adjusted to -7.5 with addition of solid NaHC03. The two solutions were mixed and allowed to stand at room temperature for 5 days. The solution was then dialyzed against water, deionized, and lyophilized. Carboxy- methylation of 90% of the histidyl and 35% of the methionyl groups was achieved. Approximately 10% of t,he s-amino groups reacted at pH 7.5. When the pH was raised to 8.5 coverage of the e-amino groups increased to 20% consistent with what was observed in an earlier report (7). The dimer content was ~57~. In an attempt to completely cover the imidazole groups the reaction was repeated in one instance with a preparation where the histidyl groups were 90% covered initially. No additional coverage of the imidazole groups was found with this more extensive treatment.

fl-dimethylaminopropionamidinated albumin was prepared by the same method used for acetamidinated albumin. Methyl p-dimethylaminopro- pionamidate.HCl, prepared by the method of Hunter and Ludwig (4), was added in three 2-g installments to 2 g BPA. Paper chromatography showed 35% of the E-amino groups covered. Dimer content was &X)0/,.

Acetylated -@- dimethylaminopropionamidinated albumin was prepared in two steps. Partial acetylation was carried out by the addition of 0.5 ml acetic anhydride to 6.25 g BPA by the method previously described. The protein was dialyzed and isolated. To 2.5 g of the isolated protein was added 5.5 g methyl p-dimethylaminopropion- amidinate.HCl in three installments, as described above. After the reaction the protein solution was dialyzed, deionized, and lyophilized. On analysis of the modified proteins it was found that 25’% of the r-amino groups were acetylated and 25’% were P-dimethylaminopropionamidinated. Dimer content was 20$&.

Cyanoethylation of albumin was carried out according to the method of Riehm and Scheraga (8). BPA (10 g) was dissolved in 800 ml 0.05 M

borate buffer pH 9.0, 52.8 ml acrylonitrile was added, and the solution stirred until homogeneous.

262 PANDE AND McMENAMY

It was then allowed to stand eight days at 4”. The solution was dialyzed against water, deionized (some protein was retained by the ion exchange column in this step), and lyophilized. Paper chromatography showed 557, of the c-amino groups covered and 50yo of the imidazole groups covered. The dimer content was 5%.

Dialysis of the modified proteins to reduce salt concentrations was always conducted using glass spacers inside the dialysis bags to introduce thin layers of solution and hasten equilibrium. Agita- tion was accomplished by suspending the mem- branes from a stirring motor assembly which ro- tated at 90 rpm and which alternated between 30- set periods of rotation and nonrotation. With changes of water outside the bag every 2 or 3 hr the salt content of the protein solution was reduced to a very low level in lo-16 hr. Following dialysis all protein solutions (except the guanidinated protein, see above) were deionized by passage through a mixed-bed resin column. The details for preparation of this column are described elsewhere (9).

The homogeneity of the protein preparation was determined: (a) by passing the preparations through a column of Sephsdex G 100 (0.9 X 130 cm) equilibrated with 0.05 M EDTA, 0.2 M NaCl, pH 6, by the method of Whitaker (10) and the dimer or aggregate content estimated; and (b) by carrying out cellulose acetate electrophoresis in 0.05 M barbiturate buffer, pH 8.6, to determine mobility alterations.

The modified amino acid residues were estimated by acid hydrolysis of 20 mg of the protein or DNP derivative of the protein (glass distilled 6 N HCl, 16 hr, llO”, in an evacuated sealed glass tube) followed by paper chromatography of the hydrolysate. The preparation of the DNP-protein was as previously described (11). Chromatography was also conducted as before except for the de- termination of the DNP amino acids. The chromatographic solvent used for separation of the DNP amino acids was isopropanol-water-30% NHIOH (80:20:1 v/v) saturated with NaCl. Chromatographic paper (S & S 539 green ribbon) was dipped in 3yo NaCl and dried prior to the hydrolysate applications. Three hours develop- ment (descending) was sufficient to separate DNP- lysine from other products. The concentrations of the amino acids and the percent modification of the amino acid residues were evaluated by earlier described methods (11). The decrease of e-DNP- lysine in the hydrolysate was taken as the amount of the e-amino groups which were covered by acetylation, acetamidination, @-dimethylamino- propionamidination, guanidination, cyanoethylation, and carboxymethylation. In some instances

the disappearance of lysine from the hydrolysates was used as a further check for the estimation of e-amino group coverage. Histidyl and methionyl modifications6 were followed by the decrease in concentrations of these two amino acids in the hydrolyzates. In all instances unmodified albumin was used as a control. The concentrations of the amino acids which were not modified serve as internal standards.6 The acetylation of the tyrosyl-OH-groups was estimated by the decrease in absorptivity at 278 rnw according to the method of Simpson et al. (6).

The binding of SCN- was studied by dialysis equilibrium at 4”, as previously described (1, 12). All experiments were conducted in 0.2 M potassium HCED which was shown earlier to have little influence on SCN- association (1). SCN- concentrations were determined by amperometry. The activity coefficient of the ligand, 7, was computed as previously described (1); it ranged from 0.75 to 0.69 in the present studies. All plots are pre- sented as S/AT versus 8, where B is the moles SCN- bound per mole albumin and A is the free concen- tration of SCN-. In the computed binding curves, included in some of the plots in order to make comparisons, the electrostatic corrections were made in the manner previously described. zp, the net charge on the albumin, was taken equal to 2s - 8. A molecular weight of 65,000 was used for albumin.

RESULTS

Bovine plasma albumin, per molecular weight of 65,000 contains 57 lysyl groups, 19 tyrosyl groups, 17 histidyl groups, 4 methionyl groups, 22 arginyl groups and one free a-amino group (13).

A one stage guanidination reaction gave a product with the E-amino groups 65% covered and a low dimer content. A two- stage reaction resulted in coverage of 80% of the E-amino groups. For practical reasons this was the maximum coverage which could

6 Values for methionyl alterations are minimal estimates. Methionine is partially regenerated on acid hydrolysis of the sulfonium compound formed by bromoacetylation.

6 We have not reported the coverage of the N- terminal amino acid aspartate in the albumin modifications. Because of instability of DNP- aspartate, hydrolysis of the proteins must be carried out under milder conditions than those used for DNP lysine analysis. DNP-aspartate analyses will be given in a subsequent manuscript where indole compound binding with the modified preparations will be reported.


be obtained and still have a product with sufficient solubility for binding studies. This material was extensively aggregated.

Acetamidination of 90% of the E-amino groups, on the other hand, gave a product with a low aggregate content which was still readily soluble.

Carboxymethylation of the histidyl groups was accomplished only to the extent of 90 %. Repeating the reaction on the same protein preparation lead to no significant increase in coverage of the histidyl groups. To test the stability of carboxymethylated histidine to acid hydrolysis, acetyl histidine was carboxymethylated, isolated, andhydrolyzed. No histidine was regenerated. The inability to carboxymethylate all histidyl groups in albumin suggests that one or two groups may be buried in the interior of the protein under the conditions in which the reaction was being carried out (7).

Acetylation with acetic anhydride easily proceeded to complete coverage of all of the e-amino groups.Acetylation by acetylimidazol covered not more than 35% of the tyrosyl groups. Part of the c-amino groups were also covered by this reagent. This protein preparation was not very soluble and on one occasion gelled in a binding study at pH 5.0.

While the mobilities were considerably different among thevariousmodified albumins the movement of the albumins as narrow zones on cellulose acetate strips indicated that heterogeneity in total net charge within the preparations was not gross in nature.

The binding of SCN- with various albumin modifications is shown in Figs. l-5. In most inst#ances in order to recognize differences the modified albumins were com- paredwith unmodified albumin studied under conditions of approximately the same net charge on the proteins (1). In studies with unmodified albumin reported elsewhere, it was found in the pH region 4.5-10.7 that binding of SCN- could be described with two sets of binding sites: k1° = 700, n = 7, and kzo = 10, Q = 90. Here k” was the intrinsic binding constant, n was the number of sites and the subscripts 1 and 2 denoted the site sets. The computed curves in the plots are derived from these parameters after al-

lowance for electrostatic effects as previously described (1). Differences in binding between modified and unmodified albumin due to the primary site alterations are largely reflected by changes in the plots at low B values (0-10)-higher g/Ay values in this region implying higher primary site binding and vice versa. Conversely, differences in secondary site binding are reflected by changes in O/AT at high 0 values (> 20) ; here again a high o/Ay value corresponds to a high secondary site binding, etc. While this latter method of comparison is ap- proximate, it nevertheless leads to conclu- sions as valid as one should draw from the data.

Acetamidination and guanidination increase rather markedly the binding constants of some of the secondary sites, whereas these modifications decrease to some extent the binding constants of the primary sites. (Fig. 1) This conclusion is necessary in order to explain the differences between points for these two preparations and those for unmodified albumin. Similar binding differences between acetamidinated and unmodified albumin were also observed in a study at pH 5.0 (not reported). The irregularities in the data points for unmodified

4115 ’ ’ ’ ’ ’ ‘-----I

FIG. 1. The bindine of SCN- with modified albumins, 0.2 M EDTA or HCED, 4”. 0, E-amino groups 80% guanidinated, pH 4.2; 0, c-amino groups 90% acetamidinated, pH 3.9; A, E-amino groups 25% acetylated and 250/, dimethylaminopropionamidinated, pH 4.1; w, unmodified albumin, pH 4.0; A., unmodified albumin, pH 3.6.


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FIG. 2. The binding of SCN- with modified albumins, 0.2 M HCED pH 5.1-5.2. 0, e-amino groups 35% B-dimethylaminopropionamidinated; 0, c-amino groups 55% cyanoethylated, imidazole 50% cyanoethylated; A, tyrosyl OH groups 30% acetylated, e-amino groups 25y0 acetylated; -a--, unmodified albumin, pH 5.2 (1); ....‘7 estimated curve for unmodified albumin with 2~ increased 20 charges (for comparison with the @- dimethylaminopropionamidinated protein data) ; ---2 estimated curve for unmodified albumin with Za decreased 10 charges (for comparison with the cyanoethylated and acetylated protein data).

albumin at pH 4.0 are attributed to the fact that they are at the position of the N-F transition (1). Such irregularities are not present at other pH values. The studies were conducted in this pH region in order to obtain the most optimal conditions (con- dition of most favorable electrostatic interaction) for observing differences in the secondary binding sites.

Acetylation of 25% of the c-amino groups followed by B - dimethylaminoproprion - amidination of 25% of the e-amino groups (Fig. 1) gave an albumin preparation with the same charge as unmodified albumin,

FIG. 3. The binding of SCN- with acetylated and acetamidinated albumin, 4”. 0, e-amino groups 100% acetylated, pH 4.2, 0.3 M HCED; 0, e-amino groups 90% acetamidinated, pH 10.2, 0.3 M HCED; ---, unmodified albumin, pH 9.7- 10.7, 0.2-0.4 M HCED (1).

This preparation binds SCN- essentially the same as unmodified albumin.

Guanidinated albumins with the e-amino groups 30 to 40% covered bind SCN- at pH 5.0-5.3 in a manner essentially in- distinguishable from that of unmodified albumin (studies not shown).

Figure 2 shows a binding study where 20 lysyl amino groups have been p-dimethylaminopropionamidinated. This reagent adds an extra positive charge with each group added to the albumin. The estimated binding curve for a charge increase of 20 fits well at D values above 20 but is somewhat too high at Iow g values. This suggests some inter- ference in binding with the modified preparation at the primary binding sites.

Figure 2 also shows a study with cyanoethylated albumin. The charge on this albumin, due to coverage of 50% of the imidazole groups and 50% of the E-amino groups by the reagent, is estimated to be 10 less than that on the unmodified albumin, The modified r-amino groups in this preparation would be expected to retain their charge at the pH of the study. The modified imidazol groups on the other hand would be expected


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FIG. 4. The binding of SCN- with carboxymethylated albumin, 0.2 M HCED. A., imidazole groups 65% covered, pH 5.2; e-., unmodified albumin, pH 7.1 (l), contains approximately the same charge as albumin with 65% of the imidazole groups carboxymethylated); 0, imidazole groups 90% covered, pH 5.1; ...., estimated curve for unmodified albumin with 2~ decreased 16 charges (for comparison with albumin with 90% of imidazole groups carboxymethylated).

to be largely uncharged (14). Comparisons with the estimated binding curve for unmodified albumin for this same charge con- dition indicate only a small reduction in primary site binding with the cyanoethylated albumin. Secondary site binding appears to be increased slightly with the cyanoethylated albumin.

The plot of SCN- binding with acetylimidazole-treated albumin (Fig. 2) is almost identical with that of the cyanoethylated protein preparation. However, the net charge on the acetylimidazole-reacted preparation is slightly higher (+5 charges) which suggests that this preparation does not bind quite as strongly as the cyanoethylated preparation.

Figure 3 reports a binding study with acetylated albumin at pH 4. This is compared with the study of unmodified albumin at pH 10. These two preparations were estimated to have approximately the same net

charge. The acetylated albumin shows a net increase in the number of primary sites from roughly 7 to 15. Secondary site binding, however, is much reduced in the acetylated albumin. Indeed if one extrapolates the binding curve for acetylated albumin to the in- tercept, approximately 40 binding sites are indicated. This is the number of positively charged groups remaining on the albumin after acetylation.

Also shown in Fig. 3 is a binding study with acetamidinated albumin at, pH 10.2. In the same manner as found in a study conducted at pH 4, binding is reduced at the primary sites but increased at the secondary sites. The acetamidinated E-amino groups are stronger bases than the unreacted e- amino groups and do not lose their charge at pH 10.2. This probably explains some of the higher binding with the acetamidinated albumin at pH 10.2. On the other hand, it is clear in studies conducted at lower pH values (where all amino groups are fully ionized in both the modified and unmodified albumins) that there is another effect which leads to increased secondary site binding with acetamidinated albumin.

Figure 4 reports binding studies of carboxymethylated albumin; 65 % coverage and 90 % coverage of the imidazole groups reduces the number of primary binding sites by approximately 2. Although imidazole groups have not been directly implicated at the primary binding site, these data indicate that some of the imidazole groups are in the region where their carboxymethylation inter- feres with binding. Secondary site binding is increased in the carboxymethylated albumin preparations.

DISCUSSION

What is perhaps the most surprising factor in these investigations is the small effect which modifications of albumin have on the association of SCN-. The binding at the primary sites is reduced only slightly by guanidination or acetamidination. Pre- sumably none of these sites were destroyed. Carboxymethylation destroyed l-3 of the primary binding sites. Acetylation did not decrease the primary binding sites; indeed, in contrast, it appeared to increase them.


The fact that the arginyl residues were the only residues not altered in the present studies, and the fact that binding at the primary sites was little affected by the modifications, supports placement of the primary sites at the arginyl residues, Earlier evidence from pH binding profiles suggests that the primary binding sites are most probably located at the arginyl residues (1). Further support for arginyl group involve- ment in the strong anion complexes comes from a demonstration by Grossberg and Pressman that these residues were present at the binding sites of antibodies with negatively charged haptens (15). The fact that in a few instances there is some decrease in the primary binding constants and the number of primary binding sites could well be due to tertiary protein structural changes inflicted by the modifying groups.

Some of the protein preparations showed moderate proportions of dimers and higher aggregates. There was no evidence that such states changed binding. Aggregation was greatest in guanidinated preparations, yet these bound almost identically to acetamidinated preparations, in which the monomer was the principal component. Unless actual unfolding of the protein occurred one might anticipate little change in the binding sites of salt ions such as SCN- upon aggregation of albumin. There could be some sites buried between the points of contact of the macromolecules. From energy considerations, on the other hand, exclusion of charged groups (presumably the sites of SCN- binding) from the aqueous environment is most unfavorable and would be expected to occur infrequently. Furthermore, unless the macromolecules are unfolded, the region of contact between aggregates is relatively small and should lead to no large loss in the total of ion binding sites.

Acetylation of albumin, although destroy- ing a large number of the secondary binding sites, nevertheless increased the affinity of SCN- at some of the remaining sites on the albumin. It is attractive to assign this increased affinity to an increase in the low dielectric shielding of the remaining charged groups on the albumin. Dielectric shielding of anionic binding sites, which has a theoreti-

cal basis, was earlier suggested as a pos- sible explanation for increased binding of SCN- with precipitated reduced albumin as compared to nonprecipitated reduced albumin (16).

An increase in binding at the secondary sites in guanidinated , acetamidinated, carboxymethylated, and cyanoethylated albumin also needs explanation. Here the adducts reduce the number of hydrogens on the charge groups and add bulk to the positively charged site. It seems, therefore, that this extra bulk increases the binding constant of the site. In two modifications (guanidination and acetamidination), the modified amino group also becomes a stronger base than previously. This might also explain a change in affinity of SCN- at the sites. The structural similarities between the guanidinium group on the arginyl residues (presumably the location of the primary binding site) and the guanidinated and acetamidinated c-amino groups is consistent with a preferred binding with this type of residue.

The net charge on the albumin, provided the latter is not unfolded, has a large but predictable influence on SCN- binding. Comparisons of albumins with approximately the same net charge have demonstrated that the binding sites, and indeed many of the binding characteristics, are not grossly changed from one albumin modification to another. The smoothed charged model term for estimation of the electrostatic effects on SCN- binding predicts reasonably satisfactorily the effect of the net protein charge on binding. This term satisfactorily ex- plained theresults of previous binding studies at different pH values with unmodified albumin (1).

The question has been raised as to whether a positively charged group on the protein is necessary for an anionic binding site. Our studies with the modified albumins are consistent with the requirement of a positive charged center at the binding site. The approximately 100 positive groups on albumin (at pH 5.0) and the estimated maximum of 100 binding sites is in agreement with a positively charge group at each binding center (1, 17). Furthermore, mechanistically


it would seem rather diflicult to place an 7.

anionic binding site at other than a positively charged position on the protein; neutraliza- tion of the charge in the low dielectric en- *. vironment of the protein would seem to require this. The interaction of anions with

9 ’

positively charged centers is also the basis 10. of anion exchange resins. 11.

1.

2.

3.

4.

5.

6.

REFERENCES 12.

MCMENAMY, R. H., MADJA, M., AND WATSON. F., J. Biol. Chem. 243, 2328 (1968). 13.

HUGHES, W. L., JR., SAROFF, H. A., AND CARNEY, A. L., J. Am. Chem. Sot. 71, 2476 (1949). 14.

WOFSY, L., AND SINGER, S. L., Biochemistry 2, 104 (1963).

HUNTER, M. J., AND LUDWIG, M. L., J. Am. Chem. Sot. 34, 3491 (1962).

15

FRAENKEI,-CONRAT, H., BEAN, R. S., AND LINEWEAVER, H., J. Biol. Chem. 177, 385 16.

(1949). SIMPSON, R. T., RIORDAN, T. F., AND VALLEE, 17.

B. L., Biochemistry 2, 616 (1963).

BANASIAK, L. J., ANDREWS, P. A., BURGNER, T. W., EYLAR, E. H., AND GURD, F. R. N., J. Biol. Chem., 238, 3307 (1963).

RIEHM, J. P., AND SCHERAGA, H. A., Biochem- istry 6, 93 (1966).

MCMENAMY, R. H., Arch. Biochem. Biophys. 122, 635 (1967).

WHITAKER, J. R., Anal. Chem. 36, 1950 (1963). MCMENAMY, R. H., AND ONCLEY, J. L., J.

Biol. Chem. 233, 1436 (1958). MCMENAMY, R. H., Anal. Biochem. 23, 122

(1968). EDSALL, J. T., AND WYMAN, J., “Biophysical

Chemistry,” Vol. I, p. 72. Academic Press, New York (1958).

MCKINNEY, L. L., UHING, E. H., SETZKORN, E. A., AND COWAN, J. C., J. Am. Chem. SOC. 73, 1641 (1951).

GROSSBERG, A. L., AND PRESSMAN, D., Bio- chemistry 7, 272, (1968).

PANDE, C. S., AND MCMENAMY, R. H., Bio- polymers 6, 1487, (1958).

SCATCHARD, G., WV, Y. V., AND SHEN, A. L., J. Am. Chem. Sot. 81, 6104 (1959).

thiocyanate binding with modified bovine plasma albumins

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