BIOPOLY MERS VOL. 6, (1968)

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Association of Thiocynate w i t h Reduced Boeine Albumin: Essential Features of the Binding Sites*

Unmodified bovint: :ilhimitt has ~ W I I found to contain 7 & 1 primary and approxi- mately 90 scwmdary SCS- biding sitc.s.1 Tht: primary sitcs are destroyed whm albumin is placed in 8.21 urea, whweas the 90 secondary sites remain largely intact. In this communicat.ion the naturc. of the secondary sites is further investigat.ed through binding studics with reduct:d SH-blockrd albumins.

The reductioii of bovine Fract.iou V albumin (Armoiir A2 1505) was carried out with freshly obtaincd thioglycolic acid (frw of thioglycolidc esters2) in 8RI urea, pH 8.5-9.0, by t.he method of Hunter and McI)uffie.3 Iteduced S-oyanoethylated albumin was prepared by addition, uiit1t.r nitrogt.11 at pH 8-9, of 1.5 g acrylonitrile to eacbh gram of albumiu reduced by thioglycolate.4.j The mixture was allowt:d to stand 4 hr in the dark thcn exhaustively di:tlyzed against water, deionized by a column of mixed-bed resins6 aud concentrated to 4%6y0 protcin by a I h f l o ultrafiltration cell (Amicon Corp.). I’aper chromatography of protein hydrolysatcs (SN HCL, 1 10”C, 16 hr) by methods earlier dtrsc*ribod7 showed no free cystine (or cysteine) and no modification Gf t-amino or imidazole groups. A vwy promiuent platinic iodine reducing zone with an ZZf valuc greater than that of cystine was present. This zone was probably S- carboxyethyl-cysteine, formed by the hydrolysis of S-cyanoethylcysteine residues. This protein was soluble in water a t pH 5.8. In 0.2111 HCElIt or 0.1M phosphate it preci- pitated a t pH values below 6. A t pH 9 it gelled. Reduced S-carboxymethylated albumin was prepared in the same manner as S-cyanoethylated albumin except that instead of acrylonitrile, 5.5 g iodoacetate was used for each gram of reduced protein. Chromatography of a hydrolysate showed the absence of cysteiiie and the presence of a faster moving zone presumably that of S-carboxymcthylcysteine. This protein was soluble in water a t pI-1 4-8; in 0.2M HCED it precipitated a t pH values below 4.5. It was soluble in 0.2111 HCEI) a t pH 5.5. The charge on reduced S-carboxymethylated albumin was greater by 35 negative groups than native albumin.

Binding studies with SCN- were conducted by the method of McMenamy et a1.l This method employed rapid dialysis equilibrium in HCED solution; the latter salt was added to minimize Donnan effects. In the present studies, 1 ml protein solution containing 1 pmole albumin and 1 ml HCEII solution (2 X con- centration were pipetted inside the dialysis bags; 3 ml of HCEI) solution containing the ligand were pipetted outside the bag. Protein concentrations were determined by refractometry8 using a An of the 0.00186 per gram protein per 100 ml water solution. (This is the same value used for nativr albumin.) The molecular weight of the modified albumin was taken as 66000. S C S - c*oiicrntrations varied from 0.0001 to 0.4M and were measured by amperometry. I)onnan correct.ions (which i n all instances were small at, the high conctmtrations of HCEI) used) were estimated in the manner earlier tloscribed.’ The moles SCS- t)oitnd~~d per molc protein, V , was estimated from the differencc between the amount of SCX- added and the amount found free in solution. The free concentration of SCK- was designated ‘1. gives a

*This investigation was supported by grants from the National Institute of General Medical Sciences, U. S. Public. Health Servire (GM 08361), and t,he Nat,ional Science Foundation GB 7224).

The cation was always K+.

A plot of ; / A versus

~N-(csrhoxymethyl)-N’-2-hydroxyethyl-N,N’-ct.hyleiiediglyririe.

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curve in which tlic: intercept O I L the i / i l axis is Zkini and that 011 the i axis is zni. l'ho association constairt for t,hc site set i is ki; ni is the nunibw of sitrs of sct i. III cits(as where the plot is a straight line (as is approximately the case i n all hut one of our studies) there is one set of sites of numbcr n1 and the slope of the line is equal to /?I.

Figure 2 shows studim with reduced S-carboxymethylatcd albumin. Binding in other than trace amounts existed only in the cases where the proteins precipitated. When the reduced albumins remained in clear solution, binding was very low or nonexistent. I n one preparation (preparation A) the precipitate tended to he somewhat transparent and not very compact. Binding did not occur a t as many sites with this preparation as with the other preparations. Solutions of this preparation were also more viscous than the other protein preparations and by the Uiaflo apparatus this protein solution could not he concentrated to the extent desired. Binding was small with a protein preparation which gelled.

It is estimated that there are a minimum of 50 binding sites with precipitated reduced S-cyanoethylated albumin and 70 with precipitated reduced S-carhoxymethylated albumin. The association constants for these sites range from 5 to 6M-'. This is to be compared with the 90 secondary binding sites on unmodified albumin which under the same ionic strength conditions have an association constant -7M-l. There was no evidence of primary site binding with any of the reduccd a lhmin preparations :md these sites can he assumed to have hcerr totally destroyed in such preparations. Allowanct:~ for electrostatic effects were not made i n the studies with reduced albumin. Theso rffects arc small for n random-coiled mncromolrrnle. However, since the protein in these studies usually had a negative charge, any correction for clertrostatic effects would have been in a direction to further increase the number of binding sites.

Some type of compactness or organiaat,ion within the peptide chain sement>s of alhumin seems required in order for the SCN- binding sites to exist. In the rron- precipitated state the random-roiled peptide chain of albumin is in free solution with prohnbly few or no fixed dimension:tl relationships hetween different parts of the chain.

Figure 1 shows the binding of SCN- with reduced S-cyanoethylated albumin.

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Y

Fig. 1. The binding of SCN- with reduced S-cyanorthylated alhumin, 0.2111 HCEII, 4°C. A, pH 5.2 (protein precipitated); A, pH 5.1 (protein precipitated) protein content 0.6 rmoles per tube; 0, pH 5.0 i n 2.8121 nrea (clear solution); 0, pH 6.0 (clear solution); a, pH 9.0 (protein gelled). In the last three studies several of the V values w?re negative. 0, pH 5.0 unmodified albumin in 8M urea.

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Fig. 2. The binding of SCN- with redured S-carboxyniethylated albumin, 0.4M A, pH 4.2 (protein preripitated); 0, 5.9 (rlear solntion, one ; value HCED 4°C.

was negative).

With the preripitated reduced albumin on the other hand energy consideration would favor the clustering together of certain groups in the peptide chain of the reduced albumin, and these configurations would be stabilized by the precipitation process. The observed binding of SCN- with unmodified albumin in 8M urea is not inconsistent with a requirement of such structural organization. Considerable organization and compactness of structure is retained with native albumin in 8M urea.9

On the basis of elertrostatic theory Shellmanlo has postulated that the ability of a protein to bind anions is due to the shielding of positive charges on the protein by the low dielectric region of the protein. Anions, presumably because of their size and low hydration, have the ability to approach the counter charge, which is buried a t the edge of or within the low dielectric region of the protein, and bind to the protein through a form of coulombic force attraction, strengthened by the low dielectric constant of the region. Shellman's proposal seems a likely model for explaining the binding observed with precipitated reduced albumins. The reduced albumin chains undoubtedly generate low dielectric regions of appreciable size in the precipitated state which are not present when the chains are in free solution. These generated low dielectric regions with adjacent positive charges (there are approximately 100 positively charged groups per molecule of reduced albumin) provide all the essential requirements for the proposed binding sites.

In some respects one might consider the binding of SCN- with reduced precipitated albumins as a coprecipitation phenomenon. SCN-, however, would need to have a greater attraction as a coprecipitant ion than the supporting electrolyte ions which are present in much higher concentration. Furthermore, the charge on the reduced albumin is usually negative in the binding studies, a fact which would not favor SCN- occlusion on a charge basis alone.

In a study not reported here, the binding of SCN- was also found not to oceur with a preparation of acetylimi- dazole-modified albumin which had gelled during the binding study. This same protein, which had 7 tyrosyl OH and approximately 30 *amino groups acetylated, bound SCN- only slightly less than unmodified albumin under conditions where it did not gel." The gelling mechanism undoubtedly introduces cross linkages between the peptide chains which gives eonsiderable rigidity to the macromolecules. Whether the anion binding sites are occupied by counter groups on the adjacent peptide chains (such as

A gelled albumin preparation did not bind SCN- (Fig. 1).

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the carboxyl groups) as a result of the gelling phenomenon, or whether rigidity of the gel matrix does not permit regeneration of the binding sites, is not known.

References

1. R. H. McMenamy, M. Madeja, and F. Watson, J . Bid . Chem., in press. 2. B. S. Harrops and E. F. Woods, Hiopolymers, 3, 595 (1965). 3. M. J. Hunter and F. C. McDuffie, J . Am. Chem. Soc., 81, 1400 (1959). 4. T. H. Plummer, Jr. and C. H. W. Hirs, J . Bid . Chem., 239, 2530 (1964). 5. L. Wiel and T. S. Seiblcs, Arch. Biochem. Biophys., 95, 470 (1961). 6. R. H. McMenamy and Y. Lee, Arch. Biochem. Riophys., 122, 635 (1067). 7. R. H. McMenamy and J. L. Oncley, J . Riol. Chem., 233, 1436 (1958). 8. J. Krasner and It. H. McMcnamy, J. Biol. Chcbm., 241, 4186 (1!)66). 9. C. McArdle, Hiochem. J., 101, 1OP (1966).

10. J. A. Schellman, J . I'hys. Chem., 57, 472 (1!153). 11. C. S. Pande and R. H. McMenamy, to be submitted for publication.

CHANDRA s. P A N D E RAPIER H. MCMENAMY

Departments of Biochemistry and Surgery State University of New York a t Buffalo Edward J. Meyer Memorial Hospital Buffalo, New York 14215.

Received February 2, 1968 Revised April 8, 1968