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Binding of sodium taurocholate by bovine serum albumin
SUMIMARY
Stu&es employing equilibrium dialysis methods were made of the binding of sodium taurocholate by bovine serum albumin. In the absence of fatty acids a maximum of one (r.or + 0.3rf mole of sodium tauroc~olate is bound per mole of alb~rn~~. The dissociation constant was found to be 8.21 . 10-5 -& 0.26 . 10-j M. The presence of I . 10-4 111sodium oleate increases the maximum stoichiometry of binding to 3 moles of bile salt per mole of bovine serum albumin. However, the fatty acid did not appreciably alter the dissociation constant.
INTRODUCTION
Observations by KCDXAN AX! ~ENDALL1 that u~co~~ngate~ bile salts are preferentially bound to albumin, when compared with other plasma proteins, suggest a specificity of interaction between these substances and the albumin molecule. A study of the mechanism and of the quantitative aspects of conjugated bile salt binding to albumin might extend o~lr understanding of the active transport of these substances by the ileumzp3,a process which presumably involves a membral~ous carrier protein with specific bile salt binding properties. This communication concerns the binding of sodium ~aurocholate by bovine serum albumin. Binding was determ~ed by equiiibrium dialysis. Furthermore the effect of a long chain fatty acid, sodium oleate, on this process was also studied, We have found that in the absence of sodium oleate one molecule of taurocholate is maximally bound per molecule of bovine serum albumin. However, the presence of sodium oleate permits the binding of 3 moles of taurocholate per mole of bovine serum albumin. MATERIALS
AND
METHODS
Eovine serum albumin, Lot ~6, was obtained from Pentex Inc., Kankakee, Ill. Sedimentation velocity showed that the sample was homogeneous. Fatty acids were removed from bovine serum albumin by the procedures of CHEP and SPECTOR et ai.j. The fatty acid free bovine serum albumin was filtered through a o.L+s-~ ~~lli~ore Biochinz.Bio&s. Acta, 231
(1971)
550-552
BINDING
OF SODIUM TAUROCHOLATE
BY ALBUMIN
551
filter to remove charcoal not separated by centrifugation. A mol. wt. of 66000 (see ref. 5) was assumed for bovine serum albumin, and an extinction coefficient of 0.667 mg-1 . cm2 at z7g rnp (see ref. 6) was used in calculations. Sodium [24JX] taurocholate was purchased from Tracer Lab, Waltham, Mass,, Lot 58-107, Alternatively the same material was synthesized and purified by the methods of NORMAN~>*. Thin layer chromatography indicated that this preparation was at least 99% pure. Oleic acid, Lot D-IA, was obtained from the Hormel Institute, Austin, Minnesota. All equilibrium dialysis experiments were carried out at room temperature in a salt solution, simulating physiological conditions, which contained 0.116 M NaC1, 0.0049 M KCI, 0.0012 M MgSO, and 0.016 M sodium phosphate, pH 7.4 (see ref. 5). Equilibrium was approached by transport of ligand across the membrane in both directions. In dialysis cells initially containing bovine serum albumin, buffer and taurocholate in the same compartment, equilibration required 24 h. When the ligand was placed in the opposite compartment 45 h were necessary for equilibration. A similar phenomenon was observed by CASSELet al.g. Consequently all samples were taken after 45 h of dialysis. In parallel experiments, sodium oleate was added to determine its influence on taurocholate binding. Sodium oleate stock solution was prepared by neutralizing oleic acid with I M NaOH, heating, and diluting with water. RESULTS AND DISCUSSION
Binding of taurocholate to bovine serum albumin in the presence and absence of sodium oleate was calculated by the following equationl:
where G is the number of moles of sodium taurocholate bound per mole of bovine serum albumin, n is the number of binding sites, K is the dissociation constant of each site and C is the molar concentration of taurocholate at equilibrium. The lines shown in Fig. I were fitted by least squares analysis of data points. The stoichiometry of binding is the reciprocal of the y-axis intercept. The dissociation constant (Kdiss) is the slope multiplied by the number of binding sites. Standard deviations of these parameters were also calculated. In the absence of fatty acid, 1.01 & 0.31 mole of sodium taurocholate is bound per mole bovine serum albumin, and Kdiss is 8.21 . IO@ f 0.26 . 10-j M. The presence of I . IO-~ M oleic acid increased the Kdiss slightly and increased the molar ratio of sodium taurocholate bound to approx. 3 (see Fig. I). The molar ratio of cholic acid bound to human serum albumin was reported to be 4 and that of deoxycholic acid was IZ (see ref. I). Three primary bovine serum albumin sites were observed for fatty acids5 and 5 were observed for the neutral ligands, octanol and decanol lo. Our results show that one mole of sodium taurocholate strongly binds to one mole of fatty acid-free bovine serum albumin, indicating a specific binding mechanism. The dissociation constant, 8 IO@ M, is of the same order of magnitude as that found for enzyme-substrate binding. Our data do not rule out the possibility of some weaker binding sites which may occur at higher concentrations of taurocholate. The increase in taurocholate binding to bovine serum albumin in the
Fig. r. Reciprocal plot of binding of sodium taurocholate by bovine serum albumin, 25’. Lines are fitted by least squares analysis of data points. Bovine serum albumin concentration is r-41. 10-j M. Vis number of moles sodium taurocholate bound per mole bovine serum albumin. C is the molar equilibrium concentration of free sodium taurocholate. Plastic equilibrium dialysis cells (Chemical Rubber Co.) containing I ml on each side of the membrane were used. Dialysis tubing from Union Carbide Corp. was boiled first in I . IO-~ M EOTA and then in 0.1 M NaHCO, for I h and washed thoroughly &I distilled water. After equilibrium dialysis, 0.1.ml samples were-added to ro ml BioSolv-toluene scintillation fluid. The above data was obtained with sodium [z4-r*Cjtaurocholate purchased from Tracer-lab. Replicate experiments performed with material synthesized in this laboratory (see text )gave comparable results.
presence of oleic acid suggests the formation of additional strong binding sitesas a result of fatty acid induced conformational changes in bovine serum albumin. Serum albumin acts physiologically as a carrier for both fatty acids and bile salts in plasma. Our observations imply that the presence of fatty acids enhances the capacity of albumin to transport bile acids. ACKNOWLEDGMENTS
This work was supported by Grant AM og58z from the Yational Institutes of Health. REFERENCES I D. RUDMAXAND F. E. KER'DALL,J.C~~~.I~~~~~., 36(1957) 538. 2 5. LACK AND 1.M. WEINER, Am. J. Physiol., zoo (1961) 313. 3 L. LACI~ AND I.M. WEIP;ER, Biochim. Biophys. Acta, 135 (1967) 1065. 4 R. F. CHEN, J.Biol. Chew., 242 (1967) 173, 5 A. A. SPECTOR, K. JOHN AND J.E.FLETcHER,J. LipidRes., 1c(136g) 56. 6 J. F. POSTER AND M. D. STERMAN, J. Am. Chem. Sm., 78 (1956) 3656. 7 A. NORMAN, Avkiv Kevnni, 8 (1956) 331. S A. NORMAN, Acta Chew. Scand., 7 (1953) 1413. g J. CASSEL, J. GALLAGHER, J. A. REYNOLDS AND J. STEINHARDT, Biochemistry, 8 (1969) 17~6. ro J. STEINHARDT AND J. A. REYNOLDS, Multiple Equilibria in Pvoteins, Academic Press, New York, 1969. B&him.
Biophys.
Acta,
231 (1971)
550-552
Stu&es employing equilibrium dialysis methods were made of the binding of sodium taurocholate by bovine serum albumin. In the absence of fatty acids a maximum of one (r.or + 0.3rf mole of sodium tauroc~olate is bound per mole of alb~rn~~. The dissociation constant was found to be 8.21 . 10-5 -& 0.26 . 10-j M. The presence of I . 10-4 111sodium oleate increases the maximum stoichiometry of binding to 3 moles of bile salt per mole of bovine serum albumin. However, the fatty acid did not appreciably alter the dissociation constant.
INTRODUCTION
Observations by KCDXAN AX! ~ENDALL1 that u~co~~ngate~ bile salts are preferentially bound to albumin, when compared with other plasma proteins, suggest a specificity of interaction between these substances and the albumin molecule. A study of the mechanism and of the quantitative aspects of conjugated bile salt binding to albumin might extend o~lr understanding of the active transport of these substances by the ileumzp3,a process which presumably involves a membral~ous carrier protein with specific bile salt binding properties. This communication concerns the binding of sodium ~aurocholate by bovine serum albumin. Binding was determ~ed by equiiibrium dialysis. Furthermore the effect of a long chain fatty acid, sodium oleate, on this process was also studied, We have found that in the absence of sodium oleate one molecule of taurocholate is maximally bound per molecule of bovine serum albumin. However, the presence of sodium oleate permits the binding of 3 moles of taurocholate per mole of bovine serum albumin. MATERIALS
AND
METHODS
Eovine serum albumin, Lot ~6, was obtained from Pentex Inc., Kankakee, Ill. Sedimentation velocity showed that the sample was homogeneous. Fatty acids were removed from bovine serum albumin by the procedures of CHEP and SPECTOR et ai.j. The fatty acid free bovine serum albumin was filtered through a o.L+s-~ ~~lli~ore Biochinz.Bio&s. Acta, 231
(1971)
550-552
BINDING
OF SODIUM TAUROCHOLATE
BY ALBUMIN
551
filter to remove charcoal not separated by centrifugation. A mol. wt. of 66000 (see ref. 5) was assumed for bovine serum albumin, and an extinction coefficient of 0.667 mg-1 . cm2 at z7g rnp (see ref. 6) was used in calculations. Sodium [24JX] taurocholate was purchased from Tracer Lab, Waltham, Mass,, Lot 58-107, Alternatively the same material was synthesized and purified by the methods of NORMAN~>*. Thin layer chromatography indicated that this preparation was at least 99% pure. Oleic acid, Lot D-IA, was obtained from the Hormel Institute, Austin, Minnesota. All equilibrium dialysis experiments were carried out at room temperature in a salt solution, simulating physiological conditions, which contained 0.116 M NaC1, 0.0049 M KCI, 0.0012 M MgSO, and 0.016 M sodium phosphate, pH 7.4 (see ref. 5). Equilibrium was approached by transport of ligand across the membrane in both directions. In dialysis cells initially containing bovine serum albumin, buffer and taurocholate in the same compartment, equilibration required 24 h. When the ligand was placed in the opposite compartment 45 h were necessary for equilibration. A similar phenomenon was observed by CASSELet al.g. Consequently all samples were taken after 45 h of dialysis. In parallel experiments, sodium oleate was added to determine its influence on taurocholate binding. Sodium oleate stock solution was prepared by neutralizing oleic acid with I M NaOH, heating, and diluting with water. RESULTS AND DISCUSSION
Binding of taurocholate to bovine serum albumin in the presence and absence of sodium oleate was calculated by the following equationl:
where G is the number of moles of sodium taurocholate bound per mole of bovine serum albumin, n is the number of binding sites, K is the dissociation constant of each site and C is the molar concentration of taurocholate at equilibrium. The lines shown in Fig. I were fitted by least squares analysis of data points. The stoichiometry of binding is the reciprocal of the y-axis intercept. The dissociation constant (Kdiss) is the slope multiplied by the number of binding sites. Standard deviations of these parameters were also calculated. In the absence of fatty acid, 1.01 & 0.31 mole of sodium taurocholate is bound per mole bovine serum albumin, and Kdiss is 8.21 . IO@ f 0.26 . 10-j M. The presence of I . IO-~ M oleic acid increased the Kdiss slightly and increased the molar ratio of sodium taurocholate bound to approx. 3 (see Fig. I). The molar ratio of cholic acid bound to human serum albumin was reported to be 4 and that of deoxycholic acid was IZ (see ref. I). Three primary bovine serum albumin sites were observed for fatty acids5 and 5 were observed for the neutral ligands, octanol and decanol lo. Our results show that one mole of sodium taurocholate strongly binds to one mole of fatty acid-free bovine serum albumin, indicating a specific binding mechanism. The dissociation constant, 8 IO@ M, is of the same order of magnitude as that found for enzyme-substrate binding. Our data do not rule out the possibility of some weaker binding sites which may occur at higher concentrations of taurocholate. The increase in taurocholate binding to bovine serum albumin in the
Fig. r. Reciprocal plot of binding of sodium taurocholate by bovine serum albumin, 25’. Lines are fitted by least squares analysis of data points. Bovine serum albumin concentration is r-41. 10-j M. Vis number of moles sodium taurocholate bound per mole bovine serum albumin. C is the molar equilibrium concentration of free sodium taurocholate. Plastic equilibrium dialysis cells (Chemical Rubber Co.) containing I ml on each side of the membrane were used. Dialysis tubing from Union Carbide Corp. was boiled first in I . IO-~ M EOTA and then in 0.1 M NaHCO, for I h and washed thoroughly &I distilled water. After equilibrium dialysis, 0.1.ml samples were-added to ro ml BioSolv-toluene scintillation fluid. The above data was obtained with sodium [z4-r*Cjtaurocholate purchased from Tracer-lab. Replicate experiments performed with material synthesized in this laboratory (see text )gave comparable results.
presence of oleic acid suggests the formation of additional strong binding sitesas a result of fatty acid induced conformational changes in bovine serum albumin. Serum albumin acts physiologically as a carrier for both fatty acids and bile salts in plasma. Our observations imply that the presence of fatty acids enhances the capacity of albumin to transport bile acids. ACKNOWLEDGMENTS
This work was supported by Grant AM og58z from the Yational Institutes of Health. REFERENCES I D. RUDMAXAND F. E. KER'DALL,J.C~~~.I~~~~~., 36(1957) 538. 2 5. LACK AND 1.M. WEINER, Am. J. Physiol., zoo (1961) 313. 3 L. LACI~ AND I.M. WEIP;ER, Biochim. Biophys. Acta, 135 (1967) 1065. 4 R. F. CHEN, J.Biol. Chew., 242 (1967) 173, 5 A. A. SPECTOR, K. JOHN AND J.E.FLETcHER,J. LipidRes., 1c(136g) 56. 6 J. F. POSTER AND M. D. STERMAN, J. Am. Chem. Sm., 78 (1956) 3656. 7 A. NORMAN, Avkiv Kevnni, 8 (1956) 331. S A. NORMAN, Acta Chew. Scand., 7 (1953) 1413. g J. CASSEL, J. GALLAGHER, J. A. REYNOLDS AND J. STEINHARDT, Biochemistry, 8 (1969) 17~6. ro J. STEINHARDT AND J. A. REYNOLDS, Multiple Equilibria in Pvoteins, Academic Press, New York, 1969. B&him.
Biophys.
Acta,
231 (1971)
550-552