Binding of branched-chain 2-oxo acids to bovine serum albumin

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Abstract
Binding of branched-chain 2-oxo acids to defatted bovine serum albumin was shown by gel chromatography and equilibrium dialysis. Equilibrium-dialysis data suggest a 2-site model for binding in Krebs-Henseleit salinr at 37.degree. C with n1 = 1 and n2 = 5. Site association constants were: 4-methyl-2- oxovalerate, k1 = 8.7 .times. 103 M-1, k2 = 0.09 .times. 103 M-1; 3-methyl-2- oxovalerate, k1 = 9.8 .times. 103 M-1, k2 = 0.08 .times. 103 M-1; 3-methyl-2- oxobutyrate, k1 = 1.27 .times. 103 M-1, k2 = < 0.05 .times. 103 M-1. Binding of 4-methyl-2-oxovalerate to defatted albumin in a phosphate-buffered saline, pH 7.4 gave the following thermodynamic parameters: primary site .**GRAPHIC**. [enthalpy change ]= 28.6kJ .cntdot. mol-1 and .**GRAPHIC**. [entropy change] = - 15.2J .cntdot. mol-1 .cntdot. K-1 .**GRAPHIC**. [free energy of binding] = - 24.0kJ .cntdot. mol-1 at 37.degree. C and secondary sites .**GRAPHIC**. = - 25.4kJ .cntdot. mol-1 and .**GRAPHIC**. = - 46.1J .cntdot. mol-1 .cntdot. K-1 .**GRAPHIC**. = - 11.2kJ .cntdot. mol-1 at 37.degree. C. Thus binding at both sites is temperature-dependent and increases with decreasing temperature. Inhibition studies suggest that 4-methyl-2-oxovalerate may associate with defatted albumin at a binding site for medium-chain fatty acids. Binding of the 2-oxo acids in bovine, rat and human plasma follows a similar pattern to binding to defatted albumin. The proportion bound in bovine and human plasma is much higher than in rat plasma. Binding to plasma protein, and not active transport, explains the high concentration of branched-chain 2-oxo acids leaving rat skeletal muscle relative to the concentration within the tissue, but does not explain the 2-oxo acid concentration gradient between plasma and liver.