Studies on the reaction between human growth hormone and oleic acid using polyacrylamide gel electrophoresis.

Through the work of U. J. Lewis and E. V. Cheever (1967, Endocrinoloyg81, 1338–1348) and U. J. Lewis, E. V. Cheever, and B. K. Seavey (1968, J. Biol. Chem.243, 260–267) it has been known for a number of years that human growth hormone (hGH), and many other proteins, reacts with unsaturated fatty acids to give rise to species with enhanced electrophoretic mobility. In view of the possible importance of this reaction in the genesis of charge isomeric protein artifacts, and for the understanding of hGH as a system of multiple isomers with distinct, and in some cases enhanced, specific activities, the nature of this reaction was investigated further. It was found that (1) the positions of oleic acid and growth hormone on polyacrylamide gel electrophoresis (PAGE) are coincident, indicating that the reaction leads to binding of the fatty acid to the protein: (2) the increment in molecular net charge on growth hormone is proportional to the molar ratio between the reactants, oleic acid and hGH; (3) the binding is noncovalent since it reverses under conditions of isoelectric focusing; (4) the reaction product has a molecular size indistinguishable from that of the reactant, hGH, by the criteria of “quantitative” PAGE (however, the reaction product exhibits an elevated negative molecular net charge in PAGE at pH 10.2); (5) the apparent isoelectric points of the hormone and its reaction products with oleic acid are indistinguishable in isoelectric focusing; (6) the interaction does not seem to involve the carboxyl charges on oleic acid since it is independent of ionic strength; (7) a noncovalent hydrophobic interaction with the protein is indicated since the range of electrophoretic mobilities exhibited by the hGH-oleic acid complex is smaller in the presence of benzyl alcohol in the gel than that exhibited by controls in it absence; (8) the reaction does not seem to involve free radical derivatives of the unsaturated fatty acid since it is not altered when the polyacrylamide gel is in a nonoxidative state; (9) the effect of the reaction with oleic acid on the tryptophan spectrum reflects only nonspecific interaction of the hormone-concomitant tryptophan perturbation.