Ligand-stabilized conformations in serum albumin.

Interaction of human serum albumin with anionic azo dyes decreases the susceptibility of the former to hydrolytic attack by five different proteases. By using methyl orange as the ligand, it is shown that the occupation of the two strong binding sites by the dye is necessary and sufficient for this effect. Different ligands decrease the digestibility of albumin to different extents when tested with the same protease. The pattern of digestion, studied by electrophoresis and gel filtration, also exhibits differences which are characteristic for the ligand used. Thus, methyl orange greatly reduces the yield of the 51,000 mol wt fragment, which regularly appears in the course of tryptic digestion in the absence of ligand. In addition, the relative quantities of the smaller fragments obtained vary with the ligand used in a characteristic manner. Analysis of the kinetics of digestion in the presence and absence of ligand suggests that the major effect of ligand is to increase the stability or to decrease the availability of hydrolyzable bonds, with little alteration of the stability of the enzyme-substrate complex. It is concluded that the conformations stabilized by ligands are different from each other and are characteristic for the ligand.