Protein substrate conformation and proteolysis.

Abstract

vailing in the absence of the stabiliziing influence. Similarly, if the shift involves no gross change in the internal organization of the molecule, optical rotatory dispersion measurements will not disclose significant differences. Sufficient sensitivity, however, cain be expected from methods which depend on the state of localized areas in the molecule. The method chosen in this study was the measurement of the rate of proteolytic cleavage of peptide bonds. It is understood, of course, that this method is unable to define any particular conformation; it merely detects conformation changes. That it can be a highly sensitive tool for this purpose will be shown by the demonstration that it canr detect conformation changes which accompany the reversible binding of a single small-molecular ligand to a protein molecule. Decreased proteolytic digestibility of some proteins in the presence of smallmolecular ligands has been repeatedly observed in the past. Thus, serum albumin was found less digestible by trypsin in the presence of Ca++ and Mn++ ions,2 and similarly, the tryptic digestibility of conalbumin and ferritin was diminished when in combination with iron. ^ 4 Increased resistance against proteolytic digestion of serum albumin in the presence of anionic detergents5 6 and of fatty acid anions7 was also reported. Several instances of increased resistance against proteolysis of enzymes in the presence of substrates, or cofactors, have been described.8 9 Greatly diminished digestibility of purified rabbit antibody in the presence of homologous hapten has recently been observed in this laboratory.'? In the present communication we report that reversible combination of serum albumin with a given smallmolecular ligand markedly reduces the digestibility of the protein by a variety of proteolytic enzymes of different specificity; it is concluded that the effect cannot be due to direct shielding of susceptible bonds by the ligand molecules, but to a transformation which may involve the entire protein molecule. Based on these observations, a suggestion is made concerning the mechanism of proteolysis. Materials and Methods.-Human serum albumin (Cutter) was deionized by passage through an Amberlite MB-3* mixed-bed resin column and exhaustively dialyzed against distilled water. Acylated human serum albumin (HSAp) was prepared by treating 100 ml of a 1% solution of HSA in water with 0.3 ml of lauroyl chloride contained in 10 ml of benzene. The solution was stirred at 25? for 5 hr and the pH was held at 9 by frequent addition of 0.1 N NaOH. After centrifugation for the removal of the organic phase and a slight amount of precipitate, the solution was dialyzed against water and subsequently pervaporated to yield a 5% protein solution. The proteolytic enzymes used were the following: trypsin (Worthington, twice crystallized), a253