Model study of hydrophobic interactions of alpha- and beta-trypsin and alpha-chymotrypsin

The hydrophobic interactions of alpha- and beta-trypsin as a function of ionic strength and pH were studied by hydrophobic chromatography. Evidence was obtained that in spite of the identical specificities and similar activities of alpha- and beta-trypsin, the cleavage of the Lys-Ser bond induces conformational changes in the neighbourhood of the active site. Over a wide range of pH and salt concentration the non-polar residues on the surface of the molecule of beta-trypsin are more exposed to an external environment than on the molecule of alpha-trypsin. In the trypsin(chymotrypsin)-inhibitor complexes the majority of hydrophobic amino acids are buried; other hydrophobic residues localized on the surface contribute only very slightly to the interaction with the chromatographic support. The retention of trypsin, chymotrypsin and their diisopropylphosphoryl derivatives on a support with flexible hydrophobic ligands bonded to the matrix through a spacer (octyl-Sepharose) was correlated with the retention on a support with hydrophobic binding sites incorporated into the rigid matrix of the resin (Spheron). The native enzymes are always more retained; this indicates that the substitution results in the shielding of the non-polar residues in the neighbourhood of the active site. The differences in the slope of individual proteins, resulting from the correlation of the retention values obtained with both supports at several sodium chloride concentrations are explained by differences in the accessibility of the surface non-polar residues in the individual proteins. In experiments with model peptides the contribution of the individual hydrophobic amino acids to the retention was investigated.