Preparation of Soluble and Insoluble Polysaccharide Conjugates of Peptides with a Free α‐Amino Group

Abstract

Synthetic peptides were covalently attached to Sepharose and water-soluble dextran of high molecular weight. In these polysaccharide conjugates, the bound peptides bear a free a-amino group. The interaction of leucine aminopeptidase with its peptide ligands Thr-Phe-Pro (substrate) or Thr-(But)-Phe-Pro (competitive inhibitor) was strikingly different in soluble dextran conjugates and insoluble Sepharose conjugates of these ligands. The affinity of the enzyme to its inhibitor Thr(Bu')-Phe-Pro was decreased by more than 100-fold upon insolubilization of the peptide on Sepharose. When the peptide was coupled to dextran ofweight-average molecular weight z 500000, the potency of the inhibition was not impaired.Leucine aminopeptidase failed to hydrolyze the Sepharose-bound substrate Thr-Phe-Pro to a measurable extent, but it released N-terminal threonine from the soluble dextran conjugate of this peptide to an extent of at least 38 % of the theory.The interference, caused by the insoluble matrix was hardly overcome, when a new type of spacer, consisting of > 80 m-alanyl residues, was inserted between matrix and ligand. The spacer which is several enzyme subunit diameters in length, gave rise to a small fraction (about 5%) of accessible ligand but did not improve specific interaction to a sufficient extent in order to enable specific binding of leucine aminopeptidase to inhibitor-, or substrate-substituted Sepharoses.