HOMOGENEOUS ENZYME-BASED BINDING ASSAY FOR STUDYING GLYCOSAMINOGLYCANINTERACTIONS WITH MACROMOLECULES AND PEPTIDES

A simple and rapid homogeneous enzyme-based binding assay is described to study the degree of interaction between glycosaminoglycans and var ious macromolecules/peptides. The method is based on the homogeneous i nhibition of a highly positively charged enzyme, acid deoxyribonucleas e II (EC 3.1.22.1), by glycosaminoglycan polyanions, such as heparin, chondroitin 4-sulfate, and dermatan sulfate. Catalytic activity of DNa se II is inhibited to nearly 100% by relatively small amounts of these glycosaminoglycan molecules. In the presence of species that bind the se polyanions, the activity of the enzyme is regained in an amount pro portional to the concentration of the species present. Thus, the relat ive binding affinities of various species with a given GAG can be asse ssed rapidly by comparing the concentration of the compound required t o reverse the enzyme inhibition to 50% of the maximum value (ED(50) va lues). The feasibility of this binding assay principle is demonstrated by measuring the ED(50) values of five macromolecules: polylysine, po lyarginine, protamine, low-density lipoprotein (LDL), and high-density lipoprotein (HDL), using heparins of different size, as well as chond roitin 4-sulfate and dermatan sulfate as the GAG polyanions. The appli cability of the assay method is further extended to study GAG-peptide interactions. A variety of small synthetic peptides (8-13 amino acid r esidues) derived from the heparin-binding domains of protamine and typ e IV collagen are used as model peptide species. Relative GAG-binding affinities of these macromolecules/peptides are compared to previous l iterature values, and data obtained via a new electrode-based titratio n method. (C) 1996 Academic Press, Inc.