Homogeneous enzyme-based binding assay for studying glycosaminoglycan interactions with macromolecules and peptides.
A simple and rapid homogeneous enzyme-based binding assay is described to study the degree of interaction between glycosaminoglycans and various macromolecules/peptides. The method is based on the homogeneous inhibition of a highly positively charged enzyme, acid deoxyribonuclease II (EC 3.1.22.1), by glycosaminoglycan polyanions, such as heparin, chondroitin 4-sulfate, and dermatan sulfate. Catalytic activity of DNase II is inhibited to nearly 100% by relatively small amounts of these glycosaminoglycan molecules. In the presence of species that bind these polyanions, the activity of the enzyme is regained in an amount proportional to the concentration of the species present. Thus, the relative binding affinities of various species with a given GAG can be assessed rapidly by comparing the concentration of the compound required to reverse the enzyme inhibition to 50% of the maximum value (ED(50) values). The feasibility of this binding assay principle is demonstrated by measuring the ED(50) values of five macromolecules: polylysine, polyarginine, protamine, low-density lipoprotein (LDL), and high-density lipoprotein (HDL), using heparins of different size, as well as chondroitin 4-sulfate and dermatan sulfate as the GAG polyanions. The applicability of the assay method is further extended to study GAG-peptide interactions. A variety of small synthetic peptides (8-13 amino acid residues) derived from the heparin-binding domains of protamine and type IV collagen are used as model peptide species. Relative GAG-binding affinities of these macromolecules/peptides are compared to previous literature values and data obtained via a new electrode-based titration method.
Guo
X
X
Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
Han
I S
IS
Yang
V C
VC
Meyerhoff
M E
ME
D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D004706:Endodeoxyribonucleases / Q000037:antagonists & inhibitors; D006025:Glycosaminoglycans / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D011506:Proteins / Q000378:metabolism*
D006025:Glycosaminoglycans; D046911:Macromolecular Substances; D011506:Proteins; D004706:Endodeoxyribonucleases; C019329:deoxyribonuclease II