Binding properties of glycosaminoglycans to lysozyme--effect of salt and molecular weight.
The cationic protein, lysozyme, is present in cartilage but its precise role in this tissue has not yet been established. This study shows that two major and structurally similar glycosaminoglycans (GAGs) of cartilage, i.e., chondroitin sulfate (CS) and hyaluronan (HA) interact with lysozyme at salt concentrations up to 40 mM. Such a low salt concentration is likely to occur in cartilage due to exclusion of microions by the high charge density of the proteoglycans (PGs). The affinity of binding to lysozyme increases with increasing molecular weight of HA and is higher for HA (Kd = 1-2 x 10(-8) M and 0.5-1 x 10(-7) M for HA of relative molecular mass of 4 x 10(5) and 5 x 10(4), respectively) than for CS (Kd = 1 x 10(-6) M). The binding displays optimal levels at around 20 mM but decreases at both lower and higher salt concentrations. This dependence of binding on salt concentration resembles that of the enzymic activity of lysozyme for its natural substrate, murein, which is structurally similar to HA/CS. The increase in binding up to 20 mM salt is characteristic for HA/CS-lysozyme interaction as such an effect was not observed in the interaction of heparin with lysozyme or of GAGs with serum albumin. Binding of HA to lysozyme was inhibited by various polyanions but not by uncharged macromolecules, indicating the electrostatic nature of the interaction. The dependence of binding on salt concentration obtained in systems where lysozyme is linked to an agarose support and the GAG is free in solution is similar to that determined when both macromolecules are free in solution; however, the number of GAG disaccharides bound per mole lysozyme increases significantly in the latter system, indicating a marked artifactual steric hindrance effect in the former.
Van Damme
M P
MP
Department of Biochemistry, Monash University, Clayton, Victoria, Australia.
Moss
J M
JM
Murphy
W H
WH
Preston
B N
BN
D002809:Chondroitin Sulfates / Q000378:metabolism; D006025:Glycosaminoglycans / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D006820:Hyaluronic Acid / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D009113:Muramidase / Q000378:metabolism*; D009844:Oligosaccharides / Q000494:pharmacology; D000071228:Polyelectrolytes; D011108:Polymers / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D012492:Salts / Q000494:pharmacology; D013329:Structure-Activity Relationship
D002809:Chondroitin Sulfates; D006025:Glycosaminoglycans; D006493:Heparin; D006820:Hyaluronic Acid; D009113:Muramidase; D009844:Oligosaccharides; D000071228:Polyelectrolytes; D011108:Polymers; D012492:Salts