An assay for bacterial and eukaryotic chondroitinases using a chondroitin sulfate-binding protein.
A simple, rapid, and reproducible microtiter-based chondroitinase (CSase) assay is reported here, based on the competition of chondroitin sulfate (CS) with immobilized hyaluronan (HA) for the binding of TSG-6 protein, the product of TNF-inducible gene 6. Although the catabolic reaction of bacterial and other prokaryotic CSase enzymes, often referred to as the chondroitin lyases, can be followed by tracking the generation of unsaturated bonds by the spectrophotometrical determination of the absorbance at 232 nm, no rapid, sensitive, and simple assay has been devised to date for measuring the activity of the vertebrate enzymes that cleave their substrate exclusively by hydrolysis. We provide data demonstrating that the CSase assay described here is suitable for the determination of the activities of both classes of enzymes. For the bacterial enzyme CSase ABC, both the determination of the absorbance at 232 nm and the assay based on TSG-6 binding are suitable using the same range of enzyme activities. However, for testicular hyaluronidase, considerably higher enzyme activities were needed to cleave CS than to cleave HA. Using the HA-binding domain of aggrecan for a comparison, we determined that the interaction between TSG-6 and chondroitin sulfate is uniquely suited for this CSase assay.
Wisniewski
Hans-Georg
HG
Department of Microbiology, School of Medicine, New York University, NY 10016, USA. hans-georg.wisniewski@med.nyu.edu
Sweet
Moshe H
MH
Stern
Robert
R
D000818:Animals; D001667:Binding, Competitive; D019921:Biotinylation; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002809:Chondroitin Sulfates / Q000737:chemistry / Q000378:metabolism*; D002810:Chondroitinases and Chondroitin Lyases / Q000032:analysis* / Q000378:metabolism; D006820:Hyaluronic Acid / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D012756:Sheep; D013053:Spectrophotometry; D013302:Streptomyces / Q000201:enzymology*; D013737:Testis / Q000201:enzymology*
D015815:Cell Adhesion Molecules; D006820:Hyaluronic Acid; D002809:Chondroitin Sulfates; D002810:Chondroitinases and Chondroitin Lyases