Spectrophotometric method for the assay of glycosaminoglycans and glycosaminoglycan-depolymerizing enzymes

Undegraded glycosaminoglycans form a complex with 1-ethyl-2-[3-(1-ethylnaphtho-[1,2-d]thiazolin-2-ylidene)-2- methylpropenyl]naphtho-[1,2-d]thiazolium bromide (Stains-all) in solution resulting in a characteristic shift in spectrum. Hyaluronic acid (a nonsulfated glycosaminoglycan) reacts with the dye to form a complex with an absorbance maximum at 650 nm, while sulfated glycosaminoglycans (chondroitin sulfates A and C, dermatan sulfate, keratan sulfate, and heparan sulfate) give rise to an increase in absorbance at 480 nm. Increases in absorbance at the appropriate wavelength are directly proportional to the concentration of glycosaminoglycan interacting with the dye. This phenomenon provided the basis for a sensitive spectrophotometric assay for the quantitative measurement of bacterial and mammalian glycosaminoglycan-depolymerizing enzymes. The basic assay method was adapted for use in 96-well microtiter trays, thus enabling large numbers of assays to be carried out simultaneously.