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.

Chondroitin sulfate depolymerase and hyaluronidase activities of viridans streptococci determined by a sensitive spectrophotometric assay.

Sensitive spectrophotometric assays for the detection of bacterial chondroitin sulfate depolymerase and hyaluronidase activities were developed by using Stains-all (1-ethyl-2-[3-(1-ethylnaphtho-[1,2-d]thiazolin-2-ylidene)-2- methylpropenyl]naphtho-[1,2-d]thiazolium bromide). Stains-all interacts with hyaluronic acid to produce a shift in the absorption spectrum with a distinct absorption peak between 620 and 660 nm, while chondroitin sulfate interacts to form a distinct shoulder between 440 and 500 nm. Assays measure undegraded substrate. A collection of 110 strains of viridans streptococci, including representatives of all the currently recognized species, was studied. Streptococcus intermedius and S. constellatus degraded hyaluronic acid, while only strains of S. intermedius, primarily isolated from brain and liver abscesses, produced chondroitin sulfate depolymerase. S. intermedius, of all the viridans streptococci, produces the widest range of glycoprotein- and glycosoaminoglycan-degrading enzymes, which may contribute to the virulence of this species.