An improved assay for the spectrophotometric determination of chondroitinase ABC activity

Abstract

Chondroitinase ABC (chABC) has evolved as a promising therapeutic for the degradation of inhibitory chondroitin sulfate proteoglycans (CSPGs) after injury to the central nervous system (CNS). The enzymatic breakdown of CSPGs by administered chABC reinstated plasticity as well as axon regeneration; leading to improved CNS recovery. Thus, in an effort to develop and evaluate treatments of CNS injury utilising chABC, the accurate detection of levels of CSPGs and more importantly activity of chABC is of paramount importance. Here we present a spectrophotometric assay suitable for accurately and quantitatively measuring chABC activity, based on the use of 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1). This novel WST-1 based assay was compared to two other assays: measurement of alkene generation at 232 nm and a 1,9 dimethyl methylene blue (DMMB) assay which have been used previously for the detection of chABC activity and CSPGs respectively. Comparison of these assays demonstrated that the WST-1 assay was quantitative, efficient, cheap and easy to perform and overall was superior in the detection of chABC activity at all concentrations when compared to the other methods tested.