Assay of collagenase activity for native triple-helical collagen using capillary gel electrophoresis with laser-induced fluorescence detection

Abstract

Three collagenase assays for two native triple-helical collagens have been developed using capillary gel electrophoresis (CGE) with laser-induced fluorescence (LIF) detection in order to discover the matrix metalloproteinases (MMPs) inhibitors. These collagenase assays include measurement of the activities of interstitial collagenase (MMP-1) and neutrophil collagenase (MMP-8) against type I collagen, and collagenase-3 (MMP-13) against type II collagen, and the enzyme activities could be readily measured by determining the 3/4 fragments produced from the cleavage of the native collagens. The highly desired sensitivity of the assays could be achieved, employing a dynamic fluorescence labeling technique with the running buffer containing 0.05% sodium dodecylsulfate and non-covalent fluorescent dye for protein, NanoOrange. The collagen, its 1/4 and 3/4 fragments of type I or II collagen could be separated and detected within the run time of 20 min by CGE mode using the gel buffer (pH 8.8) containing 4% polyacrylamide. Good linearity of the peak area of the 3/4 fragment was obtained over each assay range of collagenase (15–150 ng/tube for MMP-1, 3–30 ng/tube for MMP-8, and 1.5–30 ng/tube for MMP-13, respectively). The relative standard deviation of the peak areas of the 3/4 fragment produced from type II collagen by MMP-13 cleavage was calculated to be less than 13.4%, indicating that the assay was reproducible. Also, IC50 values of three MMPs inhibitors, which were calculated for estimation by the variation of the peak areas of the 3/4 fragments using 90 ng/tube for MMP-1, 30 ng/tube for MMP-8 or 15 ng/tube for MMP-13, were almost consistent with data from other assays. The CGE-LIF method is expected to be very useful for proteinase assay and its application to the estimation of inhibitors because this method enables an assay of collagenase activity using native substrate to be conducted without experimentally troublesome procedure such as preparation of antibody or fluorescence-labeled substrate.