An accurate, quantitative assay for collagenase activity based on the synergistic hydrolysis of collagen

✍ Scribed by Harold E. Van Wart; Michael D. Bond

• Publisher: Elsevier Science
• Year: 1982
• Tongue: English
• Weight: 639 KB
• Volume: 120
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(82)90330-x

✦ Synopsis

An accurate and quantitative collagenase assay has been developed which is based on the synergistic hydrolysis of soluble ['4CH,]collagen by thermolysin and collagenase. Thermolysin itself does not hydrolyze collagen at an appreciable rate. However, when present in collagenase assays carried out at 35°C it synergistically assists in the digestion of collagen by hydrolyzing the large collagen fragments produced by true collagenases into much smaller fragments. These smaller digestion products are much more soluble than the larger fragments produced by the primary cleavages and they remain in solution after the undigested collagen is precipitated with phosphotungstic acid. Since the assay measures the appearance of nonprecipitable '+'C, this secondary digestion by thermolysin greatly increases its sensitivity. Furthermore, the assay exhibits initial rates that are linear over a greater percentage of full hydrolysis and activities that are proportional to a wider range of collagenase concentrations than when carried out in the absence of thermolysin. In addition, it gives an accurate reading of the true collagenase activity of impure samples, since thermolysin levels out the synergistic effects of contaminating proteinases. An important feature of the assay is that it enables the calculation of a "synergistic ratio" defined as the rate observed in the presence of both thermolysin and collagenase divided by the sum of the rates obtained for each alone. This ratio reflects the degree of specificity of the collagenase and, thus, provides additional information about the enzyme.