The active site titration of proteinases by using α2-macroglobulin and high-performance liquid chromatography

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The active site titration for various proteinases relies on the development of optimal enzyme titrants for each proteinase, but these titrants are only available for a limited number of proteinases. We have described a new active site titration method applicable to various kinds of endoproteinases using small quantities of the enzymes. This method was carried out by using α 2-macroglobulin ( α 2M) as a titrant and a high-performance liquid chromatography (HPLC) system. When the proteinase solution was treated with α 2M, the active proteinase was trapped by α 2M. In this reaction α 2M does not usually complex with inactive proteinase. After the reaction of proteinase with an excess of α 2M, the reaction mixture is applied to an HPLC gel column to separate the uncomplexed enzyme from the one complexed with α 2M. The active proteinase is complexed and eluted with α 2M, but the inactive proteinase is eluted at the original elution volume. The same amount of the enzyme was also applied to the column. From the decrease of the peak height at the elution position of the uncomplexed proteinase, we can estimate the ratio between enzymatically active proteinases and total proteinases. To test the usefulness of this method, we applied this method to chymotrypsin and trypsin whose activities were predetermined by conventional active site titration, and there was good agreement between both results. With this new method, we can estimate a proteinase activity with as little as 200 ng of the enzyme, a very small amount compared with those required in conventional methods. We also studied the pH dependency of pepsin activity and did active site titrations of V8 proteinase and thermolysin by this method.