Plasma Elastase Activity Inhibition—A Microassay

Abstract

Uncontrolled tissue destruction by protease release plays a role in some pathological conditions, and an imbalance in the protease–antiprotease system is hypothesized to be an important contributing factor. Elastase, one of the matrix-degrading enzymes found in plasma, is closely associated with the elastic tissue destruction found in conditions such as abdominal aortic aneurysm (1), emphysema (2), pancreatitis (3)(4), and inflammatory conditions (5). A possible factor in the progression of these conditions may be an imbalance in the protease–antiprotease system. Excess production of protease or loss of inhibition is a factor in the alteration of the protease–antiprotease imbalance. To investigate the elastase inhibitory potential of plasma we have developed a method of evaluating the ability of plasma to inhibit purified elastase. This assay is a modification of a technique recently developed by us, which detects the primary amines that are exposed because of the enzymatic degradation of elastin substrate (6). The inhibitory effect of plasma is determined by introducing 10 μL of plasma into a reaction mixture containing succinylated elastin, and the reaction is initiated by the addition of a known amount of elastase. The inhibitory effect of plasma is evaluated by comparing the activity of elastase in the presence and absence of plasma. The inhibition assay contained 100 μg of succinylated elastin, 10 μL of plasma, and 50 μL of porcine pancreatic elastase (PPE; EC 3.4.21.36; 1 g/L made up in 50 mmol/L sodium borate buffer, pH 8.5; 297 U/mg; Calbiochem), and was made up to a final volume of 150 μL with 50 mmol/L sodium borate buffer, pH 8.5, in a 96-well microplate. [One U of enzyme will hydrolyze 1 μmol of N-Ac-(Ala)3-methyl ester per minute at 25 °C, pH 8.5.] The assay was carried out at 37 °C for 30 min. Then 50 …