Kinetic analysis of the hydrolysis of lecithin monolayers by phospholipase A

Abstract

Enzymic hydrolysis by pancreatic phospholipase A (E.C. 3.1.1.4) of L-dioctanoyl-, L-didecanoyl- and L-didodecanoyllecithin monolayers was studied under constant surface pressure by measuring the amount of substrate which disappears per unit area per unit time. The reaction is first-order with respect to the total number of substrate molecules allowing the determination of a rate constant. Apparent limitations of the monolayer techniques are often caused by diffusion problems. Experimental conditions are discussed to detect and control these difficulties. pH dependence and calcium requirements of the enzymatic reaction are similar under monolayer and bulk conditions. For all three substrates plots of velocity vs. surface pressure show bell-shaped curves with a similar maximum rate at a surface pressure of about 8 dynes/cm. This result is discussed in relation to conformational changes in the lecithin molecules. With the monolayer techniques one can determine only a minimal specific activity, because of the unknown amount of enzyme involved in the catalysis. This minimal specific activity is compared with the value obtained with lecithin micelles as substrate in similar bulk conditions. Inhibition of the enzyme by competitive inhibitors present in mixed films cannot be studied by the monolayer technique. Comparison of the monolayer and bulk methods showed that both techniques are complementary.