Enzymatic reactions on thin-layer chromatographic plates : I. Lipolysis of triglycerides and separation of products on a single plate

Abstract

A novel method for the lipase deacylation of triglycerides is described in which 0.1–0.2 ml of the enzyme solution in 1 M tris buffer (pH 8.2) containing 2–4 mg of protein is applied as a band on a 0.5-mm thick silica gel thin-layer chromatographic plate. Over this band, 1–4 mg of triglyceride in n-hexane is applied evenly and the plate is incubated at 40° for 1–2 min. The reaction is stopped by exposure to hydrogen-chloride vapour and the products are removed from the reaction zone by three consecutive developments in diethyl ether up to 2 cm from the line of application. These are resolved by re-developing the plate in n-hexane—diethyl ether—acetic acid (80:20:1.5) up to 14 cm. The sn-2-monoglyceride and sn-1,2(2,3)-diglyceride bands located by iodine vapour are extracted and the fatty acid compositions evaluated by gas-liquid chromatography for the determination of fatty acid distribution in the glyceride molecule. The method, when applied to groundnut oil, goat depot fat, body fat of shol fish ( Channa striatus) and yeast ( Rhodotorula glutinis) fat, produced representative sn-1,2(2,3)-diglycerides which can be used for the stereospecific analysis of triglyceride.