Lipase radioactive substrates

A number of methods are available for following lipase activity. Although numerous modifications and variations have been introduced, the basic methods are (1) titration of the liberated fatty acids, (2) changes in surface tension, (3) colorimetric determination of the fatty acids, (4) use of gas-liquid chromatography, and (5) use of radioactive substrates. Kuzdzal-Savoie (1980) has reviewed the subject. 

Radioactive Substrates. Koskinen et al (1969), Luhtala et al. (1970-A,B), and Scott (1965) have used labeled triglycerides as substrates for milk lipases. This method, which is extremely sensitive, requires that the acids released by lipase action be isolated uncontaminated with any tagged glycerides. It also requires the preparation of labeled substrate and, of course, counting equipment. 

Trioleoylglycerol (triolein) and olive oil are commonly used substrates, and stable emulsions may be readily obtained for use as substrate preparations. Enzyme activity can be monitored by a pH stat or analysis of released fatty acids (Brockerhoff and Jensen, 1974). The sensitivity of low-level activities may be increased with radioactive substrates. Tributyrin is commonly used because of its ease of dispersion in water, but it cannot be assumed that tributyrin hydrolysis is necessarily a measurement of true lipase activity. Triacetin is water-soluble, and although often used for lipase determinations this practice is not recommended. (A major commercial enzyme supplier markets wheat germ lipase but states on the containers that the preparation will not hydrolyze olive oil—it is, however, active on triacetin.)... 

The present work investigates the action of pancreatic lipase on spread monolayers of ethylene glycol adipate oligomers. The tritiated hydrolysis products of the uniformly labeled substrate escape into the subphase. Surface radioactivity measurements allow us to determine the substrate concentration at the interface (2, 3) and the extent of the enzymic reaction. The kinetics were investigated as a function of subphase pH and temperature and substrate length and concentration. By... 

Figure 2. Adsorption of tritiated pancreatic lipase (2 mg/l) in the presence of the substrate (0.5 mg/m2). Surface radioactivity T8Ubpha8e = 20°Cy pH8Ubpha8 ...

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