[Kinetic study of the action of pancreatic lipase on emulsified triglycerides. Enzymology assay in heterogeneous medium].

Abstract

Summary The purpose of this paper was to study the action of pancreatic lipase (glycerol ester hydrolase, EC 3.1.1.3) on emulsified triglycerides. This type of action is an interesting example of an enzymatic process taking place in an heterogeneous medium. It is shown that: 1. Lipase is adsorbed by its emulsified substrate and the initial rate ( v ) of the reaction is a function of the number of enzyme molecules adsorbed at the interface. 2. By varying the dilution of a triglyceride emulsion in bile salts in the presence of 0.1 M NaCl, linear Lineweaver-Burk representations are obtained from which the reaction paramaters V m arid K m can easily be derived. However, the abscissae of the representation, and consequently the K m values, remain undefined as long as a right substitute for the substrate concentration concept is not found. Obviously, this concept cannot be used when the substrate is in an insoluble state. 3. By using two emulsions containing particles of different size, it can be shown that v does not depend directly on the weight of the insoluble substrate. For the same weight, the same amount of lipase and the same experimental conditions. v becomes higher when the average size of the particles decreases, v depends on the area ( I ) of the interface. I can be experimentally determined with a good accuracy by two techniques : centrifugation of the emulsions in a sucrose gradient and counting of the particles according to their size in an electronic Coulter counter. 4. On the other hand, v is inversely proportional to the total volume ( A 0 ) of the emulsion. Thus, lipase adsorption may be considered as fully reversible in our experimental conditions and to take place according to Langmuir's isotherms. The “interface concentration” I / A 0 , i.e. the interfacial area in a volume unit of emulsion, is playing, in the case of lipase, the role normally devoted to the concentration of the substrate. The K m (emulsion) of lipase corresponds to a certain value of this interface concentration. This does not mean that I / A 0 is the only significant variable in the process. Other variables such as electrical charge of emulsified particles and molecular organization at the interface may also play a role. 5. It may be supposed that the well-known influence of bile salts during the intraluminar phase of triglyceride digestion is at least partly related to the ability of these substances to favour the reversibility of lipase adsorption at the interface.