PubMed · 2860528
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A J MacGilchrist, B M Frier. 1985-06-08. Testing pancreatic function.. https://doi.org/10.1016/s0140-6736(85)92834-x
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We have made a curious observation that the proteolytic enzyme, trypsin, induced a rapid and complete release of the contents of vesicles composed of dioleoylphosphatidylethanolamine (DOPE) and oleic acid (OA). Content release at 37 degrees C, monitored by the release of an entrapped fluorescence marker (calcein), was accompanied by an extensive vesicle aggregation. The lytic activity of trypsin on the vesicles depended on pH and liposome composition. The optimal pH for vesicle lysis was below pH 7.4, which was different from the optimal pH for catalytic activity of trypsin. The lytic activity of trypsin was specific for vesicles composed of DOPE and fatty acids such as OA and palmitoleic acid; vesicles composed of dioleoylphosphatidylcholine, N-methyl-DOPE, and OA, or DOPE combined with other negatively charged lipids such as phosphatidylserine and phosphatidic acid were not sensitive to trypsin. Inhibition of enzyme activity by trypsin inhibitors did not abolish the lytic activity, suggesting that the lytic activity of trypsin is not related to the catalytic activity. However, the lytic activity of trypsin on vesicles composed of DOPE and OA was inhibited in the presence of excess vesicles containing negative charges, or by a pretreatment of trypsin with acylating reagent to reduce the positive-charge content of trypsin. These data demonstrate that vesicle aggregation and lysis are the results of electrostatic interactions of positive charges on trypsin and negative charges on the vesicles. Phase separation and transition to nonbilayer phases of the vesicle lipids are likely involved.
A novel enzymatic assay method was developed for fluorogenic substrates that have significant intrinsic absorbance and fluorescence under the assay conditions. Fluorescein mono-beta-D-galactoside (FMG) was chosen as the substrate for the fluorescence enzymatic assay because of the high fluorescence of its hydrolytic product (fluorescein) and suitability of being hydrolyzed by beta-galactosidase. The fluorescence-concentration relationships for fluorescein and for FMG in both the right-angle detection mode of a fluorometer and the front-face detection mode of a fluorescence plate reader were exactly established and used to determine the kinetics of the enzyme assay. The results show that only front-face detection in the fluorescence plate reader can overcome the fluorescence concentration quenching that inevitably results from high absorbance by the intrinsically absorbing substrate in the conventional fluorometer, which utilizes right-angle detection. Only with front-face detection was the fluorescent assay of FMG hydrolysis under conditions of high optical density possible. The enzymatic measurements on the fluorescence plate reader were particularly efficient for determination of the enzyme kinetics because of the high rate of data collection. In this assay system, Michaelis-Menten constant Km and enzymatic catalysis rate k2 of FMG were determined as 117.6 microM and 22.7 mumol-(min.mg)-1, respectively. The results and methods described in this paper can be generalized for any assay using a fluorogenic substrate whether or not it has a high background absorbance.
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