β Scribed by E. Habermann; K.L. Hardt
No coin nor oath required. For personal study only.
Phospholipases A clear suspensions of egg yolk. This is also true when the substrate is incorporated into agar agar or agarose gels. Round, cleared areas develop from circumscribed enzyme depots. Their diameter is, over a wide dose range, proportional to the logarithm of the enzyme concentration applied. This parameter can be used for the measurement of phospholipases A from bee venom, Crotalus terrificus venom, and pancreas. The optimal reaction conditions have been worked out for the bee venom enzyme.
Under these conditions, 7.515 pg of the bee venom enzyme and 30-60 ng of pancreatic and Crotalus phospholipase A are still detectable. Incubation at 50Β°C increases the cleared areas and eliminates simultaneously less resistant enzymes, such as trypsin and chymotrypsin.
The specificity of the test for phospholipase A can be further improved by incorporation of human erythrocytes into the egg yolk plates. Sensitivity remains unchanged.
Phospholipase C produces turbid areas in egg yolk plates. Their diameter can be used for the quantitation of the enzyme. Low activities of phospholipase B, together with highly active phospholipase A, can be followed in the egg yolk plate since phospholipase B renders the once cleared zones centrally turbid. With lysolecithin plates, phospholipase B can be measured by this dimming effect.
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