STUDIES ON PHOSPHOLIPASE A2 (PLA2) INHIBITOR FROM Echis ocellatus SERUM AND ITS EFFECT ON NEUROTOXIC PLA2 AND NON TOXIC SECRETORY PLA2
STUDIES ON PHOSPHOLIPASE A2 (PLA2) INHIBITOR FROM Echis ocellatus SERUM AND ITS EFFECT ON NEUROTOXIC PLA2 AND NON TOXIC SECRETORY PLA2
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Date
2014-08
Authors
ADAMUDE, Fatima Amin
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Abstract
The most effective and acceptable therapy for snakebite victims is the immediate administration
of antivenin which is limited by problems of hypersensitivity reactions in some individuals and
its inability to resolve the local effects of the venom. The aim of this study was to investigate the
presence and specificity of Phospholipase A2 Inhibitor from Echis ocellatus serum towards snake
venom neurotoxic PLA2 and mammalian secretory PLA2. Phospholipase A2 (EC.3.1.1.4) was
isolated and partially purified from the venom of Echis ocellatus (carpet viper). A two step
purification process on DEAE-cellulose and Sephadex G-75 column chromatography gave a
specific activity of 53.17μmol/min/mg protein, 16.36 purification fold and 43.11% recovery.
Initial velocity studies for the determination of kinetic constants using L-α- Lecithin as substrate
revealed a KM and Vmax of 1.4mgml-1 and 4.5μmolmin-1 respectively. A protein (Phospholipase
A2 Inhibitor from Echis ocellatus Serum (PIES)) isolated and purified from E. ocellatus serum
inhibited the carpet viper PLA2 enzyme in a dose dependent manner. A two step purification
process on Sephadex G-200 column and DEAE- cellulose chromatography gave 2 active
fractions that inhibited the venom PLA2 by 78%. The result from SDS-PAGE showed the
inhibitor to be a 24.98kDa protein and its kinetic study revealed a mixed pattern of inhibition on
the carpet viper PLA2 with an estimated Ki values of 3.8%(v/v) to 7.3%(v/v). Membrane
stabilizing and protective ability of PIES was recorded by its potential to reduce hemolysis due
to venom PLA2 from 81.20 % to 35.30 % in vitro. Coagulant potentials of PIES were also seen in
its ability to restore plasma coagulation time to less than a minute. Interestingly, PIES does not
affect the enzymatic activity of mammalian secretory PLA2 but strongly inhibits PLA2 activity of
carpet viper in this study. In conclusion, the present study shows that PIES may be a promising
candidate for future development of a novel antivenin drug.
Description
A THESIS SUBMITTED TO THE SCHOOL OF POSTGRADUATE STUDIES,
AHMADU BELLO UNIVERSITY ZARIA, NIGERIA, IN PARTIAL FULFILMENT FOR
THE AWARD OF MASTERS OF SCIENCE DEGREE IN BIOCHEMISTRY
DEPARTMENT OF BIOCHEMISTRY
FACULTY OF SCIENCE
AHMADU BELLO UNIVERSITY, ZARIA
AUGUST, 2014
Keywords
STUDIES,, PHOSPHOLIPASE A2, (PLA2) INHIBITOR,, chis ocellatus,, SERUM,, EFFECT,, NEUROTOXIC PLA2,, NON TOXIC,, SECRETORY PLA2