Isolation and
Characterization of Antithrombin Peptides from the
Saliva of Malaysian Leeches
Mohamed Alaama1*,
ABM Helal Uddin1*, Abbas Mohammad Ghawi2,
Ahmed Merzouk3, Abdualrahman Mohammed
Abdualkader¹, and Mohamed bin Awang4
¹Department of
Pharmaceutical Chemistry, 2Department of
Basic Medical Science, Faculty of Pharmacy,
International Islamic University Malaysia (IIUM), 25200
Kuantan, Malaysia, 3Biopep Solutions Inc,
Vancouver, BC, Canada, 4Faculty of Pharmacy, CUCMS,
Cyberjaya, Selangor.
*For correspondence:
Email:
hass83pharm@yahoo.com,
abmhelal@iium.edu.my
Received: 11 May 2013
Revised accepted: 23
February 2014
Tropical
Journal of Pharmaceutical Research, April 2014;
13(4):
553-557
http://dx.doi.org/10.4314/tjpr.v13i4.10
Abstract
Purpose: To isolate and characterize
the antithrombin compounds of Malaysian leeches’ saliva
collection (LSC) for use as anticoagulant proteins and
peptides.
Method: Reversed phase - high
performance liquid chromatography (RP-HPLC) was used to
isolate all proteins from LSC. All isolated proteins
were tested for anticoagulant activity by applying two
tests, thrombin time test and the inhibition of the
amidolytic activity of thrombin on the chromogenic
substrate (S-2238). The molecular weights of isolated
active compounds were identified using tricine sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE).
Results: More than 40 proteins were
isolated from LSC using RP-HPLC. Two compounds (protein
1 and protein 2) were found to be active as they
increased thrombin time by 30.26 and 36.75%,
respectively and inhibited the amidolytic activity of
thrombin. as evaluated by measuring the conversion of
the chromogenic substrate (S-2238). Decrease in the
conversion of the substrate (S-2238) by 31.10 and
41.61%, respectively was observed. The molecular weights
of the proteins (6.289 and 14.255 kDa, respectively) had
no similarity with other leech species.
Conclusion: Two antithrombin proteins
with anticoagulant properties were isolated from the
saliva of Malaysian leeches using one-step isolation
procedure. The proteins did not show any similarity to
previous isolated proteins from leech.
Keywords: Leech, Saliva, Antithrombin,
Anticoagulant, Peptides, Molecular weight
