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Detection and purification of protein hirudin from local leech using a gel filtration chromatography

It has long been known that local leech contain a substance with anticoagulant properties. In 1984, the blood-thinning agent (anticoagulant) hirudin was isolated for the first time from leech salivary glands (Weinnberg, 1994). The purification of hirudin, protein profiling and protein quantification...

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Main Authors: Zulhisyam Abdul Kari @ Abdullah, Ahmad Anwar Mohamed Ismail, Ibrahim Che Omar
Format: Non-Indexed Article
Language:English
Published: Universiti Malaysia Kelantan 2012
Online Access:http://discol.umk.edu.my/id/eprint/7835/1/Sbizhub00512110810020.pdf
http://discol.umk.edu.my/id/eprint/7835/
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spelling my.umk.eprints.78352022-05-23T15:15:41Z http://discol.umk.edu.my/id/eprint/7835/ Detection and purification of protein hirudin from local leech using a gel filtration chromatography Zulhisyam Abdul Kari @ Abdullah Ahmad Anwar Mohamed Ismail Ibrahim Che Omar It has long been known that local leech contain a substance with anticoagulant properties. In 1984, the blood-thinning agent (anticoagulant) hirudin was isolated for the first time from leech salivary glands (Weinnberg, 1994). The purification of hirudin, protein profiling and protein quantification from different part of leech are reported. The result shows the sample from leech head was gave the highest result in protein concentration with value of 69.33 µg/ml compared with the sample from the whole body and leech body which was 36.67 µg/ml and 26.67 µg/ml, respectively. Sample from leech head was used for purification by gel permeation chromatography on Sephadex G-75. Fractions which contain significant amounts of protein with absorbance value more than 0.2 at 595 nm were tested on their anti-coagulant activity. The result shows that that the peak number 3 (Number of fraction= 23, optical density (O.D) = 0.256, protein concentration = 82.00 µg/ml) gave the longest time of blood to clot (123.33±15.28 minutes) and the clotted blood was not contracted up to 380.00±17.32 minutes compared with peak number 2 (Number of fraction= 21, optical density (O.D) = 0.293, protein concentration = 94.33 µg/ml) and 5 (Number of fraction= 36, optical density (O.D) = 0.214, protein concentration = 68.00 µg/ml ) where the blood was clot in 13.33±2.89 and 23.33±2.89 minutes and the clotted blood was not contracted up to 24.33±0.58 and 41.67±2.89 minutes, respectively. Crude protein and eluted fractions which contain significant amounts of protein with the longest time of blood to clot were subjected to SDS-PAGE analysis. The result shows that the molecular weight of protein hirudin was about 11.36 kDa. From the purification by Sephadex G-75 Gel Filtration profiles of leech crude extract from different parts of body indicates that anti-coagulant agent (protein hirudin) was only exist in leech head (salivary glands). Universiti Malaysia Kelantan 2012 Non-Indexed Article NonPeerReviewed text en http://discol.umk.edu.my/id/eprint/7835/1/Sbizhub00512110810020.pdf Zulhisyam Abdul Kari @ Abdullah and Ahmad Anwar Mohamed Ismail and Ibrahim Che Omar (2012) Detection and purification of protein hirudin from local leech using a gel filtration chromatography. Journal of Bioentrepreneurship, 2 (1). pp. 44-53. ISSN 1231-8704
institution Universiti Malaysia Kelantan
building Perpustakaan Universiti Malaysia Kelantan
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Malaysia Kelantan
content_source UMK Institutional Repository
url_provider http://umkeprints.umk.edu.my/
language English
description It has long been known that local leech contain a substance with anticoagulant properties. In 1984, the blood-thinning agent (anticoagulant) hirudin was isolated for the first time from leech salivary glands (Weinnberg, 1994). The purification of hirudin, protein profiling and protein quantification from different part of leech are reported. The result shows the sample from leech head was gave the highest result in protein concentration with value of 69.33 µg/ml compared with the sample from the whole body and leech body which was 36.67 µg/ml and 26.67 µg/ml, respectively. Sample from leech head was used for purification by gel permeation chromatography on Sephadex G-75. Fractions which contain significant amounts of protein with absorbance value more than 0.2 at 595 nm were tested on their anti-coagulant activity. The result shows that that the peak number 3 (Number of fraction= 23, optical density (O.D) = 0.256, protein concentration = 82.00 µg/ml) gave the longest time of blood to clot (123.33±15.28 minutes) and the clotted blood was not contracted up to 380.00±17.32 minutes compared with peak number 2 (Number of fraction= 21, optical density (O.D) = 0.293, protein concentration = 94.33 µg/ml) and 5 (Number of fraction= 36, optical density (O.D) = 0.214, protein concentration = 68.00 µg/ml ) where the blood was clot in 13.33±2.89 and 23.33±2.89 minutes and the clotted blood was not contracted up to 24.33±0.58 and 41.67±2.89 minutes, respectively. Crude protein and eluted fractions which contain significant amounts of protein with the longest time of blood to clot were subjected to SDS-PAGE analysis. The result shows that the molecular weight of protein hirudin was about 11.36 kDa. From the purification by Sephadex G-75 Gel Filtration profiles of leech crude extract from different parts of body indicates that anti-coagulant agent (protein hirudin) was only exist in leech head (salivary glands).
format Non-Indexed Article
author Zulhisyam Abdul Kari @ Abdullah
Ahmad Anwar Mohamed Ismail
Ibrahim Che Omar
spellingShingle Zulhisyam Abdul Kari @ Abdullah
Ahmad Anwar Mohamed Ismail
Ibrahim Che Omar
Detection and purification of protein hirudin from local leech using a gel filtration chromatography
author_facet Zulhisyam Abdul Kari @ Abdullah
Ahmad Anwar Mohamed Ismail
Ibrahim Che Omar
author_sort Zulhisyam Abdul Kari @ Abdullah
title Detection and purification of protein hirudin from local leech using a gel filtration chromatography
title_short Detection and purification of protein hirudin from local leech using a gel filtration chromatography
title_full Detection and purification of protein hirudin from local leech using a gel filtration chromatography
title_fullStr Detection and purification of protein hirudin from local leech using a gel filtration chromatography
title_full_unstemmed Detection and purification of protein hirudin from local leech using a gel filtration chromatography
title_sort detection and purification of protein hirudin from local leech using a gel filtration chromatography
publisher Universiti Malaysia Kelantan
publishDate 2012
url http://discol.umk.edu.my/id/eprint/7835/1/Sbizhub00512110810020.pdf
http://discol.umk.edu.my/id/eprint/7835/
_version_ 1763303897588301824
score 13.250246

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