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Research Article

ScienceAsia 31 (2005): 055-063 |doi: 10.2306/scienceasia1513-1874.2005.31.055


Peroxidase from Hevea brasiliensis (B.H.K) Mull. Arg. Leaves and its Applications

Chatchamon Daengkanit and Wallie Suvachittanont*


ABSTRACT: Peroxidase from Hevea brasiliensis leaves (RBP) was purified by ammonium sulfate precipitation, followed by DEAE-Sephacel ion exchange chromatography and gel filtration on Sephadex G-75. RBP was conjugated to anti-rabbit IgG using three different cross-linkers : periodate, glutaraldehyde and sulfo-SMCC. The RBP-antibody conjugates were purified in a Sephadex G-200 column and the RBP-anti-rabbit IgG conjugate from the periodate oxidation was found to retain a higher peroxidase activity (68%) than when crosslinked by the glutaraldehyde (9.7%) and sulfo-SMCC (5.4%) methods. Its molecular weight (MW) is 348 kDa as determined by gel filtration chromatography in a Sephadex G-200 column. RBP-anti-human IgG conjugate prepared by the periodate oxidation method gave high yield of protein and also high peroxidase activity, as found in the case of an RBP-anti-rabbit IgG conjugate. The RBP-anti-rabbit IgG conjugate was detected using dot blot technique. The RBP-anti-rabbit IgG conjugate can be used as a secondary antibody in the western blot technique to detect vitellogenin, a phosphoglycoprotein in mullet plasma, and HMG-CoA synthase, an enzyme in C-serum of rubber latex. Its optimal dilution is 1:200 using commercial HRP-anti-rabbit IgG conjugate as positive control. The purified RBP can also be used as a coupling enzyme for cholesterol assay.

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Department of Biochemistry, Faculty of Science, Prince of Songkla University, Hat Yai, 90112, Thailand.

* Corresponding author, E-mail: wallie.s@psu.ac.th

Received 26 Aug 2004, Accepted 1 Dec 2004