Purification, Peptide Sequencing and Study of Antiproliferative activity of Laccase against Liver...
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Purification, Peptide Sequencing and Study of Antiproliferative Activity of
Laccase against Liver cancer cell line Hep G2 and human breast cancer cell line
MCF 7
Author
Antik Kiron Bose
See Affilations
Senior Scientist and Project Advisor, Fred Hutchinson Cancer Research Center,
1100 Fairview Avenue N, Seattle, WA 98109
Summary
Three isozymes of laccase LCC 1, LCC 2 and LCC 3 have been purified from Pleurotus ostreatus strain V-184
using Toyopearl DEAE-650 Isoelectric focusing, ConA-sepharose 4B chromatography, Superdex-75 gel filtration, PD10
desalting column and Vydac C-18 Reverse phase HPLC chromatography with 950, 1220 and 935 folds of purification for
LCC 1, 2 and 3 respectively. The native molar mass of LCC 1 and 2 heterodimer was calculated to be 130Kda by gel
filtration and by 6% SDS-PAGE bands of 65, 60 and 80Kda were obtained for LCC 1, 2 and 3 respectively. Number of SH
groups in LCC 1 was calculated to be 4.68 and its full peptide sequencing gave a 533aa protein which was deposited in
Protein public database Uniprot with Acc. no. Q12739. IC50 of LCC 1 on Hepatocellular Carcinoma human (Hep G2) and
human brest cancer cell lines (MCF 7) were found to be 2.8 and 3.3 µM respectively. 1 to 5µM Menadione with 4.6µM
LCC 1 was found to kill Hep G2 cells by production of hydroxyl radical from semiquinone produced by laccase catalysed
Quinone-redox cycle. For elucidation of mode of action of laccase on killing Hep G2 cells; a patent has been registered
with application no. 3299/DELP/2011 A at CGPDTM, West Bengal. The temperature and pH optima for all isozymes were
found to be 30˚C and 5.0 respectively. Spectral analysis showed presence of Type 3 and Type 1 Cu (II) in LCC 1 and
reduction of Type 1 during catalysis of Guaiacol. KM and Vmax for 3 isozymes with Guaiacol was found to be 84.034µM,
714.25 U/min; 74.074µM, 204.0816 U/min; 158.73µM, 714.3U/min respectively for LCC 1, 2 and 3.
Introduction
Laccases, EC 1. 10. 3. 2, p-diphenol: dioxygen oxidoreductase, are part of a larger group of enzymes termed the multi-
copper enzymes, which includes among others ascorbic acid oxidase and ceruloplasmin. Laccase was first described by
Yoshida (1883) and was characterized as a metal containing oxidase by Bertrand(1985). This makes it one of the oldest
enzymes ever described.