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.