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 M respectively. 1 to 5M Menadione with 4.6M 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 30C 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 84.034M, 714.25 U/min; 74.074M, 204.0816 U/min; 158.73M, 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 multicopper 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.