A SPECTROPHOTOMETRIC ASSAY FOR LACCASE USING O-TOLIDINE

A spectrophotometric method for measuring laccase activity using o-tol idine has been developed. Oxidation of o-tolidine by laccase to a blue colored product corresponded with increases in absorbancies at 366 an d 630 nm. This oxidation reaction and increases in absorbance at 366 a nd 630 nm could also be mimicked using hypochlorite, periodate and UV light in place of laccase. After a lag period, the assay was linear in absorbance with time, although the duration of linear region appeared to be affected by the pH. When assayed from 0.025 to 7 mM tolidine, m aximum oxidation of substrate occurred using 3 mM o-tolidine. Oxidatio n of o-tolidine exhibited a pH dependency and showed an apparent pH op timum at approximately 5,0. The utility of this assay was shown by det ermining laccase activity in various fungal extracts.