use another buffer its normal of colour

try to use 25 mM succinate (from succinate acid or Na-succinate, Fluka), pH 4.5 or 0.1 M citrate-phosphate buufer ph 4.0.

in my work i used 0.1 M citrate-phosphate buufer ph 4.0. and the color substrate solution was faint green and when incubate it with laccase enzyme the color will changed from faint green to dark green according the amount of units of enzyme per ml per min. below the protocol of laccase assay according method of Niku-Paavola et al. (1988). I used it through my work:

Reagents:

1. ABTS reagent  (Boehringer) 275 mg/25 ml in distilled water

2. 25 mM succinate (from succinate acid or Na-succinate, Fluka), pH 4.5 (adjusted with NaOH)

Equipment:

Spectrophotometer, temperature controlled cuvette holder and possibility to perform kinetic measurements

1.15 ml of the enzyme dilution (diluted to succinate buffer) is combined with 0.35 ml ABTS reagent in a disposable cuvette. The absorbance increase is followed at 436 nm (e = 29,3 mM-1cm-1) for usually 2-3 minutes. (in my work the reading was taken after 10 min of incubation. it dependent on the best time required top obtain the maximum activity.  The spectrofotometer is zeroed with the ABTSzero sample, which contains succinate buffer (1.15 ml) and ABTS-reagent (0.35 ml).

The activity is calculated according to the formula:

activity (nkat/mL)= 0.371 x dilution factor x dAbs

where dAbs is the absorbance increase within 2 minutes.

Note that enzyme dilution should be chosen so that dAbs/min will not increase 0.50.

Reference:

Niku-Paavola, M.-L., Karhunen, E., Salola, P. ja Raunio V., Lignolytic enzymes of the white-rot fungus Phlebia radiata, Biochem.J. 254 (1988) 877-884.