Effect of subzero incubation on fluoride binding by laccase

We have refined a useful incubation method for preparing adducts of tree laccase with inhibitor anions; however, the technique should have wider application. The procedure involves incubating a previously frozen aqueous solution at subzero temperature. Factors influencing the amount of adduct that forms include the nature of the buffer, pH changes that occur at subzero temperatures and the presence of glycerol. In the absence of a glassing agent like glycerol, the phase separation and solute pooling that occur with ice formation help drive adduct formation. The results reveal several important factors to consider in designing or interpreting low-temperature spectroscopic investigations.