EFFECT OF SUBZERO INCUBATION ON FLUORIDE BINDING BY LACCASE

We have refined a useful incubation method for preparing adducts of tr ee laccase with inhibitor anions; however, the technique should have w ider application. The procedure involves incubating a previously froze n aqueous solution at subzero temperature. Factors influencing the amo unt of adduct that forms include the nature of the buffer, pH changes that occur at subzero temperatures and the presence of glycerol. In th e absence of a glassing agent like glycerol, the phase separation and solute pooling that occur with ice formation help drive adduct formati on. The results reveal several important factors to consider in design ing or interpreting low-temperature spectroscopic investigations.