CHALCONE ISOMERASE FROM CITRUS-SINENSIS - PURIFICATION AND CHARACTERIZATION

A chalcone isomerase was purified to apparent homogeneity from develop ing fruit tissue of Citrus sinensis by gel filtration, hydrophobic int eraction chromatography and anion exchange chromatography. A single pr otein band was observed on SDS-PAGE gels, corresponding to a M(r) of 2 7 800. In addition, the M(r) of the native protein was determined as 2 7 200 using analytical gel filtration. The enzyme exhibited micro-hete rogeneity on native PAGE gels. Enzyme staining indicated the presence of four activity bands. Four protein bands were also observed on isoel ectric focusing gels with pI values of 6.55, 6.38, 6.35 and 6.04. Char ge-heterogeneity could not be ascribed to glycosylation as the enzyme was not found to be a glycoprotein. The enzyme catalysed isomerization was found to be optimal at pH 7.4; and an energy of activation of 31. 9 kJ mol(-1) was calculated. The enzyme cyclized only naringenin chalc one of all the aglycone substrates tested and a K-m of 4 mu M and V-ma x of 67 mkat kg(-1) were determined for this substrate. No activity wa s observed with chalcone glycosides. The turnover number was 1.87 sec( -1) and the catalytic efficiency 4.62 x 10(5) M(-1)sec(-1). Competitiv e inhibition of enzyme activity was observed with naringenin (K-i = 18 0 mu M), quercetin (K-i =45 mu M) and morin (K-i = 30 mu M).