PURIFICATION OF AN UDP-GLUCOSE - FLAVONE, 7-O-GLUCOSYLTRANSFERASE, FROM SILENE-LATIFOLIA USING A SPECIFIC INTERACTION BETWEEN THE ENZYME AND PHENYL-SEPHAROSE

An UDP-glucose:flavonoid, 7-O-glucosyltransferase, from Silene latifol ia was isolated from petals and purified 450-fold using a combination of gel-filtration. affinity chromatography and anion-exchange chromato graphy. Affinity chromatography on a phenyl-Sepharose CL-4B column in combination with elution with the substrate, isovitexin (6-C-glucosyla pigenin), was an especially effective purification step. A purificatio n factor between 10 and 20 could be reached using this column. A possi ble mechanism for the specific interaction of the enzyme with the phen yl-Sepharose will be discussed. This method of purification may also b e applicable to other enzymes which use aromatic compounds as substrat es. On a SDS-PAGE gel a band of 54 kDa, which co-purified with enzyme activity, could be detected in the purest fraction.