Purification of an UDP-glucose:flavone, 7- O-glucosyltransferase, from Silene latifolia using a specific interaction between the enzyme and phenyl-sepharose

• Published in: FEBS letters Vol. 330; no. 1; pp. 36 - 40
• Main Authors: Vellekoop, P., Lugones, L., van Brederode, J.
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 06.09.1993
• Subjects: Affinity chromatography; Chromatography, Affinity; Chromatography, Gel; Chromatography, Ion Exchange; DTT, dithiothreitol; EG, ethylene-glycol; EGME, ethylene-glycol monoethyl ether; Electrophoresis, Polyacrylamide Gel; Enzyme purification; Flavonoid; Glucosyltransferase; Glucosyltransferases - isolation & purification; Glucosyltransferases - metabolism; Phenyl-Sepharose: Silene; Plants - enzymology; PMSF, phenylmethylsulfonyl fluoride; Sepharose - analogs & derivatives; Sepharose - metabolism; TCA, trichloro acetic acid; UDPG, uridine diphosphate glucose; Phenyl-Sepharose: Silene; PMSF, phenylmethylsulfonyl fluoride; TCA, trichloro acetic acid; Enzyme purification; Affinity chromatography; EG, ethylene-glycol; UDPG, uridine diphosphate glucose; EGME, ethylene-glycol monoethyl ether; Flavonoid; Glucosyltransferase; DTT, dithiothreitol
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(93)80914-G

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