A COLORIMETRIC ASSAY FOR XYLOGLUCAN-ENDOTRANSGLYCOSYLASE FROM GERMINATING-SEEDS

One of the key enzymes involved in the breakdown of reserve xyloglucan in seeds of some dicotyledonous plants during germination is the spec ific endo-beta-(1,4)-glucanase. The enzyme operates predominantly by a transglycosylic mechanism, i.e., by random splitting the beta-(1,4)-l inked polyglucose backbone of xyloglucan molecules and rejoining the n ewly created reducing ends by beta-(1,4) glycosidic bonds to nonreduci ng ends of other xyloglucan molecules or xyloglucan subunit oligosacch arides. For this reason, the enzyme is regarded primarily as xylogluca n-endotransglycosylase (XET). Since almost no net formation of reducin g ends occurs in the course of transglycosylation, the conventional re ductometric methods used for the assessment of glycanase activities ar e not applicable for detection and determination of XET activity. The described colorimetric assay is based on the property of xyloglucan-de rived subunit oligosaccharides (DP 5-10) to stimulate selectively the breakdown of xyloglucan by endotransglycosylation while serving as add itional glycosyl accepters. The depolymerization of xyloglucan in the course of reaction is followed colorimetrically by measuring the disap pearance of the blue-green-colored iodine:xyloglucan complex. The tran sglycosylase activity is calculated as the difference of activities me asured in the presence of stimulating xyloglucan-derived oligosacchari des and in their absence. The advantages of the described colorimetric method include its low cost, simplicity, speed, and the possibility t o analyze multiple samples simultaneously. (C) 1995 Academic Press, In c.