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 specific endo-beta-(1,4)-glucanase. The enzyme operates predominantly by a transglycosylic mechanism, i.e., by random splitting the beta-(1,4)-linked polyglucose backbone of xyloglucan molecules and rejoining the newly created reducing ends by beta-(1,4) glycosidic bonds to nonreducing ends of other xyloglucan molecules or xyloglucan subunit oligosaccharides. For this reason, the enzyme is regarded primarily as xyloglucan-endotransglycosylase (XET). Since almost no net formation of reducing ends occurs in the course of transglycosylation, the conventional reductometric methods used for the assessment of glycanase activities are not applicable for detection and determination of XET activity. The described colorimetric assay is based on the property of xyloglucan-derived subunit oligosaccharides (DP 5-10) to stimulate selectively the breakdown of xyloglucan by endotransglycosylation while serving as additional glycosyl acceptors. The depolymerization of xyloglucan in the course of reaction is followed colorimetrically by measuring the disappearance of the blue--green-colored iodine:xyloglucan complex. The transglycosylase activity is calculated as the difference of activities measured in the presence of stimulating xyloglucan-derived oligosaccharides and in their absence. The advantages of the described colorimetric method include its low cost, simplicity, speed, and the possibility to analyze multiple samples simultaneously.