A reliable reducing end assay for chito-oligosaccharides

The enzymatic degradation of chitin typically yields a mixture of chito-oligosaccharides of different size. This degradation process can be followed by measuring the production of reducing ends. We show that the widely used Schalesʹ procedure does not permit accurate determination of reducing ends since the signal increases with the length of the chito-oligosaccharides. In contrast, the 3-methyl-2-benzothiazolinone hydrazone (MBTH) method gives the same absorbance per reducing end, irrespective of the length of the chito-oligosaccharide. To our knowledge the MBTH method is the most sensitive reducing end assay for the quantification of chito-oligosaccharides, detecting concentrations down to 5 μM. This method was successfully applied to monitor the enzymatic degradation of chito-oligosaccharides and beta chitin.