| The primary purpose of this study was to (1) develop methods for the analysis of and (2) provide information on the chemical nature of reducing ends in typical cellulose substrates used for the study of cellulolytic enzymes. The studies were designed such that values obtained for cellulose substrates were compared with those obtained for a series of soluble cellooligosaccharides. The initial phase of the study tested the validity of using established colorimetric reducing sugar assays, developed for the measurement of reducing sugars in solution, for the quantification of reducing ends on insoluble substrates. The results demonstrate that published methods give widely differing values for the number of reducing ends per unit weight cellulose. The Cu++ -based assay, using bicinchoninic acid (BCA) as a color yielding chelator of Cu+, is shown to provide values that appear most consistent the properties of the substrates. A method was developed using the Cu ++-BCA reagent, following a mild sodium borohydride treatment, to provide an estimate of the number of solvent accessible reducing ends on insoluble substrates. The kinetics of sodium borohydride reduction of reducing ends on crystalline cellulose, amorphous cellulose and soluble cellooligosaccharides were compared in order to ascertain the relative reactivity of these reducing ends. The apparent second order rate constants for the reduction of reducing ends associated with the crystalline celluloses were significantly lower than those for the reduction of reducing ends associated with either the insoluble amorphous celluloses or the soluble cellooligosaccharides. These results indicate the reducing ends associated with crystalline celluloses are not extended out from the surface as though mimicking solution phase reducing ends. The relevance of this, as well as the other results, to the behavior of cellulolytic enzymes is discussed. The final phase of the study was the demonstration of both a reducing sugar-based and a viscosity-based assay for the detection of a prototypical polysaccharide depolymerizing glycosyl hydrolase, polygalacturonase. |
