Based on nitrogen factors (NF) determined for two commercial cellulases produced by the fungus Trichodenna reesei, a nitrogen analyzer was used to directly estimate the amount of enzyme adsorbed on biomass solids, rather than relying on the difference between initial and final dissolved protein as typically practiced. For adsorption of cellulase at 4 degrees C where reactions are minimal, the results for this method agreed closely with those indirectly determined by the usual BCA measurements of dissolved protein. But for hydrolysis at 50 degrees C, the BCA method indicated that much less protein was adsorbed on the solids compared to the NF method. Because the reagents used in conventional methods are very sensitive to reducing sugars, reagents, chemicals, and especially lignin components released during hydrolysis of lignocellulosic biomass, the result was a high amount of free protein in solution which indirectly affected the amount of protein adsorbed on the solid. Another important advantage of this approach compared to conventional methods is that material balances on protein can be closed. Application of the NF method revealed that sugars and especially cellobiose strongly inhibit cellulase binding, and cellulase binding was completely shut down at cellobiose concentrations greater than 150 g/L. However, this binding inhibition can be reversed using a higher ratio of substrate concentration to inhibitors. (C) 2008 Elsevier Inc. All rights reserved.