7-57: Investigations into the variability of protein assays against glycosylated proteins

Accurate protein concentration determination is of utmost importance for determination of kinetics and specific activities. Given this, an understanding of protein-specific factors which affect the accuracy of various protein assays is crucial for researchers and enzyme producers alike.

There are many methods for determining protein concentrations by today’s researchers.  Of the many assays currently used, the Bradford method has found increasing favor within industry to become the standard methodology.   While the Bradford method has many advantages, it’s interactions with glycosylated cellulases is not well understood. As the method utilizes site-specific dye binding for quantification, problems may arise if reactive sites are sterically blocked by carbohydrates. Because of this, protein concentrations may be underestimated, and thus, a cellulase preparation may be assigned an artificially high specific activity. 

The current study utilizes multiple protein concentration assays in order to find the most accurate method for glycosylated proteins, such as fungal cellulases.  The Lowery method, Bradford, bicinchoninic acid (BCA), Pierce 660nm, and UV280 were all applied to predetermined concentrations of model proteins and cellulases with varying degrees of and types of glycosylation.