An accurate UV/visible method to quantify proteins and nzymes:Impact of aggregation, buffer concentration and the nature of the standard

Abstract

A simple, fast and low cost UV/visible based method to quantify proteins or enzymes is presented. This method avoids some drawbacks found using conventional techniques. Representative proteins and enzymes such as bovine serum albumin (BSA), insulin (Ins.), Rhizomucor meihei lipase (RML), Candida rugosa lipase (CRL) and horseradish peroxidase (HRP) were assayed as model. Experiments revealed that the aggregation of the enzyme/protein molecules in aqueous solution was the main cause of inaccurate results obtained with the simple UV/visible method. It was determined that aggregation of proteins/ enzymes in aqueous solutions follows a reversible mechanism that could be reverted by simple magnetic stirring treatment. The results achieved within this study warn about common error sources in protein quantification by UV/visible based methods and clearly shows the magnitude of the mistakes that could be achieved if aggregation and other factors are not considered.