Measurement of the onset temperature of irreversible inactivation of proteins using FITC as a fluorescent reporter

Abstract

Proteins can be inactivated by high temperature commonly encountered during manufacture, delivery, and storage. Therefore, an accurate determination of the onset temperature (T_on) at which proteins are irreversibly inactivated is of great importance to both protein science and the pharmaceutical industry. The commonly used techniques for monitoring the process of protein denaturation including intrinsic fluorescence spectroscopy, CD, and DSC are often not sensitive enough to reflect T_on at which proteins start to loss their bioactivities. In this study, we found that T_on of irreversible inactivation of lysozyme and trypsin can be more accurately determined by analysis of the heating and cooling fluorescence-temperature courses (FTCs) of proteins labeled with FITC fluorescence dye, compared with the intrinsic fluorescence reporter. Moreover, we have evaluated the storage buffers for trypsin using this method. We speculate that this method could find practical utility in biopharmaceutical high-throughput screening for evaluation of stabilizing buffers and enzyme formulation.