THE APPLICATION OF CAPILLARY ELECTROPHORESIS FOR MONITORING EFFECTS OF EXCIPIENTS ON PROTEIN CONFORMATION

Studies were conducted to assess the utility of free solution capillar y electrophoresis (CE) for monitoring the effects of selected excipien ts on the thermal denaturation of a model protein (Ribonuclease A, RNa se A) at low pH. Thermal denaturation/unfolding experiments were condu cted via temperature-controlled CE using a run buffer of 20 mM citric acid in the pH range of 2.3-3.1, with a marker peptide incorporated to correct for temperature-induced changes in endoosmotic flow. The effe cts of selected excipients on the thermal unfolding of RNase A were th en evaluated by adding either sorbitol, sucrose, polyethylene glycol 4 00 (PEG 400) or 2-methyl-2,4-pentanediol (MPD) to the electrophoretic run buffer (pH 2.3). Confirmatory denaturation experiments were conduc ted under the same solution conditions using circular dichroism (CD) s pectropolarimetry. Using temperature-controlled CE, an increase in sol ution pH from 2.3 to 2.7 and 3.1 resulted in an increase in transition temperatures of RNase A by approximately 8 and 13 degrees C, respecti vely. Similar shifts in transition temperatures were observed when the rmal denaturation transitions were monitored by far-UV CD. Sorbitol (0 .55-1.1 M) and sucrose (0.55 M) each shifted the denaturation transiti on temperatures of RNase A to higher values: whereas PEG 400 and MPD h ad minimal effect on the unfolding transition midpoint at the concentr ations evaluated (0.55 M for each). The observed changes in the transi tion temperatures for RNase A as a function of pH and selected excipie nts were similar when measured by either CE or far-UV CD. These result s support the utility of CE for monitoring the effects of neutral exci pients on the thermal denaturation of a model protein under selected c onditions. The widespread utility of the technique may be limited by t he narrow temperature range of most commercial CE instruments and the need to use extreme pH conditions to monitor the complete denaturation transition. (C) 1998 Elsevier Science B.V. All rights reserved.