Protein Polarimetry, Perfected: Specific Rotation Measurement for the Refracto-Polarimetric Detection of Cryptic Protein Denaturation

Protein polarimetry has been evaluated as a simple and straightforward technique to detect the cryptic denaturation of exemplary proteins. The general rules of rotation vs. amino acid and structural composition and the respective knowledge gaps were reviewed, and the specific rotation of cystine was determined in 4 M NaCl solution as [α]D20 = –302.5°. The specific rotations at 589 nm and 436 nm and the ratio were measured for several model proteins, some purified plasma-derived proteins and for three monoclonal antibodies. The immunoglobulin G concentrates all showed a narrow ratio range likely characteristic for this protein class. Heat denaturation experiments were conducted at temperatures between 50 and 85 °C both for short-time (10 min) and for prolonged periods of heat exposure (up to 210 min). Denaturation by heat resulted not only in the known levorotatory shift, but also in a shift in the specific rotation ratio. The stabilizing effect of fatty acids in bovine serum could be demonstrated by this parameter. Polarimetry thus appears to be a particularly sensitive and simple method for the characterization of the identity and the thermal stability of proteins and should therefore be added again as a complimentary method to the toolbox of protein chemistry.