E.A. Kostina, M.A.Proskurnin, A.D.Khrycheva, E.V. Proskurnina, Yu.A. Vladimirov

Recently, IR spectroscopy has become one of the key methods for structure analysis of biological molecules, namely, proteins. Nevertheless, the principles of quantitation of proteins are established not enough to solve many research problems in medicine and biology and to determine proteins in complex mixtures. The aim of our paper was to develop a simple IR procedure for protein quantitation in small volumes using albumins and heme proteins as examples with detection limits as small as 1 mg/mL. We studied the possibility of protein quantitation in solid films adsorbed on plates made of CaF2. The novelty of our decision is in use of pure protein films without polymers, surfactants, and other structure-forming agents. We examined cytochrome c, bovine and human serum albumins, human and rat hemoglo-bins. For calibration, we used main characteristic absorption bands of proteins Amide I (1543 cm-1), Amide II (1655 cm-1), and Amide III (1457 cm-1), as well as absorption bands 1396 cm-1 and 3294 cm-1, the latter bands have not been used for analytical determination of proteins with IR spectroscopy. We paid much at-tention to the reproducibility as well as the selection of the conditions providing the certain quantity of water in a sample, which did not interfere with the quantitation and provided the protein structure intact. With variance analysis, we have optimized the sample preparation conditions, which provided the maximum sensitivity and reproducibility, low background signals, and native protein structure. The detection limits for all the proteins studied were as small as 100-400 µg/mL. The sensitivity corre-lates well with the number of functional groups in the proteins studied which form the signal. The sensitivity of the procedure is better than of the existing analogs, the sample preparation is simpler. The standard deviation for protein quantitation of concentrations 1-15 mg/mL was no less than 0.1.