Fourier-Transform Infrared (FTIR) Spectroscopy Transmission Technique for Analysis of Characteristic Infrared Bands of Peptide Linkage using Dried Proteins

Abstract

Protein activity is well dependent on its conformational structure and thus secondary structure prediction and determination of proteins are vital requirements. FTIR is rapidly coming up for protein structural studies, as an alternative to circular dichroism. However, FTIR spectra are primarily recorded using protein samples prepared in water, organic solvents, deuterium oxide, and membranes. FTIR spectra are thus dominated by the solvent spectrum and subtraction of the solvent spectrum introduces severe spectral distortions. In aqueous protein solutions, a strong water signal overlaps amide I bands (C=O stretching; 1700-1600 cm−1), thus masking most of the protein secondary structure information. Further, most of the purified proteins including protein drug formulations are more stable in the solid state than in the liquid state. Hence there is a need to explore the use of FTIR for various physical states of proteins. In the present study, we showed that protein secondary structure can be analyzed in a dried state; using potassium bromide (KBr) pellet method for dried known proteins (bovine serum albumin, proteinase K, fibrinogen etc). KBr pellet method gave excellent spectra for all the used dried proteins; shown by the appearance of characteristic infrared bands of peptide linkage (Amide A, B, I-VI) without much interference from water, buffers, stabilizing factors, co-factors etc. We also found that KBr pellet method can analyze a range of protein types; globular to fibrous. Thus KBr method is a great tool for prediction/determination of the secondary structure of unknown proteins and quality control of solid-state protein drug formulations.