2ND DERIVATIVE INFRARED-SPECTROSCOPY AS A NONDESTRUCTIVE TOOL TO ASSESS THE PURITY AND STRUCTURAL INTEGRITY OF PROTEINS

Second derivative infrared (IR) spectroscopy can be used as a quick, e asy, reproducible, cost-effective, non-destructive tool by which to ev aluate the purity and structural integrity of samples of water-soluble proteins from a variety of sources. For this study, second derivative IR spectra were collected at ambient conditions for aqueous (D2O) sol utions of seven different commercial samples of the same enzyme, porci ne pancreatic elastase (2.0 to 3.8 mg protein/100 mu L D2O, pD = 5.4 t o 9.1). As with other globular proteins possessing a large fraction of beta-structure, the amide I' region [1700-1620 cm(-1)] of the second derivative IR spectra for each of the seven elastase samples exhibits a characteristic pair of bands: one of weak intensity appears near 168 4 cm(-1); the other close to 1633 cm(-1) is moderate-to-strong. Howeve r, one of the seven samples shows a striking decrease in the observed intensities of the amide I' bands relative to the 1516 cm(-1) absorpti on, along with the appearance of a strong, new band at 1614 cm(-1). Th ese intensity disparities strongly suggest that this sample is of much lower quality than the others and clearly has an appreciable proporti on of the protein present in a non-native state. In addition, minor di fferences evident in the position and relative intensity of some indiv idual amide I' bands among the seven spectra imply that subtle variati ons exist in the conformation of the peptide backbone of the seven sam ples. For two of the samples, these small, but reproducible, changes s eem to be correlated with marked losses of enzyme activity. Finally, b ands outside the amide I' region may prove useful in assessing sample purity and identifying non-protein contaminants.