Second derivative infrared spectroscopy as a non-destructive tool to assess the purity and structural integrity of proteins.
Second derivative infrared (IR) spectroscopy can be used as a quick, easy, reproducible, cost-effective, non-destructive tool by which to evaluate 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) solutions of seven different commercial samples of the same enzyme, porcine pancreatic elastase (2.0 to 3.8 mg protein/100 microL D2O, pD = 5.4 to 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 1684 cm-1; the other close to 1633 cm-1 is moderate-to-strong. However, one of the seven samples shows a striking decrease in the observed intensities of the amide I' bands relative to the 1516 cm-1 absorption, along with the appearance of a strong, new band at 1614 cm-1. These intensity disparities strongly suggest that this sample is of much lower quality than the others and clearly has an appreciable proportion of the protein present in a non-native state. In addition, minor differences evident in the position and relative intensity of some individual amide I' bands among the seven spectra imply that subtle variations exist in the conformation of the peptide backbone of the seven samples. For two of the samples, these small, but reproducible, changes seem to be correlated with marked losses of enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Byler
D M
DM
Department of Chemistry and Physical Science, Philadelphia College of Textiles and Science, Pennsylvania 19144-5497, USA.
Wilson
R M
RM
Randall
C S
CS
Sokoloski
T D
TD
D000818:Animals; D004586:Electrophoresis; D010196:Pancreatic Elastase / Q000737:chemistry; D010194:Pancreatin; D011506:Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D013055:Spectrophotometry, Infrared
D011506:Proteins; D010194:Pancreatin; D010196:Pancreatic Elastase