ISSN:
1573-904X
Keywords:
elastase
;
protein conformation
;
protein structure
;
protein purity
;
infrared (IR)
;
second derivative spectra
;
SDS-PAGE electrophoresis
;
enzyme activity
Source:
Springer Online Journal Archives 1860-2000
Topics:
Chemistry and Pharmacology
Notes:
Abstract 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 µL D2O, pD = 5.4 to 9.1). As with other globular proteins possessing a large fraction of β-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−1is 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. Finally, bands outside the amide I′ region may prove useful in assessing sample purity and identifying non-protein contaminants.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1023/A:1016273122944
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