Structural analysis of pigeon lens crystallins by near-infrared Fourier transform Raman spectroscopy.
Crystallins from pigeon eye lenses were isolated and purified by gel-permeation and anion-exchange chromatographies and characterized by gel electrophoresis, amino acid analysis and Raman spectroscopy. alpha- and beta-Crystallins could be obtained in relatively pure forms by single-step size-exclusion chromatography whereas an extra step of ion-exchange chromatography was needed for the separation of delta crystallin from the beta-crystallin fraction. In contrast to most characterized vertebrate species, a large amount of glycogen is eluted as a high molecular form in the first peak of gel filtration column. Structural analyses of total crude soluble proteins and purified alpha-, beta- and delta-crystallin fractions were made with respect to their amino acid compositions and characteristic near-IR Fourier-transform Raman spectra. The results indicate that the major secondary structures of alpha- and beta-crystallins are mainly anti-parallel beta-pleated sheet in nature as judged by the Raman signals at 1242 (amide III) and 1669-1670 cm-1 (amide I) whereas delta-crystallin consists of a significant content of alpha-helices as evidenced by the Raman signal at 1657-1660 cm-1 (amide I). The low intensity of S-S disulfide stretching vibration at 508-510 cm-1 coupled with the presence of S-H stretching at 2560-2580 cm-1 for alpha-, beta- and delta-crystallin pointed to the fact that sulfhydryl groups in most crystallins are resistant to air oxidation during the process of homogenization and protein extraction. It is also found that the relative Raman signal intensities of Tyr, Phe, and Trp residues in purified crystallins correlate very well with the data obtained from amino acid analysis. Especially noteworthy is the demonstrated usefulness of applying Raman techniques in the detection of the microenvironments of the aromatic amino acids such as Tyr and Trp in the native crystallins, which may prove useful in the study of contribution of these aromatic residues to crystallin packing and stability.
Chiou
S H
SH
Laboratory of Crystallin Research, National Taiwan University, Taipei.
Chen
W
W
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002853:Chromatography, Liquid; D010856:Columbidae; D003459:Crystallins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005583:Fourier Analysis; D011487:Protein Conformation; D013055:Spectrophotometry, Infrared; D013059:Spectrum Analysis, Raman
D000596:Amino Acids; D003459:Crystallins