The disulfide content of calf gamma-crystallin.
The disulfide content of calf gamma-crystallin polypeptides has been investigated. The gamma-crystallin fraction of the soluble lens proteins was separated into five distinct polypeptides and characterized by isoelectric focusing, amino acid composition, and N-terminal sequence analysis to 25 residues. It has been demonstrated that 7 cysteines are present in gamma II, 4 to 5 cysteines in gamma IIIa, gamma IIIb, and gamma IV, and 6 cysteines in gamma I (beta s). Reduction of the total gamma-crystallin fraction with DTT resulted in an increase of approximately 1 to 1.5 mol of free SH per mole of protein. This increase in sulfhydryls was demonstrated to be contributed primarily by gamma II, the major polypeptide representing 50% of the total gamma-crystallin, which showed an increase of approximately 2.5 mol of sulfhydryl per mole of protein upon reduction. Insignificant disulfide content was present in gamma III and gamma IV and only a slight amount of disulfide was found in gamma I (beta s). The observed increase in sulfhydryl content upon reduction was not due to the presence of mixed disulfides of 2-mercaptoethanol, glutathione, or cysteine. The data are consistent with approximately 1 mol of intramolecular disulfide per mole of protein being present in gamma II. X-ray crystallography of gamma II has shown that the spatial location of Cys18 and Cys22 in the tertiary structure permits disulfide bond formation. Sequence analysis of the four major polypeptides of gamma-crystallin, gamma II, gamma IIIa, gamma IIIb, and gamma IV indicates that only gamma II has both Cys18 and Cys22. Cys18 is present in gamma IIIa, gamma IIIb, and gamma IV but Cys22 is replaced by His22. It is probable that the lack of disulfide in gamma IIIa, gamma IIIb, and gamma IV is due to the absence of Cys22.
McDermott
M J
MJ
Department of Ophthalmology, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Gawinowicz-Kolks
M A
MA
Chiesa
R
R
Spector
A
A
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003459:Crystallins / Q000032:analysis*; D003545:Cysteine / Q000032:analysis; D004220:Disulfides / Q000032:analysis*; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction
D003459:Crystallins; D004220:Disulfides; D003545:Cysteine