Dehydroalanine crosslinks in human lens.
This study was conducted to develop a methodology for the purification and detection of histidinoalanine, lanthionine and lysinoalanine in the lens tissue. Cataractous and aged human lens proteins were hydrolysed and fractionated by using anion-exchange chromatography. The fraction containing the bulk of dehydroalanine crosslinks was derivatized with dansyl chloride and then separated and quantified by means of RP-HPLC. The spectral and chromatographic properties of all three substances purified and quantified in this study were identical to those of their synthesized counterparts. Histidinoalanine and lanthionine were the most abundant dehydroalanine crosslinks in both water-soluble and water-insoluble lens proteins. Histidinoalanine levels in water-soluble proteins from the cataractous lenses of Indian origin were 6.2-fold higher than those in water-soluble proteins from normal lenses (1.68+/-0.75 vs 0.26+/-0.06 nmol/mg protein; p<0.001). In water-insoluble proteins, they were 2.2-fold higher in cataractous lenses compared with normal lenses (1.59+/-0.76 vs 0.73+/-0.17 nmol/mg protein; p<0.01). Lanthionine levels were significantly higher in water-insoluble proteins of cataractous lenses when compared to non-cataractous lenses (2.5+/-1.68 vs 0.95+/-0.08 nmol/mg protein; p<0.03). Unlike histidinoalanine, this crosslink appears to accumulate in relatively high concentrations in water-soluble lens proteins; its concentration was 9-fold higher than histidinoalanine from the same proteins (0.26+/-0.06 HAL vs 2.34+/-0.76 LAN nmol/mg protein; p<0.0004). The concentration of lysinoalanine was in the picomolar range and in cataractous lens proteins only.
Linetsky
Mikhail
M
Mason Eye Institute, University of Missouri, Columbia, MO 65212, USA. linetskym@health.missouri.edu
Hill
J M W
JM
LeGrand
Roy D
RD
Hu
F
F
D000328:Adult; D000368:Aged; D000375:Aging / Q000378:metabolism; D000409:Alanine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000302:isolation & purification; D002386:Cataract / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003459:Crystallins / Q000032:analysis* / Q000302:isolation & purification; D004151:Dipeptides / Q000032:analysis / Q000302:isolation & purification; D006801:Humans; D007908:Lens, Crystalline / Q000737:chemistry*; D008241:Lysinoalanine / Q000032:analysis / Q000302:isolation & purification; D008875:Middle Aged; D012995:Solubility; D013440:Sulfides
D003459:Crystallins; D004151:Dipeptides; D013440:Sulfides; C034124:histidinoalanine; D008241:Lysinoalanine; C015102:dehydroalanine; C001520:lanthionine; D000409:Alanine