Proximity of sulfhydryl groups in lens proteins. Excimer fluorescence of pyrene-labeled crystallins.
Lens proteins labeled with the -SH-specific reagents N-(1-pyrene)-maleimide (PM) and N-(1-pyrene)-iodo-acetamide (PIA) exhibited pyrene excimer fluorescence around 480 nm. Among the gamma-fractions, only gamma II showed excimer band at room temperature with both probes PM and PIA. As the temperature increased, PM-labeled gamma IIIA, gamma IIIB, and gamma IV also began to exhibit excimer around 55 degrees C, which did not disappear at a very high temperature (85 degrees C). With PIA, gamma IIIA and gamma IVA did not show excimer at any temperature. The beta-crystallins, on the other hand, revealed a very strong excimer/monomer intensity ratio at room temperature, which decreased with an increase in temperature. Life-time measurements indicated a difference in the micro-environments around the labeled -SH residues. The origin of the excimer band as well as temperature effects on this band have been explained on the basis of intra- and inter-molecular interaction among the Cys residues in the lens proteins. The temperature-dependent CD studies further indicated retention of thermodynamic stability of the crystallins after chemical modifications. Both PM and PIA could be used conveniently to probe -SH proximity, determine the ease and extent of disulfide formation, and monitor the dynamics of lens protein conformation, all of which are critically important with regard to cataract formation.
Sen
A C
AC
Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114.
Chakrabarti
B
B
D000818:Animals; D002417:Cattle; D003459:Crystallins / Q000302:isolation & purification / Q000378:metabolism; D005456:Fluorescent Dyes; D007460:Iodoacetamide / Q000031:analogs & derivatives* / Q000494:pharmacology*; D007461:Iodoacetates / Q000494:pharmacology*; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology*; D008958:Models, Molecular; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000032:analysis*; D013439:Sulfhydryl Reagents / Q000494:pharmacology*; D013816:Thermodynamics; D014961:X-Ray Diffraction
D003459:Crystallins; D005456:Fluorescent Dyes; D007461:Iodoacetates; D008301:Maleimides; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; C029358:N-(1-pyrenyl)iodoacetamide; C003944:N-(3-pyrene)maleimide; D007460:Iodoacetamide