Spectroscopic studies on the riboflavin-sensitized conformational changes of calf lens α-crystallin

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Abstract

The change in conformation of calf lens α-crystallin by oxidation in the presence of the photosensitizer riboflavin and light has been investigated. Near-UV circular dichroism (CD) spectrum, absorption spectrum, tryptophan fluorescence yield and fluorescence lifetime of the SH-specific fluorescent probe, N-iodoacetyl-N′-(5-sulfo-1-naphtyl) ethylenediamine (1,5-IAEDANS), were significantly altered by irradiation in the presence of RF. In the initial stages of photolysis (1-2 hr), a slight degradation of the protein to lower molecular weight peptides was observed. Upon increased photolysis, intersubunit cross-linking to dimers and other high molecular weight species was observed. To determine the effects of cross-linking on the accessibility of the cysteine residues of the protein, lifetime quenching studies on the IAEDANS-labeled α-crystallin were performed. A decrease in the quenching constant (kq) in the photolysed sample indicates that the labeled SH groups are less susceptible to collisional quenching, which requires contact between the quencher and the excited state of the fluorophore, due to steric inhibition in the cross-linked protein. Cross-linking and the rate of loss of tryptophan fluorescence of α-crystallin diminished under anaerobic conditions and increased when D2O was used in the medium for irradiation. Use of inhibitors and quenchers of active species of oxygen suggests that photo-oxidation probably occurs via the action of singlet oxygen as well as substrate-sensitizer complexation.

Original languageEnglish
Pages (from-to)531-544
Number of pages14
JournalExperimental eye research
Volume46
Issue number4
DOIs
StatePublished - Apr 1988
Externally publishedYes

Keywords

  • 5,5′-dithiobis (2-nitrobenzoic acid)
  • CD
  • DTNB
  • IAEDANS
  • MB
  • Methylene Blue
  • N-iodoacetyl-N′-(5-sulfo-1-naphthyl) ethylenediamine
  • RF
  • SH
  • SOD
  • acrylamide quenching
  • circular dichroism
  • conformation
  • crystallins
  • cysteine
  • disulfide
  • fluorescence
  • lens
  • lifetime
  • oxidation
  • photo-oxidation
  • photosensitizer
  • protein
  • riboflavin
  • singlet oxygen
  • sulfhydryl
  • superoxide dismutase
  • unfolding

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