Tyrosine 54 and tryptophan 108 of streptavidin are photolabelled by N-(2-nitro-5-azidobenzoyl)- N′-(d-biotinyl)-1,4-diaminobutane and N-(4-azidophenyl)- N′-(d-biotinyl)-1,4-diaminobutane, respectively. Isolation, spectrophotometric characterization and sequence analysis of photolabelled peptides

Abstract

Two new photoreactive biotin derivatives, N-(2-nitro-5-azidobenzoyl)- N′-(d-biotinyl)-1,4-diaminobutane (I) and N-(4-azidophenyl)- N′-(d-biotinyl)-1,4-diaminobutane (II), have been synthesized and used for a photoaffinity labelling of streptavidin at a molar ratio of 1:1 reagent to streptavidin subunit. Photolabelled streptavidin preparations are subjected to extensive digestion by proteinase K, and the mixture of peptides obtained is fractionated by reversed-phase HPLC. The fractions containing the peptides photolabelled by the photobiotin I are identified by UV spectroscopy (λ = 360 nm) and the fractions containing the peptides photolabelled by the photobiotin II are characterized by their interaction with streptavidin alkaline phosphatase conjugate. The photolabelled peptides are analysed by spectrophotometric absorbance and submitted to sequence analysis by automatic Edman's degradation. The sequence of the peptide photolabelled by photobiotin I is found to be SerArgTyr * ValLeu, corresponding to residues 52–56 of the streptavidin sequence, where Tyr * designates modified Tyr-54. The sequences of the peptides photolabelled by photobiotin II are found to be ThrGlnTrp *LeuLeu (residues 106–110) and Trp *LeuLeu (residues 108–110), where Trp * designates modified Trp-108.