Optical detection of triplet-state magnetic resonance studies on individual tryptophan residues of serum albumin: correlation between phosphorescence and zero-field splittings.
Cyanogen bromide cleavage of bovine serum albumin (BSA) yields two fragments, N (1-183) and C (184-582), containing 183 and 399 amino acid residues, respectively. Each in each fragment are characterized in this study by phosphorescence and optically detected magnetic resonance spectroscopy, and the results are compared with those of the intact albumin. Trp-134 in fragment N is located in a hydrophobic environment in the interior of the protein, as reflected by its red-shifted phosphorescence and characteristic zero-field splittings. The spectral properties of Trp-212 in fragment C suggest its location in a partially buried, inhomogeneous environment. They show great similarity to those of human serum albumin, which contains a single Trp at position 214. The Trp phosphorescence 0,0-bands of fragments C and N are fitted with Gaussian functions by computer, and their relative contributions to the phosphoresence 0,0-band of BSA are adjusted to fit the observed BSA 0,0-band. The wavelength dependence of the [D[-[E[ transition frequencies of fragments N and C is then weighted by their 0,0-band intensity, taking into account differences in spin alignment, and summed to predict the peak frequency of the [D[-[E[ band profile as a function of phosphorescence wavelength for the intact BSA. Good agreement between predicted and observed behavior of [D[-[E[ vs. wavelength for the intact protein provides strong evidence for the additivity of the phosphorescence and ODMR spectra of the individual Trp sites in BSA. We find that Trp-134 and Trp-212 have wavelength-independent and wavelength-dependent zero-field splittings, respectively.
D003488:Cyanogen Bromide; D006801:Humans; D008163:Luminescent Measurements; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments; D011487:Protein Conformation; D012709:Serum Albumin; D012710:Serum Albumin, Bovine; D014364:Tryptophan
D010446:Peptide Fragments; D012709:Serum Albumin; D012710:Serum Albumin, Bovine; D014364:Tryptophan; D003488:Cyanogen Bromide