A variety of one- and two-dimensional 1H-NMR methods have been applied to the study of defatted 66.5-kDa bovine serum albumin in solution.
- 1The majority of the protons gave rise to broad unresolved resonances and spectral enhancement methods for one-dimensional spectra were investigated in detail. A combination of exponential and sine-bell functions was particularly effective.
- 2The presence of contaminating glycoproteins in some commercial samples of bovine serum albumin was readily detectable from their N-acetyl resonances at about 2.1 ppm.
- 3The release of bound Cys (from mixed disulphide at Cys34) was observed after addition of dithiothreitol.
- 4Through the use of two-dimensional shift-correlated spectroscopy, assignments of some 80 spin systems to amino acid type were made.
- 5The pKa of the N-terminal Asp was measured as 7.8 (0.1 M phosphate buffer. 310 K).
- 61H NMR spectra of bovine, human, porcine and rat serum albumins have been compared. Using sequence comparisons, specific assignments have been made for the N-terminal residues of bovine (Asp-Thr-His), human (Asp-Ala-His), porcine (Asp-Thr-Tyr) and rat (Glu-Ala-His) albumins, and for Thr189, Tyr155 and His59/377 of bovine albumin.
- 7These NMR data suggest that certain local regions of bovine serum albumin are highly mobile yet structured in solution, and demonstrate that the application of both one- and and two-dimensional NMR methods will allow more detailed investigations of structural transitions in serum albumins induced by, for example, pH, drug and metal binding.
- 1 D and 2D
one- and two-dimensional
- BSA, HSA
PSA and RSA, bovine, human, porcine and rat serum albumin
BSA purified by affinity chromatography
homonuclear shift-correlated spectroscopy
double-quantum-filtered phase-sensitive COSY
homonuclear Hartmann-Hahn correlated spectroscopy
pH meter reading in 2H2O
- P5 and P95
5th and 95th percentile
- T1 and T2
longitudinal and transverse relaxation times