Proton NMR studies of bovine serum albumin

Assignment of spin systems

Authors


Correspondence to P. J. Sadler, Department of Chemistry, Birkbeck College, University of London, Gordon House, 29 Gordon Square, London WC1H OPP, England

Abstract

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.
Abbreviations
1 D and 2D

one- and two-dimensional

BSA, HSA

PSA and RSA, bovine, human, porcine and rat serum albumin

BSA2

BSA purified by affinity chromatography

COSY

homonuclear shift-correlated spectroscopy

DQF-COSY

double-quantum-filtered phase-sensitive COSY

HOHAHA

homonuclear Hartmann-Hahn correlated spectroscopy

pH*

pH meter reading in 2H2O

P5 and P95

5th and 95th percentile

T1 and T2

longitudinal and transverse relaxation times