Proton NMR studies of bovine serum albumin. Assignment of spin systems.
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. 1. The 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. 2. The 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. 3. The release of bound Cys (from mixed disulphide at Cys34) was observed after addition of dithiothreitol. 4. Through the use of two-dimensional shift-correlated spectroscopy, assignments of some 80 spin systems to amino acid type were made. 5. The pKa of the N-terminal Asp was measured as 7.8 (0.1 M phosphate buffer, 310 K). 6. 1H 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. 7. These 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.
Sadler
P J
PJ
Christopher Ingold Laboratories, Birkbeck College London, England.
Tucker
A
A
D000595:Amino Acid Sequence; D000596:Amino Acids; D000818:Animals; D002417:Cattle; D003545:Cysteine; D004220:Disulfides; D006023:Glycoproteins / Q000737:chemistry; D006801:Humans; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008969:Molecular Sequence Data; D011487:Protein Conformation; D051381:Rats; D012709:Serum Albumin / Q000737:chemistry / Q000235:genetics; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D013552:Swine
D000596:Amino Acids; D004220:Disulfides; D006023:Glycoproteins; D012709:Serum Albumin; D012710:Serum Albumin, Bovine; D006859:Hydrogen; D003545:Cysteine