Ligands containing heavy atoms: perturbation of phosphorescence of a tryptophan residue in the binding site of wheat germ agglutinin.
Information on the structure of binding sites of wheat germ agglutinin was obtained on the basis of fluorescence and phosphorescence changes of tryptophan residues induced by the binding of several thiomercuribenzoate derivatives of glycosides. The thiomercuribenzoate derivatives bind selectively to wheat germ agglutinin in the same way as the corresponding sugars. Using the thiomercuribenzoate of di-N-acetyl-beta-chitobiose, it was found that: (i) the fluorescence of tryptophan residues was drastically quenched at both 298 and 77 K; (ii) the phosphorescence intensity was strongly enhanced at 77 K; (iii) the phosphorescence lifetime was markedly decreased. A similar effect was observed with the thiomercuribenzoate of N-acetyl-beta-D-glucosamine. These changes were completely reversed upon addition of 1-O-methyl-di-N-acetyl-beta-chitobioside. The thiomercuribenzoate of beta-D-glucose had no effect at all, and the thiomercuribenzoate of tri-N-acetyl-beta-chitotriose had a limited effect. These results are interpreted as a specific heavy atom effect due to a close contact between one tryptophan residue of the protein and the heavy atom of the bound ligand. They are consistent with the view that: (i) binding sites of wheat germ agglutinin may be divided in three subsites, A, B, and C; (ii) a tryptophan residue is in the binding site at subsite C; and (iii) this residue and the ligand are in close contact. This new method, using the enhancement of spin-orbit coupling due to the selective perturbation induced in a tryptophan residue by a ligand containing a heavy atom, has proved to be suitable for locating the tryptophan residue in the binding site of wheat germ agglutinin and can probably be extended to other sugar-binding proteins.
D001665:Binding Sites; D006601:Hexoses / Q000378:metabolism*; D037102:Lectins; D008024:Ligands; D008163:Luminescent Measurements; D009844:Oligosaccharides / Q000378:metabolism*; D009941:Organomercury Compounds / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014364:Tryptophan
D006601:Hexoses; D037102:Lectins; D008024:Ligands; D009844:Oligosaccharides; D009941:Organomercury Compounds; D014364:Tryptophan
Biochemistry. 1975 Feb 11;14(3):558-63
234245
Eur J Biochem. 1975 Jun 16;55(1):257-62
809273
Arch Biochem Biophys. 1977 Jan 30;178(2):625-30
836049
J Mol Biol. 1977 Apr 25;111(4):439-57
871318
Eur J Biochem. 1977 Sep 15;79(1):275-83
913420
Eur J Biochem. 1976 Sep 15;68(2):563-72
976273
Eur J Biochem. 1976 Sep 15;68(2):573-80
976274
Biochimie. 1976;58(10):1247-53
1009178
Nature. 1975 Jul 31;256(5516):414-6
1170502
Eur J Biochem. 1975 Dec 15;60(2):555-67
1204655
Proc Natl Acad Sci U S A. 1969 Mar;62(3):994-1001
4308100
Proc Natl Acad Sci U S A. 1972 Aug;69(8):2198-202
4340750
FEBS Lett. 1974 Sep 15;46(1):224-8
4419603
FEBS Lett. 1974 Sep 15;46(1):229-32
4419604
Eur J Biochem. 1974 Aug 15;47(1):5-14
4434989
Biochem J. 1973 Jan;131(1):155-62
4737292
Biochem Biophys Res Commun. 1973 Dec 19;55(4):1340-6
4771999
J Biol Chem. 1974 May 25;249(10):3116-22
4830237
Biochem Biophys Res Commun. 1974 May 7;58(1):13-9
4831061
Proc Natl Acad Sci U S A. 1970 Nov;67(3):1390-7
5274466
Proc Natl Acad Sci U S A. 1963 Oct;50:613-9
14077487
Proc Natl Acad Sci U S A. 1967 Feb;57(2):359-66
16591478