The role of tryptophan residues and hydrophobic interaction in the binding of riboflavin in egg-yolk flavoprotein.
Egg-yolk flavoprotein has 7.2 tryptophan residues exposed, while the apoprotein shows an apparent exposure of 80 percent of these (5.7 residues) with dimethylsulphoxide as the perturbant. In the apoprotein at pH 6.9 only 4 groups are perturbed to ethylene glycol, 3.2 to glycerol and 1.4 to sucrose. Diminishing estimates of exposure obtained with increasing molecular diameter of the perturbant suggests that part of indole chromophores of apoprotein are located in "crevices" of the protein molecule. The apoprotein was treated with 2-hydroxy-5-nitrobenzyl bromide, H2O2 and N-bromosuccinimide under conditions designed to accomplish modification of tryptophan residues. Five to six of the eight tryptophans present in the protein were modified. Under these conditions the apoprotein completely looses its capacity for binding riboflavin and the fluorescent intensity of the protein at 360 nm is quenched at the same time to about 80 percent of its initial value. The presence of nonpolar amino acid residues on the surface of the apoprotein suggested the importance of hydrophobic interactions as the dominant factor controlling the binding of riboflavin. The hydrophobic probes Indocyanine green and 4-benzoylamide-4-aminostilbene-2,2-disulphonic acid bound to the apoprotein giving equimolar complexes with dissocation constants, KD 6.5-10(-7) M and 1.8-10(-6) M, respectively, Addition of an equimolar amount of riboflavin quantitatively displaced these dyes from their complexes with apoprotein as shown by spectrophotometric and spectrofluorometric studies.
D000596:Amino Acids / Q000032:analysis; D001059:Apoproteins; D001593:Benzyl Compounds; D001665:Binding Sites; D004527:Egg Proteins; D004530:Egg Yolk; D005260:Female; D005420:Flavoproteins; D006861:Hydrogen Peroxide; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009574:Nitro Compounds; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D012256:Riboflavin; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013388:Succinimides; D014364:Tryptophan / Q000032:analysis
D000596:Amino Acids; D001059:Apoproteins; D001593:Benzyl Compounds; D004527:Egg Proteins; D005420:Flavoproteins; D009574:Nitro Compounds; D012997:Solvents; D013388:Succinimides; D014364:Tryptophan; D006861:Hydrogen Peroxide; D012256:Riboflavin