Fourth-derivative spectrophotometry analysis of tryptophan environment in proteins. Application to melittin, cytochrome c and bacteriorhodopsin.
Fourth-derivative spectrophotometry is applied to the analysis of solvent effects on the spectral transitions of N-acetyl-L-tryptophan amide, as well as of its mixtures with N-acetyl-L-tyrosine ethyl ester. These compounds were analyzed in different media. It was found that the position of the longest-wavelength minimum of the fourth-derivative spectrum is mainly determined by the nature of the Trp environment, with a minor contribution from that of Tyr. A geometrical parameter is also defined, which depends on both Trp and Tyr environments. The simultaneous consideration of both parameters allows an estimation to be made of the Trp environment. The method is applied to the study of conformational changes of three well-characterized proteins: melittin from bee venom, cytochrome c from horse heart and bacteriorhodopsin from Halobacterium halobium. The results obtained are found to be in accordance with the known conformational properties of these proteins. In addition, the fourth-derivative operation completely eliminates the interference from the near-ultraviolet bands of the prosthetic groups.
D000818:Animals; D001436:Bacteriorhodopsins / Q000302:isolation & purification*; D001514:Bee Venoms / Q000032:analysis / Q000302:isolation & purification*; D002338:Carotenoids / Q000302:isolation & purification*; D055598:Chemical Phenomena; D002621:Chemistry; D003574:Cytochrome c Group / Q000302:isolation & purification*; D006217:Halobacterium / Q000378:metabolism; D006736:Horses; D008555:Melitten / Q000302:isolation & purification*; D009206:Myocardium / Q000201:enzymology; D012997:Solvents; D013053:Spectrophotometry / Q000379:methods; D014364:Tryptophan / Q000302:isolation & purification*
D001514:Bee Venoms; D003574:Cytochrome c Group; D012997:Solvents; D008555:Melitten; D002338:Carotenoids; D001436:Bacteriorhodopsins; D014364:Tryptophan