RESUMO
Carbonmonoxyhemoglobin prepared from protein isolated from rabbits maintained on a diet supplemented with 4-fluorophenylalanine (Phe (4F)) has been studied by fluorine NMR spectroscopy. Substitution of Phe(4F) appears to take place randomly at the sixteen nonequivalent phenylalanine positions of the globins; examination of hybrid hemoglobins in which only one type of globin chain contained the fluorinated amino acid, as well as changes in the spectrum upon exposure to oxygen, aided in the assignment of fluorine resonances from the alpha- or beta-globin chains. The effects of modification of Cys-beta-93 with a spin label and variation of pH and sample temperature on the spectrum were also examined. These data in association with theoretical estimates of aromatic ring current and van der Waals effects on chemical shifts were used to support tentative assignments of several signals observed to specific amino acid residues. Evidence suggesting the presence of two conformational forms of the protein, possibly due to disorder of the heme groups, is described.
Assuntos
Carboxihemoglobina , Fenilalanina/análogos & derivados , p-Fluorfenilalanina/sangue , Animais , Flúor , Concentração de Íons de Hidrogênio , Substâncias Macromoleculares , Espectroscopia de Ressonância Magnética , Física Nuclear , Multimerização Proteica , Coelhos , TemperaturaRESUMO
This paper describes a rapid method for the determination of low levels of 4-fluorophenylalanine biosynthetically incorporated into proteins. Precolumn derivatization with phenylisothiocyanate followed by reverse-phase high-performance liquid chromatography enables determination of the amino acid composition of the protein as well as accurate detection of a 1-3% substitution of phenylalanine by fluorophenylalanine.