Phenylalanine ionizing groups

There are two catalytically active residues in pepsin Asp-32 and Asp-215. Their ionizations are seen in the pH-activity profile, which has an optimum at pH 2 to 3, and which depends upon the acidic form of a group of pKa 4.5 and the basic form of a group of pKa 1.1.160,161 The pKa values have been assigned from the reactions of irreversible inhibitors that are designed to react specifically with ionized or un-ionized carboxyl groups. Diazo compounds—such as A-diazoacetyl-L-phenylalanine methyl ester, which reacts with un-ionized carboxyls—react specifically with Asp-215 up to pH 5 or so (equation 16.28).162-164 Epoxides, which react specifically with ionized carboxyls, modify Asp-32 (equation 16.29). 

The aromatic amino acids also have fluorescence emissions when excited by light in the UV range. Table Bl.3.3 gives the excitation wavelength, fluorescence emission wavelength, and quantum yield (Q) for tryptophan, tyrosine, and phenylalanine. The quantum yield is the ratio of photons emitted to photons absorbed. Typically, phenylalanine fluorescence is not detected in the presence of tyrosine and tryptophan due to low Q. Furthermore, tyrosine fluorescence is nearly completely quenched if the tyrosine residue is ionized or near an amino group, a carboxyl group, or a tryptophan residue (Teale, 1960 Freifelder, 1982). Therefore, tryptophan fluorescence is what is customarily measured. 

The surprising feature of the absorption spectrum of phenylalanine is that it is sensitive to acid and alkali. This indicates that an appreciable change in the vibrational levels of the benzyl chromophor occurs, due to inductive effects brought about by ionization of the carboxyl or amino group. As will be shown later, such an influence on the vibrational levels can be shown to occur when an aromatic amino acid is combined in peptide linkage (Section VI, 2). The absorption curves of phenylalanine in acid and alkali are shown in Fig. 3. 

In proteins, three amino acids, tryptophan, tyrosine and phenylalanine, are responsible for the U.V. absorption, c in proteins is detemiined at the maximum (278 nm) (Fig. 1.13) and thus, concentrations of proteins are calculated by measuring the absorbance at this wavelength. Cystine and the ionized sulfhydryl groups of cysteine absorb also in this region but much weakly than the three aromatic amino acids. Ionization of the sulfhydiyl group induces an increase in the absorption and an appearance of a new peak around 240 nm. The imidazole group of histidine absorbs in the 185-220 nm region. Finally, important absorption of the peptide bonds occurs at 190 nm. 

The difference spectrum of phenylalanine closely resembles a first derivative of the spectrum. A portion of the difference spectrum, due to the ionization of the carboxyl group, is shown in Fig. 118. Using Eq. (VI-41) and the data of Fig. 118, Donovan et al. (1961) have calculated a shift of 1.5 A in the central vibrational band. A shift of 5 A has been observed for this band by Beaven and Holiday (1952) when protons are dissociated from both groups on the molecule. 

Three of the twenty common amino acids exhibit fluorescence. These are phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp). Phenylalanine, as the name implies, consists of alanine with a benzene ring attached. It is weakly fluorescent at 2f = 282 nm = 260 nm) and cannot be detected in the presence of Tyr or Trp. Tyrosine is phenolic and fluoresces = 275 nm 2f = 303 nm) with much greater intensity than Phe just as phenol fluoresces far more intensely than benzene. The phenolic group, which ionizes at pH above 10, introduces the need to control pH in the assay. The phenolate form of... 

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