Tyrosinate fluorescence

Tyrosine Fluorescence and Phosphorescence from Proteins and Polypeptides... 

Figure 1.3. Tyrosine and tyrosinate fluorescence and phosphorescence emission spectra with 280-nm excitation. Curve 1 tyrosine fluorescence in water, pH 7. Curve 2 tyrosinate fluorescence in 0.01 M NaOH. 25 Curve 3 tyrosine phosphorescence in 50% (v/v) ethylene glycol/water/24 Curve 4 tyrosinate phosphorescence in 50% (v/v) ethylene glycol/0.01 A/NAOH/24. ...

Cowgill pointed out that there are essentially two distinct quenching processes of tyrosine fluorescence resulting from association with the peptide bond.(3) Tyrosines affected by these mechanisms are classified in Table 1.3 as... 

Most of the research on tyrosine fluorescence and phosphorescence in polypeptides and proteins has involved steady-state measurements. This is understandable when one considers that only recent developments have... 

Tyrosine fluorescence emission in proteins and polypeptides usually has a maximum between 303 and 305 nm, the same as that for tyrosine in solution. Compared to the Stokes shift for tryptophan fluorescence, that for tyrosine appears to be relatively insensitive to the local environment, although neighboring residues do have a strong effect on the emission intensity. While it is possible for a tyrosine residue in a protein to have a higher quantum yield than that of model compounds in water, for example, if the phenol side chain is shielded from solvent and the local environment contains no proton acceptors, many intra- and intermolecular interactions result in a reduction of the quantum yield. As discussed below, this is evident from metal- and ionbinding data, from pH titration data, and from comparisons of the spectral characteristics of tyrosine in native and denatured proteins. 

The H2a-H2b histone dimer also has strong salt-dependent conformational properties, with a transition near 0.5 M NaCl.(93) Above 0.5 M NaCl, the tyrosine fluorescence emission becomes less quenchable by Cs+, and the dimer structure becomes more compact. 

In an investigation of the physical basis of the interaction of histones with DNA, De Petrocellis et al.< 95 > have examined the effect of phosphate ions on histone HI. Binding results have shown that there are high-affinity sites for phosphate ions. In addition, phosphate ions were found to perturb the absorption spectra of HI and quench tyrosine fluorescence. Binding of the phosphate group resulted in positive difference absorption bands near 275 and 293 nm, which are similar to those produced at acid and alkaline pH, respectively. 

A protein induced after coliphage N4 infection has been studied. Although it has one or two tryptophans, its intrinsic fluorescence is dominated by the ten tyrosines/1111 Tryptophan fluorescence is seen after denaturing the protein. Upon binding to single-stranded DNA, the tyrosine fluorescence is quenched. This signal has been used to demonstrate that the binding affinity is very dependent on salt concentration and is also very sensitive to the nucleotide sequence. 

The way in which calcium binding changes the tyrosine fluorescence of calmodulin was initially a controversial issue. In 1980, Seamon(n5) examined the binding of Ca2+ and Mg2+ by NMR and concluded that both Tyr-99 and Tyr-138 were perturbed by the first two calcium ions. Although Tyr-138 was also perturbed by the binding of the fourth calcium, both residues appeared to be associated with high-affinity domains (III and IV). 

The fluorescence of the two tyrosine residues in bovine testes calmodulin was investigated by Pundak and Roche.(123) Upon excitation at 278 nm, a second emission, in addition to tyrosine fluorescence, was observed at 330-355 nm, which they characterized as being due to tyrosinate fluorescence. The tyrosinate fluorescence appeared to be from Tyr-99, which has an anomalously low pKa of about 7 for the phenol side chain. Pundak and Roche(123) reasoned that since tyrosinate emission is apparently not being seen in other species of calmodulin, it is possible that the bovine protein contains a carboxylate side chain in domain III which is amidated in other species. They further argued that the tyrosinate emission from bovine testes calmodulin arises from direct excitation of an ionized tyrosine residue. This tyrosinate fluorescence is discussed in more detail in Section 1.5.2. 

The physical dimensions and dynamics of calmodulin have also been investigated by tyrosine fluorescence. To learn about the internal mobility of calmodulin, Lambooy et al 1 and Steiner et al measured the steady-state fluorescence anisotropy of the tyrosine. Since the average correlation... 

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