Energy transfer in poly-l-tyrosine. V. Fluorescence polarization studies

Knopp, James A.; Bosch, Jeroen; Longworth, J. W.

doi:10.1016/0005-2795(69)90065-8

Cited by 13 publications

(3 citation statements)

References 18 publications

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“…For 290-nm polarized excitation, the apoazurin emission polarization is 0.340 ± 0.005, not very different from the reported values of 0.37 ± 0.01 (Knopp et al, 1969) and 0.36 ± 0.01 (corrected to polarized excitation condition from Helene et al, 1968) for A'-acetyl-L-tyrosine amide and tyrosine, respectively, and much larger than 0.25 for RNase and 0.19 for insulin (corrected to polarized excitation condition from Weber, 1960). In native azurin, the polarization decreases slightly to 0.320 ± 0.007 for 290 nm excitation.…”

Section: Discussioncontrasting

confidence: 83%

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Tyrosine emission in the tryptophanless azurin from Pseudomonas fluorescens

Uğurbil¹,

Bersohn²

1977

Biochemistry

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A strain of Pseudomonas fluorescens contains an azurin with no tryptophan and two tyrosines. This protein is interesting because it allows one to study both the structure of azurin and the emission of tyrosines in proteins. Comprehensive measurements were carried out including spectrophotometric and fluorimetric titration, fluorescence quantum yield, fluorescence polarization, and I- quenching. In the copper-containing protein, almost independent of the copper ion oxidation, the fluorescence quantum yield is approximately 60% of that of the apoprotein. The latter has the remarkable property that its quantum yield is even greater than free tyrosine. The two tyrosines in the metalloprotein have different pKa's, 10.75 and 12.78, but there is only one average pKa, 10.9 in the apoprotein. The polarization of the fluorescence at 310 nm (290-nm excitation) is 0.32 for the metalloproteins and 0.34 for the apoprotein. I- hardly quenches the fluorescence. The conclusion is that the two tyrosines are inaccesible to the solvent, located in nonpolar environments, larger than or equal to 20 A apart, and not adjacent to the disulfide bridge.

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Section: Discussioncontrasting

confidence: 83%

“…The reduced and oxidized azurin polarizations are similar within experimental error. The emission of the apoprotein is polarized to a higher degree than that of the native azurin in the oxidized and reduced forms and is not much different from values observed for /V-acetyl-L-tyrosine amide (Knopp et al" 1969).…”

Section: Resultsmentioning

confidence: 60%

Tyrosine emission in the tryptophanless azurin from Pseudomonas fluorescens

Uğurbil¹,

Bersohn²

1977

Biochemistry

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“…The fluorescence polarizations were measured with an instrument built by Knopp et al (1969). The exciting light was monochromatic.…”

Section: Methodsmentioning

confidence: 99%

Transfer of singlet energy within trypsin

Ghiron¹,

Longworth²

1979

Biochemistry

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Transfers of singlet energy within trypsin were investigated by measuring the fluorescence absorption anisotropy of its tryptophan residues. A ratio of the anisotropy of trypsin to that for N-acetyl-L-tryptophanamide was determined between 306 and 250 nm. The ratio had an average value of 0.7, whether the trypsin anisotropy was measured at 228 of 296 K. However, trypsin dissolved in 5 M guanidine hydrochloride showed little fluorescence depolarization at 228 K (the anisotropy ratio was approximately equal to 0.9). Thus, there is an extensive conformation-dependent energy transfer between tryptophans in trypsin. The ratio of anisotropies of tyrpsin at 304--270 nm was used to estimate energy transfer from tyrosine to tryptophan. Ratios of 1.8 and 1.7 were obtained at 296 K for the native and guanidinium-unfolded enzyme, respectively. The comparable value for N-acetyl-L-tryptophanamide was 1.7. This indicates that there is little transfer from tyrosine to tryptophan in trypsin at 296 K. As confirmation, the excitation wavelength dependencies of the indole fluorescence quantum yield were the same for native and unfolded trypsin. When experiments were performed at 228 K, the 304--270-nm anisotropy ratios were 2.6 for native and 2.1 for unfolded trypsin at pH2. This indicates that the efficiency of energy transfer from tyrosine to tryptophan increases at low temperatures. A photochemical source of error in the quantitation of the efficiency of energy transfer from tyrosine to tryptophan is also described.

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Luminescence of Polypeptides and Proteins

Longworth

1971

Excited States of Proteins and Nucleic Acids

155

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Energy transfer in poly-l-tyrosine. V. Fluorescence polarization studies

Cited by 13 publications

References 18 publications

Tyrosine emission in the tryptophanless azurin from Pseudomonas fluorescens

Tyrosine emission in the tryptophanless azurin from Pseudomonas fluorescens

Transfer of singlet energy within trypsin

Luminescence of Polypeptides and Proteins

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