Spectroscopy and Fluorescence Quenching of Tyrosine in Lima Bean Trypsin/Chymotrypsin Inhibitor and Model Peptides

Liu, Xiao‐Yuan; Cottrell, Kazuo Ota; Nordlund, Thomas M.

doi:10.1111/j.1751-1097.1989.tb02902.x

Cited by 8 publications

(11 citation statements)

References 30 publications

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“…1 ). The absorption maxima were consistent with relevant examples from the literature [ 11 , 16 ]. The fluorescence emission maxima were 9 nm higher than a literature example (Leu-Tyr-Leu), probably as a result of differences in the conditions under which the measurements were made.…”

Section: Resultssupporting

confidence: 89%

Quantification of a peptide standard using the intrinsic fluorescence of tyrosine

Preston

Phillips

2016

Anal Bioanal Chem

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Absolute quantification of peptides is typically achieved using amino acid analysis, elemental analysis or derivatisation chemistry. Impurities, if present, may be accounted for using analytical high-performance liquid chromatography (HPLC) with detection of the peptide bond ultraviolet (UV) absorbance. To do this, peak areas from a UV chromatogram are used to estimate percentage purity on a mass basis, and this purity value is used as a correction. However, because the approach assumes that UV absorbance is uniformly proportional to mass, the result may be only semi-quantitative. Here, an alternative approach involving HPLC with detection of intrinsic tyrosine fluorescence is described. The fluorescence properties of a 21-residue synthetic peptide corresponding to an S-carbamidomethylated tryptic fragment of human serum albumin were characterised, and a method involving quantification relative to a non-peptidic calibrant, N-acetyl-l-tyrosine ethyl ester, was established. The method was used to quantify the thiol form of the peptide, and the results were compared with a parallel analysis involving derivatisation of the same material with Ellman’s reagent. When differences in fluorescence response (analyte versus calibrant) were accounted for, the measurements obtained via the two methods were in good agreement. Contributions from peptidic impurities were also considered, and their influence on the validity of the conclusions was evaluated. Despite some ambiguities introduced by the impurities, and the identification of some other potential sources of error, the results demonstrate that use of Tyr fluorescence is a promising solution to the challenging problem of absolute peptide quantification.Graphical AbstractA low-molecular-weight tyrosine derivative was used as a generic fluorescence calibrant for the quantification of a 21-residue peptideElectronic supplementary materialThe online version of this article (doi:10.1007/s00216-016-9334-1) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 89%

Quantification of a peptide standard using the intrinsic fluorescence of tyrosine

Preston

Phillips

2016

Anal Bioanal Chem

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“…It is known from long ago [27] that tyrosine residue free in solution (pK a 10.6) or in peptides and proteins (pK a 9.6–10.4) [28] exhibits pK a due to ionization of the tyrosine phenolic hydroxyl. The latter was shown to depend on protein environment [23], and may vary from 9.6 to 10.4 due to interaction with protein interior.…”

Section: Resultsmentioning

confidence: 99%

Fluorescence Anisotropy of Tyrosinate Anion Using One-, Two- and Three-Photon Excitation

Kierdaszuk

2012

J Fluoresc

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We examined the emission spectra and steady-state anisotropy of tyrosinate anion fluorescence with one-photon (250–310 nm), two-photon (570–620 nm) and three-photon (750–930 nm) excitation. Similar emission spectra of the neutral (pH 7.2) and anionic (pH 13) forms of N-acetyl-L-tyrosinamide (NATyrA) (pKa 10.6) were observed for all modes of excitation, with the maxima at 302 and 352 nm, respectively. Two-photon excitation (2PE) and three-photon excitation (3PE) spectra of the anionic form were the same as that for one-photon excitation (1PE). In contrast, 2PE spectrum from the neutral form showed ~30-nm shift to shorter wavelengths relative to 1PE spectrum (λmax 275 nm) at two-photon energy (550 nm), the latter being overlapped with 3PE spectrum, both at two-photon energy (550 nm). Two-photon cross-sections for NATyrA anion at 565–580 nm were 10 % of that for N-acetyl-L-tryptophanamide (NATrpA), and increased to 90 % at 610 nm, while for the neutral form of NATyrA decreased from 2 % of that for NATrpA at 570 nm to near zero at 585 nm. Surprisingly, the fundamental anisotropy of NATyrA anion in vitrified solution at −60 °C was ~0.05 for 2PE at 610 nm as compared to near 0.3 for 1PE at 305 nm, and wavelength-dependence appears to be a basic feature of its anisotropy. In contrast, the 3PE anisotropy at 900 nm was about 0.5, and 3PE and 1PE anisotropy values appear to be related by the cos6θ to cos2θ photoselection factor (approx. 10/6) independently of excitation wavelength. Attention is drawn to the possible effect of tyrosinate anions in proteins on their multi-photon induced fluorescence emission and excitation spectra as well as excitation anisotropy spectra.

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“…The single tyrosine in LBTI has an abnormally high of > 11.5, most likely due to interactions with the protein. (173) Citrate fluorescence quenching studies of LBTI showed complex behavior. Based on simplifying assumptions, the quenching data also suggest that the tyrosine residue is shielded from solvent.…”

Section: Protease Inhibitorsmentioning

confidence: 99%

“…Based on simplifying assumptions, the quenching data also suggest that the tyrosine residue is shielded from solvent. (173) 1.5.1.5. Other Proteins l.5.l.5a.…”

Section: Protease Inhibitorsmentioning

confidence: 99%

“…(210) An additional emission band near 350 nm has been observed for lima bean trypsin inhibitor (LBTI). (173) The authors discussed both the possibility of contamination by tryptophan and excited-state tyrosinate formation. Since this 350-nm emission has a tyrosine-like excitation spectrum that is slightly shifted compared to that of the major 302-nm emission, it is also possible that the tyrosine residue in a fraction of the LBTI molecules could be hydrogen bonded.…”

Section: Fluorescence Of Tyrosinatementioning

confidence: 99%

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Tyrosine Fluorescence and Phosphorescence from Proteins and Polypeptides

Ross

Laws

Rousslang

et al.

Topics in Fluorescence Spectroscopy

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The fluorescence and phosphorescence of proteins and polypeptides is the sum of the contributions from the three aromatic amino acids tryptophan, tyrosine, and phenylalanine. The work on protein and polypeptide luminescence prior to 1971 has been reviewed in detail by Longworth. (1) Another fine account of the early work, emphasizing tryptophan and tyrosine, is the monograph by Konev. (2) An excellent review on tyrosine fluorescence in proteins and model peptides, for the period up to 1975, is given by Cowgill. (3) In 1984, Creed (4) reviewed the photophysics and photochemistry of tyrosine and its simple derivatives, including a thorough coverage of steady-state fluorescence and a brief discussion of triplet-state properties, but did not include any work on proteins or polypeptides.The first quantitative studies of the excited-state properties of the three aromatic amino acids were carried out in the 1950s. The low-temperature phosphorescence of the aromatic amino acids was initially observed by Debye and Edwards (5) in 1952, and phosphorescence emission spectra were reported by Steele and Szent-Gyorgyi (6) in 1957. In 1953, Weber (7) postulated that the fluorescence of the aromatic amino acids should occur in the near-ultraviolet region of the electromagnetic spectrum. In 1956, independently and almost simultaneously, Duggan and Udenfriend (8) and Shore and Pardee (9) reported the results of their investigations of protein fluorescence. At the same time,

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Spectroscopy and Fluorescence Quenching of Tyrosine in Lima Bean Trypsin/Chymotrypsin Inhibitor and Model Peptides

Cited by 8 publications

References 30 publications

Quantification of a peptide standard using the intrinsic fluorescence of tyrosine

Quantification of a peptide standard using the intrinsic fluorescence of tyrosine

Fluorescence Anisotropy of Tyrosinate Anion Using One-, Two- and Three-Photon Excitation

Tyrosine Fluorescence and Phosphorescence from Proteins and Polypeptides

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