A rigorous and convenient method of correcting for the wavelength variation of the instrument response function in time correlated photon counting fluorescence decay measurements is described. The method involves convolution of a modified functional form F̃s of the physical model with a reference data set measured under identical conditions as the measurement of the sample. The method is completely general in that an appropriate functional form may be found for any physical model of the excited state decay process. The modified function includes a term which is a Dirac delta function and terms which give the correct decay times and preexponential values in which one is interested. None of the data is altered in any way, permitting correct statistical analysis of the fitting. The method is readily adaptable to standard deconvolution procedures. The paper describes the theory and application of the method together with fluorescence decay results obtained from measurements of a number of different samples including diphenylhexatriene, myoglobin, hemoglobin, 4′, 6-diamidine–2-phenylindole (DAPI), and lysine–trytophan–lysine.

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. Fluorescence attributable to the tyrosinate form of the amino acid tyrosine, previously only observed at p H > pK(So) = 10.3 where tyrosinate exists in the ground state, has been observed at neutral p H in the presence of high buffer base concentrations. This observation is consistent with the large shift in pK(S1) predicted from absorption measurements and confirn~s that proton transfer is indeed a mechanism by which carboxylate ions quench tyrosine fluorescence. The dependence of the fluorescence quantum yields of tyrosine and tyrosinate o n p H does not fit a simple excited state acid-base equilibrium model but a more complicated system where carboxylate is also capable of simultaneously quenching tyrosine fluorescence by a mechanism not involving proton transfer. Kinetic analysis of the system allows calculation of pK(S,) = 4.2 for tyrosine. The quantum yield of tyrosinate fluorescence can be appreciably higher than that normally measured at alkaline p H where a separate quenching mechanism niust operate. These results have significance in the interpretation of the fluorescence properties of proteins.DAVID MICHAEL RAYNER, DONALD THEODORE KRAJCARSKI et ARTHUR GUSTAV SZABO. Can. J. Chem. 56, 1238Chem. 56, (1978.On a observe de la fluorescence attribuable a la fornie tyrosinate d e I'acide anline tyrosine, observe anterieurement uniquement 2 des p H > pK(So) = 10.3 auquel le tyrosinate existe dans son &tat fondamental, a un p H neutre en presence de concentrations ClevCes de tampons basiques. Cette observation est en accord avec un grand deplacement dans le pK(S,) qili peut Ctre predit a partir de mesures d'absorption et confirme que le transfert de proton est d e fait un mecanisme par lequel les ions carboxylates piegent la fluorescence de la tyrosine. Le fait que les rendements quantiques de la fluorescence de la tyrosine et du tyrosynate dependent du p H ne cadre pas avec un niodele acide-base a I'tquilibre d'un etat excite simple niais avec un systime plus complique dans lequel le carboxylate est aussi capable de pitger simultantment la fluorescence de la tyrosine par un mecanisme n'inipliquant pas un transfert de proton. Une analyse cinetique du systeme permet de calculer le pK(S1) = 4.2 pour la tyrosine. Le rendement quantique de la fluorescence du tyrosinate peut &tre beaucoup plus eleve que celui mesure normalement au p H alcalin quand un mecanisme de piegeage separe peut itre en optration. Ces resultats ont une signification dans I'interpretation des proprietes de fluorescence des proteines.[Traduit par le journal] Introduction In the course of our studies of the fluorescence properties of proteins and amino acids and their structural implications we were struck by the lack of attention paid in recent reviews (1, 2) t o the excited state acid-base properties of tyrosine. As a substituted phenol tyrosine would be expected to show similar behaviour to 2-napthol, Weller's (3) classic example of a compound which is far more acidic in the excited state than the ground state (pK(So) = 9.46, pK(S,) '= 2.8...

Steady-state and time-resolved fluorescence spectroscopy were employed in the study of the structure and interactions of alpha-MSH (alpha-melanocyte-stimulating hormone) and its analogs, [Nle4,D-Phe7]-alpha-MSH (MSH-I) and Ac-[Nle4,Asp5,D-Phe7,Lys10]-alpha-MSH(4-10)-NH2 (MSH-II). In aqueous buffer, the fluorescence parameters of the single tryptophan of alpha-MSH and MSH-I were similar and did not allow any distinction between these molecules. On the other hand, the tryptophan fluorescence of MSH-II was notably different, reflecting its cyclic lactam turn structure. In the presence of acidic lipid vesicles, the fluorescence properties of the peptides were different, indicating structural changes on incorporation of the peptide into the liquid-crystalline phase of the lipid. No evidence of interaction was observed in the presence of the neutral lipid dimyristoylphosphatidylcholine (DMPC). The association constants for lipid-peptide interactions were compared for binding isotherms which either neglected or accounted for electrostatic effects through Gouy-Chapman potential functions. The relative order of association constants in either treatment was MSH-II > MSH-I > alpha-MSH. These results parallel the reported biological activities that show increased potencies and prolongation of response for the analogs, MSH-II and MSH-I, as compared to the native hormone, alpha-MSH. Time-resolved fluorescence results showed that the fluorescence decay of melanotropins is best described by triple-exponential kinetics. In the lipid-peptide complex, there was a change in the relative concentrations of the components, with the intermediate-lifetime component predominating compared to those in solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract— The steady state and time resolved fluorescence of the drug and chromosomal staining agent, 4′,6‐diamidino‐2‐phenylindole dihydrochloride, DAPI, was examined under different solvent conditions. In solutions between pH 3 and pH 9 the fluorescence spectral maximum of DAPI was found at 460 nm. The fluorescence decayed with double exponential kinetics, with decay times of 2.86 and 0.144 ns, at all wavelengths below 550 nm. At 550 nm single exponential decay kinetics with a lifetime of 0.153 ns was observed. The fluorescence spectrum could be resolved into two components, the 2.86 ns component having a spectral maximum near 450 nm and the 0.144 ns component having a spectral maximum near 490 nm. The results are rationalized in terms of there being two different configurations of DAPI, one of which undergoes a rapid protonation of the indole ring by proton transfer from the 6‐amidinium group in the excited singlet state. The 0.144 ns component is assigned as the fluorescence from the excited state of the protonated indole ring. The results provide an explanation of the fluorescence enhancement in DAPI‐nucleic acid complexes.