Pulsefluorimetry of tyrosyl peptides

The fluorescence quantum yield and the fluorescence decay of aqueous solutions of derivatives containina a single tyrosine residue have been measured at different pH. In these derivatives tyrosine was substituted on its amino end (series I) or/and, on its carboxyl end (series II), by acyl, amino or amino acyl groups. The fluorescence decays of series I derivatives are monoexponential regardless to the ionization state of their amino group. Upon deprotonation of the α-amino group, the quantum yields and the lifetimes increase in the case of dipeptides, and slightly decrease, for the tripeptides. The quantum yield and the lifetime increase with the side chain length of the aliphatic residue adjacent to the tyrosine residue, (the fluorescence of Val Tyr anion being identical to that of free Tyrosine). Quite different is the behavior of series II derivatives: their decays at pH 5.5 must be described by two exponential terms, one of them decaying with a short time constant (about 0.5 ns) and little side chain effect is observed. The fluorescence intensity increases upon deprolonalion of the α-amino proup (though to a lesser extent than for series I derivatives); a nearly monoexponential decay is observed at basic pH for dipeptides. but not for tyrosine amide, amide or dipeptides, or tripeptides. The following interpretation of our results is proposed: fluorescence quenching occurs in molecular conformations in which a peptide carbonyl can come in contact with the phenolic chromophore. This condition depends mainly on the value of the angle x₁ which determines the conformation of the tyrosyl residue around its C_α-C_β bond. It appears that the rotamer in which quenching occurs are not the same for series I and series II derivatives, which can explain the different behavior of these two kinds of compounds. The interpretation of the fluorescence properties is developed taking into account on one side the relative population of the rotamers in the ground state, which is given by studies of crystals and of solutions, and on the other side the possibility of an exchange between these rotamers during the excited state time. In this scheme the protonated α-amino groups would act to reinforce the quenching efficiency of the carbonyl. At last it is found that the radiative lifetime of the phenolic chromophore is the same for all the compounds studies.     

Influence of alkyl group on amide nitrogen atom on fluorescence quenching of tyrosine amide and N-acetyltyrosine amide

The steady-state and time-resolved fluorescence spectroscopy was applied to determine the influence of an alkyl substituent(s) (methyl or ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, or t-butyl) on amide nitrogen atom on photophysical properties of tyrosine and N-acetyltyrosine amides in water. Generally, the amide group strongly quenches the fluorescence of tyrosine, however, the size and number of substituents on amide nitrogen atom modify the quenching process only in small degree. The fluorescence intensity decays of all amides studied are bi-exponential. The contribution of both components (alphai) to the fluorescence decay undergoes irregular change. An introduction of alkyl substituent on amide nitrogen atom causes an increase of the fluorescence lifetime of tyrosine derivative compared to the unsubstituted amide for both N-acetyltyrosine and tyrosine with the protonated amino group. Calculated, basing on the fluorescence quantum yield (QY) and average lifetime, the radiative rate constants (kf) are similar, which indicates that the substituent(s) does not have substantial influence on radiative process of the deactivation of the excited state of the phenol chromophore for all compounds studied regardless the amino group status as well as the number and type of substituent (linear or branched). The comparison of the ground-state rotamer populations of tyrosine amides and N-acetyltyrosine amides with different alkyl substituent on amide nitrogen atom obtained from 1H NMR with the value of pre-exponential factors indicates that not the rotamer populations, but specific hydration of a whole molecule of the amino acid including chromophore and amino acid moiety, seems to be the main reason of the heterogenous fluorescence intensity decay of tyrosine derivatives.     

Effect of caffeine on G2 repair and its reversion by adenosine

Chromosome damage induced in root meristems of Allium cepa L. by an 18-h treatment with 5-aminouracil (AU) was enhanced by 2-h pulses with 5 mM caffeine, the most effective pulse being given from the 8th to the 10th h after AU. Chromosome damage was detected by the percentage of anaphases or telophases showing chromosomal aberrations. This caffeine effect was partially reversed by adenosine. We suggest that this caffeine anti-repair effect could be due to a competition between methylxanthine and an adenine-nucleotide derivative (ATP?) required for some step(s) of G₂-prophase repair.     

Effect of caffeine and adenosine on G2 repair: mitotic delay and chromosome damage

Proliferating plant cells treated during the late S period with 5-aminouracil (AU), give the typical response that DNA-damaging agents induce, characterized by: an important mitotic delay, and a potentiation of the chromosome damage by caffeine post-treatment. The study of labelled prophases, after a tritiated thymidine pulse, allowed evaluation of the mitotic delay induced by AU as well as its reversion by caffeine, while chromosome damage was estimated by the percentage of anaphases and telophases showing chromosomal aberrations. Post-treatment with adenosine alone has shown no effect on mitotic delay or chromosomal damage. However, when cells after AU were incubated in caffeine plus adenosine, the chromosome damage potentiation was abolished without affecting the caffeine action on mitotic delay. As a consequence, we postulate that caffeine could have two effects on G2 cells with damaged DNA: the first, to cancel their mitotic delay and the second to inhibit some DNA-repair pathway(s). Only this last effect could be reversed by adenosine.     

Autoradiographic study of the effect of 5-aminouracil on the S phase and mitosis of barley root meristems

The effect of 5-aminouracil on the S phase and mitosis in root meristems of barley embryos cultivated in the liquid nutrient solution was followed. Embryos were cultivated in different concentrations of 5-aminouracil (200 ppm, 400 ppm and 750 ppm) for 48 h. The drug postponed the onset of mitosis. In the lowest concentration used, synchronization was observed even in the presence of 5-aminouracil. In higher concentrations, mitosis was suppressed irregularly with increasing concentration. 5-aminouracil slowed down the rate of DNA synthesis during S phase and prolonged the S phase, as measured by the utilization of [³H] thymidine. The drug does not influence considerably the entry of cells into the S phase. The transition from G₂ to mitosis is delayed in the presence of 5-aminouracil, especially in higher concentrations. After prolonged treatment with 5-aminouracil, all the effects of the drug on the mitotic cycle decrease continuously.     

A study of H-bonding of 3- and 5-substituted 6-aminouracils in duplex and triplex structures

All possible dimers of the title modified bases with native nucleobases [10 dimers from 3-methylated 6-aminouracils (3sau) and 20 from 5-methyled 6-aminouracils (5sau), respectively have been calculated by ab initio method (Hartree-Fock method, 3 21G basis set). We have found two potential duplexes of 5sau and three possible duplexes of 3sau. Altogether seven dimers containing one or two bifurcating H-bonds have been found. Later on, five triplexes from ten possible calculated dimers have been found. In two of them the amino group of 6-aminouracil moiety takes part in H-bonding and there are H-bonds, too, between the first and third base of the triplexes causing an extra stabilization.     

Conformational dynamics of unfolded peptides as a function of chain length: a frequency domain fluorescence approach

The fluorescence emission decays of single-tryptophan-containing peptides of different chain lengths in their unfolded state were investigated in the frequency domain. The data were analyzed using different functions, i.e., exponential fit and probability-density functions of different shape. We found that unimodal Lorentzian distributions best describe the fluorescence decays. This finding agrees with the point of view, now broadly accepted, that rapid motions exist in polypeptides. As a consequence of this flexibility, a large variety of conformations, with an unequal perturbation of tryptophan in its excited state, is generated. The lifetime distribution center was independent of the length of the polypeptide chain but strongly related to the nature of the amino acid residues located in the proximity of the tryptophan in the primary structure. The full width at half maximum, W, of the lifetime distribution was found to be related to the length of unfolded polypeptide by the empirical logarithmic relationship W = 0.83 log n, where n indicates the number of residues. For short peptides, a single lifetime or a narrow range of lifetimes is observed because of the fast relaxation of the tryptophanyl environment. On peptide lengthening, the spectrum of conformations, which the peptide can assume, increases; this causes a complex fluorescence decay represented by a lifetime distribution. For long polypeptide chains, the motions of the regions far from tryptophan do not significantly perturb the chromophore environment.     

Induction of premature mitosis in S-blocked onion cells

Onion root-tip cells were blocked at S-phase by treating them with 5-aminouracil (5AU). These cells were then further treated with caffeine/2-aminopurine (Caf/2AP) or a combination of both in the presence of 5AU. These tyrosine kinase inhibitors were able to induce premature mitosis in the S-blocked cells as evident from the breaks and gaps in the metaphase chromosomes and the presence of laggards and fragments in anaphase. Immunofluorescence showed normal spindle formation in these cells. Immunoblotting of cyclin B revealed that the level of cyclin B was slightly higher in the recovered and treated samples than the S-blocked one. The level of p(34)was found to be almost equal in all three samples as expected. We failed to observe any significant difference in the level of p(34)containing phosphorylated tyrosine. Such premature induction of mitosis by the purine derivatives has also been reported in BHK cells. However, those cells failed to progress through mitosis. A comparative analysis indicates that the plant cells and the animal cells, perhaps, follow identical pathway for the initiation of mitosis. The possible causes for differential behaviour in mitotic progression in these cells have been discussed.     

Spectroscopic Studies of the Supramolecular Interactions Between Uracil and 5-Hydroxy-6-Methyluracil with Bovine Serum Albumin and its Bilirubin Complex

Using fluorescence and absorption spectroscopy the interaction of bovine serum albumin and its bilirubin complex with uracil and 5-hydroxy-6-methyluracil in phosphate buffer at pH 7.4 was investigated. The parameters of forming intermolecular complexes (binding constants, quenching rate constants, the radius of the quenching sphere and etc.) were determined. The interaction between serum albumin and uracils is carried out by the static quenching of protein fluorescence and has predominantly hydrophobic character. Using synchronous fluorescence spectroscopy the influence of uracil and 5-hydroxy-6-methyluracil on the conformational changes of the protein molecule was studied. Uracils effectively binds to bilirubin-albumin complex compared to free protein, which is caused by the interaction with tetrapyrrolic pigment in macromolecular complex. Molecular docking calculations also being presented.     

Resolution of the lifetimes and correlation times of the intrinsic tryptophan fluorescence of human hemoglobin solutions using 2 GHz frequency-domain fluorometry

We used 2 GHz harmonic content frequency-domain fluorescence to measure the intensity and the anisotropy decays from the intrinsic tryptophan fluorescence from human hemoglobin (Hb). The tryptophan intensity decays are dominated by a short-lived component which accounts for 35-60% of the total steady state intensity. The decay time of this short component varies from 9 to 27 ps and this component is sensitive to the ligation state of Hb. Our error analyses indicate the uncertainty is about +/- 3 ps. The intensity decays also show two longer lived components near 0.7 and 8 ns, which are probably due either to impurities or to Hb molecules in conformations which do not permit energy transfer. The anisotropy decays indicate the tryptophan residues in Hb are highly mobile, with apparent correlation times near 55 ps.     

Synthesis and spectroscopic studies on the new Schiff base derived from the 1:2 condensation of 2,6-diformyl-4-methylphenol with 5-aminouracil (BDF5AU) and its transition metal complexes. Influence on biologically active peptides-regulating aminopeptidases

The synthesis, spectroscopic (IR, 1H and 13C NMR, UV-Vis-NIR, EPR), magnetic measurements and biological studies of a number of complexes of Co(II), Ni(II), Cu(II), Zn(II), Cd(II), Au(III) and Hg(II) of the Schiff base derived from the 1:2 condensation of 2,6-diformyl-4-methylphenol and 5-aminouracil, ((5-[[(3-[[(2,4-dioxopyrimidin-5(1H,3H)-yl)imino]methyl]-2-hydroxy-5-methylphenyl)methylene]amino]pyrimidine-2,4(1H,3H)-dione, hereafter denoted as BDF5AU) are reported. In all cases, the complexes appear to be monomeric. The deprotonated ligand in the phenolic oxygen atom shows a tridentate coordination mode through the two azomethine nitrogen atoms and the phenolic oxygen atom. The coordination of the neutral ligand takes place through the phenolic oxygen atom and one azomethine nitrogen atom and the carbonylic oxygen atom in fourth position of one uracil ring. The biological properties of some perchlorate complexes on the activity of some neutral, acid, basic and omega aminopeptidases (AP) are assayed, demonstrating a general inhibitory effect. Neutral and basic AP are mainly inhibited by Cu(II), Ni(II) and Cd(II) complexes, although tyrosyl-AP is activated by Zn(II) complex. Glutamyl-AP but not aspartyl-AP is inhibited by all the complexes assayed excepting Zn(II) complex. Finally, omega AP is inhibited by Ni(II) and Cd(II) complexes.     

Kinetics of tryptophan fluorescence enhancement in myofibrils during ATP hydrolysis

The mechanism of ATP hydrolysis in myofibrils can be studied by following the time course of tryptophan fluorescence. Stoichiometric quantities of ATP produce an enhancement of the tryptophan fluorescence in stirred suspensions of rabbit psoas myofibrils at pCa greater than 7. Approximately 1 mol of ATP/myosin head is required to obtain the maximum fluorescence enhancement of 4-6%. Upon the addition of quantities of ATP greater than 1 mol/mol of myosin head, the fluorescence rapidly increases to a steady state, which lasts for a period that is proportional to the amount of ATP added. The fluorescence then decays to the initial level with a half-time of approximately 40 s at 20 degrees C. Hydrolysis of [gamma-32P]ATP at pCa greater than 7 in myofibrils has an initial burst of approximately 0.7 mol/mol of myosin head that is followed by a constant rate of hydrolysis. The duration of the steady state hydrolysis is identical to the duration of the enhancement of tryptophan fluorescence. A lower limit of 5 X 10(5) M-1 S-1 was obtained for the second order rate constant of the fluorescence enhancement by ATP. At pCa of 4, the duration of the fluorescence enhancement is one-tenth to one-twentieth as long as at pCa greater than 7; this is consistent with the increased steady state rate of ATP hydrolysis at higher calcium concentrations. The time course of the fluorescence enhancement observed in myofibrils during ATP hydrolysis is qualitatively and quantitatively similar to that observed with actomyosin-S1 in solution. These results suggest that the kinetic mechanism of ATP hydrolysis that has been well established by studies of actomyosin-S1 in solution also occurs in myofibrils.     

Conformation heterogeneity in proteins as an origin of heterogeneous fluorescence decays, illustrated by native and denatured ribonuclease T1

We examined the frequency-domain intensity decays of the intrinsic tryptophan fluorescence (Trp-59) from ribonuclease T1 (EC 3.1.27.3) (RNAase T1). At pH 5.5 in the native state (below 30 degrees C), the intensity decay of the single tryptophan residue is a single-exponential process. Conditions which result in protein unfolding were found to induce more complex intensity decays. At temperatures above 40 degrees C, or in the presence of guanidine hydrochloride, the intensity decays became obviously double exponential. In general, the main effect of temperature or guanidine was to induce a second subnanosecond component in the intensity decay. The increased complexity of the decays could not be explained by a unimodal distribution of decay times. These results indicate that conformational dispersion of protein structure can be one origin of the multi-exponential decays which are generally observed for protein fluorescence.     

Studies of the fluorescence from tryptophan in melittin

The fluorescence lifetime and rotational correlation time of the tryptophan residue in melittin, as both a monomer and tetramer, have been measured between pH 6 and 11. The fluorescence decays are non-exponential and give lifetimes of 0.7±0.1 ns and 3.1±0.1 ns. This emission is consistent with a model in which the tryptophan residue is in slightly different environments in the protein. In a dilute solution of monomer the mean fluorescence lifetime is 2.3±0.1 ns, below pH 10, but falls to 1.7 ns at higher pH. In contrast, the melittin tetramer has a mean fluorescence lifetime of only 2.2 ns at pH 6, which falls to 1.9 ns by pH 8, and falls again above pH 10 to the same value as in monomeric melittin. The behaviour between pH 6 and 8 is explained as the quenching of the Trp residue by lysine groups, which are near to the Trp in the tetramer but in the monomer, are too distant to quench. Fluorescence anisotropy decays show that the Trp residue has considerable freedom of motion and the range of wobbling motion is 35±10° in the tetramer     

Synthesis of substituted 5-fluoro-5,6-dihydropyrimidines.

The reaction of 5-substituted uracils with fluorine in acetic acid and other solvents and the following treatment with different alcohols yielded the corresponding 5-fluoro-5,6-substituted-5,6-dihydropyrimidines. Thymine gave 5-fluoro-5-methyl-6-alkoxy-5,6-dihydropyrimidines. 5-Halogeno uracils and 5-nitrol uracil were converted into 5-fluoro-5-halogeno-6-hydroxy-5,6-dihydropyramidines and the 5-nitroanalogue, respectively. The structures of the compounds were confirmed by mass spectrometry.     

Intramolecular and intermolecular hydrogen-bonding effects on photophysical properties of 2'-aminoacetophenone and its derivatives in solution.

Effects of intra- and intermolecular hydrogen-bonds on the photophysical properties of 2'-aminoacetophenone derivatives (X-C6H4-COCH3) having a substituted amino group (X) with different hydrogen-bonding ability to the carbonyl oxygen (X: NH2(AAP), NHCH3(MAAP), N(CH3)2(DMAAP), NHCOCH3(AAAP), NHCOCF3(TFAAP)) are investigated by means of steady-state and time-resolved fluorescence spectroscopy and time-resolved thermal lensing. Based on the photophysical parameters obtained in aprotic solvents with different polarity and protic solvents with different hydrogen-bonding ability, the characteristic photophysical behavior of the 2'-aminoacetophenone derivatives is discussed in terms of hydrogen-bonding and n,pi*-pi,pi* vibronic coupling. The dominant deactivation process of AAP and MAAP in nonpolar aprotic solvents is the extremely fast internal conversion (k(ic)= 1.0 x 10(11) s(-1) for AAP and 3.9 x 10(10) s(-1) for MAAP in n-hexane). The internal conversion rates of both compounds decrease markedly with increasing solvent polarity, suggesting that vibronic interactions between close-lying S1(pi,pi*) and S2(n,pi*) states lead to the large increase in the non-radiative decay rate of the lowest excited singlet state. It is also suggested that for MAAP, which has a stronger hydrogen-bond as compared to AAP, an intramolecular hydrogen-bonding induced deactivation is involved in the dissipation of the S1 state. For DMAAP, which cannot possess an intramolecular hydrogen-bond, the primary relaxation mechanism of the S1 state in nonpolar aprotic solvents is the intersystem crossing to the triplet state, whereas in protic solvents very efficient internal conversion due to intermolecular hydrogen-bonding is induced. In contrast, the fluorescence spectra of AAAP and TFAAP, which have an amino group with a much stronger hydrogen-bonding ability, give strongly Stokes-shifted fluorescence, indicating that these compounds undergo excited-state intramolecular proton transfer reaction upon electronic excitation.     

THE MECHANISM OF 5-AMINOURACIL-INDUCED SYNCHRONY OF CELL DIVISION IN VI CIA FABA ROOT MERISTEMS

Cessation of mitosis was brought about in Vicia faba roots incubated for 24 hours in the thymine analogue, 5-aminouracil. Recovery of mitotic activity began 8 hours after removal from 5-aminouracil and reached a peak at 15 hours. If colchicine was added 4 hours before the peak of mitoses, up to 80 per cent of all cells accumulated in mitotic division stages. By use of single and double labeling techniques, it was shown that synchrony of cell divisions resulted from depression in the rate of DNA synthesis by 5-aminouracil, which brought about an accumulation of cells in the S phase of the cell cycle. Treatment with 5-aminouracil may have also caused a delay in the rate of exit of cells from the G₂ period. It appeared to have no effect on the duration of the G₁ period. When roots were removed from 5-aminouracil, DNA synthesis resumed in all cells in the S phase. Although thymidine antagonized the effects of 5-aminouracil, an exogenous supply of it was not necessary for the resumption of DNA synthesis, as shown by incorporation studies with tritiated deoxycytidine.     

Some cytological aspects of antagonism in synthesis of nucleic acid

Summary The effects of solutions of benzimidazole and 5-aminouracil onAllium cepa root meristems immersed in them have been studied cytologically. Benzimidazole and 5-aminouracil, analogues of purines and pyrimidines respectively, reduce the number of nuclear divisions being initiated. A marked decline in frequency of cells in mitosis occurs at the beginning of treatment with benzimidazole (300 p.p.m.) but not until 6 or more hours later with 5-aminouracil (125 p.p.m.).After treatment with 5-aminouracil, small interstitial portions of chromosomes are Feulgen-negative and become extended during anaphase into thin strands. The distal portion of these chromosomes may lag during anaphase and contribute to malformations of the surface of the sister nuclei and to micronuclei-like bodies lying along their adjacent surfaces. However, stickiness resulting in chromosome bridges was not observed. After treatments with benzimidazole, chromosomes are more contracted at metaphase and anaphase than in the control. At the concentrations used, neither component was observed to affect the spindle mechanism of the divisions in progress at the beginning of treatment nor of the divisions which escape inhibition.Reversal of these effects by the introduction of metabolites which these analogues resemble structurally, was investigated. Adenine sulphate (800 p.p.m.) acts antagonistically to benzimidazole (300 p.p.m.) by delaying the marked decline in frequency of mitoses by more than 6 hrs. By 12 hrs. the frequency of cells in mitoses was very low; thus, reversal is not permanent. Half the inhibition of growth caused by 5-aminouracil (50 p.p.m.) is reversed by thymine (50 p.p.m.) for the 24 hrs. of the test period. When 5 p.p.m. folic acid was added to this mixture, root growth exceeded that of the controls. Feulgen-negative gaps, however, are still present in chromosomes at anaphase.Estimations of Feulgen content of nuclei to determine the effect of these treatments on DNA synthesis were made by means of a microphotometer. In control roots there are two classes of nuclei at interphase according to the amount of DNA per nucleus. The amount doubles during interphase before the next division of the nuclei in the root meristem. In roots treated with 5-aminouracil this doubling is inhibited, so that interphase nuclei with the lower amount of DNA accumulate. In roots treated with benzimidazole the relative frequencies of the two classes of nuclei is unaltered; however, mitotic activity is inhibited. This compound apparently affects not only processes concerned with doubling the amount of DNA per nucleus but also processes necessary to the division itself. There was, however, no unquestionable evidence of a nucleus being reconstituted after it had started a division.Contribution from the Program in Cytology, Department of Botany University of Wisconsin, Madison, supported in part by grants to Dr.C. Leonard Huskins from the American Cancer Society, Rockefeller Foundation, and Research Committee of the Graduate School with funds supplied by the Wisconsin Alumni Research Foundation.     

Conformational effects on tryptophan fluorescence in cyclic hexapeptides

The peptide bond quenches tryptophan fluorescence by excited-state electron transfer, which probably accounts for most of the variation in fluorescence intensity of peptides and proteins. A series of seven peptides was designed with a single tryptophan, identical amino acid composition, and peptide bond as the only known quenching group. The solution structure and side-chain chi(1) rotamer populations of the peptides were determined by one-dimensional and two-dimensional (1)H-NMR. All peptides have a single backbone conformation. The -, psi-angles and chi(1) rotamer populations of tryptophan vary with position in the sequence. The peptides have fluorescence emission maxima of 350-355 nm, quantum yields of 0.04-0.24, and triple exponential fluorescence decays with lifetimes of 4.4-6.6, 1.4-3.2, and 0.2-1.0 ns at 5 degrees C. Lifetimes were correlated with ground-state conformers in six peptides by assigning the major lifetime component to the major NMR-determined chi(1) rotamer. In five peptides the chi(1) = -60 degrees rotamer of tryptophan has lifetimes of 2.7-5.5 ns, depending on local backbone conformation. In one peptide the chi(1) = 180 degrees rotamer has a 0.5-ns lifetime. This series of small peptides vividly demonstrates the dominant role of peptide bond quenching in tryptophan fluorescence.