UV light spectral response of photosynthetic photochemical efficiency in alpine mosses

Aims Bryophytes play an important role in primary production in harsh alpine environment. As other alpine plants, the alpine bryophytes are often exposed to stronger UV radiation than lowland plants. Plants growing under high UV radiation may differ from those from low UV regimes in their physiological response to UV radiation. We were to (i) test the hypothesis and to address whether and/or how alpine bryophytes differ in photosynthetic photochemical characteristics in response to UV light and (ii) understand the potential effects of UV radiation on photosynthetic photochemical process in alpine bryophytes.Methods We examined the maximum quantum efficiency of photosystem II (PSII) photochemistry (F v /F m) for two alpine bryophyte species, Distichium inclinatum and Encalypta alpine, from a Kobresia humilis meadow and a Kobresia tibetica wetland, respectively, in Haibei, Qinghai (37°29′N, 101°12′E, altitude 3?250 m), and for a lowland bryophyte, Polytrichum juniperinum, under different spectrum of UV light. Biological spectral weighting function (BSWF) was obtained to evaluate the effect of UV light on the physiological response in these species.Important findings1)?The maximum quantum efficiency of photosystem II photochemistry (F v /F m) declined linearly with the increase of radiation dose in wavelengths from 250 to 420 nm. The effect of UV radiation on F v /F m decreased with higher rate from 250 to 320 nm and from 400 to 420 nm than in UVA range. 2)?The three species from different ecosystems contrasting in altitudes showed similar pattern of UV effectiveness. In comparison with other species reported so far, the moss BSWF was among those with the most modest decrease trend with spectrum effect of UV light 50 times higher at 250 than at 420 nm. 3)?Under the scenario of 16% reduction of stratospheric ozone, the integrated effectiveness from 290 to 345 nm increased only 5%, suggesting that the photochemical activity of the bryophyte PSII is likely to insensitive to O 3 depletion.     

Characteristics of the tertiary structure of histone H1 from the calf thymus

By optical methods it has been previously shown that the globular "head" of histone H1 forms a hydrophobic cavity containing Tyr72. The latter is screened from the polar water surrounding and its intramolecular mobility is drastically hindered. As a consequence of the alteration in the micromilieu are a long wave shift (lambda max = 279,5 nm) and a more pronounced longwave absorption spectra, higher anisotropy (A = 0,11), augmented quantum yield of fluorescence (approximately 0,2) and a decrease of the Stern-Volmer constant for Hl at fluorescence quenching by acrylamide. It was found that changes in fluorescence intensity of histones are connected with alterations in the quantum yield of fluorescence at lambda exc = = 265 nm, but not at lambda exc = 280 nm. The changes in fluorescence intensity at light excitation 280 nm (F280) and 265 nm (F265) are in good accordance with shift delta E286 in differential absorption spectra. Introduction of parameter Cf = F280/F265 allows to study shifts of excitation spectra instead of shifts in absorption spectra of histones. This method has certain advantages, since it permits investigations with lower protein concentrations and in turbid solutions. The data obtained allow to draw out Tyr72 of histone Hl into a special class of fluorescent-tyrosyls, that differ in properties from those of other tryptophandevoided proteins: RNAse, insulin and core-histones H2A, H2B, H3 and H4.     

Role of Tyr348 in Tyr385 radical dynamics and cyclooxygenase inhibitor interactions in prostaglandin H synthase-2

Both prostaglandin H synthase (PGHS) isoforms utilize a radical at Tyr385 to abstract a hydrogen atom from arachidonic acid, initializing prostaglandin synthesis. A Tyr348-Tyr385 hydrogen bond appears to be conserved in both isoforms; this hydrogen bonding has the potential to modulate the positioning and reactivity of the Tyr385 side chain. The EPR signal from the Tyr385 radical undergoes a time-dependent transition from a wide doublet to a wide singlet species in both isoforms. In PGHS-2, this transition results from radical migration from Tyr385 to Tyr504. Localization of the radical to Tyr385 in the recombinant human PGHS-2 Y504F mutant was exploited in examining the effects of blocking Tyr385 hydrogen bonding by introduction of a further Y348F mutation. Cyclooxygenase and peroxidase activities were found to be maintained in the Y348F/Y504F mutant, but the Tyr385 radical was formed more slowly and had greater rotational freedom, as evidenced by observation of a transition from an initial wide doublet species to a narrow singlet species, a transition not seen in the parent Y504F mutant. The effect of disrupting Tyr385 hydrogen bonding on the cyclooxygenase active site structure was probed by examination of cyclooxygenase inhibitor kinetics. Aspirin treatment eliminated all oxygenase activity in the Y348F/Y504F double mutant, with no indication of the lipoxygenase activity observed in aspirin-treated wild-type PGHS-2. Introduction of the Y348F mutation also strengthened the time-dependent inhibitory action of nimesulide. These results suggest that removal of Tyr348-Tyr385 hydrogen bonding in PGHS-2 allows greater conformational flexibility in the cyclooxygenase active site, resulting in altered interactions with inhibitors and altered Tyr385 radical behavior.     

Spectral tuning and evolution of primate short-wavelength-sensitive visual pigments

The peak sensitivities (λ(max)) of the short-wavelength-sensitive-1 (SWS1) pigments in mammals range from the ultraviolet (UV) (360-400 nm) to the violet (400-450 nm) regions of the spectrum. In most cases, a UV or violet peak is determined by the residue present at site 86, with Phe conferring UV sensitivity (UVS) and either Ser, Tyr or Val causing a shift to violet wavelengths. In primates, however, the tuning mechanism of violet-sensitive (VS) pigments would appear to differ. In this study, we examine the tuning mechanisms of prosimian SWS1 pigments. One species, the aye-aye, possesses a pigment with Phe86 but in vitro spectral analysis reveals a VS rather than a UVS pigment. Other residues (Cys, Ser and Val) at site 86 in prosimians also gave VS pigments. Substitution at site 86 is not, therefore, the primary mechanism for the tuning of VS pigments in primates, and phylogenetic analysis indicates that substitutions at site 86 have occurred at least five times in primate evolution. The sole potential tuning site that is conserved in all primate VS pigments is Pro93, which when substituted by Thr (as found in mammalian UVS pigments) in the aye-aye pigment shifted the peak absorbance into the UV region with a λ(max) value at 371 nm. We, therefore, conclude that the tuning of VS pigments in primates depends on Pro93, not Tyr86 as in other mammals. However, it remains uncertain whether the initial event that gave rise to the VS pigment in the ancestral primate was achieved by a Thr93Pro or a Phe86Tyr substitution.     

The stacking tryptophan of galactose oxidase: a second-coordination sphere residue that has profound effects on tyrosyl radical behavior and enzyme catalysis

The function of the stacking tryptophan, W290, a second-coordination sphere residue in galactose oxidase, has been investigated via steady-state kinetics measurements, absorption, CD and EPR spectroscopy, and X-ray crystallography of the W290F, W290G, and W290H variants. Enzymatic turnover is significantly slower in the W290 variants. The Km for D-galactose for W290H is similar to that of the wild type, whereas the Km is greatly elevated in W290G and W290F, suggesting a role for W290 in substrate binding and/or positioning via the NH group of the indole ring. Hydrogen bonding between W290 and azide in the wild type-azide crystal structure are consistent with this function. W290 modulates the properties and reactivity of the redox-active tyrosine radical; the Y272 tyrosyl radicals in both the W290G and W290H variants have elevated redox potentials and are highly unstable compared to the radical in W290F, which has properties similar to those of the wild-type tyrosyl radical. W290 restricts the accessibility of the Y272 radical site to solvent. Crystal structures show that Y272 is significantly more solvent exposed in the W290G variant but that W290F limits solvent access comparable to the wild-type indole side chain. Spectroscopic studies indicate that the Cu(II) ground states in the semireduced W290 variants are very similar to that of the wild-type protein. In addition, the electronic structures of W290X-azide complexes are also closely similar to the wild-type electronic structure. Azide binding and azide-mediated proton uptake by Y495 are perturbed in the variants, indicating that tryptophan also modulates the function of the catalytic base (Y495) in the wild-type enzyme. Thus, W290 plays multiple critical roles in enzyme catalysis, affecting substrate binding, the tyrosyl radical redox potential and stability, and the axial tyrosine function.     

Rapid kinetics investigations of peracid oxidation of ferric cytochrome P450cam: nature and possible function of compound ES

Previously, we reported spectroscopic properties of cytochrome P450cam compound I, (ferryl iron plus a porphyrin π-cation radical (Fe^IV = O/Por⁺)), as well as compound ES (Fe^IV = O/Tyr) in reactions of substrate-free ferric enzyme with m-chloroperbenzoic acid [T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300-9]. Compound ES arises by intramolecular electron transfer from nearby tyrosines to the porphyrin π-cation radical of Compound I, and has been characterized by rapid-freeze-quench-Mössbauer/EPR spectroscopy; the tyrosyl radical was assigned to Tyr96 for wild type or to Tyr75 for the Tyr96Phe variant [V. Schünemann, F. Lendzian, C. Jung, J. Contzen, A.L. Barra, S.G. Sligar, A.X. Trautwein, J. Biol. Chem. 279 (2004) 10919–10930]. Here we report rapid-scanning stopped-flow studies of the reactions of peracids with substrate-free ferric Y75F, Y96F, and Y96F/Y75F P450cam variants, showing how these active site changes influence electron transfer from nearby tyrosines and affect formation of intermediates. Curiously, rates of generation of Compounds I and ES for both single mutants were not very different from wild type. Contrasting with the earlier EPR results, the Y96F/Y75F variant was also shown to form an ES-like species, but more slowly. When substrate is not present, or is improperly bound, compound I rapidly converts to compound ES, which can be reduced to form H₂O and ferric P450, thus avoiding the modification of nearby protein groups or release of reactive oxygen species.     

Interaction of resveratrol and genistein with nucleic acids

Resveratrol (RES) and genistein (GEN) are the dietary natural products known to possess chemopreventive property and also the ability to repair DNA damage induced by mutagens/carcinogens. It is believed that the therapeutic activity of these compounds could be primarily due to their interaction with nucleic acids but detailed reports are not available. We here explore the interaction of these drugs with nucleic acids considering DNA and RNA as a potential therapeutic target. The interaction of RES and GEN has been analysed in buffered solution with DNA [saline sodium citrate (SSC)] and RNA [tris ethylene diammine tetra acetic acid (TE)] using UV-absorption and Fourier transform infrared (FTIR) spectroscopy. The UV analysis revealed lesser binding affinity with nucleic acids at lower concentration of RES (P/D = 5.00 and 10.00), while at higher drug concentration (P/D = 0.75, 1.00 and 2.50) hyperchromic effect with shift in the lambda(max) is noted for DNA and RNA. A major RES-nucleic acids complexes was observed through base pairs and phosphate backbone groups with K = 35.782 M(-1) and K = 34.25 M(-1) for DNARES and RNA-RES complexes respectively. At various concentrations of GEN (P/D = 0.25, 0.50, 0.75, 1.00 and 2.50) hyperchromicity with shift in the lambda(max) from 260-->263 nm and 260--> 270 nm is observed for DNA-GEN and RNA-GEN complexes respectively. The binding constant (from UV analysis) for GEN-nucleic acids complexes could not be obtained due to GEN absorbance overlap with that of nucleic acids at 260 nm. Nevertheless a detailed analysis with regard to the interaction of these drugs (RES/GEN) with DNA and RNA could feasibly be understood by FTIR.     

Recruitment of a foreign quinone into the A1 site of photosystem I. Altered kinetics of electron transfer in phylloquinone biosynthetic pathway mutants studied by time-resolved optical, EPR, and electrometric techniques

Interruption of the menA or menB gene in Synechocystis sp. PCC 6803 results in the incorporation of a foreign quinone, termed Q, into the A(1) site of photosystem I with a number of experimental indicators identifying Q as plastoquinone-9. A global multiexponential analysis of time-resolved optical spectra in the blue region shows the following three kinetic components: 1) a 3-ms lifetime in the absence of methyl viologen that represents charge recombination between P700(+) and an FeS(-) cluster; 2) a 750-microseconds lifetime that represents electron donation from an FeS(-) cluster to methyl viologen; and 3) an approximately 15-microseconds lifetime that represents an electrochromic shift of a carotenoid pigment. Room temperature direct detection transient EPR studies of forward electron transfer show a spectrum of P700(+) Q(-) during the lifetime of the spin polarization and give no evidence of a significant population of P700(+) FeS(-) for t 相似文献   

An ultraviolet photoacoustic spectroscopy study of the interaction between Lys49-phospholipase A2 and amphiphilic molecules

We have used near ultraviolet photoacoustic spectroscopy (PAS) over the wavelength range 240-320 nm to investigate the complex formed between the homodimeric bothropstoxin-I, a lysine-49-phospholipase A2 from the venom of Bothrops jararacussu (BthTx-I), with the anionic amphiphile sodium dodecyl sulfate (SDS). At molar ratios>10, the complex developed a significant light scatter, accompanied by a decrease in the intrinsic tryptophan fluorescence intensity emission (ITFE) of the protein, and an increase in the near UV-PAS signal. Difference PAS spectroscopy at SDS/BthTx-I ratios<8 were limited to the region 280-290 nm, suggesting initial SDS binding to the tryptophan 77 located at the dimer interface. At SDS/BthTx-I ratios>10, the intensity between 260 and 320 nm increases demonstrating that the more widespread tyrosine and phenylalanine residues contribute to the SDS/BthTx-I interaction. PAS signal phase changes at wavelengths specific for each aromatic residue suggest that the Trp77 becomes more buried on SDS binding, and that protein structural changes and dehydration may alter the microenvironments of Tyr and Phe residues. These results demonstrate the potential of near UV-PAS for the investigation of membrane proteins/detergent complexes in which light scatter is significant.     

Spectroscopic investigations of intermediates in the reaction of cytochrome P450(BM3)-F87G with surrogate oxygen atom donors

Rapid mixing of substrate-free ferric cytochrome P450_BM3–F87G with m-chloroperoxybenzoic acid (mCPBA) resulted in the sequential formation of two high-valent intermediates. The first was spectrally similar to compound I species reported previously for P450_CAM and CYP 119 using mCPBA as an oxidant, and it featured a low intensity Soret absorption band characterized by shoulder at 370 nm. This is the first direct observation of a P450 compound I intermediate in a type II P450 enzyme. The second intermediate, which was much more stable at pH values below 7.0, was characterized by an intense Soret absorption peak at 406 nm, similar to that seen with P450_CAM [T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300–20309]. Double mixing experiments in which NADPH was added to the transient 406 nm-absorbing intermediate resulted in rapid regeneration of the resting ferric state, with the flavins of the flavoprotein domain in their reduced state. EPR results were consistent with this stable intermediate species being a cytochrome c peroxidase compound ES-like species containing a protein-based radical, likely localized on a nearby Trp or Tyr residue in the active site. Iodosobenzene, peracetic acid, and sodium m-periodate also generated the intermediate at 406 nm, but not the 370 nm intermediate, indicating a probable kinetic barrier to accumulating compound I in reactions with these oxidants. The P450 ES intermediate has not been previously reported using iodosobenzene or m-periodate as the oxygen donor.     

Synthesis and conformational studies of ribooligonucleotides which contain an alternating C-G sequence and show unusual circular dichroism spectra

The poly [r(C-G)] duplex shows an unusually large negative CD band in the long wavelength region. In order to elucidate this phenomenon, r(C-G-C-G) and r(C-G-C-G-C-G) were synthesized by a phosphotriester method and their properties were examined by UV, CD, 1H and 31P NMR spectroscopy. These ribooligomers form self-duplexes at low temperature, the CD spectra of which show negative bands at around 290 nm and positive bands at around 265 nm. The results of 1H nuclear Overhauser effect experiments, 1H chemical shift-temperature profiles of base protons, and the sharp singlet observed for all H1' protons are consistent with a normal A-RNA structure but not with a Z-DNA like structure. The CD-temperature profiles and 31P NMR spectra support this conclusion. These results indicate that RNA duplexes with an alternating C-G sequence can give an unusually large negative CD band in the long wavelength region despite their right-handed helical structure.     

Identification of Tyr(290(6.58)) of the human gonadotropin-releasing hormone (GnRH) receptor as a contact residue for both GnRH I and GnRH II: importance for high-affinity binding and receptor activation

Molecular modeling showed interactions of Tyr (290(6.58)) in transmembrane domain 6 of the GnRH receptor with Tyr (5) of GnRH I, and His (5) of GnRH II. The wild-type receptor exhibited high affinity for [Phe (5)]GnRH I and [Tyr (5)]GnRH II, but 127- and 177-fold decreased affinity for [Ala (5)]GnRH I and [Ala (5)]GnRH II, indicating that the aromatic ring in position 5 is crucial for receptor binding. The receptor mutation Y290F decreased affinity for GnRH I, [Phe (5)]GnRH I, GnRH II and [Tyr (5)]GnRH II, while Y290A and Y290L caused larger decreases, suggesting that both the para-OH and aromatic ring of Tyr (290(6.58)) are important for binding of ligands with aromatic residues in position 5. Mutating Tyr (290(6.58)) to Gln increased affinity for Tyr (5)-containing GnRH analogues 3-12-fold compared with the Y290A and Y290L mutants, suggesting a hydrogen-bond between Gln of the Y290Q mutant and Tyr (5) of GnRH analogues. All mutations had small effects on affinity of GnRH analogues that lack an aromatic residue in position 5. These results support direct interactions of the Tyr (290(6.58)) side chain with Tyr (5) of GnRH I and His (5) of GnRH II. Tyr (290(6.58)) mutations, except for Y290F, caused larger decreases in GnRH potency than affinity, indicating that an aromatic ring is important for the agonist-induced receptor conformational switch.     

Fluorescence and folding properties of Tyr mutant tryptophan synthase alpha-subunits from Escherichia coli

The fluorescence of tyrosine has been used to monitor a folding process of tryptophan synthase alpha-subunit from Escherichia coli, because this protein has 7 tyrosines, but not tryptophan. Here to assess the contribution of each Tyr to fluorescence properties of this protein during folding, mutant proteins in which Tyr was replaced with Phe were analyzed. The result shows that a change of Tyr fluorescence occurring during folding of this protein is contributed to approximately 40% each by Tyr(4) and Tyr(115), and to the remaining approximately 20% by Tyr(173) and Tyr(175). Y173F and Y175F mutant proteins showed an increase in their fluorescence intensity by approximately 40% and approximately 10%, respectively. These increases appear to be due to multiple effects of increased hydrophobicity, quenching effect of nearby residue Glu(49), and/or energy transfer between Tyrs. Two data for Y173F alpha-subunit of urea-induced unfolding equilibrium monitored by UV and fluorescence were different. This result, together with ANS binding and far UV CD, shows that folding intermediate(s) of Y173F alpha-subunit, contrary to that of wild-type, may contain self-inconsistent properties such as more buried hydrophobicity, highly quenched fluorescence, and different dependencies on urea of UV absorbance, suggesting an ensemble of heterogeneous structures.     

Purification and properties of a novel nucleotide-hydrolysing enzyme (5''-nucleotidase) from Boophilus microplus.

Conformational changes in the beta-subunit of the bovine brain Ca2+-binding protein S100b (S100-beta) accompanying Ca2+ binding were investigated by analysis of the spectroscopic properties of the single tyrosine residue (Tyr17 beta) and flow-dialysis binding experiments. S100-beta binds Ca2+ sequentially at two sites to change the conformation of the protein. The first Ca2+ ion binds to site II beta, a typical Ca2+-binding site in the C-terminal region, and it does not significantly perturb the proximal environment of Tyr17 beta. After the first site is occupied, another Ca2+ ion binds to the N-terminal Ca2+-binding site, I beta, and strengthens a hydrogen bond between Tyr17 beta and a neighbouring carboxylate acceptor group, which results in a large increase in the Tyr17 beta fluorescence spectrum half-width and a positive absorption and c.d. signal between 290 and 275 nm. Ca2+ binding to the S100b.Zn2+6 complex, studied by flow-dialysis and fluorescence measurements showed that, although Zn2+ ions increase the affinity of S100b protein for Ca2+, the Ca2+-binding sequence was not changed. Tb3+ (terbium ion) binding studies on the S100b.Zn2+6 complex proved that Tb3+ antagonizes only Ca2+ binding site II beta and confirmed the sequential occupation of Ca2+-binding sites on the S100b.Zn2+6 complex.     

Replacement of tyrosine residues by phenylalanine in cytochrome P450cam alters the formation of Cpd II-like species in reactions with artificial oxidants

Our previous rapid-scanning stopped-flow studies of the reaction of substrate-free cytochrome P450cam with peracids [Spolitak et al. (2005) J Biol Chem 280:20300-20309; (2006) J Inorg Biochem 100:2034-2044] spectrally characterized compound I [ferryl iron plus a porphyrin pi-cation radical (Fe(IV) = O/Por(+))], as well as Cpd ES (Fe(IV) = O/Tyr.). In the present studies, we report how the substitutions in Y75F, Y96F, and Y96F/Y75F P450cam variants permit the formation of a species we attribute to Cpd II (Fe(IV) = O) in reactions with peracids and cumene hydroperoxide. These variants produce changes in hydrogen bonding patterns and increased hydrophobicity that affect the ratio of heterolytic to homolytic pathways in reactions with cumene hydroperoxide, resulting in a shift of this ratio from 84/16 for WT to 72/28 for the Y96F/Y75F double mutant. Various ways of generating the Cpd II-like species were explored, and it was possible, especially with the more hydrophobic variants, to generate large fractions of the P450cam variants as Cpd II. The Cpd II-like species is ineffective at hydroxylating camphor, but can be readily reduced by ascorbate (as well as other peroxidase substrates) to ferric P450cam, which could then bind camphor to form the high-spin heme. The difference in the spectral properties of Cpd ES and Cpd II was rationalized as possibly being due to different states of protonation.     

Independent tyrosyl contributions to the CD of Ff gene 5 protein and the distinctive effects of Y41H and Y41F mutants on protein-protein cooperative interactions

The gene 5 protein (g5p) of the Ff virus contains five Tyr, individual mutants of which have now all been characterized by CD spectroscopy. The protein has a dominant tyrosyl 229-nm L(a) CD band that is shown to be approximately the sum of the five individual Tyr contributions. Tyr41 is particularly important in contributing to the high cooperativity with which the g5p binds to ssDNA, and Y41F and Y41H mutants are known to differ in dimer-dimer packing interactions in crystal structures. We compared the solution structures and binding properties of the Y41F and Y41H mutants using CD spectroscopy. Secondary structures of the mutants were similar by CD analyses and close to those derived from the crystal structures. However, there were significant differences in the binding properties of the two mutant proteins. The Y41H protein had an especially low binding affinity and perturbed the spectrum of poly[d(A)] in 2 mM Na(+) much less than did Y41F and the wild-type gene 5 proteins. Moreover, a change in the Tyr 229 nm band, assigned to the perturbation of Tyr34 at the dimer-dimer interface, was absent in titrations with the Y41H mutant under low salt conditions. In contrast, titrations with the Y41H mutant in 50 mM Na(+) exhibited typical CD changes of both the nucleic acid and the Tyr 229-nm band. Thus, protein-protein and g5p-ssDNA interactions appeared to be mutually influenced by ionic strength, indicative of correlated changes in the ssDNA binding and cooperativity loops of the protein or of indirect structural constraints.     

Spectroscopic and electrochemical studies on structural change of plastocyanin and its tyrosine 83 mutants induced by interaction with lysine peptides

Interactions of wild-type and Tyr83 mutant (Y83F, Y83S, Y83L, and Y83H) plastocyanins (PCs) with lysine peptides as models for the PC interacting site of cytochrome f have been studied by absorption, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopies and electrochemical measurements. The spectral and electrochemical properties of PCs corresponded well with each other; species having a longer wavelength maximum for the S(Cys) pi --> Cu 3d(x)()()2(-)(y)()()2 charge transfer (CT) band observed around 600 nm and a stronger intensity for the 460-nm absorption band exhibited stronger intensities for the positive Met --> Cu 3d(x)()()2(-)(y)()()2 and negative His pi(1) --> Cu 3d(x)()()2(-)(y)()()2 circular dichroism (CD) bands at about 420 and 470 nm, respectively, a lower average nu(Cu)(-)(S) frequency, a smaller |A( parallel)| EPR parameter, and a higher redox potential, properties all related to a weaker Cu-S(Cys) bond and a more tetrahedral planar geometry for the Cu site. Similarly, on oligolysine binding to wild-type and several Tyr83 mutant PCs, a longer absorption maximum for the 600-nm CT band, a stronger intensity for the 460-nm absorption band, stronger 420-nm positive and 470-nm negative CD bands, and a lower average nu(Cu)(-)(S) frequency were observed, suggesting that PC assumes a slight more tetrahedral geometry on binding of oligolysine. Since changes were observed for both wild-type and Tyr83 mutant PCs, the structural change due to binding of oligolysine to PC may not be transmitted through the path of Tyr83-Cys84-copper by a cation-pi interaction which is proposed for electron transfer.     

Dose response, wavelength dependence and rate of excision of ultraviolet radiation-induced pyrimidine dimers in mouse skin DNA

The yield of thymine-containing dimers produced in mouse skin DNA in vivo by 290 nm ultraviolet radiation was shown to increase with dose up to around 2000 J/m2 and subsequently at a much slower rate up to 8000 J/m2. The study of wavelength dependence of dimer formation in skin indicated that 290 nm was the most effective wavelength of those investigated, followed by 300, 280 and 260 nm, with 310 nm being by far the least effective. A reduction in the number of dimers present in skin DNA was shown to occur by 24 h post-irradiation in a dose-dependent manner. A significant percentage of the dimers was, however, found to persist in the skin until at least 72 h post-irradiation.     

Diffuse reflectance spectroscopy of fibrous proteins

UV-visible diffuse reflectance (DR) spectra of the fibrous proteins wool and feather keratin, silk fibroin and bovine skin collagen are presented. Natural wool contains much higher levels of visible chromophores across the whole visible range (700-400?nm) than the other proteins and only those above 450?nm are effectively removed by bleaching. Both oxidative and reductive bleaching are inefficient for removing yellow chromophores (450-400?nm absorbers) from wool. The DR spectra of the four UV-absorbing amino acids tryptophan, tyrosine, cystine and phenylalanine were recorded as finely ground powders. In contrast to their UV-visible spectra in aqueous solution where tryptophan and tyrosine are the major UV absorbing species, surprisingly the disulphide chromophore of solid cystine has the strongest UV absorbance measured using the DR remission function F(R)(∞). The DR spectra of unpigmented feather and wool keratin appear to be dominated by cystine absorption near 290?nm, whereas silk fibroin appears similar to tyrosine. Because cystine has a flat reflectance spectrum in the visible region from 700 to 400?nm and the powder therefore appears white, cystine absorption does not contribute to the cream colour of wool despite the high concentration of cystine residues near the cuticle surface. The disulphide absorption of solid L: -cystine in the DR spectrum at 290?nm is significantly red shifted by ~40?nm relative to its wavelength in solution, whereas homocystine and lipoic acid showed smaller red shifts of 20?nm. The large red shift observed for cystine and the large difference in intensity of absorption in its UV-visible and DR spectra may be due to differences in the dihedral angle between the crystalline solid and the solvated molecules in solution.     

Intrinsic fluorescence of B and Z forms of poly d(G-m5C).poly d(G-m5C), a synthetic double-stranded DNA: spectra and lifetimes by the maximum entropy method.

A study has been made of the fluorescence of poly d(G-m5C).poly d(G-m5C), a synthetic double-stranded DNA, in buffered neutral aqueous solution at room temperature, excited by synchrotron radiation at 280 nm and 250 nm and by a frequency-doubled pulse dye laser at 290 nm. Exciting at 280 nm, the B form shows a uni-modal UV spectrum with lambdaf(max) approximately 340 nm. The Z form has in addition a visible emission lambdaf(max) at 450 nm. The spectral positions remain unchanged on exciting at 250 nm but the relative intensities change considerably. Decay profiles have been obtained at 360 nm and 450 nm for both the B and Z forms and have been analyzed by fitting to a pseudo-continuous distribution of 100 (and occasionally 200) exponentials, ranging from 10 ps to 20 ns, by optimizing the 'entropy' of the signal (the method of maximum entropy). We find the mean lifetimes for both wavelengths of emission and for both structural forms fall into three well-separated regions in the ranges indicated tau1 approximately 0.04-0.21 ns, tau2 approximately 0.9-1.26 ns, and tau3 approximately 5.1-6.5 ns. The UV emission, from its spectral position and half-width, correlates with monomeric emission from m5C (and from C for poly d(G-C)). However the lifetime tau1 is approximately 2 orders of magnitude longer than the monomers and points to an involvement of protonated guanosine (GH+, tauf approximately 200 ps) in the overall absorption/emission sequence. In the UV the tau3 emission is predominant, with fractional time-integrated emission approximately 86% for B DNA and approximately 64% for Z. We suggest it results from exciton (stacked) absorption followed by dissociative emission. For Z DNA the visible (450 nm) emission is dominated by a tau3 species (approximately 91%) with a lifetime of 6.5 ns and we suggest it represents a hetero-excimer emission consequent upon absorption by the strongly overlapped base-stacking, which differs from that in B DNA. The weak emission corresponding to tau2 is made more apparent by scanned gated detection of the emission from laser excitation (290 nm) of single-crystal d(m5C-G)3. A central role is attributed to the tight stacking of the bases in the Z form which correlates with enhanced hypochromism at 250 nm vs. 280 nm and with the reversal of the fluorescence intensity ratios UV-visible between these wavelengths.