Photo control of enzyme activity of alpha-amylase.

Photo control of enzyme activity was performed by attaching a photochromic spiropyran compound to α-amylase. Modified α-amylase exhibited reverse photochromism in water: a colored form in the dark and a colorless form under light irradiation, which indicated that bound spiropyran possessed a hydrophilic structure (an open-ring form) in the dark and a hydrophobic structure (a closed-ring form) under light irradiation. The activity of modified α-amylase under light irradiation was extremely retarded as compared with that determined in the dark. The photo-induced change of the activity reversibly occurred in accordance with the photochromism of bound spiropyran. The mechanism of the photo control is discussed.     

Fluorescent dye monitoring of mitochondrial changes associated with malignant cell transformation

The fluorescent dyes, rhodamine 6G and 123, which specifically stain mitochondria, were used to examine changes in mitochondria that follow malignant transformation. The spatial distribution and shapes of mitochondria differ in untransformed and malignant-transformed cells. In untransformed C3H/10T1/2 clone 8 cells, the mitochondria were distributed radially around the nucleus, and each had a fibrous shape. In chemically transformed MCA clone 16 cells, the mitochondria were distributed randomly in the cytoplasm, and each was shaped like a short rod. Another important mitochondrial change after malignant transformation was the change in the time course of fluorescence emission from the rhodamine present in the mitochondria. A slow increase in fluorescence, which was instantaneous at the time of excitation irradiation occurred in untransformed but not in transformed cells. This slow fluorescence emission, peculiar to untransformed cells was affected by proton ionophore but not by calcium ionophore treatment. The difference in the time courses of fluorescence emission for untransformed and transformed cells may reflect differences in the quenching of the dye fluorescence. The data reported provide evidence that mitochondria are affected by malignant cell transformation.     

A highly selective fluorescent chemosensor for Cu2+: synthesis and properties of a rhodamine B‐containing diarylethene

A diarylethene bearing a triazole‐linked rhodamine B unit was synthesized. Its fluorescent emission was significantly enhanced in the presence of protons or Cu²⁺ due to transformation from the pirocyclic form to open‐ring form. The fluorescence was quenched sequentially upon irradiation with 297 nm light based on the intramolecular fluorescence resonance energy transfer mechanism. In an acetonitrile: water binary solvent (1: 1 v /v), the compound showed significant fluorescent enhancement for Cu²⁺ compared with a wide range of tested metal ions with a fast response and a limit of detection of 2.86 × 10^?8 mol L^?1. Using Cu²⁺ and UV light as the chemical inputs, and fluorescence intensity at 597 nm as the output, a logic gate was developed at the molecular level. Moreover, the compound can be used with a high accuracy to detect Cu²⁺ in a natural water sample.     

Design and synthesis of a novel fluorescence probe for Zn based on the spirolactam ring-opening process of rhodamine derivatives

The spirolactam ring-opening process of rhodamine derivative is one of the most useful mechanisms for controlling fluorescence properties. However, the open/closed equilibrium reaction of rhodamine spirolactam has not been well characterized. Therefore, we examined the relationship between the spirolactam ring-opening process of rhodamine derivatives and the structure of the xanthene moiety. Based on the results of this investigation, we selected a candidate xanthene moiety for a Zn²⁺ sensor, and successfully developed a new fluorescence probe for Zn²⁺.     

Role of the retinal hydrogen bond network in rhodopsin Schiff base stability and hydrolysis

Little is known about the molecular mechanism of Schiff base hydrolysis in rhodopsin. We report here our investigation into this process focusing on the role of amino acids involved in a hydrogen bond network around the retinal Schiff base. We find conservative mutations in this network (T94I, E113Q, S186A, E181Q, Y192F, and Y268F) increase the activation energy (E(a)) and abolish the concave Arrhenius plot normally seen for Schiff base hydrolysis in dark state rhodopsin. Interestingly, two mutants (T94I and E113Q) show dramatically faster rates of Schiff base hydrolysis in dark state rhodopsin, yet slower hydrolysis rates in the active MII form. We find deuterium affects the hydrolysis process in wild-type rhodopsin, exhibiting a specific isotope effect of approximately 2.5, and proton inventory studies indicate that multiple proton transfer events occur during the process of Schiff base hydrolysis for both dark state and MII forms. Taken together, our study demonstrates the importance of the retinal hydrogen bond network both in maintaining Schiff base integrity in dark state rhodopsin, as well as in catalyzing the hydrolysis and release of retinal from the MII form. Finally, we note that the dramatic alteration of Schiff base stability caused by mutation T94I may play a causative role in congenital night blindness as has been suggested by the Oprian and Garriga laboratories.     

The position of QB in the photosynthetic reaction center depends on pH: a theoretical analysis of the proton uptake upon QB reduction

Electrostatics-based calculations have been performed to examine the proton uptake upon reduction of the terminal electron acceptor Q(B) in the photosynthetic reaction center of Rhodobacter sphaeroides as a function of pH and the associated conformational equilibrium. Two crystal structures of the reaction center were considered: one structure was determined in the dark and the other under illumination. In the two structures, the Q(B) was found in two different positions, proximal or distal to the nonheme iron. Because Q(B) was found mainly in the distal position in the dark and only in the proximal position under illumination, the two positions have been attributed mostly to the oxidized and the reduced forms of Q(B), respectively. We calculated the proton uptake upon Q(B) reduction by four different models. In the first model, Q(B) is allowed to equilibrate between the two positions with either oxidation state. This equilibrium was allowed to vary with pH. In the other three models the distribution of Q(B) between the proximal position and the distal position was pH-independent, with Q(B) occupying only the distal position or only the proximal position or populating the two positions with a fixed ratio. Only the first model, which includes the pH-dependent conformational equilibrium, reproduces both the experimentally measured pH dependence of the proton uptake and the crystallographically observed conformational equilibrium at pH 8. From this model, we find that Q(B) occupies only the distal position below pH 6.5 and only the proximal position above pH 9.0 in both oxidation states. Between these pH values both positions are partially occupied. The reduced Q(B) has a higher occupancy in the proximal position than the oxidized Q(B). In summary, the present results indicate that the conformational equilibrium of Q(B) depends not only on the redox state of Q(B), but also on the pH value of the solution.     

Photochromic polypeptides as synthetic models of biological photoreceptors: a spectroscopic study.

L-Glutamic acid polypeptides containing photochromic nitrospiropyran bound to the side chains at various percentages ("local" concentration) have been synthesized and investigated as possible artificial models of biological photoreceptors. Absorption and fluorescence spectroscopy have been utilized to investigate the photophysical and photochemical properties of nitrospiropyrans, both inserted in the polypeptide chain and in solution as "free" dye. Conformational variations produced by dark storage and light exposure of the photochromic polypeptides have been studied by means of circular dichroism. Dark-kept "free" dyes in hexafluoro-2-propanol solution in the merocyanine form ("open" form) give rise to molecular aggregates, which have been characterized as merocyanine dimers. The equilibrium constant between the monomer and the dimer, K, and their molar extinction coefficients, epsilon, at several wavelengths have been determined. Fluorescence measurements on "free" and polypeptide-bound nitrospiropyrans suggest that the dimerization process between merocyanines is favored when the photochromic units are inserted in the polypeptide chain and that under these conditions an efficient energy transfer from the monomer (donor) to the dimer (acceptor) occurs. By varying "local" as well as total nitrospiropyran concentration, it has been shown that the dimeric species result from intermolecular interactions between photochromic groups inserted in the same polypeptide chain. The alpha-helix --> random coil transition of the polypeptide structure after dark storage has eventually been shown to be the result of the dimerization process and not of the dark isomerization per se from the "closed" spiropyran form to the "open" merocyanine form of the dye.     

Melting of a self-complementary DNA minicircle. Comparison of optical melting theory with exchange broadening of the nuclear magnetic resonance spectrum

Melting curves are calculated for the 16-base-pair duplex DNA sequence 5' GTATCCGTACGGATAC 3' linked on the ends by TTTT single-strand loops. The equilibrium statistical thermodynamic theory of DNA melting is modified to include effects of end-loops on the melting transition. An excellent fit of the experimental melting curve in 0.2 M-NaCl is obtained using two adjustable parameters, one for end-loop formation and the other for formation of the complete 40-base single-strand loop. The best-fit calculated melting curve permits evaluation of these parameters. The free energy to close a TTTT end-loop is 2.12 kcal/mol (1 cal = 4.184 J). A TTTT end-loop or hairpin loop is significantly more stable than an internal loop of comparable size sandwiched between two helical regions, even after allowing for the different stacking contributions. Reasons for this increased stability are presented. The loop free energy of the 40-base single-strand open minicircle is evaluated to be +1.27 kcal/mol, thus favoring the melting of two end-loops into the large open minicircle. The present results are compared with those of others for d(T-A) oligomers. The sequence TTTT forms a more stable end-loop, or hairpin, than TATA by about 2.0 kcal/mol. Theoretical rate constants for the proton-transfer step in the standard hydrogen-exchange model are calculated by extending the theory of diffusion-controlled reactions to take account of the electrostatic potential of the DNA. The predicted ratios of rate constants for different pairs of catalysts exchanging an A.T proton agree satisfactorily with the available experimental data for a 14-base-pair linear duplex, which confirms the diffusion-control of the proton-transfer step. Data presented here for the 16 base-pair duplex of the minicircle are consistent with catalysis-limited exchange in which the proton-transfer step is likewise diffusion-controlled. Under catalysis-limited conditions, the imino proton exchange rates are predicted from the catalytic rate constants, prevailing buffer catalyst concentrations, and the equilibrium constants to form the unstacked open state of optical melting theory. The observed exchange rates of the A.T base-pairs show no sign of the strong predicted end-melting trend, and exceed the predicted values by factors of 10 to 400. Moreover, the succession of "melting" in the nuclear magnetic resonance line-broadening deviates from that predicted by optical melting theory.(ABSTRACT TRUNCATED AT 400 WORDS)     

pH Dependence of the photocycle kinetics of the E46Q mutant of photoactive yellow protein: protonation equilibrium between I1 and I2 intermediates,chromophore deprotonation by hydroxyl uptake,and protonation relaxation of the dark state

The kinetics of the photocycle of PYP and its mutants E46Q and E46A were investigated as a function of pH. E46 is the putative donor of the chromophore which becomes protonated in the I(2) intermediate. For E46Q we find that I(2) is in a pH-dependent equilibrium with its precursor I(1)' with a pK(a) of 8.15 and n = 1. From this result and from experiments with pH indicator dyes, we conclude that in the I(1)' to I(2) transition one proton is taken up from the external medium. The pK(a) of 8.15 is that of the surface-exposed chromophore in the equilibrium between I(1)' and I(2) and is close to that of the phenolate group of p-hydroxycinnamic acid. The pH-dependent I(1)'/I(2) equilibrium with associated H(+) uptake is reminiscent of the M(I)/M(II) equilibrium in the formation of the signaling state of rhodopsin. Well above this pK(a) no I(2) is formed and I(1)' returns in a pH-independent manner to the initial state P. The decay rate for the return to P via I(2) is between pH 4 and pH 8, exactly proportional to the hydroxide concentration (first order), and the deprotonation of the chromophore in this transition occurs by hydroxide uptake. Well above the pK(a) of 8.15 the apparent rate constant for the return to P is constant due to the branching from I(1)'. Complementary measurements with the pH indicator dye cresol red at pH 8.3 show that the remaining PYP molecules that still cycle via I(2) take up one proton in the formation of I(2). Together, these observations provide compelling evidence that during the photocycle the chromophore in E46Q is protonated and deprotonated from the external medium. For the yellow form of the mutant E46A the apparent rate constant for the return to P is also linear in [OH(-)] below about pH 8.3 and constant above about pH 9.5, with a pK(a) value of 8.8 for I(1)', suggesting a similar mechanism of chromophore protonation/deprotonation as in E46Q. For wild type qualitatively similar observations were made: the amplitude of I(2) decreased at alkaline pH, I(1)' and I(2) were in equilibrium, and I(1)' decayed together with the return to P. Chromophore hydrolysis prevented, however, an accurate determination of the pK(a) of I(1)'. We estimate that its value is above 11. The ground state P is in the dark in a pH-dependent equilibrium with a low-pH bleached form P(bl) with protonated chromophore. The pK(a) values for these equilibria are 4.8 and 7.9 for E46Q and E46A, respectively. When the pH is close to these pK(a)'s, the kinetics of the photocycle contains additional components in the millisecond time range. Using pH-jump stopped-flow experiments, we show that these contributions are due to the relaxation of the P/P(bl) equilibrium which is perturbed by the rapid decrease in the P concentration caused by the flash excitation of P. The condition for the occurrence of this effect is that the relaxation time of the P/P(bl) equilibrium is faster than the photocycle time.     

1H NMR and circular dichroism studies of the B and Z conformations of the self-complementary deoxyhexanucleotide d(m5C-G-C-G-m5-C-G): mechanism of the Z-B-coil transitions

The double-helical conformations of d(m5-C-G-C-G-m5-C-G) in aqueous solution were studied by circular dichroism and 1H NMR spectroscopy. In 0.1 M NaCl, only the B form is detected whereas the Z form is strongly predominant in 3 M NaCl. In the presence of 2 M NaCl, two resonance signals corresponding to the B and Z duplexes were observed for each proton below 50 degrees C, indicating a slow exchange between B and Z. However, the B-Z exchange becomes intermediate or fast in the 55-80 degrees C temperature interval. By contrast the exchange between B helix and single-stranded (or coil) forms is much faster for the same temperature conditions. The Z form is only detectable when the coil form is practically absent. With decreasing temperature the B form decreases in favor of the Z form. From proton line-width measurements under various experimental conditions, it was also shown that Z exchanges only with B, while the latter also exchanges with the single-stranded form (S): Z in equilibrium B in equilibrium S. The enthalpy value is about 8 +/- 1 kcal/mol for the B-Z transition and about 40 +/- 2 kcal/mol for the B-S dissociation (2 M NaCl solution). The activation energy is about 47 +/- 2 kcal/mol for the Z----B and 39 +/- 2 kcal/mol for the B----Z reaction. Very good agreement between the experimental results and computed data (based on the above kinetic reaction model) was found for the B, Z, and coil proportions. The B-Z transition of methylated d(C-G)n oligomers is only possible when the Watson-Crick hydrogen bonds between the CG base pairs are firmly maintained; otherwise, the transformation from B to Z would not occur, and B-S dissociation would take place instead.     

In Vivo Properties of Membrane-bound Phytochrome

After a 3-minute irradiation with red light, which saturates the phototransformation from the red light-absorbing form of phytochrome to the far red light absorbing form of phytochrome, about 40% of the phytochrome extractable from hooks of etiolated squash seedlings (Cucurbita pepo L. cv. Black Beauty) can be pelleted as Pfr at 17,000g after 30 minutes. Dark controls yield only 2 to 4% pelletable phytochrome in the form Pr. If a dark period intervenes between red irradiation and extraction, the bound Pfr gradually loses its photoreversibility. The time course for this destruction parallels the time course for phytochrome destruction in vivo following saturating red irradiation. The soluble fraction of phytochrome remains constant. These results suggest that in squash seedlings phytochrome destruction is related exclusively to the fraction which becomes membrane-bound. The induction of phytochrome binding by red light is not completely reversible by far red. In plants given saturating red followed immediately by saturating far red light, 12% of the phytochrome is found in the bound fraction as Pr if the phytochrome extraction is immediate. If a dark period intervenes between red-far red treatment and extraction, the bound phytochrome is released within 2 hours. A model of the binding properties of phytochrome, based on molecular interaction at the membrane is proposed, and possible consequences for the mechanism of action of phytochrome are discussed.     

Ionized and wobble base-pairing for bromouracil-guanine in equilibrium under physiological conditions. A nuclear magnetic resonance study on an oligonucleotide containing a bromouracil-guanine base-pair as a function of pH

A one and two-dimensional nuclear magnetic resonance study of a non-selfcomplementary oligonucleotide containing a central 5-bromouracil-guanine pair is reported. For these two bases three types of hydrogen bonding schemes could exist; wobble, rare tautomer and ionized. The two-dimensional spectra of non-exchangeable protons together with one-dimensional spectra recorded in water show that at pH 7.0 the predominant species is a right-handed B-form DNA in which the brU.G pair has wobble geometry. On raising the pH we observe a transition monitored by proton chemical shift changes for the brU.G and adjacent base-pairs. The mid-point of the transition was observed at pH 8.6. Spectra recorded at pH 9.8 show that the helix remains intact with B form conformation. It is shown that this high pH form has an ionized brU.G base-pair now in Watson-Crick geometry. Thus under physiological conditions an equilibrium exists between wobble and ionized structures.     

Photogravitropic equilibrium in Avena coleoptiles: fluence rate-response relationships and dependence on dark adaptation and clinostating

Phototropism of Avena coleoptiles was measured in response to blue-light irradiation lasting between 2 and 24 h. During this time the coleoptiles established a bending angle of photogravitropic equilibrium that was dependent on the time of irradiation and also on the pretreatment in light or darkness prior to stimulation. The absolute threshold for the photogravitropic equilibrium in response to blue light was 10(-8) micromol m(-2) s(-1). Photon fluence rate-response curves, which were generated after several hours of dark adaptation, had a characteristic shape with a prominent optimum in the middle of the dynamic range. Curves which were generated without prior dark adaptation displayed no such optimum. Clinostating dark-adapted coleoptiles caused an increase of sensitivity and responsiveness during a 2-h period of unilateral irradiation. The advantages and the drawbacks of long-term irradiation experiments for the investigation of phototropism and the generation of action spectra are discussed.     

1H nuclear magnetic resonance studies of the conformation and environment of nucleotides bound to pig heart NADP+-dependent isocitrate dehydrogenase

The binding of coenzymes, NADP+ and NADPH, and coenzyme fragments, 2'-phosphoadenosine 5'-(diphosphoribose), adenosine 2',5'-bisphosphate, and 2'-AMP, to pig heart NADP+-dependent isocitrate dehydrogenase has been studied by proton NMR. Transferred nuclear Overhauser enhancement (NOE) between the nicotinamide 1'-ribose proton and the 2-nicotinamide ring proton indicates that the nicotinamide-ribose bond assumes an anti conformation. For all nucleotides, a nuclear Overhauser effect between the adenine 1'-ribose proton and 8-adenine ring proton is observed, suggesting a predominantly syn adenine--ribose bond conformation for the enzyme-bound nucleotides. Transferred NOE between the protons at A2 and N6 is observed for NADPH (but not NADP+), implying proximity between adenine and nicotinamide rings in a folded enzyme-bound form of NADPH. Line-width measurements on the resonances of free nucleotides exchanging with bound species indicate dissociation rates ranging from less than 7 s-1 for NADPH to approximately 1600 s-1 for adenosine 2',5'-bisphosphate. Substrate, magnesium isocitrate, increases the dissociation rate for NADPH about 10-fold but decreases the corresponding rate for phosphoadenosine diphosphoribose and adenosine 2',5'-bisphosphate about 10-fold. These effects are consistent with changes in equilibrium dissociation constants measured under similar conditions. The 1H NMR spectrum of isocitrate dehydrogenase at pH 7.5 has three narrow peaks between delta 7.85 and 7.69 that shift with changes in pH and hence arise from C-4 protons of histidines. One of those, with pK = 5.35, is perturbed by NADP+ and NADPH but not by nucleotide fragments, indicating that this histidine is in the region of the nicotinamide binding site. Observation of nuclear Overhauser effects arising from selective irradiation at delta 7.55 indicates proximity of either a nontitrating histidine or an aromatic residue to the adenine ring of all nucleotides. In addition, selective irradiation of the methyl region of the enzyme spectrum demonstrates that the adenine ring is close to methyl side chains. The substrate magnesium isocitrate produces no observable differences in these protein--nucleotide interactions. The alterations in enzyme--nucleotide conformation that result in changes in affinity in the presence of substrate must involve either small shifts in the positions of amino acid side chains or changes in groups not visible in the proton NMR spectrum.     

Structural and biochemical characterization of a fluorogenic rhodamine-labeled malarial protease substrate

Activation of the proenzyme form of the malarial protease PfSUB-1 involves the autocatalytic cleavage of an Asp-Asn bond within the internal sequence motif (215)LVSADNIDIS(224). A synthetic decapeptide based on this sequence but with the N- and C-terminal residues replaced by cysteines (Ac-CVSADNIDIC-OH) was labeled with 5- or 6-isomers of iodoacetamidotetramethylrhodamine (IATR). The doubly labeled peptides have low fluorescence because of ground-state, noncovalent dimerization of the rhodamines. Cleavage of either peptide by recombinant PfSUB-1 results in dissociation of the rhodamine dimers, which abolishes the self-quenching and consequently leads to an approximately 30-fold increase in the fluorescence. This spectroscopic signal provides a continuous assay of proteolysis, enabling quantitative kinetic measurements to be made, and has also enabled the development of a fluorescence-based assay suitable for use in high-throughput screens for inhibitors of PfSUB-1. The structure of the rhodamine dimer in the 6-IATR-labeled peptide was shown by NMR to be a face-to-face stacking of the xanthene rings. Time-resolved fluorescence measurements suggest that the doubly labeled peptides exist in an equilibrium consisting of rhodamines involved in dimers (closed forms) and rhodamines not involved in dimers (open forms). These data also indicate that the rhodamine dimers fluoresce and that the associated lifetimes are subnanosecond.     

Signaling states of rhodopsin. Retinal provides a scaffold for activating proton transfer switches

The G-protein-coupled receptor rhodopsin is activated by photoconversion of its covalently bound ligand 11-cis-retinal to the agonist all-trans-retinal. After light-induced isomerization and early photointermediates, the receptor reaches a G-protein-dependent equilibrium between active and inactive conformations distinguished by the protonation of key opsin residues. In this report, we study the role of the 9-methyl group of retinal, one of the crucial steric determinants of light activation. We find that when this group is removed, the protonation equilibrium is strongly shifted to the inactive conformation. The residually formed active species is very similar to the active form of normal rhodopsin, metarhodopsin II. It has a deprotonated Schiff base, binds to the retinal G-protein transducin, and is favored at acidic pH. Our data show that the normal proton transfer reactions are inhibited in 9-demethyl rhodopsin but are still mandatory for receptor activation. We propose that retinal and its 9-methyl group act as a scaffold for opsin to adjust key proton donor and acceptor side chains for the proton transfer reactions that stabilize the active conformation. The mechanism may also be applicable to related receptors and may thus explain the partial agonism of certain ligands.     

Measuring the functionality of the mitochondrial pumping complexes with multi-wavelength spectroscopy

The proton pumps of the mitochondrial electron transport chain (ETC) convert redox energy into the proton motive force (ΔP), which is subsequently used by the ATP synthase to regenerate ATP. The limited available redox free energy requires the proton pumps to operate close to equilibrium in order to maintain a high ΔP, which in turn is needed to maintain a high phosphorylation potential. Current biochemical assays measure complex activities far from equilibrium and so shed little light on their function under physiological conditions. Here we combine absorption spectroscopy of the ETC hemes, NADH fluorescence spectroscopy and oxygen consumption to simultaneously measure the redox potential of the intermediate redox pools, the components of ΔP and the electron flux in RAW 264.7 mouse macrophages. We confirm that complex I and III operate near equilibrium and quantify the linear relationship between flux and disequilibrium as a metric of their function under physiological conditions. In addition, we quantify the dependence of complex IV turnover on ΔP and the redox potential of cytochrome c to determine the complex IV driving force and find that the turnover is proportional to this driving force. This form of quantification is a more relevant metric of ETC function than standard biochemical assays and can be used to study the effect of mutations in either mitochondrial or nuclear genome affecting mitochondrial function, post-translation changes, different subunit isoforms, as well as the effect of pharmaceuticals on ETC function.     

Induction of anthocyanin formation and of enzymes related to its biosynthesis by UV light in cell cultures of Haplopappus gracilis

Only UV light below 345 nm stimulates anthocyanin formation in dark grown cell suspension cultures of Haplopappus gracilis. A linear relationship between UV dose and flavonoid accumulation, as found previously with parsley cell cultures, was not observed with the H. gracilis cells. Only continuous irradiation with high doses of UV was effective. Drastic increases in the activities of the enzymes phenylalanine ammonia-lyase, chalcone isomerase and flavanone synthase were observed under continuous UV light. The increase in enzyme activities paralleled anthocyanin formation.     

Spectroscopic study of the dark interaction and of the photoreaction between a new monofunctional psoralen : 3-carbethoxy psoralen, and DNA

3-carbethoxypsoralen (3-CPs) is a new linear psoralen derivative. Its dark interaction and photoreaction with DNA has been studied and compared with that of a well known bifunctional psoralen: 8-methoxypsoralen (8-MOP). 3-CPs is able to form in the dark a non covalent complex with native DNA. After irradiation of this complex with UV-A light (365 nm) 3-CPs is able to link covalently to DNA. Heat denaturation and renaturation patterns of treated DNA clearly show that, in contrast to 8-MOP, 3-CPs does not form DNA interstrand cross links. Fluorescence studies show that the photobinding of 3-CPs gives rise to the formation of monoadducts involving the 4′,5′ double bond of this molecule.