Design, synthesis and antiproliferative properties of oligomers with chromophore units linked by amide backbones

The preparation of oligomers made up of several chromophore units as compounds with potential fluorescent and antiproliferative properties is described. Specifically, chromophore units with protected-amino groups and one carboxylic group are described, together with methods to assemble these units using peptide chemistry. Some of these compounds have antiproliferative activity.     

Polarized absorption picosecond kinetics as a probe of energy transfer in phycobilisomes of Synechococcus 6301

The energy transfer between C-phycocyanin chromophores in intact phycobilisomes of Synechococcus 6301 is shown to lead to an anisotropy relaxation with a lifetime of 10 ± 2 ps. However, due to the molecular order within the hexameric units of C-phycocyanin the anisotropy does not decay to zero. The Förster dipole-dipole mechanism of energy transfer can qualitatively explain these data provided that there is no back transfer of excitation energy and that the chromophore distribution is non-random. The rate of energy transfer in phycobilisomes between C-phycocyanin and allophycocyanin can best be described by a double exponential with lifetimes of 12 ± 3 and 84 ± 8 ps.     

A nomogram for deconvolution of single exponential fluorescence decays.

An extremely rapid technique for deconvolving single exponential luminescence decay data is described that involves essentially no mathematical manipulation of the experimental data. The method permits "real time" measurement of deconvolved luminescence lifetimes with conventional pulsed, lifetime-fluorometers and phosphorimeters. The method assumes that the true luminescence decay of the chromophore is accurately represented by a single exponential decay function.     

A highly sensitive chromogenic microtiter plate assay for plasminogen activators which quantitatively discriminates between the urokinase and tissue-type activators

A simple and highly sensitive chromogenic microtiter plate assay for plasminogen activators is described. The assay is based on plasmin cleavage of the synthetic tripeptide plasmin substrate H-D-norleucyl-hexahydrotyrosyl-lysine p-nitroaniline, which yields the yellow chromophore p-nitroanilide. Production of the latter compound is then quantitated spectrophotometrically at 405 nm on an ELISA plate reader. Linearity of the assay can be achieved over at least four orders of magnitude in a single experiment (0.01-100 milliPloug units) with appropriate incubation times. Capitalizing on tissue-type plasminogen activator's dependence on fibrin for enzymatic activity, the selective use of soluble fibrin products allows discrimination between urokinase and tissue-type activator. The utility of this aspect of the assay for the analysis of complex samples containing both types of plasminogen activators is demonstrated.     

Conformational studies of the human complex-forming glycoprotein, heterogeneous in charge: protein HC

Human complex-forming glycoprotein, heterogeneous in charge, is a single polypeptide chain widely distributed in physiological fluids. The conformation of the protein has been studied with attention to the secondary and tertiary structures. Circular dichroism and predictive methods from the amino acid sequence have been employed for the characterization of the secondary structure. This is composed of 20% alpha-helix, 21% beta-structure, 29% beta-turns, 30% aperiodic conformation, and an average number of residues per helical segment of nine. Titration of the protein indicated the existence of two groups for the tyrosine residues, each of them composed of three and five residues. The four tryptophan residues of the molecule are located in two different polarity microenvironments, according to the fluorescence studies. These observations are corroborated by studying the hydropathic profile of the protein. From this study, three different domains are observed in the protein, one of them being exposed and containing the main part of the unordered structure of the molecule. The chromophore naturally associated with the protein has been resolved in three fluorescent units not dependent on the protein conformation. These bands have been observed centered around 290, 360, and 410 nm, which do not correspond to any described chromophore.     

A modified equilibrium dialysis method for studying fatty acid binding to proteins

A modified equilibrium dialysis method is described which is suitable for investigating the binding of fatty acids in the form of aqueous micellar dispersions to proteins. The method uses a permeant chromophore which complexes reversibly with free fatty acid within the dialysis bag. The concentration outside the dialysis bag is determined spectrophotometrically. Binding of oleic acid to bovine serum albumin is given as an example. A simplified analysis of fatty acid binding is given and used to indicate the potential of the method.     

Potentiation of BCNU cytotoxicity by molecules targeting abasic lesions in DNA.

We describe the synthesis, DNA binding measurements and pharmacological properties of a series of new heterodimeric molecules, in which a 2,6-diaminopurine is linked to a 9-aminoacridine chromophore. The linking chain contains a central N,N'-disubstituted guanidine, connected to the two chromophores by polymethylenic units of variable length.     

Detection and estimation of 3,4-dehydroproline

A method has been described for the detection and estimation of 3,4-dehydroproline using initial oxidation with isatin or H2O2 and subsequent reaction with p-dimethylamino benzaldehyde (Ehrlich reagent). The method is sensitive enough to detect as low as 0.6 micrograms of 3,4-dehydroproline/sq. cm on paper chromatogram. 3,4-dehydroproline could also be estimated by the classical H2O2-oxidation procedure employed for hydroxyproline apparently yielding the same chromophore on a molar basis. However, when estimated by the chloramine-T oxidation method, its sensitivity was only 1/100th of that of 4-hydroxyproline. The usefulness of these procedures in the detection and estimation of 3,4-dehydroproline has been described.     

Kinetic study of de novo chromophore maturation of fluorescent proteins

Green fluorescent protein (GFP) has a chromophore that forms autocatalytically within the folded protein. Although many studies have focused on the precise mechanism of chromophore maturation, little is known about the kinetics of de novo chromophore maturation. Here we present a simple and efficient method for examining the de novo kinetics. GFP with an immature chromophore was synthesized in a reconstituted cell-free protein synthesis system under anaerobic conditions. Chromophore maturation was initiated by rapid dilution in an air-saturated maturation buffer, and the time course of fluorescence development was monitored. Comparison of the de novo maturation rates in various GFP variants revealed that some folding mutations near the chromophore promoted rapid chromophore maturation and that the accumulation of mutations could reduce the maturation rate. Our method will contribute to the design of rapidly maturing fluorescent proteins with improved characteristics for real-time monitoring of cellular events.     

Dynamics of protein and chromophore structural changes in the photocycle of photoactive yellow protein monitored by time-resolved optical rotatory dispersion

The dynamics of the PYP photocycle have been studied using time-resolved optical rotatory dispersion (TRORD) spectroscopy in the visible and far-UV spectral regions to probe the changes in the chromophore configuration and the protein secondary structure, respectively. The changes in the secondary structure in PYP upon photoisomerization of the chromophore can be described by two exponential lifetimes of 2 +/- 0.8 and 650 +/- 100 ms that correspond to unfolding and refolding processes, respectively. The TRORD experiments that follow the dynamics of the chromophore report three exponential components, with lifetimes of 10 +/- 3 micros, 1.5 +/- 0.5 ms, and 515 +/- 110 ms. A comparison of the kinetic behaviors of the chromophore and protein shows that during the decay of pR(465) an initial relaxation that is localized to the chromophore hydrophobic pocket precedes the formation of the chromophore and protein structures found in pB(355). In contrast, the protein and chromophore processes occur with similar time constants during inactivation of the signaling state.     

Solution structure of a novel chromoprotein derived from apo-neocarzinostatin and a synthetic chromophore

The natural complex Neocarzinostatin comprises a labile chromophore noncovalently bound to an 11.2 kDa protein. We present the first high-resolution structure of a novel complex derived from the recombinant apoprotein bound to a non-natural synthetic chromophore. Fluorescence and nuclear magnetic resonance spectroscopy were used to probe the strength and location of binding. Binding occurred in a location similar to that observed for the chromophore in the natural Neocarzinostatin complex, but with a distinct orientation. These results provide structural evidence that the apoprotein can readily accommodate small druglike entities, other than the natural chromophore within its binding cleft. The clinical use of the natural complex described by others, together with the results reported here, suggests potential applications for small molecule binding by apo-Neocarzinostatin.     

Effect of high pressure and reversed micelles on the fluorescent proteins

Two physico-chemical perturbations were applied to ECFP, EGFP, EYFP and DsRed fluorescent proteins: high hydrostatic pressure and encapsulation in reversed micelles. The observed fluorescence changes were described by two-state model and quantified by thermodynamic formalism. ECFP, EYFP and DsRed exhibited similar reaction volumes under pressure. The changes of the chemical potentials of the chromophore in bis(2-ethylhexyl)sulfosuccinate (AOT) micelles caused apparent chromophore protonation changes resulting in a fluorescence decrease of ECFP and EYFP. In contrast to the remarkable stability of DsRed, the highest sensitivity of EYFP fluorescence under pressure and in micelles is attributed to its chromophore structure.     

Assay of inorganic phosphate in the mild pH range, suitable for measurement of glycogen phosphorylase activity

A spectrophotometric assay of orthophosphate at mild pH is described. Phosphomolybdate complex is reduced by ascorbic acid at pH 5.0 in the presence of Zn2+. The chromophore produced is measured at 850 nm. The method is simple and sensitive, and the reaction proceeds normally in the presence of "interfering substances" such as metal-chelating agents and thiol compounds. The method could be widely employed for the assay of phosphate-releasing reaction from labile organic phosphates, such as for the assay of glycogen phosphorylase.     

An improved method for the automated determination of meta- and iso-saccharinic acids

Manual and automated spectrophotometric methods are described for the determination of 3-deoxyhexonic and 3-deoxy-2-C-hydroxymethylpentonic acids. The method utilises the chromophores formed by condensation of barbituric acid with the products of oxidation with periodate. This method obviates the need for solvent extraction required when using 2-thiobarbituric acid for chromophore production.     

Structural insight into DNA-assembled oligochromophores: crystallographic analysis of pyrene- and phenanthrene-modified DNA in complex with BpuJI endonuclease

The use of the DNA duplex as a supramolecular scaffold is an established approach for the assembly of chromophore aggregates. In the absence of detailed structural insight, the characterization of thus assembled oligochromophores is, today, largely based on solution-phase spectroscopy. Here, we describe the crystal structures of three DNA-organized chromophore aggregates. DNA hybrids containing non-nucleosidic pyrene and phenanthrene building blocks were co-crystallized with the recently described binding domain of the restriction enzyme BpuJI. Crystal structures of these complexes were determined at 2.7, 1.9 and 1.6 Å resolutions. The structures reveal aromatic stacking interactions between pyrene and/or phenanthrene units within the framework of the B-DNA duplex. In hybrids containing a single modification in each DNA strand near the end of the duplex, the two polyaromatic hydrocarbons are engaged in a face-to-face stacking orientation. Due to crystal packing and steric effects, the terminal GC base pair is disrupted in all three crystal structures, which results in a non-perfect stacking arrangement of the aromatic chromophores in two of the structures. In a hybrid containing a total of three pyrenes, crystal lattice induced end-to-end stacking of individual DNA duplexes leads to the formation of an extended aromatic π-stack containing four co-axially arranged pyrenes. The aromatic planes of the stacked pyrenes are oriented in a parallel way. The study demonstrates the value of co-crystallization of chemically modified DNA with the recombinant binding domain of the restriction enzyme BpuJI for obtaining detailed structural insight into DNA-assembled oligochromophores.     

Electrostatic interactions at charged lipid membranes. Measurement of surface pH with fluorescent lipoid pH indicators.

The 5-dimethylaminonapthalene-1-sulfonyl (dansyl) chromophore attached to the polar head groups of lipids has been used as a fluorescent lipoid pH indicator to evaluate the interfacial pH in lipid-water lamellar systems prepared from negatively charged lipids. The pH in the vicinity of the charged lipid bilayers is different from the pH of the bulk aqueous phase and the difference is a function of the electrolyte concentration in the aqueous phase and of the lipid packing in the bilayer. At a fixed electrolyte concentration in the aqueous phase, the observed interfacial pH is 0.6 to 0.7 pH units lower above the thermal phase transition of the lipid than it is below this temperature. A quantitative interpretation of the results is given on the basis of the Gouy-Chapman theory. The results indicate that the dansyl chromophore is located in front of the charged surface and its distance from this surface increases with a decrease in lipid packing.     

Monitoring of collagen and collagen fragments in chromatography of protein mixtures

A new method for the detection of collagen and collagen peptides in the presence of other proteins is described. The procedure is based on alkaline hydrolysis of the proteins and monitoring of the free hydroxyproline after chromophore formation. The method is quick and sensitive and the color yield of hydroxyproline is not affected by chromatography solvents. As all steps of the assay take place in a single test tube the method is suitable for batch processing of column fractions. Due to the high sensitivity of the assay it can be used for the analysis of collagen and collagen peptides in extracts of small tissue samples or biopsies.     

Reconstitution of Escherichia coli DNA photolyase with various folate derivatives

DNA photolyase from Escherichia coli contains both flavin and pterin. However, the isolated enzyme is depleted with respect to the pterin chromophore (0.5 mol of pterin/mol of flavin). The extinction coefficient of the pterin chromophore at 360 nm is underestimated by a method used in earlier studies which assumes stoichiometric amounts of pterin and flavin. The extinction coefficient of the pterin chromophore, determined on the basis of its (p-aminobenzoyl)polyglutamate content (epsilon 360 = 25.7 x 10(3) M-1 cm-1), is in good agreement with that expected for a 5,10-methenyltetrahydrofolate derivative. Also consistent with this structure, the pterin chromophore could be reversibly hydrolyzed to yield a 10-formyltetrahydrofolate derivative or reduced to yield a 5-methyltetrahydrofolate derivative. The isolated enzyme could be reconstituted with various folate derivatives to yield enzyme that contained equimolar amounts of pterin and flavin. Similar results were obtained in reconstitution studies with the natural pterin chromophore, with 5,10-methenyltetrahydrofolate, and with 10-formyltetrahydrofolate. The results show that the polyglutamate moiety, previously identified in the natural chromophore, is not critical for binding. Reconstitution with the natural pterin chromophore did not affect catalytic activity. The latter is consistent with our previous studies which show that, although the pterin chromophore acts as a sensitizer in native enzyme, it is not essential for dimer repair which can occur at the same rate under saturating light with flavin (1,5-dihydro-FAD) as the only chromophore.     

Chromophore configuration of pharaonis phoborhodopsin and its isomerization on photon absorption.

The configuration of the retinylidene chromophore in pharaonis phoborhodopsin (ppR) and its changes during the photoreaction cycle were investigated by means of a chromophore extraction method followed by HPLC analysis. The ppR has an all-trans chromophore, and unlike bacteriorhodopsin, it exhibits no dark isomerization of the chromophore. Irradiation of a ppR sample in the presence of 10 mM hydroxylamine, at which concentration a negligible amount of ppR was bleached, caused the formation of 90% 13-cis- and 10% all-trans-retinal oximes. Because the ppR sample under the continuous irradiation was a mixture containing original ppR, ppRM, and a small amount of ppRO, the above results showed that the chromophores of ppRM and ppRO are in a 13-cis form and an all-trans form, respectively. Therefore, the all-trans chromophore of ppR is isomerized to the 13-cis form on photon absorption, and it is thermally reisomerized to the all-trans form on the conversion process from ppRM to ppRO. The extracted retinal oximes from ppR and ppRO were mainly the 15-syn form, while that from ppRM was mainly the 15-anti form. This fact indicated that the attack of hydroxylamine on the chromophore is stereoselective owing to the unique structure of the chromophore binding site near the Schiff base region of the chromophore.     

Depth of immersion of fluorescent chromophores in biomembranes studied by quenching with nitroxide radical

A novel method has been developed for measuring depth of immersion of a fluorescent chromophore in biological matrices, such as biomembranes. The method is based on dynamic quenching of chromophore fluorescence by a nitroxide probe freely diffused in solution. Theoretical considerations and experimental evidences relating to the method are discussed. The proposed method was applied to investigation of lecithin liposomes and membranes from Bacillus subtilis modified by the photochrome-fluorescent probe 4,4'-dimethylaminocyanostilbene.