Syntheses and DNA binding of new cationic porphyrin-tetrapeptide conjugates

Recently cationic porphyrin-peptide conjugates were synthesized to enhance the cellular uptake of porphyrins or deliver the peptide moiety to the close vicinity of nucleic acids. DNA binding of such compounds was not systematically studied yet.We synthesized two new porphyrin-tetrapeptide conjugates which can be considered as a typical monomer unit corresponding to the branches of porphyrin-polymeric branched chain polypeptide conjugates. Tetra-peptides were linked to the tri-cationic meso-tri(4-N-methylpyridyl)-mono-(4-carboxyphenyl)porphyrin and bi-cationic meso-5,10-bis(4-N-methylpyridyl)-15,20-di-(4-carboxyphenyl)porphyrin. DNA binding of porphyrin derivatives, and their peptide conjugates was investigated with comprehensive spectroscopic methods. Titration of porphyrin conjugates with DNA showed changes in Soret bands with bathocromic shifts and hypochromicities. Decomposition of absorption spectra suggested the formation of two populations of bound porphyrins.Evidence provided by the decomposition of absorption spectra, fluorescence decay components, fluorescence energy transfer and induced CD signals reveals that peptide conjugates of di- and tricationic porphyrins bind to DNA by two distinct binding modes which can be identified as intercalation and external binding. Tri-cationic structure and elimination of negative charges in the peptide conjugates are preferable for the binding. Our findings provide essential information for the design of DNA-targeted porphyrin-peptide conjugates.     

Analysis of porphyrin carboxylic acids in biological fluids by high-performance liquid chromatography

A method for the rapid and sensitive fluorometric analysis of porphyrin carboxylic acids by reverse-phase high-performance liquid chromatography is described. Separation of free porphyrin carboxylic acids was carried out with a microparticulate octadecylsilane column with elution by a gradient of methanol in phosphate buffer containing tetrabutylammonium hydroxide. Separation and quantitation of di-, tri-, tetra-, penta-, hexa-, hepta-, and octacar-boxylic porphyrins was achieved within 25 min at picomolar concentrations. The method is also capable of separating the type I and type III isomers of tetracarboxylic through hexacarboxylic porphyrins. By using a stopped flow technique, one can record fluorescence excitation and emission spectra of porphyrin carboxylic acids. This method is directly applicable to biological fluids such as urine, plasma, red cell lysates, or medium or extracts from cell culture.     

Porphyrin, chlorin, and bacteriochlorin isothiocyanates: useful reagents for the synthesis of photoactive bioconjugates

A novel method for conjugating porphyrins and related molecules to proteins has been developed. The method, which involves synthesizing porphyrins, chlorins, and bacteriochlorins bearing a single amine-reactive isothiocyanate group represents a facile system for protein labeling with these photoactive species. Problems associated with the noncovalent binding of porphyrins to proteins are highlighted, and a method for purifying conjugates to yield exclusively covalently bound porphyrin protein species is demonstrated. Biological activity of porphyrin-bovine serum albumin conjugates formed and purified by these methods is demonstrated using laser scanning confocal microscopy.     

Single-stranded DNA modification by an oligonucleotide-phthalocyanine Fe(II) conjugate: the kinetic features and the process mechanism

Catalytic oxidative modification of a single-stranded DNA with hydrogen peroxide and molecular oxygen in the presence of a conjugate containing an oligonucleotide complementary to the DNA fragment and tetra-4-carboxyphthalocyanine Fe(II) was studied. The conjugate examined was found to be active in the reaction of oxidative DNA cleavage in the presence of hydrogen peroxide, like the earlier studied oligonucleotide conjugates containing metallocomplexes tetra-4-carboxyphthalocyanine Co(II) and 2,4-di-[2-(2-hydroxyethyl)]deuteroporphyrin IX Fe(III) generating active oxygen forms. The new conjugate was more active in the case of oxidation with molecular oxygen. Kinetic features and optimal regimes of DNA oxidation with hydrogen peroxide were found.     

Synthesis and spectroelectrochemistry of N-cobaltacarborane porphyrin conjugates

N-Substituted porphyrins are well-known for the distortion they exhibit of the porphyrin plane through the sp(3) hybridization of one of the pyrrolenic units. They have served as model compounds in investigations of many biochemical processes. In this paper, we developed an efficient route to N-substituted porphyrins, and report the synthesis of a series of new N-substituted cobaltacarborane-porphyrins containing one or two cobaltabisdicarbollide anions linked by (CH(2)CH(2)O)(2) chains to either the core porphyrin nitrogens or to a meso-aminophenyl group. These conjugates show different degrees of distortion of the porphyrin macrocycle, which affect their spectroscopic and electrochemical properties. In particular, the core N-substituted conjugates show significant fluorescence quenching in comparison with the noncore substituted macrocycles. The X-ray structures of two targeted core N-cobaltacarborane porphyrin conjugates are presented. The electrochemical and spectroelectrochemical properties of these porphyrin conjugates were investigated; while the peripheral N-substituted cobaltacarboranylporphyrins undergo three reversible reductions and three reversible oxidations (two attributed to the porphyrin and one to the Co(III) cluster), the core N-substituted porphyrins exhibit complicated electrochemical behavior with coupled chemical reactions.     

β-d-Galactosidases immobilized on soluble matrices: Kinetics and stability

Three β-d-galactosidases (β-d-galactoside galactohydrolase, EC 3.2.1.23) from different origins have been immobilized on sucrose-polyacrolein and sucrose sulphate-polyacrolein. This gave enzyme conjugates insoluble in the immobilization medium but which could be made soluble by reduction with sodium borohydride before use. The optimum conditions for both copolymer synthesis and the immobilization reaction were investigated. I.r. and ¹³C n.m.r. spectroscopy were used to follow the sulphation and the copolymerization reaction. The characteristics of the enzyme conjugates were compared with those of the free enzymes: the V_max values of the enzyme conjugates were lower than those of the corresponding free enzymes, whilst the K_m values were similar. The thermal stability of the enzyme conjugates depended on the enzyme origin, while their pH stability was in all cases higher than that of the free enzymes. These data suggest some advantages in using enzyme immobilization supports which can be made soluble after separation of the immobilized enzyme without altering the enzyme characteristics.     

Quantitative analysis of unconjugated and conjugated bile acids in duodenal fluid by densitometry after paper electrophoresis

A new paper electrophoretic method for the separation of bile acids into five groups, (1) unconjugated, (2) glycine conjugates and (3) taurine conjugates, and (4) and (5) the respective monosulfates, is described. Rapid and accurate qualitative and quantitative estimations of each group are obtained by densitometry after internal standardization and phosphomolybdate color development. The technique can be done in the routine clinical laboratory and is useful for the detection of diseases affecting the enterohepatic circulation of bile acids.     

Affinity separation using an Fv antibody fragment-"smart" polymer conjugate

Poly(N-isopropylacrylamide), or PNIPAAm, is considered a "smart" polymer because it sharply precipitates when heated above a critical temperature, about 32 degrees C in water, and redissolves when cooled. Conjugates made of PNIPAAm and IgG antibodies also exhibit the same critical temperature behavior. Interestingly, antigens that are complexed with these conjugates can also be phase-separated along with the conjugates. In this work, we conjugated PNIPAAm for the first time to the immunoglobulin Fv fragment, the smallest fragment of an antibody that still retains the antigenic affinity of the whole antibody. For our studies, we used an Fv fragment that strongly binds hen egg white lysozyme (HEL). The purified Fv fragment-polymer conjugate precipitated at the same temperature as did the pure polymer. After addition of the conjugate to a mixture containing HEL and after thermal separation of the conjugate at 37 degrees C, the amount of HEL in solution was reduced by as much as 80%. We were able to demonstrate the reversibility of the separation through three cycles of precipitation and dissolution. It was also possible to recover free HEL by thermal separation of the conjugate in the presence of an eluant, 50 mM diethylamine. The conjugate can then be recycled for second use. In conclusion, immunoseparations can be performed using smart polymer conjugates made with just the variable domains of an antibody. Unlike whole antibodies, fragments of antibodies can be produced in Escherichia coli, allowing easier genetic engineering of the antibody and tailoring of the conjugate.     

High-performance liquid chromatographic analysis of porphyrins and their isomers with radial compression columns

Porphyrin methyl esters and the isomers of uroporphyrin and heptacarboxylic porphyrin were separated by high-performance liquid chromatography. Isocoproporphyrin was also separated from coproporphyrin. By slight modifications to the solvent mixture, the separation of all biological polycarboxylic porphyrins was achieved. These separations were made possible through the high efficiency of 10- or 5-μm particle-size Radial-PAK cartridges, which have been used in the separation of porphyrins in various excreta and tissues in a number of porphyrias.     

Simultaneous quantification of phytohormones in fermentation extracts of Botryodiplodia theobromae by liquid chromatography–electrospray tandem mass spectrometry

Fermentation broth and biomass from three strains of Botryodiplodia theobromae were characterized by high performance liquid chromatography–electrospray tandem mass spectrometry (HPLC–ESI–MS/MS) method, in order to quantify different phytohormones and to identify amino acid conjugates of jasmonic acid (JA) present in fermentation broths. A liquid–liquid extraction with ethyl acetate was used as sample preparation. The separation was carried out on a C18 reversed-phase HPLC column followed by analysis via ESI–MS/MS. The multiple reaction monitoring mode was used for quantitative measurement. For the first time, indole-3-acetic acid, indole-3-propionic acid, indole-3-butyric acid and JA were identified and quantified in the ethyl acetate extracts from the biomass, after the separation of mycelium from supernatant. The fermentation broths showed significantly higher levels of JA in relation to the other phytohormones. This is the first report of the presence of gibberellic acid, abscisic acid, salicylic acid and the cytokinins zeatin, and zeatin riboside in fermentation broths of Botryodiplodia sp. The presence of JA-serine and JA-threonine conjugates in fermentation broth was confirmed using HPLC-ESI tandem mass spectrometry in negative ionization mode, while the occurrence of JA-glycine and JA-isoleucine conjugates was evidenced with the same technique but with positive ionization. The results demonstrated that the used HPLC–ESI–MS/MS method was effective for analysing phytohormones in fermentation samples.     

Distribution of cell surface saccharides on pancreatic cells

We describe here a simple, general procedure for the purification of a variety of lectins, and for the preparation of lectin-ferritin conjugates of defined molar composition and binding properties to be used as probes for cell surface saccharides. The technique uses a “universal” affinity column for lectins and their conjugates, which consists of hog sulfated gastric mucin glycopeptides covalently coupled to agarose. The procedure involes: (a) purification of lectins by chromatography of aqueous extracts of seeds or other lectin-containing fluids over the affinity column, followed by desorption of the desired lectin with its hapten suge; (b) iodination of the lectin to serve as a marker during subsequent steps; (c) conjugation of lectin to ferritin with glutaraldehyde; (d) collection of active lectin-ferritin conjugates by affinity chromatography; and (e) separation of monomeric lectin-ferritin conjugates from larger aggregates and unconjugated lectin by gel chromatography. Based on radioactivity and absorbancy at 310 nm for lectin and ferritin, respectively, the conjugates consist of one to two molecules of lectin per ferrritin molecule. Binding studies of native lectins and their ferritin conjugates to dispersed pancreatic acinar cells showed that the conjugation procedure does not significantly alter either the affinity constant of the lectin for its receptor on the cell surface or the number of sites detected.     

Isocratic separation of estrogen conjugates on DEAE-sephadex.

Mixtures of estrogen conjugates containing estrone-3-glucosiduronate, 17β-estradiol-3-glucosiduronate, 17β-estradiol-17-glucosiduronate, estrone-3-sulfate and 17β-estradiol-3-sulfate have been separated on DEAE-Sephadex resin by isocratic elution using NaCI concentrations ranging from 0.05M to 0.4M. The results indicate that an NaCI gradient is not necessary for the Chromatographic separation of these estrogen conjugates. An NaCl concentration of 0.05M was adequate to separate the various monoglucosiduronates and sulfates. Isocratic elution of the columns with or without a possible stepup in the salt concentration was shown to give a higher resolution of estrogen conjugates in a more convenient volume than a gradient elution. For ideal chromatography of estrogen conjugates on a DEAE-Sephadex column, isocratic elution with 0.05 or 0. IM NaCl is preferred for the separation of monoglucosiduronates and 0.2 or 0.25M NaCl for the separation of sulfate conjugates. Contrary to current expectations, the molarity at which a particular conjugate elutes in a gradient mode does not bear a consistent relationship to the structure of the conjugate. However, the holdback volume in the isocratic mode may be used for identification purposes. When holdback volume was plotted against molarity, separate curves were obtained for each of the above mentioned conjugates. Tests of fit were carried out using a number of models. The best fit was obtained using the simple model $\text{y}\text{=}\text{a}\text{+}\text{b}\text{1}\text{x}$ where the independent variable, x, is the molarity; the dependent variable, y, is the volume and a and b are the intercept and slope respectively. Each curve fitted the model, but the values for a and b were significantly different. Using this model, a simple and predictable relationship between molarity and holdback volume can be demonstrated for each of the estrogen conjugates. The advantages of the isocratic mode of elution over gradient elution are discussed.     

酶标记抗人IgA单克隆抗体及其在Epstein-Barr病毒免疫酶技术中的应用

继成功地建立了4株分泌高效价抗人IgA McAb杂交瘤细胞株后,我们将制备出的McAb用于改进鼻咽癌早期诊断中的IgA/VCA和IgA/EA免疫酶技术,并探索了高效价抗人IgA McAb的纯化、辣根过氧化物酶(HRP)标记及有效保存的方法,结果令人满意。 采用饱和硫酸铵沉淀法及Sephadex A-50吸附法对来自细胞培养上清液和腹水的     

Studies of interactions of porphyrins with transfer RNA by high-resolution NMR

The interactions of tetra-4N-methylpyridyl porphyrin and its zinc(II), copper(II) and manganese(III) complexes with brewer's yeast type V phenylalanine specific tRNA have been evaluated by high-resolution NMR. Differences in chemical shifts have been noted for three proton resonances in response to the presence of small quantities of the free base and the zinc and copper complexes. The protons giving rise to these signals are located on bases T54 and psi 55, both of which are involved in the primary intraloop and interloop hydrogen bonds that hold the D and T psi C loops together in the tertiary structure. In addition, broadening of specific resonances due to hydrogen bonding protons in the D stem at low ratios of porphyrin to tRNA indicates that the association of porphyrins increases the rate of imino proton exchange. The titration of the tRNA with the manganese(III) complex did not reveal shifts or specific broadening comparable to the other porphyrins at low ratios. The changes induced in the NMR spectrum of tRNA by porphyrins define their site of interaction with the polynucleotide. This site, at the outside of the elbow-bend in the tRNA 'L', is different from the locus of binding in tRNA for other classical DNA intercalators. Furthermore, a new mode of binding may be involved that is neither intercalative nor simply electrostatic.     

Characterization of covalent protein conjugates using solid-state 13C NMR spectroscopy.

Cross-polarization magic-angle spinning (CPMAS) 13C NMR spectroscopy has been used to characterize covalent conjugates of alachlor, an alpha-chloroacetamide hapten, with glutathione (GSH) and bovine serum albumin (BSA). The solid-state NMR method demonstrates definitively the covalent nature of these conjugates and can also be used to characterize the sites of hapten attachment to proteins. Three different sites of alachlor binding are observed in the BSA system. Accurate quantitation of the amount of hapten covalently bound to GSH and BSA is reported. The solid-state 13C NMR technique can easily be generalized to study other small molecule/protein conjugates and can be used to assist the development and refinement of synthetic methods needed for the successful formation of such protein alkylation products.     

A reversed-phase high-performance liquid chromatography method for analysis of monoclonal antibody-maytansinoid immunoconjugates

A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed for detection and quantification of free maytansinoid drug in disulfide-linked conjugates between monoclonal antibodies and the maytansinoid drug DM1 (MAb-DM1). Mobile phases and gradient conditions were optimized for separation of several DM1-related free drug species from MAb-DM1 conjugates. The selectivity, linearity, and reproducibility of the method are reported. Reduction of the disulfide-linked DM1 followed by RP-HPLC allowed estimation of purity of MAb-linked DM1 as well as recovery of L-DM1. The method was also used to estimate drug per MAb ratios, which were consistent with those determined by UV spectroscopy.     

In vitro interaction of macrocyclic photosensitizers with intact mitochondria: a spectroscopic study

Six water-soluble macrocyclic photosensitizers, the members of two groups of expanded porphyrins (metallotexaphyrins and free-base sapphyrins) containing hydrophilic substituents and meso-tetra(4-sulfonatophenyl)-porphyrin, were tested by UV-Vis absorption and resonance Raman spectroscopy in the in vitro binding experiments with intact mitochondria isolated from swine liver. Studied macrocycles showed markedly different affinity to mitochondria. The highest uptake was observed for sapphyrin-sugar conjugate and metallotexaphyrins. Sapphyrin-polyamine conjugates exhibit something less affinity to mitochondria, while the porphyrin of anionic character showed very low mitochondrial uptake. Obtained spectroscopic results confirm that the binding process altered the self-aggregation degree of expanded porphyrins.     

Separation of estrogen conjugates by high pressure liquid chromatography.

High pressure liquid chromatography using a prepacked commercial strong anion exchanger column (mu Partisil 10 SAX, 25 cm x 4.6 mm) was used to separate a mixture of eight estrogen conjugates. Chromatographic conditions using a 0.01 M potassium phosphate or 0.1 M NaCl as solvent in the isocratic mode are described for the separation of estrone glucosiduronate, 17beta-estradiol-3-glucosiduronate, 17beta-estradiol-17-glucosiduronate, estriol-3-glucosiduronate, estriol-16alpha-glucosiduronate, estriol-17-glucosiduronate, estrone sulfate and 17beta-estradiol-3-sulfate. This system gives high resolution of the estrogen conjugates in small eluent volumes in less than 30 min. The advantages of this high pressure liquid chromatographic system over other methods of separation are discussed.     

Preparation of protein conjugates via intermolecular hydrazone linkage

Proteins can be modified at their amino groups under gentle conditions to contain an average of three to six aryl aldehyde or acyl hydrazide groups. These two types of modified proteins at about 10 microM concentration condense with each other at pH approximately 5 to form conjugates linked by hydrazone bonds. Under proper conditions conjugates mainly of dimers and trimers in size or, if desired, higher oligomers can be obtained. The conjugates can be dissociated to their individual protein components by an exchange reaction with an excess of acetyl hydrazide. The reversible hydrazone bonds of conjugates can be reduced with NaCNBH3 to give stable hydrazide bonds. The stability of protein-hydrazone conjugates was found to be significantly greater than that of the model compound, the N-acetylhydrazone of p-carboxybenzaldehyde. This difference is believed to result from the presence of multiple hydrazone linkages in protein conjugates.     

Preparation of reproducible alkaline phosphatase-antibody conjugates for enzyme immunoassay using a heterobifunctional linking agent

Conjugates of alkaline phosphatase (AP) and mouse monoclonal immunoglobulins G (IgG) were prepared by means of the heterobifunctional linker, N-succinimidyl 3-(2-pyridyldithio)-propionate. The efficiency of such conjugates can be improved by optimizing the degree of substitution of IgG and AP. We have determined conditions yielding better performing conjugates than those synthesized by methods described previously. Moreover, the results obtained with the technique presented here are quite reproducible with all four monoclonal antibodies tested.