A new technique for staining catecholic residues in biological samples

This technique for localizing catecholic residues in biological samples is based on the condensation of Besthorn's hydrazone (3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) with quinone residues obtained by the oxidation of catechols in the presence of ammonia. The product is a dark pink MBTH-quinone compound. This method is very sensitive and positive to catechol even at the 0.05 microgram level and the final product is chemically stable.     

Selective oxidation in vitro by myeloperoxidase of the N-terminal amine in apolipoprotein B-100.

In contrast to the multiple low abundance 2,4-dinitrophenylhydrazine-reactive tryptic peptides formed by oxidation of LDL with reagent HOCl in vitro, myeloperoxidase-catalyzed oxidation produces a dominant product in considerably greater yield and selectivity. This modified peptide had a single amino-terminal sequence corresponding to amino acids 53-66 of apolipoprotein B-100 (apoB-100), but its mass spectra indicated a significantly higher mass than could be reconciled with simple modifications of this peptide. Subsequent studies indicate that this product appears to result from N-chlorination of the N-terminal amino group of apoB-100 and dehydrohalogenation to the corresponding imine, which may form the hydrazone derivative directly, or after hydrolysis to the ketone. The methionine residue is oxidized to the corresponding sulfoxide, and the primary sequence peptide (residues 1-14 of apoB-100) is linked by the intramolecular disulfide bond between C-12 and C-61 to the peptide composed of residues 53-66, as we have observed previously (Yang, C-Y., T. W. Kim, S. A. Weng, B. Lee, M. Yang, and A. M. Gotto, Jr. 1990. Proc. Natl. Acad. Sci. USA. 87: 5523-5527) in unmodified LDL. The selective oxidation by myeloperoxidase of the N-terminal amine suggests strong steric effects in the approach of substrate to the enzyme catalytic site, an effect that may apply to other macromolecules and to cell surface molecules.     

Quantitative analysis of N-sulfated, N-acetylated, and unsubstituted glucosamine amino groups in heparin and related polysaccharides

A colorimetric procedure for quantitative determination of free and substituted glucosamine amino groups in heparin and related polysaccharides has been developed. The total content of hexosamine amino groups is determined by a modification of the method of Tsuji et al. (1969, Chem. Pharm. Bull. 17, 1505-1510); this method involves acid hydrolysis under conditions effecting complete removal of N-acetyl and N-sulfate groups, deaminative cleavage with nitrous acid, and colorimetric analysis of the resultant anhydromannose residues by reaction with 3-methyl-2-benzothiazolinone hydrazone (MBTH). N-sulfated glucosamine residues are cleaved selectively by treatment with nitrous acid at pH approximately 1.5 (J. E. Shively, and H.E. Conrad, 1976, Biochemistry 15, 3932-3942) and quantitated by the MBTH reaction. Under carefully controlled conditions, deamination at pH approximately 1.5 is highly specific for N-sulfated glucosamine residues, but an excess of reagent causes some cleavage of residues with unsubstituted amino groups as well. Deaminative cleavage at pH approximately 4.5 results in preferential degradation of unsubstituted glucosamine residues, but some cleavage (5-8%) of N-sulfated residues also occurs. However, analysis of the content of N-sulfated residues by the specific pH 1.5 procedure allows appropriate corrections to be made. From the value for total hexosamine content and the sum of N-sulfated and unsubstituted residues, the content of N-acetylated residues is calculated by difference. The modified deamination procedures, in combination with product analysis by the MBTH reaction, have been applied to several problems commonly encountered in the analysis and characterization of heparin.     

Hydrazone formation of 2,4-dinitrophenylhydrazine with pyrroloquinoline quinone in porcine kidney diamine oxidase

Homogeneous diamine oxidase (EC 1.4.3.6) from porcine kidney was treated with the inhibitor 2,4-dinitrophenylhydrazine (DNPH). The coloured compounds formed were detached with pronase and purified to homogeneity. When the reaction with DNPH was conducted under an O2 atmosphere, the product (obtained in a yield of 55%) was the C(5)-hydrazone of pyrroloquinoline quinone (PQQ) and DNPH, as revealed by its chromatographic behaviour, absorption spectrum and 1H-NMR spectrum. Only 6% of this hydrazone was formed under air, the main product isolated being an unidentified reaction product of DNPH with the enzyme. Porcine kidney diamine oxidase is the second mammalian enzyme shown to have PQQ as its prosthetic group. In view of the requirements for hydrazone formation with DNPH, it is incorrect to assume that inhibition of this type of enzymes with common hydrazines is simply due to blocking of the carbonyl group of its cofactor.     

Preparation of well-defined protein conjugates using enzyme-assisted reverse proteolysis

A two-step approach to the production of well-defined protein conjugates is described. In the first step, a linker group, carbohydrazide, having unique reactivity (a hydrazide group) is attached specifically to the carboxyl terminus by using enzyme-catalyzed reverse proteolysis. Since the hydrazide group exists nowhere else on the protein, specificity is assured in a subsequent chemical reaction (formation of a hydrazone bond) of the modified protein with a molecule (chelator, drug, or polypeptide) carrying an aldehyde or keto group. The product is sufficiently stable at neutral pH, no reduction of the hydrazone bond being necessary for the hydrazones described. Protein modification is thus restricted to the carboxyl terminus and a homogeneous product results. With insulin as a model, conditions are described for producing such well-defined conjugates in good yields. The use of other linker groups besides carbohydrazide, and applications of these techniques to antibody fragments, are discussed.     

Determination of erythrocyte surface sialic acid residues by a new colorimetric method.

The 3-methyl-2-benzothiazolone hydrazone method has been applied to the determination of erythrocyte membrane sialic acid residues. This method requires a mild oxidation of sialic acid which results in the formation of analogs. Their separation by chromatography, after labeling, allows the choice of the best conditions for this oxidation. The concomitant liberated formaldehyde is determined. This method requires no prior release of sialic acid as opposed to the periodate-thiobarbituric method of Warren (1959, J. Biol. Chem., 234, 1971–1975). These two methods have been compared.     

The active site structure of quinohemoprotein amine dehydrogenase inhibited by p-nitrophenylhydrazine

Quinohemoprotein amine dehydrogenase (QH-AmDH) catalyzes the oxidative deamination of aliphatic and aromatic amines. The enzyme from Pseudomonas putida has an alpha beta gamma heterotrimeric structure with two heme c groups in the largest alpha subunit, and a novel quinone cofactor [cysteine tryptophylquinone (CTQ)] and hitherto unknown internal cross-bridges in the smallest gamma subunit. The crystal structure of the enzyme in the complex with the inhibitor [p-nitrophenylhydrazine (pNPH)] has been determined at a 2.0 A resolution.(1) The hydrazone of the cofactor with the inhibitor was nicely modeled into the omit electron density map, identifying the C6 carbonyl group as the reactive site of the cofactor. The Asp33 gamma is unambiguously determined as the catalytic base to abstract the alpha-proton from a substrate, because N beta atom of the inhibitor corresponding to the C alpha atom of the substrate amine is neighbored to Asp33 gamma. The bound inhibitor is completely enclosed in the active site pocket formed by the residues from the beta- and gamma-subunits. The cofactor-inhibitor adduct may be predominantly in the hydrazone with the azo form as a minor component. The binding of the inhibitor causes minor but important conformational changes in the residues surrounding the active site. The inhibitor may have access to the active site pocket through the water-filled crevice between the beta- and gamma-subunits.     

Group X aldehyde dehydrogenases of Pseudomonas aeruginosa PAO1 degrade hydrazones

Hydrazones are natural and synthetic compounds containing a C=N-N moiety. Here we found that the opportunistic pathogen Pseudomonas aeruginosa PAO1 produced NAD(+)- or NADP(+)-dependent hydrazone dehydrogenase (HDH), which converts hydrazones to the corresponding hydrazides and acids rather than to the simple hydrolytic product aldehydes. Gene cloning indicated that the HDH is part of the group X aldehyde dehydrogenase (ALDH) family, which is distributed among bacteria, although the physiological roles of the ALDH family remain unknown. The PAO1 strain upregulated HDH in the presence of the hydrazone adipic acid bis(ethylidene hydrazide) (AEH). Gene disruption of the HDH-encoding hdhA (PA4022) decreased growth rates in culture medium containing AEH as the sole carbon source, and this effect was more obvious in the double gene disruption of hdhA and its orthologous exaC (PA1984), indicating that these genes are responsible for hydrazone utilization. Recombinant proteins of group X ALDHs from Escherichia coli, Paracoccus denitrificans, and Ochrobactrum anthropi also acted as HDHs in that they produced HDH activity in the cells and degraded hydrazones. These findings indicated the physiological roles of group X ALDHs in bacteria and showed that they comprise a distinct ALDH subfamily.     

Cross-linking between 16S ribosomal RNA and protein S4 in Escherichia coli ribosomal 30S subunits effected by treatment with bisulfite/hydrazine and bromopyruvate

Cytosine in nucleic acids can be modified by treatment with a mixture of bisulfite and hydrazine. The reaction is specific for single-stranded regions of nucleic acids and the product is N4-aminocytosine. Bromopyruvate has been used for alkylation of protein SH groups and through its 2-oxo group it can form a hydrazone with N4-aminocytosine. Escherichia coli ribosomal 30S subunits were treated with 1 M sodium bisulfite + 2 M hydrazine in the presence of 10 mM MgCl2 at pH 7.0 and 37 degrees C for 30 min. By this treatment, 2.4 cytosine residues/molecule 16S rRNA were derivatized into N4-aminocytosines. 35S-labeled 30S subunits were modified in this way and then treated with 10 mM bromopyruvate at pH 8.0 and 37 degrees C for 5 min. Analysis in sodium dodecyl sulfate/sucrose density gradient centrifugation showed co-sedimentation of a part of the 35S radioactivity with the RNA. The co-sedimentation was dependent on both the bisulfite/hydrazine and the bromopyruvate treatments. The RNA-protein complex was prepared from unlabeled 30S subunits. The protein portion was labeled with 125I, the RNA portion was digested with nucleases, and then the hydrazone linkage between the protein and oligonucleotides was cleaved by treatment with 0.2 M HCl. The oligonucleotides formed were removed by dialysis and the protein was identified as S4 by two-dimensional electrophoresis and by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The results indicate that the cysteinyl residue of protein S4 at position 31 from the N-terminus is located close to a cytosine residue which is non-base-paired and easily accessible by the externally present bisulfite/hydrazine reagent.     

A Rapid Method for Detection of Tyrosinase Activity in Electrophoresis

This rapid and sensitive method for localizing tyrosinase in polyacrylamide slab gels is based on the condensation of Bestthorn's hydrazone (3 methyl-4-benzothiazolinone hydrazone hydrochloride) with the quinone obtained by enzymatic oxidation of phenol. Both monophenolase and diphenolase activities are localized by this method.     

A rapid method for detection of tyrosinase activity in electrophoresis

This rapid and sensitive method for localizing tyrosinase in polyacrylamide slab gels is based on the condensation of Bestthorn's hydrazone (3 methyl-2-benzothiazolinone hydrazone hydrochloride) with the quinone obtained by enzymatic oxidation of phenol. Both monophenolase and diphenolase activities are localized by this method.     

Role of the interactions between the active site base and the substrate Schiff base in amine oxidase catalysis. Evidence from structural and spectroscopic studies of the 2-hydrazinopyridine adduct of Escherichia coli amine oxidase

2-Hydrazinopyridine (2HP) is an irreversible inhibitor of copper amine oxidases (CAOs). 2HP reacts directly at the C5 position of the TPQ cofactor, yielding an intense chromophore with lambda(max) approximately 430 nm (adduct I) in Escherichia coli amine oxidase (ECAO). The adduct I form of wild type (WT-ECAO) was assigned as a hydrazone on the basis of the X-ray crystal structure. The hydrazone adduct appears to be stabilized by two key hydrogen-bonding interactions between the TPQ-2HP moiety and two active site residues: the catalytic base (D383) and the conserved tyrosine residue (Y369). In this work, we have synthesized a model compound (2) for adduct I from the reaction of a TPQ model compound (1) and 2HP. NMR spectroscopy and X-ray crystallography show that 2 exists predominantly as the azo form (lambda(max) at 414 nm). Comparison of the UV-vis and resonance Raman spectra of 2 with adduct I in WT, D383E, D383N, and Y369F forms of ECAO revealed that adduct I in WT and D383N is a tautomeric mixture where the hydrazone form is favored. In D383E adduct I, the equilibrium is further shifted in favor of the hydrazone form. UV-vis spectroscopic pH titrations of adduct I in WT, D383N, D383E, and 2 confirmed that D383 in WT adduct I is protonated at pH 7 and stabilizes the hydrazone tautomer by a short hydrogen-bonding interaction. The deprotonation of D383 (pKa approximately 9.7) in adduct I resulted in conversion of adduct I to the azo tautomer with a blue shift of the lambda(max) to 420 nm, close to that of 2. In contrast, adduct I in D383N and D383E is stable and did not show any pH-dependent spectral changes. In Y369F, adduct I was not stable and gradually converted into a new species with lambda(max) at approximately 530 nm (adduct II). A detailed mechanism for the adduct I formation in WT has been proposed that is consistent with the mechanism proposed for the oxidation of substrate by CAOs but addresses some key differences in the active site chemistry of hydrazine inhibitors and substrate amines.     

Benzophenone semicarbazone protection strategy for synthesis of aza-glycine containing aza-peptides

Aza-glycine has been incorporated into peptide mimics as a tool for studying the active conformation and characterizing structure-function relationships for activity. Side reactions, such as intramolecular cyclizations to form hydantoins and oxadiazalones, have, however, inhibited efforts to make activated aza-Gly residues in solution using carbamate protection. Herein, we describe efficient incorporation of aza-glycine into aza-peptides using diphenyl hydrazone protection. Hydrazone acylation with p-nitrobenzyl chloroformate provided the protected aza-Gly activated ester, which was used to acylate a set of amino ester and amino acids to provide aza-Gly-Xaa aza-dipeptide fragments for peptide synthesis. Removal of the hydrazone protection was performed under acidic conditions to provide the hydrochloride salt of the aza-Gly residue for subsequent elongation of the aza-peptide chain using standard coupling conditions. A proof of concept for the use of benzophenone protection has been established by the synthesis of an aza-peptide analog of a potent activator of caspase 9 in cancer cells.     

Tuning the antiproliferative activity of biologically active iron chelators: characterization of the coordination chemistry and biological efficacy of 2-acetylpyridine and 2-benzoylpyridine hydrazone ligands

2-Pyridinecarbaldehyde isonicotinoyl hydrazone (HPCIH) and di-2-pyridylketone isonicotinoyl hydrazone (HPKIH) are two Fe chelators with contrasting biological behavior. HPCIH is a well-tolerated Fe chelator with limited antiproliferative activity that has potential applications in the treatment of Fe-overload disease. In contrast, the structurally related HPKIH ligand possesses significant antiproliferative activity against cancer cells. The current work has focused on understanding the mechanisms of the Fe mobilization and antiproliferative activity of these hydrazone chelators by synthesizing new analogs (based on 2-acetylpyridine and 2-benzoylpyridine) that resemble both series and examining their Fe coordination and redox chemistry. The Fe mobilization activity of these compounds is strongly dependent on the hydrophobicity and solution isomeric form of the hydrazone (E or Z). Also, the antiproliferative activity of the hydrazone ligands was shown to be influenced by the redox properties of the Fe complexes. This indicated that toxic Fenton-derived free radicals are important for the antiproliferative activity for some hydrazone chelators. In fact, we show that any substitution of the H atom present at the imine C atom of the parent HPCIH analogs leads to an increase in antiproliferative efficacy owing to an increase in redox activity. These substituents may deactivate the imine R–C=N–Fe (R is Me, Ph, pyridyl) bond relative to when a H atom is present at this position preventing nucleophilic attack of hydroxide anion, leading to a reversible redox couple. This investigation describes novel structure–activity relationships of aroylhydrazone chelators that will be useful in designing new ligands or fine-tuning the activity of others. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Accessibility of the carbohydrate moiety of membrane-bound rhodopsin to enzymatic and chemical modification

Galactose was specifically inserted into the carbohydrate moiety of rhodopsin by incubating retinal disk membranes with UDP-galactose: N-acetylglucosamine galactosyltransferase. The stoichiometry of labeling ranged from 1.2 to 1.8 (average = 1.5) residues of galactose per molecule of rhodopsin, indicating that some or all of the oligosaccharide chains of membrane-bound rhodopsin are readily accessible to enzymatic modification. These modified membranes were treated with galactose oxidase to generate an aldehyde at the C-6 position of the inserted galactose units. The enzymatically-oxidized membranes were then reacted with dansyl hydrazide to yield a fluorescent hydrazone which is sufficiently stable to permit spectroscopic analysis. This procedure for the specific attachment of a spectroscopic probe should be applicable to a wide variety of membrane glycoproteins.     

Mapping protein surface accessibility via an electron transfer dissociation selectively cleavable hydrazone probe

A protein's surface influences its role in protein-protein interactions and protein-ligand binding. Mass spectrometry can be used to give low resolution structural information about protein surfaces and conformations when used in combination with derivatization methods that target surface accessible amino acid residues. However, pinpointing the resulting modified peptides upon enzymatic digestion of the surface-modified protein is challenging because of the complexity of the peptide mixture and low abundance of modified peptides. Here a novel hydrazone reagent (NN) is presented that allows facile identification of all modified surface residues through a preferential cleavage upon activation by electron transfer dissociation coupled with a collision activation scan to pinpoint the modified residue in the peptide sequence. Using this approach, the correlation between percent reactivity and surface accessibility is demonstrated for two biologically active proteins, wheat eIF4E and PARP-1 Domain C.     

Hydrazinolysis of monosaccharides: a two-step synthesis of chiral pentane-1,2,3-triols from pentoses and observations on the hydrazinolysis of glycoproteins and glycopeptides

1,2-Dideoxyalditols, the corresponding 1-alkenes, and 1-deoxyalditols are formed in various proportions from d-glucose, d-mannose, l-arabinose, and d-xylose by the action of refluxing hydrazine. Sequential hydrazinolysis, catalytic hydrogenation, and chromatography afford a route to 1,2-dideoxyalditols. For example, 1,2-dideoxy-l-erythro-pentitol is formed from l-arabinose in 42% yield, and d-xylose is a source of 1,2-dideoxy-d-threo-pentitol (50%). Under the conditions (anhydrous hydrazine at 100° for 30 h in the absence of air) used by Montreuil for the hydrazinolysis of glycoproteins and glycopeptides, no 1,2-dideoxyalditol was formed; degradation was incomplete, there being some aldose hydrazone present. Under Kochetkov's hydrazinolysis conditions (105° for 10 h with hydrazinium sulphate), less degradation occurred and the product from d-galactose was identified as 1-deoxy-d-tagatose hydrazone.     

Synthesis and properties of new rubomycin derivatives

Condensation of rubomycin (daunorubicin) with respective hydrazides yielded novel substituted hydrazones: 13-cyanoacetyl hydrazone rubomycin, 13-L-phenylalanyl hydrazone rubomycin, 13-BOC-3-(uracilyl-1)-DL-alanyl hydrazone rubomycin and 13-BOC-3-(adenylyl-9)-DL-alanyl hydrazone rubomycin. With successive treatment of rubomycin with hydrazine hydrate and respective ketones novel asymmetric azines were prepared: 13-cyclopentylidene hydrazone rubomycin, 13-alpha,alpha'-dimethyl-cyclopentylidene hydrazone rubomycin and 13-(1-phenylethylidene-1) hydrazone rubomycin. 14-Adenylyl-N9-rubomycin was synthesized by interaction of 14-bromorubomycin with adenine and hydrogenation of its analog, 14-N-imidazolyl rubomycin by sodium borhydrite yielded 13-dihydro-14-N-imidazolyl rubomycin. There was observed correlation between the antimicrobial activity of the derivatives against B. mycoides and their cytostatic effect on the cells of murine leukemia NK/LI. The high in vitro activity of 13-cyclopentylidene hydrazone rubomycin showed satisfactory correlation with the results of the study on the antitumor effect in animals.     

Covalently bound pyrroloquinoline quinone is the organic prosthetic group in human placental lysyl oxidase.

Treatment of purified human placental lysyl oxidase with 2,4-dinitrophenylhydrazine (DNPH) resulted in a large spectral change and inhibition of enzyme activity. Proteolytic degradation of the derivatized enzyme yielded only one single coloured product, which was spectrally and chromatographically identical with the C-5 hydrazone of PQQ (pyrroloquinoline quinone) and DNPH. Since this represents the first example of a PQQ-containing enzyme in man, possible implications of the finding are discussed.     

Iron chelators of the pyridoxal isonicotinoyl hydrazone class Part I. Ionisation characteristics of the ligands and their relevance to biological properties

The orally effective iron chelator, pyridoxal isonicotinoyl hydrazone (PIH), and five analogues, pyridoxal benzoyl hydrazone (PBH), pyridoxal p-methoxybenzoyl hydrazone ((PpMBH), pyridoxal m-fluorobenzoyl hydrazone (PmFBH), 3-hydroxy- isonicotinaldehyde isonicotinoyl hydrazone (IIH) and salicylaldehyde isonicotinoyl hydrazone (SIH) were synthesised and characterised and their acid dissociation constants measured by glass electrode potentiometry and UV—Vis spectrophotometry. Analysis of the data showed that at physiological pH all of the ligands are predominantly (av. 80%) in the form of the neutral molecule, allowing passage through cell membranes and access to intracellular iron pools. The results are discussed in the context of the development of an orally effective iron chelator for clinical use.