Kinetic analysis of the fluorescence reaction of histamine with orthophthalaldehyde

Histamine reacts with orthophthalaldehyde (OPA) in an alkaline medium to form an unstable fluorescent adduct (F_base). Acidification of the solution gives a stable adduct (F_acid). In order to elucidate the mechanism of this fluorescence reaction, a kinetic study of this reaction was carried out. Although F_base was believed to be the precursor of F_acid, it was shown not to be the precursor of F_acid owing to the effects of the reaction time in an alkaline medium and OPA concentration on the yields of F_base and F_acid. The kinetic analysis of the formation and degradation of F_base revealed the pathway of the fluorescence reaction. On the basis of the results obtained in this study, the mechanism of the fluorescence reaction is proposed.     

Improved fluorometric assay of histamine: condensation with O-phthalaldehyde at -20 degrees C

Histamine reacts with OPT at an alkaline pH giving rise to fluorescent conjugation products. Optimum fluorophore formation was observed at pH 12.5 after 10 hr under nitrogen at ?20°C, i.e., in the frozen state. After acidification with sulfuric acid to pH 2.5 the resulting fluorescence, read at room temperature, was stable for hours. The procedure now measures as little as 1 ng histamine/ml and is much more specific than the conventional fluorometric assay. Spermidine did not interfere with the assay of histamine, and histidine only if present in great excess over histamine. It could be shown that with deproteinized extracts of rat gastric mucosa, histamine could be estimated without further purification, which means saving a lot of time and labor.     

Improved fluorometric assay of histamine: Condensation with O-phthalaldehyde at −20°C

Histamine reacts with OPT at an alkaline pH giving rise to fluorescent conjugation products. Optimum fluorophore formation was observed at pH 12.5 after 10 hr under nitrogen at −20°C, i.e., in the frozen state. After acidification with sulfuric acid to pH 2.5 the resulting fluorescence, read at room temperature, was stable for hours. The procedure now measures as little as 1 ng histamine/ml and is much more specific than the conventional fluorometric assay. Spermidine did not interfere with the assay of histamine, and histidine only if present in great excess over histamine. It could be shown that with deproteinized extracts of rat gastric mucosa, histamine could be estimated without further purification, which means saving a lot of time and labor.     

Development of an assay for histamine using automated high-performance liquid chromatography with electrochemical detection

Previous studies have measured histamine by derivatization with o-phthaldialdehyde (OPA) and mercaptoethanol (ME), followed by reversed-phase HPLC separation and electrochemical detection. The derivatization product, however, was very unstable. In the present study, inclusion of less polar solvents (e.g., acetonitrile or tetrahydrofuran) in the OPA/ME derivatization reaction produced an OPA/ME-histamine product that was stable for many hours. Changes of the HPLC mobile phase (increasing its ionic strength and pH and including triethylamine) dramatically improved the chromatography and reduced the histamine detection limit to <0.1 pmol. The modified assay was suitable for batchwise manual derivatization of histamine samples followed by their automated analysis by HPLC with an automatic injector.     

Allisonella histaminiformans gen. nov., sp. nov. A novel bacterium that produces histamine,utilizes histidine as its sole energy source,and could play a role in bovine and equine laminitis

When cattle and horses are fed large amounts of grain, histamine can accumulate in the gastrointestinal tract, and this accumulation can cause an acute inflammation of the hooves (laminitis). When ruminal fluid from dairy cattle fed grain supplements was serially diluted in anaerobic MRS medium containing histidine (50 mM), histamine was detected at dilutions as high as 10(-7). The histidine enrichments were then transferred successively in an anaerobic, carbonate-based medium (50 mM histidine) without glucose. The histamine producing bacteria could not be isolated from the rumens of cattle fed hay; however, histamine producing bacteria could be isolated the feces of cattle fed grain and the cecum of a horse. All of the histamine producing isolates had the same ovoid morphology. The cells stained Gram-negative and were resistant to the ionophore, monensin (25 microM). The doubling time was 110 min, and the yield was 1.5 mg cell protein per mmol histidine. The G+C content was 46.8%. Lysine was the only other amino acid used, but lysine did not allow growth if histidine was absent. Because carbohydrate and organic acid utilization was not detected, it appeared that the isolates used histidine decarboxylation as their sole mechanism of energy derivation. 16s rRNA gene sequencing indicated that the isolates were most closely related to low G+C Gram-positive bacteria (firmicutes), but similarities were < or = 94%. Because the most closely related bacteria (Dialister pneumonsintes, Megasphaera elsdenii and Selenomonas ruminantium) did not produce histamine from histidine, we propose that these histamine producing bacteria be assigned to a new genus, Allisonella, as Allisonella histaminiformans gen. nov., sp. nov. The type strain is MR2 (ATCC BAA610, DSM 15230).     

Spin trapping artifacts in DMSO

Spin-trapping experiments in alkaline aqueous dimethyl sulfoxide (DMSO) solution using sodium 3,5-dibromo-4-nitrosobenzenesulfonate (DBNBS) yielded a strong signal of the sulfur trioxide anion radical adduct. This radical adduct is identical to that obtained by the oxidation of sulfite with horseradish peroxidase/hydrogen peroxide and subsequent spin trapping with DBNBS. This radical adduct is very stable, and satellite peaks of the natural abundance 13C and 33S could be obtained. Apparently, under alkaline conditions DMSO decomposes in air to form the sulfur trioxide anion radical. A comparison with a recent publication shows that this DMSO-derived radical adduct has been misassigned as a uniquely stable spin adduct of superoxide (Ozawa and Hanaki (1986) Biochem. Biophys. Res. Commun. 136, 657-664).     

Analysis of amino acids: neurochemical application

For high sensitivity analysis of neuroactive amino acids, liquid chromatography employing precolumn derivatisation with o-phthalaldehyde (OPA) is suitable for several reasons. The OPA reagent is non-fluorescent per se, the reaction occurs rapidly in alkaline aqueous solutions and forms highly fluorescent derivatives with primary amines.     

Immunocytochemical colocalization of histamine, histidine decarboxylase, 5-hydroxytryptamine and tyrosine hydroxylase in the superior cervical ganglion of the rat

Summary The coexistence of histamine, histidine decarboxylase (the enzyme synthesizing histamine), 5-hydroxytryptamine and tyrosine hydroxylase (the rate-limiting enzyme in catecholamine synthesis), was studied in the rat superior cervical ganglion with the indirect immunofluorescence method. Possible colocalization was examined by staining consecutive sections with two different antibodies, or alternatively in the same section by eluting the first antibody with a mild solution containing potassium permanganate and sulphuric acid, and by staining the same section with another antibody. It was shown that tyrosine hydroxylase immunoreactivity was found both in large principal nerve cells and in small cells, which on the basis of their size and high nucleus—cytoplasm ratio corresponded to small intensely fluorescent (SIF) cells. Histamine, histidine decarboxylase and 5-hydroxytryptamine immunoreactivities were observed only in SIF cells. Those SIF cells which were immunoreactive for histamine, histidine decarboxylase or 5-hydroxytryptamine also contained tyrosine hydroxylase immunoreactivity. On the other hand, all tyrosine hydroxylase-immunoreactive SIF cells were also immunoreactive for histidine decarboxylase or 5-hydroxytryptamine. Some of the SIF cells, which were non-reactive for histamine, were immunoreactive for tyrosine hydroxylase.     

Generation of a proton motive force by histidine decarboxylation and electrogenic histidine/histamine antiport in Lactobacillus buchneri.

Lactobacillus buchneri ST2A vigorously decarboxylates histidine to the biogenic amine histamine, which is excreted into the medium. Cells grown in the presence of histidine generate both a transmembrane pH gradient, inside alkaline, and an electrical potential (delta psi), inside negative, upon addition of histidine. Studies of the mechanism of histidine uptake and histamine excretion in membrane vesicles and proteoliposomes devoid of cytosolic histidine decarboxylase activity demonstrate that histidine uptake, histamine efflux, and histidine/histamine exchange are electrogenic processes. Histidine/histamine exchange is much faster than the unidirectional fluxes of these substrates, is inhibited by an inside-negative delta psi and is stimulated by an inside positive delta psi. These data suggest that the generation of metabolic energy from histidine decarboxylation results from an electrogenic histidine/histamine exchange and indirect proton extrusion due to the combined action of the decarboxylase and carrier-mediated exchange. The abundance of amino acid decarboxylation reactions among bacteria suggests that this mechanism of metabolic energy generation and/or pH regulation is widespread.     

A microplate fluorescence assay for DAPA aminotransferase by detection of the vicinal diamine 7,8-diaminopelargonic acid

7,8-Diaminopelargonic acid (DAPA) aminotransferase is an enzyme of the biotin biosynthetic pathway that plays an essential role in Mycobacterium tuberculosis virulence. Inhibition of this enzyme is a potential strategy to combat this microorganism, the causative agent of tuberculosis. To identify new inhibitors as potential drugs, a simple enzymatic assay for high-throughput screening (HTS) is needed. Several methods for measuring DAPA aminotransferase activity are already available. However, requirements for their implementation for HTS are tedious. We describe here a microplate fluorescence assay for DAPA aminotransferase that is simple, cheap, and sensitive, allowing linear detection of DAPA in the range of 20 nM to 50 μM. The principle of the method is the direct detection in the enzymatic reaction mixture of the vicinal diamine DAPA derivatized with ortho-phthalaldehyde (OPA) and 2-mercaptoethanol (2ME). The assay was validated with the known inhibitor desmethyl-KAPA (8-amino-7-oxopelargonic acid) and adapted to microplate for HTS. The structure of the stable fluorescent adduct formed between a vicinal primary diamine and OPA in the presence of 2ME was characterized by mass spectrometry and nuclear magnetic resonance spectroscopy.     

Histidine decarboxylase of Lactobacillus 30a: function and reactivity of sulfhydryl groups.

Two classes of sulfhydryl groups in histidine decarboxylase from Lactobacillus 30 a can be differentiated by their reaction with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Five cysteinyl residues (class I) of the native enzyme are titrated by DTNB as the pH of the reaction medium is increased from 6.5 to 7.5; the pH-rate profile for their reaction is described by a pKa of 9.2. An additional five thiol groups (class II) are titrated only when denaturing agents are added above neutral pH. Histidine decarboxylase is completely inactivated by DTNB in a kinetically second-order process (Kapp = 660 +/- 20 M-1 min-1 at pH 7.6 and 25 degrees C) which occurs coincident with and at the same rate as modification of the five class-I SH groups of the enzyme, i.e., one thiol group per pyruvoyl prosthetic group. The competitive inhibitors, histamine and imidazole, markedly enhanced the reactivity of these cysteinyl residues toward DTNB; this enhancement is accompanied by a concomitant increase in the rate of inactivation. A single SH group in each of the five catalytic units of histidine decarboxylase is thus implicated as being critical for the expression of enzymatic activity.     

Structural study reveals that ser-354 determines substrate specificity on human histidine decarboxylase

Histamine is an important chemical mediator for a wide variety of physiological reactions. l-Histidine decarboxylase (HDC) is the primary enzyme responsible for histamine synthesis and produces histamine from histidine in a one-step reaction. In this study, we determined the crystal structure of human HDC (hHDC) complexed with the inhibitor histidine methyl ester. This structure shows the detailed features of the pyridoxal-5'-phosphate inhibitor adduct (external aldimine) at the active site of HDC. Moreover, a comparison of the structures of hHDC and aromatic l-amino acid (l-DOPA) decarboxylase showed that Ser-354 was a key residue for substrate specificity. The S354G mutation at the active site enlarged the size of the hHDC substrate-binding pocket and resulted in a decreased affinity for histidine, but an acquired ability to bind and act on l-DOPA as a substrate. These data provide insight into the molecular basis of substrate recognition among the group II pyridoxal-5'-phosphate-dependent decarboxylases.     

Nucleophilic addition to the 9 position of 9-phenylcarboxylate-10-methylacridinium protects against hydrolysis of the ester

The chemiluminescent reaction of an acridinium ester (AE) requires addition of peroxide to the 9 position of the acridinium ring. The addition of a hydroxide ion to the 9 position of an acridinium ester to form the carbinol adduct has also been well documented. We have observed a similar addition of other nucleophiles to the acridinium ring to form an acridan adduct. The adduct formed with bisulphite has been particularly well-characterized for rate of formation, rate of reversion, and reaction equilibrium. The formation of an adduct (other than H₂O₂) has been demonstrated to decrease significantly the reactivity of the adjacent ester bond to alkaline hydrolysis. The resulting, more stable adduct is very useful when the acridinium ester is used as a label in DNA probe-based assays. The adduct is highly resistant to hydrolysis under the conditions often desired for DNA probe-based assays (high temperature, elevated pH, extended storage).     

Fluorometric determination of urea by flow injection analysis.

Urea was determined using fluorometry with flow injection analysis. O-phthalaldehyde (OPA) reacts with enzymatically generated ammonia and sulfite in alkaline medium to give a highly fluorescent compound that has an excitation wavelength of 372 nm and an emission wavelength of about 430 nm. The method is more selective to ammonia than the one which uses mercaptoethanol in place of the sulfite. Urease was immobilized to a Pall Immunodyne membrane which is commercially available. The immobilization occurs through covalent bonding which results in a highly stable enzyme preparation. The enzymatic membrane was fitted in a 5 cm long, 0.125 inch o.d. Teflon tubing which served as the enzymatic reactor. The system is difficult to use for the analysis of urea in serum because some compounds normally present in serum fluoresce at the same wavelength. This results in higher values for urea. If the reaction system is to be used for the evaluation of urea in serum, a blank should be run so that urea concentration can be calculated by difference.     

Analysis of the decarboxylation step in mammalian histidine decarboxylase. A computational study

We report a hybrid quantum mechanics/molecular mechanics theoretical study on the reaction mechanism of mammalian histidine decarboxylase that allows us to obtain valuable insights on the structure of the cofactor-substrate adduct (external aldimine) in the active site of rat histidine decarboxylase. By means of molecular dynamics simulations, we traced the potential of mean force corresponding to the decarboxylation reaction of the adduct both in the active site of the enzyme and in aqueous solution. By comparing this process in both media, we have identified the key electrostatic interactions that explain the lowering of the free energy barrier in the enzyme. Our analysis also offers a validation of Dunathan's hypothesis (Dunathan, H. (1966) Proc. Natl. Acad. Sci. U. S. A. 55, 712-716) regarding the role of stereoelectronic effects in the enzymatic decarboxylation process.     

Elimination by formaldehyde of interference with 3-methylhistidine determination: Application of the method to the study of muscle protein degradation

The fluorescent adduct of histidine, but not of 3-methylhistidine, with orthophthalaldehyde is destroyed by reaction with formaldehyde. This is used as the basis for an improved method of detection of 3-methylhistidine in the column effluent of an amino acid analyser. The method is capable of detecting free 3-methylhistidine in physiological fluids in which its analysis by chromatographic methods was previously very difficult due to insufficiently good resolution between histidine and 3-methylhistidine. The method has been used to detect changes of 3-methylhistidine concentration in intramuscular water and in plasma in a variety of circumstances of altered myofibrillar degradation rate.     

Adduct formation between sulfite and the flavin of phototrophic bacterial flavocytochromes c. Kinetics of sequential bleach, recolor, and rebleach of flavin as a function of pH.

The kinetics of sulfite adduct formation with the bound flavin in flavocytochromes c from the purple phototrophic bacterium Chromatium vinosum and the green phototrophic bacterium Chlorobium thiosulfatophilum have been investigated as a function of pH. Both species of flavocytochrome c rapidly react with sulfite to form a flavin sulfite adduct (k = 10(3)-10(5) M-1 s-1) which is bleached at 450-475 nm and has associated charge-transfer absorbance at 660 nm. The rate constant for adduct formation in flavocytochrome c is 2-4 orders of magnitude faster than for model flavins of comparable redox potential and is likely to be due to a basic residue near the N-1 position of the flavin, which not only raises the redox potential but also stabilizes the negatively charged adduct. There is a pK for adduct formation at 6.5, which suggests that the order of magnitude larger rate constant at pH 5 as compared to pH 10 in flavocytochrome c is due the influence of another positive charge, possibly a protonated histidine residue. The adduct is indefinitely stable at pH 5 but decomposes (the flavin recolors) in a first-order process accelerating above pH 6 (at pH 10, k = 0.1 s-1). The pK for recoloring is 8.5, which is suggestive of a cysteine sulfhydryl. On the basis of the observed pK and available chemical information, we believe that recoloring is due to a secondary effect of the reaction of sulfite with a protein cystine disulfide, which is adjacent to the flavin.(ABSTRACT TRUNCATED AT 250 WORDS)     

邻苯二甲醛(OPA)法检测铝吸附疫苗中抗原含量

目的:建立快速测定铝胶吸附的疫苗中抗原含量的OPA荧光检测法。 方法:利用邻苯二甲醛(OPA)在2-巯基乙醇存在下与氨基酸的N端或L-谷氨酸侧链反应,在460nm处生成荧光衍生物的原理,建立了无需进行抗原蛋白提取的检测方法,并对该方法的特异性、线性、精密度、回收率、重复性进行考察。同时,配合钠十二烷基的硫酸盐-聚丙烯酰胺凝胶电泳法(SDS-PAGE),研究在配制过程中抗原蛋白与佐剂是否分离。结果: OPA荧光法,在抗原蛋白含量为0.02~0.16mg/ml时,线性良好,准确度达90%~115%,批内和批间相对标准偏差≤15%。电泳结果显示,CpG的加入可能会导致少量抗原蛋白从铝胶中解离。结论:该方法快速、准确、灵敏度较高,重复性好,可以应用于含铝胶疫苗制剂的实验室甚至生产质控过程。     

Comparison of differential plating media and two chromatography techniques for the detection of histamine production in bacteria

The bacterial enzyme histidine decarboxylase (Hdc) catalyses the conversion of histidine into histamine. This amine is essential for the biosynthesis of iron chelators (siderophores) and is an important cause of food poisoning after consumption of fish contaminated with histamine-producing bacteria. In this work we compared different methods for detecting histamine secreted by different bacterial strains. The presence of histamine in the culture supernatant of Vibrio anguillarum, which produces Hdc and secretes the histamine-containing siderophore anguibactin, was detected by thin-layer chromatography. Similar results were obtained using the culture supernatant of the Acinetobacter baumannii 19606 prototype strain that secretes the histamine-containing siderophore acinetobactin. Conversely, histamine was not detected in the culture supernatant of an isogenic V. anguillarum Hdc mutant and the A. baumannii 8399 strain that secretes a catechol siderophore different from anguibactin and acinetobactin. These results were confirmed by capillary gas chromatography/mass spectrometry. However, all these strains tested positive for histamine secretion when cultured on differential plating media containing histidine and a pH indicator, which were specifically designed for the detection of histamine-producing bacteria. The pH increase of the medium surrounding the bacterial colonies was however drastically reduced when the histidine-containing medium was supplemented with peptone, beef extract, and glucose. The histidine-containing culture supernatants of the A. baumannii and V. anguillarum strains showed an increase of about two units of pH, turned purple upon the addition of cresol red, and contained high amounts of ammonia. Escherichia coli strains, which are Hdc negative and do not use histidine as a carbon, nitrogen, and energy source, gave negative results with the differential solid medium and produced only moderate amounts of ammonia when cultured in the presence of excess histidine. This study demonstrates that, although more laborious and requiring some expensive equipment, thin-layer and gas chromatography/mass spectrometry are more accurate than differential media for detecting bacterial histamine secretion. The results obtained with these analytical methods are not affected by byproducts such as ammonia, which are generated during the degradation of histidine and produce false positive results with the differential plating media.     

Conformation and polarity of the active site of xylanase I from Thermomonospora sp. as deduced by fluorescent chemoaffinity labeling. Site and significance of a histidine residue.

A fluorescent chemoaffinity label o-phthalaldehyde (OPTA) was used to ascertain the conformational flexibility and polarity at the active site of xylanase I (Xyl I). The kinetics of inactivation of Xyl I with OPTA revealed that complete inactivation occurred due to the binding of one molecule of OPTA to the active site of Xyl I. The formation of a single fluorescent isoindole derivative corroborated these findings. OPTA has been known to form a fluorescent isoindole derivative by crosslinking the proximal thiol and amino groups of cysteine and lysine. The involvement of cysteine in the formation of a Xyl I-isoindole derivative has been negated by fluorometric and chemical modification studies on Xyl I with group-specific reagents and by amino-acid analysis. The kinetic analysis of diethylpyrocarbonate-modified Xyl I established the presence of an essential histidine at or near the catalytic site of Xyl I. Modification of histidine and lysine residues by diethylpyrocarbonate and 2,4,6-trinitrobenzenesulfonic acid, respectively, abolished the ability of the enzyme to form an isoindole derivative with OPTA, indicating that histidine and lysine participate in the formation of the isoindole complex. A mechanism for the reaction of OPTA with histidine and lysine residues present in the protein structure has been proposed. Experimental evidence presented here suggests for the first time that the active site of Xyl I is conformationally more flexible and more easily perturbed in the presence of denaturants than the molecule as a whole. The changes in the fluorescence emission maxima of a model compound (isoindole adduct) in solvents of different polarity were compared with the fluorescence behaviour of the Xyl I-isoindole derivative, leading to the conclusion that the active site is located in a microenvironment of low polarity.