Fluorometric determination of histamine with OPT: optimum reaction conditions and tests of identity

Histamine reacts with OPT at an alkaline pH giving fluorescent conjugation products. Optimum fluorophore formation was observed at pH 12.5 after 40 min reaction at 0°C under continuous gassing with nitrogen. After acidification with sulfuric acid to pH 2–5 the fluorescence was stable for hours. Reagent blanks were reduced by the lowering of the reaction temperature and by decreasing the amount of OPT added. These modifications permitted the determination of 2 ng histamine/ml and gave far better reproducibility than the original procedure of Shore, Burkhalter, and Cohn. The fluorometric assay for histamine is nonspecific; the major interfering OPT-reactive tissue component is believed to be spermidine. Specificity was secured by adding formaldehyde before acidification, thus abolishing the fluorescence of histamine but not that of spermidine, or by adding CdCl₂ or SrCl₂ together with OPT, thus preventing the formation of the spermidine fluorophore but not that of the histamine fluorophore.     

Fluorometric determination of spermidine using o-phthalaldehyde: optimum reaction conditions and tests of identity

Spermidine and histamine react with o-phthalaldehyde at alkaline pH, giving rise to fluorescent conjugation products that are stabilized by acidification to pH 2–4. The reaction has been used in the fluorometric assay of both these compounds. In the present study, the reaction conditions have been analyzed with the purpose of improving the sensitivity as well as the specificity of the spermidine assay. The sensitivity was improved by carrying out the condensation at pH 11.0–11.6 for 2 min at 100°C. Under these conditions, the lowest measurable amount of spermidine was 10 ng/ml, and the fluorescence of histamine (below 1–2 μg/ml) was nonmeasurable. Boiling the samples for 1 hr after the acidification did not affect the spermidine fluorescence but abolished residual histamine fluorescence. The spermidine fluorescence failed to develop in the presence of CdCl₂ or SrCl₂, whereas the histamine fluorescence was unaffected. Crude extracts of rat brain gave fluorescence readings similar to those of butanol extracts, suggesting that extensive purification of tissue sperimidine prior to assay is not always necessary.     

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.     

Polyamine cytochemistry

Summary o-Phtalaldehyde (OPT) reacts with a number of biologically important molecules, including the polyamines, spermidine and spermine. By systematically varying reaction conditions with respect to temperature, pH, concentration and length of exposure to the reagent, using both model systems and tissues, we have succeeded in constructing a cytochemical OPT-method specific for spermidine and spermine. The method detects cell types known to contain these polyamines, including growing and neoplastic cells. The staining pattern obtained with the OPT method is identical to that obtained with the formaldehyde-fluorescamine (FF) technique recently shown to be specific for spermidine and spermine. In contrast to the FF technique, the OPT method can be used for staining suspensions of isolated cells and may hence be employed in studies using fluorescence-activated cell sorting (FACS). Preliminary such studies show a pronounced decrease in cellular OPT-induced fluorescence, paralleled by a decrease in content of polyamines, after treatment with the polyamine biosynthesis inhibitor -difluoromethylornithine (DFMO). In contrast, cells simultaneously treated with DFMO+spermidine show pronounced increases in their spermidine content and parallel increases in their OPT-induced fluorescence. Availability of methods selectively demonstrating polyamines at the cellular and subcellular level is expected to aid our understanding of polyamine functions in normal growth and cancer.     

Some factors that influence the nonenzymatic glycation of peptides and polypeptides by glyceraldehyde

The rate of reaction of glyceraldehyde with a series of peptides was found to be dependent on their amino acid composition, sequence, and chain length. The presence of a histidine near the NH₂-terminal increased the rate of glycation, whereas the presence of a carboxyl group near the reaction site led to a decrease in reaction rate. In general, tripeptides reacted faster than dipeptides, and dipeptides reacted faster than amino acids. Sodium phosphate and 2,3-diphosphoglycerate enhanced the rate of reaction of glyceraldehyde with all the dipeptides tested. Sodium chloride inhibited the reaction in phosphate buffer, but not in HEPES buffer. The NH₂-terminal heptapeptide from the -chain of human hemoglobin A (HbA), where histidine is the second residue, reacted with glyceraldehyde faster than the NH₂-terminal hexapeptide from the -chain. The glycation of tetrameric human Hb by glyceraldehyde was found to be dependent on the ligation state of the protein since deoxy-HbA reacted about 50% more with glyceraldehyde than did liganded HbA. The enhanced glycation of deoxy HbA was mainly attributable to the more extensive reaction at the NH₂-terminal of the -chain. The presence of a histidine adjacent to the NH₂-terminal at this site may facilitate the Amadori rearrangement. The glycation of horse Hb in which the second residue is glutamine was not increased under anaerobic conditions.     

Isotopic microassay of histamine, histidine, histidine decarboxylase and histamine methyltransferase in brain tissue

Abstract— Microassays are described for histamine, histidine, and the activities of the enzymes histidine decarboxylase (EC 4.1.1.22) and histamine niethyltransferase (EC 2.1.1.8) in brain tissue. The enzymic-isotopic microassay for histamine is based on the methylation of tissue histamine by added histamine methyl-transferase and [¹⁴C]- or [³H]-labelled S-adenosyl-l -methionine. In a double-isotopic form of the assay, a tracer of [³H]histamine is employed along with [¹⁴C]S-adenosyl-l -methionine, and the ratio [¹⁴C]:[³H] reflects the amount of histamine in the sample. Because the methylation of histamine is uniform in brain samples studied, a single isotopic assay with [³H]S-adenosyl-l -methionine as the methyl donor is possible and increases sensitivity, so that 10 pg of tissue histamine can be estimated reliably. The assay for histidine involves decarboxylation of histidine by a bacterial histidine decarboxylase and measurement of the histamine formed by the enzymicisotopic procedure. In the histidine decarboxylase assay, histamine synthesized from added histidine is measured. The assay for histamine methyltransferase involves measuring the formation of [¹⁴C]methylhistamine with [¹⁴C]S-adenosyl-l -methionine serving as the methyl donor.     

4-Nitro-L-histidine as a substrate for histidine ammonia-lyase: the role of beta-hydrogen acidity in the rate-limiting step.

The K_m for the interaction of 4-nitro-L-histidine with histidine ammonia-lyase (reduced enzyme, pH 8.0) is comparable to that for L-histidine, while V_max is $\text{1}\text{8}$ that for the natural substrate. With the analog, addition of Cd⁺² effects a small decrease in K_m but fails to alter V_max; the normal deuterium isotope effect for removal of the β-hydrogen (1.5–2.0) is eliminated; and enzyme-catalyzed incorporation of solvent tritium into substrate occurs to a much greater extent than into histidine. Thus, the nitro group increases the acidity of the β-hydrogen and the stability of the conjugate carbanion to such a degree that CH bond cleavage now precedes rate-limiting CN bond cleavage.     

Solid phase synthesis of somatostatin-28

The synthesis of ovine hypothalamic somatostatin-28 (Ser-Ala-Asn-Ser-Asn-Pro-Ala-Met-Ala-Pro-Arg-Glu-Arg-Lys-Ala-Gly-$\text{Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys}$-OH) has been accomplished by solid phase methodology. The structure of the synthetic material was verified by: (1) direct sequence analysis with a Beckman 89°C sequencer, (2) correlation of the amino acid analyses of the isolated tryptic peptide fragments with their theoretical compositions, and (3) comparison, using high performance liquid chromatography, of the synthetic methionine-sulfoxide and methionine-sulfone modified NH₂-terminal peptides (residues 1–11) with the corresponding tryptic fragment from somatostatin-28.     

Accurate and efficient method for quantification of urinary histamine by gas chromatography negative ion chemical ionization mass spectrometry

Because of the recognized inaccuracy and unreliability of currently available methods for the quantification of histamine in biological fluids, a method for quantification of urinary histamine by stable isotope dilution assay with negative ion chemical ionization mass spectrometry has been developed. Following the addition of [²H₄]histamine to 1 ml of urine, histamine is extracted into butanol, back-extracted into HCl, derivatized to the pentafluorobenzyl derivative (CH₂C₆F₅)₃-histamine, extracted into methylene chloride, and then quantified with negative ion chemical ionization mass spectrometry by selected ion monitoring of the ratio of ions $\text{m}\text{z}$$\text{430}\text{434}$. Twenty samples can be assayed in 2 days. Precision of the assay is ±2.7% and the accuracy is 97.6%. Lower limits of sensitivity are approximately 100–500 fg injected on-column. This assay provides a very sensitive, accurate, and efficient method for the quantification of histamine in human urine.     

Cyanamide mediated syntheses of peptides containing histidine and hydrophobic amino acids

Summary Using the model of a primitive earth evaporation pond, the synthesis of three histidyl peptides in yields of up to 11% was demonstrated when aqueous solutions of histidine, leucine, ATP, cyanamide, and MgCl₂ were evaporated and heated for 24 h at 80°C. In addition, peptides were formed in yields of up to 56%, 35%, and 21%, respectively for phenylalanine, leucine, and alanine when aqueous solutions of the appropriate amino acid were evaporated and heated with cyanamide and one or more of the following components: ATP, AMP, 4-amino-5-imidazole carboxamide, or MgCl₂. The greatest peptide yield occurred at pH 3. But peptide formation was demonstrated for a system of Leu, cyanamide, and MgCl₂ adjusted to pH 7 with NH₄OH.Peptide synthesis was also studied in the presence of CaCl₂, ZnCl₂, different adenosine nucleotides, and UTP to compare their effects on peptide synthesis. The optimum conditions for cyanamide mediated peptide synthesis were also studied in terms of pH, reaction time, reaction temperature, and cyanamide concentration. The major side product in nearly all reactions studied appears to be an amino acid-cyanamide adduct. Peptides were analyzed and identified by thin layer chromatography, acid hydrolysis, and enzymatic degradation.     

Response of human and chick kidney adenylate cyclase to biologically active synthetic fragments of human parathyroid hormone

The 34-amino acid NH₂-terminal fragment of human parathyroid hormone synthesized according to the sequence described by Niall $\text{et al.}$ (1) is approximately 140 times more potent than the fragment synthesized according to Brewer $\text{et al}$ (2) in activating human renal cortex adenylate cyclase. The potencies of the two peptides, relative to the effect of MRC standard bovine parathyroid hormone preparation $\text{67}\text{342}$ in this system, were 5600 ± 600 (S.E.M.) units/mg and 40 ± 5 units/mg respectively. The potencies of the more active peptide and the corresponding bovine parathyroid hormone sequence were similar in this system and also in assays based upon the production of cyclic AMP by chick kidney both $\text{in vivo}$ and $\text{in vitro}$.     

Arginyl-tRNA-protein transferase in eucaryotic protists

Soluble extracts of $\text{Saccharomyces cerevisiae}$ and $\text{Blastocladiella emersonii}$ were found to catalyze the specific transfer of arginine from a mixture of [¹⁴C] aminoacyl-tRNAs into protein. Arginine transfer was stimulated by bovine serum albumin. Glu-Ala, Asp-Ala and cystinyl-$\text{bis}$-Ala inhibited incorporation into protein, whereas dipeptides with other NH₂-terminal residues linked to alanine did not. These results indicate the presence of an enzyme in eucaryotic protists with the same donor and acceptor specificity as mammalian arginyl-tRNA-protein transferase.     

1H-N.m.r. and c.d. investigation of the histidine side chain conformation in small peptides

Three series of model peptides containing histidine have been examined by ¹H-n.m.r. and c.d. spectroscopy: X-His peptides with X = Gly, Ala, Leu; His-X peptides with X = Gly, Ala, Leu, Ser, Lys, Phe, Tyr; and Pro-His-X peptides with X = Gly; Ala; Leu; Val; Phe; Tyr, C.d. spectra were obtained for pH values between 1 and 11 to give titration curves [θ] vs. pH; ¹H-n.m.r. spectra were recorded at four selected pH values corresponding to defined ionic species. ¹H-n.m.r. spectra in Me₂SO of the NH₃⁺, Imid⁺, COO^? ionic state (pH 4.5) were also obtained. The histidine side chain conformation in the various peptides and the changing ionic states is reflected in the ³J_αβ,β′ coupling constants, the Δδ ββ′ anisochrony values and the c.d. histidine chromophore contribution at 215 nm, and qualitative and semiquantitative correlations can be established between these parameters. Whereas the histidine side chain conformation is quite different in each of the three series, and varies with the ionic state and environment, it is practically identical for each peptide within a series: the nature of the X-residue does not exert any influence on the histidine side chain conformational behaviour. Thus, the classical rotamer distribution R I > R II > R III which is due to steric factors is usually observed unless specific intramolecular interactions such as hydrogen or ionic bonds override these.     

The amino terminal sequences of acid proteases-human pepsin and gastricsin and the protease of Rhizopus chinensis.

The amino acid sequences near the amino termini of human pepsin (34 residues) and gastricsin (24 residues) and the acid protease from $\text{Rhizopus chinensis}$ (27 residues) have been determined using automated Edman degradation. From these results three additional observations were made. First, two structural variants have been observed for human gastricsin and for the $\text{Rhizopus}$ protease. Both cases are apparently genetic in origin. Second, a stretch of sequence in the $\text{Rhizopus}$ protease, residues 14 to 26, is highly homologous to the known sequence of porcine pepsin at the region of residues 11 to 23. Third, the sequences of the NH₂-terminal region of human pepsin and gastrisin are homologous.     

Nitrate reductase from Penicilliumchrysogenum: Kinetic mechanism at sub-optimum pH

The steady-state kinetics of the NADPH + FAD-dependent reduction of nitrate by nitrate reductase from $\text{Penicillium}\text{chrysogenum}$ was studied at pH 6.18. At this sub-optimum pH, V_max was about 83 units × mg protein^?1 compared with 225 units × mg protein^?1 at pH 7.20. All initial velocity reciprocal plot patterns at pH 6.18 as well as the NADP⁺/nitrate product inhibition pattern were intersecting. In contrast, the NADP(H)/nitrate plots at pH 7.20 were parallel (Renosto, F. $\text{et}\text{al.}$ J. Biol. Chem. $\text{256}$, 8616, 1981). A major effect of lowering the assay pH was to change the K_m for FAD from 0.17 μM at pH 7.20 to 4 μM at pH 6.18. The results suggest that nitrate reductase has a steady-state random kinetic mechanism in which k_cat in the forward direction at pH 7.20 (ca. 375 sec^?1) is greater that k_off for the dissociation of one or more substrates. Several observations suggest that k_off for FAD is extremely small at pH 7.20.     

A microassay for mammalian histidine decarboxylase.

This report describes a microassay procedure for mammalian histidine decarboxylase (HDC) based on the measurement of [¹⁴C]O₂ formed from l-[1-¹⁴C]histidine. This assay is particularly useful for quick measurement of HDC activity both in microgram quantities of cell or tissue extract and in tissues that contain significant levels of endogenous histamine.Using this assay, we have shown that the pH optimum, K_m and thermolability of HDC are similar for extracts prepared both from normal rat peritoneal mast cells and from the Furth mouse mastocytoma. HDC activity could be detected in homogenates prepared from 10⁵ rat mast cells, and it was expressed on a per cell basis. Mast cell HDC activity varied with the strain of rat from which the cells were obtained and with the season when they were assayed.     

Chiroptical properties of fluorescamine condensation compounds with dipeptides in situ

The free amino group of a dipeptide reacts efficiently with fluores-camine (FLURAM^®) to form pyrrolinone-type chromophores with long wavelength absorption maxima in the 380 nm region. A simple test tube procedure is described which allows $\text{in situ}$ determination of the absolute configuration of the NH₂-terminal amino acids of the dipeptides based on the chiroptical properties of their chromophoric derivatives.     

Role of histidine residues in ovine lutropin: effects on steroidogenic activity.

The modification of histidine residues of ovine pituitary lutropin by rose bengal sensitized photooxidation has been investigated. The destruction of an average of one histidine out of six lead to 90% loss of biological activity as examined by the $\text{in}\text{vitro}$ steroidogenic response in the rat Leydig cell essay. Further modification of an average 2 – 3 histidine residues reduced the biological activity to less than 1% of the native lutropin. The modified lutropin was incapable of inhibiting the native lutropin induced steroidogenesis. Gel filtration experiments and polyacrylamide disc gel electrophoresis patterns indicated that no dissociation of the molecule into subunits occurred. This is the first report on the essentiality of the histidine residue for the activity of lutropin.     

Effect of cyclic AMP on RNA and protein synthesis in Candida albicans.

Sequence analysis by the automated Edman degradation shows that dopamine $\text{β}$-hydroxylase (dopamine $\text{β}$-monooxygenase; EC 1.14.17.1) from bovine adrenal medulla contains equal amounts of NH₂-terminal alanine and serine residues. The sequence data are in agreement with the proposal that this enzyme consists of two types of polypeptide chains which are identical in the NH₂-terminal ends, except that one of the chains lack the NH₂-terminal tripeptide Ser-Ala-Thr.