- Pyridoxamine-5-phosphate enzyme-linked immune mass spectrometric assay substrate for linear absolute quantification of alkaline phosphatase to the yoctomole range applied to prostate specific antigen
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There is a need to measure proteins that are present in concentrations below the detection limits of existing colorimetric approaches with enzyme-linked immunoabsorbent assays (ELISA). The powerful enzyme alkaline phosphatase conjugated to the highly specific bacterial protein streptavidin binds to biotinylated macromolecules like proteins, antibodies, or other ligands and receptors with a high affinity. The binding of the biotinylated detection antibody, with resulting amplification of the signal by the catalytic production of reporter molecules, is key to the sensitivity of ELISA. The specificity and amplification of the signal by the enzyme alkaline phosphatase in ELISA together with the sensitivity of liquid chromatography electrospray ionization and mass spectrometry (LC-ESI-MS) to detect femtomole to picomole amounts of reporter molecules results in an ultrasensitive enzyme-linked immune mass spectrometric assay (ELIMSA). The novel ELIMSA substrate pyridoxamine-5-phosphate (PA5P) is cleaved by the enzyme alkaline phosphatase to yield the basic and hydrophilic product pyridoxamine (PA) that elutes rapidly with symmetrical peaks and a flat baseline. Pyridoxamine (PA) and 13C PA were both observed to show a linear relationship between log ion intensity and quantity from picomole to femtomole amounts by liquid chromatography-electrospray ionization and mass spectrometry. Four independent methods, (i) internal 13C isotope PA dilution curves, (ii) internal 13C isotope one-point calibration, (iii) external PA standard curve, and (iv) external 13C PA standard curve, all agreed within 1 digit in the same order of magnitude on the linear quantification of PA. Hence, a mass spectrometer can be used to robustly detect 526 ymol of the alkaline phosphatase streptavidin probe and accurately quantify zeptomole amounts of PSA against log linear absolute standard by micro electrospray on a simple ion trap.
- Florentinus-Mefailoski, Angelique,Marshall, John G.
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- Interaction between Pyridoxal Hydrochloride and L-α-Asparagine in Comparison to L-α- and D-α-Aspartic Acids
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Abstract: The kinetics and mechanism of condensation of pyridoxal hydrochloride with L-α-asparagine, L?α- and D-α-aspartic acids are analyzed via UV spectroscopy and polarimetry. It is found that L?α?asparagine containing α-NH2 and γ-NH2/
- Pishchugin,Tuleberdiev
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- Kinetic and mechanism of reactions of L-α-glutamic acid and L-Glutamine with pyridoxal
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The kinetics and mechanisms of condensation of pyridoxal with L-α-glutamic acid and L-glutamine were studied by UV spectroscopy and polarimetry. L-α-Glutamic acid reacts with pyridoxal to form a Schiff base whose subsequent hydrolysis gives rise to pyridoxamine and α-ketoglutaric acid. The reaction of Lglutamine with pyridoxal involves the Γ-NH 2 group and affords a Schiff base whose subsequent hydrolysis gives rise to pyridoxamine and L-α-glutamic acid.
- Pishchugin,Tuleberdiev
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p. 1362 - 1366
(2014/10/15)
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- Glycation Cross-link Breakers to Increase Resistance to Enzymatic Degradation
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The present invention relates to a method to treat a grafts, implant, scaffold, and constructs, including allografts, xenografts, autografts, and prosthetics comprising collagen, with an inhibitor of collagen cross-links and/or advanced glycation endproducts (AGE), in order to alleviate the mechanical weakness induced by the cross-links The invention also provides for kits for use in the operating theater during autograft, allograft or xenograft procedures, or for preparing allograft, xenografts or prosthetics that have not been already treated prior to packaging. The kit comprises a first agent or agents that inhibit collagen cross-links and/or advanced glycation endproducts, instructions for use, optionally a wash or rinse agent, and a device for containing the graft and first agent.
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- SUBSTITUTED PYRIDOXINE-LACTAM CARBOXYLATE SALTS
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The present invention provides salt adducts comprising at least one positively charged moiety being a pyridoxine or a derivative thereof and at least one carboxylated 5- to 7-membered lactam ring, optionally additionally substituted, methods of their preparation, and pharmaceutical compositions and medicaments comprising them. Salt adducts of the invention and compositions comprising them may be used to in the treatment of diseases or disorders associated with or inflicted by alcohol consumption.
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Page/Page column 11-12
(2011/12/13)
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- Engineering Mesorhizobium loti pyridoxamine-pyruvate aminotransferase for production of pyridoxamine with l-glutamate as an amino donor
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Pyridoxamine-pyruvate aminotransferase (PPAT), a novel pyridoxal 5′-phosphate-independent aminotransferase, reversibly catalyzes the transfer of an amino group between pyridoxamine and pyruvate to generate pyridoxal and l-alanine. The enzyme can be used for synthesis of pyridoxamine, a promising candidate for prophylaxis and treatment of diabetic complications. A disadvantage of PPAT for industrial application to the synthesis is that it requires an expensive amino acid l-alanine as an amino donor. Here, mutated PPATs with a high activity toward 2-oxoglutarate (and hence toward l-glutamate) were prepared by a rational design plus random mutagenesis of the wild-type PPAT because l-glutamate is readily and economically available. The PPAT(Y35H/V70R/F247C) showed 9.1-fold lower Km and 4.3-fold higher kcat values than those of the wild-type PPAT. The model of the complex of mutated PPAT and pyridoxyl-l-glutamate showed that γ-carboxyl group of l-glutamate was hydrogen-bound with an imidazole group of His35. The production of pyridoxamine from pyridoxal with transformed Escherichia coli cells expressing the mutated PPAT did not correlate with the kcat value or catalytic efficiency of the mutated PPAT but with Km value at a low level. E. coli cells expressing the PPAT(M2T/Y35H/V70K/E212G) could be used for in vitro conversion of pyridoxal into pyridoxamine at 30 °C with l-glutamate as an amino donor.
- Yoshikane, Yu,Tamura, Asuka,Yokochi, Nana,Ellouze, Khalil,Yamamura, Eitora,Mizunaga, Hanae,Fujimoto, Noboru,Sakamoto, Keiji,Sawa, Yoshihiro,Yagi, Toshiharu
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experimental part
p. 104 - 110
(2011/02/23)
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- Chemical transformations of the condensation products of pyridoxal with L-α-alanine and D-α-alanine
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The kinetics and mechanism of the reactions of pyridoxal with L- and D-α-alanine were studied. Under comparable conditions, the condensation of L- and D-α-alanines with pyridoxal includes three kinetically different steps. The first fast step is addition
- Pishchugin,Tuleberdiev
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body text
p. 117 - 120
(2009/05/30)
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- Chemical transformations of pyridoxal and pyridoxal 5′-phosphate condensation products with amino acids
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The mechanism of chemical transformations of pyridoxal and pyridoxal 5′-phosphate condensation products with amino acids is studied by kinetic measurements. The Schiff bases are shown to be fairly stable in neutral media. In acid media, the Schiff bases a
- Pishchugin,Tuleberdiev
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p. 1225 - 1229
(2008/12/22)
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- METHODS FOR THE SYNTHESIS OF PYRIDOXAMINE
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The invention provides non-oxidative methods for the large scale manufacture of pyridoxamine (I) (4-aminomethyl-3-hydroxy-5-hydroxymethyl-2-methylpyridine): Formula (I), and salts thereof. The invention also provides intermediate compounds for the synthesis of pyridoxamine, as well as compositions and methods for the treatment and/or prevention of conditions associated with the formation of post-Amadori advanced glycation end-products.
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Page/Page column 43
(2008/06/13)
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- Production of pyridoxal phosphate by a mutant strain of Schizosaccharomyces pombe.
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Conditions for extracellular production of vitamin B6 compounds (B6), especially pyridoxal 5'-phosphate (PLP) by Schizosaccharomyces pombe leul strain were examined. The productivity was dependent on concentration of L-leucine in the culture medium: 30 mg/l gave the highest concentrations of total B6 and PLP. The viable cells harvested at different growth phases showed different productivity: middle and late exponential phase cells showed the highest productivity of total B6 and PLP, respectively. D-Glucose (1%, w/v) among other sugars gave the best productivity. Supplementation of air and ammonium sulfate significantly increased extracellular production of PLP. Superoxide anion producers, menadione and plumbagin, and H202 increased the productivity of PLP. Cycloheximide inhibited the increase of PLP by the oxidative stress and, in contrast, increased pyridoxine.
- Chumnantana,Hirose,Baba,Yagi
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p. 1789 - 1795
(2007/10/03)
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- Transaminative Desilylation of (Aminomethyl)trimethylsilane and Transitory Inactivation of Plasma Amine Oxidase
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(Aminomethyl)trimethylsilane (AMTMS), a compound which has previously been used as a probe for electron-transfer oxidation of amines, undergoes transaminative desilylation by active carbonyl reagents and induces a mechanism-based inactivation of the quinone-containing plasma amine oxidase.
- Wang, F.,Venkataraman, B.,Klein, M. E.,Sayre, L. M.
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p. 6687 - 6689
(2007/10/02)
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- PHOTODECARBOXYLATION OF PYRIDOXAL-α-AMINO ACID SCHIFF BASES. INTERMEDIATE SPECIES INDUCED BY FLASH EXCITATION
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Pyridoxal Schiff bases of α-amino acids, phenylalanine and α-aminoisobutyric acid underwent photochemical decarboxylation in methanol to give pyridoxamine and carbonyl compounds after hydrolysis.Flash photolysis of the Schiff bases induced transient absorptions which were assigned to the decarboxylated quinonoid intermediates.
- Kurauchi, Yoshiaki,Ohga, Kazuya,Morita, Shigeru,Nagamura, Toshihiko,Matsuo, Taku
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p. 349 - 352
(2007/10/02)
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- Pyridoxamine and 2-Oxalopropionic Acid in Aqueous Systems: Conditional Imine Formation Constants and Rate Constants of Vitamin B6 Catalyzed Decarboxylation
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The conditional Schiff base formation constants (Kc) of pyridoxamine (PM) and 2-oxalopropionic acid (OPA) were determined spectrophotometrically in basis aqueous solutions.These equilibrium constants exhibit pH dependence with maximum ketimine formation occuring near pH 9.0 (Kc = 9.6).The kinetics of vitamin B6 catalyzed decarboxylation of OPA have been studied spectrophometrically, and the observed rate constant of decarboxylation (kobsd) exhibited pH dependence with the maximum occuring at approximatly the pH corresponding to maximum ketimine formation (kobsd = 15.6 * 10-4 s-1 at 1.01 * 10-4 M OPA and 1.00 * 10-5 M PM).Microscopic rate constants (kSB) for the decarboxylation of the Schiff base species present in solution also showed pH dependence with the greatest catalytic activity at pH 7.0 (kSB = 38.4 s-1) and the least at pH 12.47 (kSB = 0.2 s-1).The relative magnitudes of the kSB's determined are discussed in terms of the various protonated forms of the OPA:PM ketimine,and the corresponding pKa's have been estimated from the rate constant-pH profiles.
- Kubala, Gregory,Martell, Arthur E.
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p. 449 - 455
(2007/10/02)
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