Phosphates, identification

Wu Y, Glimcher MJ, Rey C, Ackerman JL (1994) A unique protonated phosphate group in bone mineral not present in synthetic calcium phosphates. Identification by phosphoms-31 sohd-state NMR spectroscopy. J Mol Biol 244 423-435... 

Analysis. Excellent reviews of phosphate analysis are available (28). SoHds characterization methods such as x-ray powder diffraction (xrd) and thermal gravimetric analysis (tga) are used for the identification of individual crystalline phosphates, either alone or in mixtures. These techniques, along with elemental analysis and phosphate species deterrnination, are used to identify unknown phosphates and their mixtures. Particle size analysis, surface area, microscopy, and other standard soHds characterizations are useful in relating soHds properties to performance. SoHd-state nmr is used with increasing frequency. 

Definitive identification of lysine as the modified active-site residue has come from radioisotope-labeling studies. NaBH4 reduction of the aldolase Schiff base intermediate formed from C-labeled dihydroxyacetone-P yields an enzyme covalently labeled with C. Acid hydrolysis of the inactivated enzyme liberates a novel C-labeled amino acid, N -dihydroxypropyl-L-lysine. This is the product anticipated from reduction of the Schiff base formed between a lysine residue and the C-labeled dihydroxy-acetone-P. (The phosphate group is lost during acid hydrolysis of the inactivated enzyme.) The use of C labeling in a case such as this facilitates the separation and identification of the telltale amino acid. 

Figure 12.2 Cliromatograms of an ABS copolymer sample (a) microcolumn SEC ti ace (b) capillaiy GC trace of peak x . Peak identification is as follows 1, C,4 alkanes 2, C,5 alkanes 3, C 8 alkanes 4, nonylphenol 5, palmitic acid 6, styrene-acetonitrile firmer 7, stearic acid 8, styrene-acetonitrile trimer 9, Irganox 1076 10, fiinonylphenyl phosphate 11, Ethanox 330. Reprinted with permission from Ref. (12).

Probably all adenylyl cyclases are inhibited competitively by substrate analogs, which bind at the site and to the enzyme configuration with which cation-ATP binds (cf Fig. 4). One of the best competitive inhibitors is (3-L-2, 3 -dideoxy adenosine-5 -triphosphate ( 3-L-2, 3 -dd-5 -ATP Table 4) [4], which allowed the identification of the two metal sites within the catalytic active site (cf Fig. 4) [3]. This ligand has also been labeled with 32P in the (3-phosphate and is a useful ligand for reversible, binding displacement assays of adenylyl cyclases [4]. The two inhibitors, 2, 5 -dd-3 -ATP and 3-L-2, 3 -dd-5 -ATP, are comparably potent... 

The phosphotriesterase from Pseudomonas diminuta was shown to catalyze the enantioselective hydrolysis of several racemic phosphates (21), the Sp isomer reacting faster than the Rp compound [65,66]. Further improvements using directed evolution were achieved by first carrying out a restricted alanine-scan [67] (i.e. at predetermined amino acid positions alanine was introduced). Whenever an effect on activity/ enantioselectivity was observed, the position was defined as a hot spot. Subsequently, randomization at several hot spots was performed, which led to the identification of several highly (S)- or (R)-selective mutants [66]. A similar procedure was applied to the generation of mutant phosphotriesterases as catalysts in the kinetic resolution of racemic phosphonates [68]. 

Table 2.1 HPLC capacity factors for secbuto-barbitone and vinbarbitone with an octadecyl silyl stationary phase and mobile phases of methanoiyO.l M sodium dihydrogen phosphate (40 60) at (a) pH 3.5, and (b) pH 8.5. From Moffat, A.C. (Ed.), Clarke s Isolation and Identification of Drugs, 2nd Edn, The Pharmaceutical Press, London, 1986. Reproduced by permission of The Royal Pharmaceutical Society...

Sprenger, G.A. et al.. Identification of a thiamin-dependent synthase in Escherichia coli required for the formation of the 1-deoxy-D-xylulose 5-phosphate precursor to isoprenoids, thiamin, and pyridoxol, Proc. Natl. Acad Sci. USA 94, 12857, 1997. Lange, B.M. et al., A family of transketolases that directs isoprenoid biosynthesis via a mevalonate-independent pathway, Proc. Natl. Acad Sci. USA 95, 2100, 1998. Lois, L.M. et al., Cloning and characterization of a gene from Escherichia coli encoding a transketolase-like enzyme that catalyzes the synthesis of D-1- deoxyxylulose 5-phosphate, a common precursor for isoprenoid, thiamin, and pyridoxol biosynthesis, Proc. Natl. Acad. Sci. USA 95, 2105, 1998. 

Murphy Ml, LM Siegel, H Kamin (1973) Reduced nicotinamide adenine dinucleotide phosphate-sulfite reductase of enterobacteria. II. Identification of a new class of heme prosthetic group an iron-tetrahydroporphyrin (isobacteriochlorin type) with eight carboxylic acid groups. J Biol Chem 248 2801-3814. 

Arkowitz RA, RH Abeles (1989) Identification of acetyl phosphate as the product of clostridial glycine reductase evidence for an acyl enzyme intermediate. Biochemistry 28 4639-4644. 

Although commonly formed from endogenous material, the occurrence of synovial crystals formed following environmental exposure to exogenous agents is indicated by the identification of both aluminium phosphate and aluminium silicate particulates (Netter etal., 1983, 1991). It is noteworthy in this context that arthritic symptoms have been reported following the... 

Figure 11 Separation of anti-inflammatories by CZE at various pHs in a 40-cm polyacrylamide-coated (left) and a 70-cm uncoated (right) capillary Experimental conditions 275 V/cm UV = 215 nm buffers 20 mM borate-100 mM boric acid, pH 8.4 (46 pA) 30 mM phosphate-9 mM borate, pH 7.0 (70 pA) 80 mM MES-30 mM Tris, pH 6.1 (20 pA) peak identification 1 = naproxen, 2 = ibuprofen, 3 = tolmetin. (From Wainwright, A., /. Microcol Sep., 2, 166, 1990. With permission.)...

Successful combination of a chromatographic procedure for separating and isolating additive components with an on-line method for obtaining the IR spectrum enables detailed compositional and structural information to be obtained in a relatively short time frame, as shown in the case of additives in PP [501], and of a plasticiser (DEHP) and an aromatic phenyl phosphate flame retardant in a PVC fabric [502], RPLC-TSP-FTIR with diffuse reflectance detection has been used for dye analysis [512], The HPLC-separated components were deposited as a series of concentrated spots on a moving tape. HPLC-TSP-FTIR has analysed polystyrene samples [513,514], The LC Transform has also been employed for the identification of a stain in carpet yarn [515] and a contaminant in a multiwire cable [516], HPLC-FTIR can be used to maintain consistency of raw materials or to characterise a performance difference. 

Major components Chemical formula Chemical structure Identification numbers Tri-o-cresyl phosphate C2iH2i04P (RO) (RO) (RO) P=0 Predominantly saturated hydrocarbons predominantly in the range C15 through C30 Hydrocarbons predominantly in the range C11 through C20... 

Lombardo P, Egry IJ. 1979. Identification and gas-liquid chromatographic determination of aryl phosphate residues in environmental samples. J Assoc Off Anal Chem 62 47-51. 

Nomier AA, Abou-Donia MB. 1986. Studies on the metabolism of the neurotoxic tri-ort/20-cresyl phosphate Synthesis and identification by infrared, proton nuclear magnetic resonance and mass spectrometry of five of its metabolites. Toxicology 38 1-13. 

Yang SM, Thieme RA, von Meyerinck L, et al. 1990. Identification of isopropylated phenyl phosphates in rabbit bile. Biomed Environ Mass Spectrom 19 573-576. 

H8. Hirono, A., Kuhl, W., Gelbart, T., Forman, L., Fairbanks, V. F., and Beutler, E., Identification of the binding domain for NADP of human glucose-6-phosphate dehydrogenase by sequence analysis of mutants. Proc. Natl. Acad. Sci. U.S.A. 86,10015-10017 (1989). 

V12. Vives Corrons, J. L. 1., Rovira, A., Pujades, A., Vulliamy, T., and Luzzatto, L., Molecular heterogeneity of glucose-6-phosphate dehydrogenase (G6PD) in Spain and identification of two new base substitutions in the G6PD gene, Blood 84 (Suppl. 1), 551a (1994). 

Figure 2.7. Identification ofphosphoproteins by site-specific chemical modification. A. Method of Zhou et al. (2001) involves trypsin digest of complex protein mixture followed by addition of sulfhydryl groups specifically to phosphopeptides. The sulfhydryl group allows capture of the peptide on a bead. Elution of the peptides restores the phosphate and the resulting phosphopeptide is analyzed by tandem mass spectrometry. B. Method of creates a biotin tag in place of the phosphate group. The biotin tag is used for subsequent affinity purification. The purified proteins are proteolyzed and identified by mass spectrometry.

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