PubMed · 5725199
The source did not provide an abstract. Follow the original record for more information.
Explore connections, maps & timelines
T D Lavery. 1968. A simplified method for estimating plasma uric acid.. https://doi.org/10.1016/0009-8981(68)90070-3
Cite the original work for its findings. Save a collection to share your selection of sources.
KEEP EXPLORING
Amino acid (AA) profiles from body fluids such as blood and urine are clinical indicators for diagnosing metabolic and hepatic diseases. Current quantitative methods, such as liquid chromatography-mass spectrometry (LC-MS) with isotopically labelled internal standards (ISs), are costly and technically demanding. This study proposes a cost-efficient alternative using structural isomers as ISs in a direct liquid infusion (DLI) tandem mass spectrometry (MS/MS) approach. The method leverages chimeric spectra and fragment intensity ratios to quantify AAs, demonstrating high linearity and precision even with a 3D ion trap mass analyser. This approach offers a viable strategy for AA quantification in preventive medicine, particularly for screening metabolic diseases such as phenylketonuria, diabetes, and liver dysfunction.
To provide quantitative information on the sites that promote polymerization of sickle hemoglobin (HbS) after formation of the initial hydrophobic bond involving Val-6(beta) [E6V(beta)] and also to provide hemoglobins with an enhanced polymerization that could be used in a mouse model for sickle cell anemia, we have expressed recombinant double, triple, and quadruple HbS mutants with substitutions on both the alpha- and beta-chains, E6V(beta)/E121R(beta), D75Y(alpha)/E6V(beta)/E121R(beta) and D6A(alpha)/D75Y(alpha)/E6V(beta)/E121R(beta). These recombinant hemoglobins were extensively characterized by high-performance liquid chromatography analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, amino acid analysis, and mass spectroscopy. They retained the functional properties of the Hb tetramer and polymerized in a linear manner at progressively lower Hb concentration as a function of the degree of substitution, suggesting that these remote sites (alphaD6A, alphaD75Y, and betaE121R) on the alpha- and beta-chains exhibit additive, enhanced polymerization properties. The quadruple mutant has a polymerization concentration close to that of the purified SAD hemoglobin from transgenic mouse red blood cells consisting of HbS, Hb Antilles, and Hb D-Punjab. Normal mouse Hb increases the polymerization concentration of each mutant. Thus, the general approach of using recombinant Hbs as described here should prove useful in elucidating the quantitative aspects of the mechanism of HbS polymerization and in identifying the contribution of individual sites to the overall process. The strategy described here demonstrates the feasibility of a systematic approach to achieve future recombinant HbS mutants that could provide a new generation of the transgenic mouse model for sickle cell anemia.
We present evidence that the size of an active site side chain may modulate the degree of hydrogen tunneling in an enzyme-catalyzed reaction. Primary and secondary kH/kT and kD/kT kinetic isotope effects have been measured for the oxidation of benzyl alcohol catalyzed by horse liver alcohol dehydrogenase at 25 degrees C. As reported in earlier studies, the relationship between secondary kH/kT and kD/kT isotope effects provides a sensitive probe for deviations from classical behavior. In the present work, catalytic efficiency and the extent of hydrogen tunneling have been correlated for the alcohol dehydrogenase-catalyzed hydride transfer among a group of site-directed mutants at position 203. Val-203 interacts with the opposite face of the cofactor NAD+ from the alcohol substrate. The reduction in size of this residue is correlated with diminished tunneling and a two orders of magnitude decrease in catalytic efficiency. Comparison of the x-ray crystal structures of a ternary complex of a high-tunneling (Phe-93 --> Trp) and a low-tunneling (Val-203 --> Ala) mutant provides a structural basis for the observed effects, demonstrating an increase in the hydrogen transfer distance for the low-tunneling mutant. The Val-203 --> Ala ternary complex crystal structure also shows a hyperclosed interdomain geometry relative to the wild-type and the Phe-93 --> Trp mutant ternary complex structures. This demonstrates a flexibility in interdomain movement that could potentially narrow the distance between the donor and acceptor carbons in the native enzyme and may enhance the role of tunneling in the hydride transfer reaction.