Activation and enzymatic characterization of recombinant human kallikrein 8.

Human kallikrein 8 (hK8), whose gene was originally cloned as the human ortholog of a mouse brain protease, is known to be associated with diseases such as ovarian cancer and Alzheimer's disease. Recombinant human pro-kallikrein 8 was activated with lysyl endopeptidase-conjugated beads. Amino-terminal sequencing of the activated enzyme demonstrated the cleavage of a 9-aa propeptide from the pro-enzyme. The substrate specificity of activated hK8 was characterized using synthetic fluorescent substrates. hK8 showed trypsin-like specificity, as predicted from sequence analysis and enzymatic characterization of the mouse ortholog. All synthetic substrates tested containing either arginine or lysine at P1 position were cleaved by hK8. The highest kcat/Km value of 20x10(3)M-1 s-1 was observed with Boc-Val-Pro-Arg-7-amido-4-methylcoumarin. The activity of hK8 was inhibited by antipain, chymostatin, and leupeptin. The concentration for 50% inhibition by the best inhibitor, antipain, was 0.46 microM. The effect of different metal ions on the enzyme activity was analyzed. Whereas Na+ had no effect on hK8 activity, Ni2+ and Zn2+ decreased the activity and Ca2+, Mg2+, and K+ had a stimulatory effect. Ca2+ was the best activator, with an optimal concentration of approximately 10 microM

Study of the stoichiometry and the kinetics of the Jaffé reaction with a picrate ion-selective electrode

Potentiometric studies with the picrate ion-selective electrode indicate that the species formed as the product of reaction between alkaline picrate and creatinine in the Jaffé reaction is a 1:1 complex. Kinetic studies indicate that the forward reaction is first order with respect to picrate, creatinine, and hydroxide concentration. The second-order rate constant k was found to be in the range 9.1-10.4 M-2 sec-1 at 27 °C and μ = 1.00, with creatinine or picrate in excess, k increases with increasing μ and temperature. An activation energy of 10.1 kcal/mol was calculated for the Jaffé reaction, with creatinine in excess. © 1983

Potentiometric determination of some common alkaloids with a picrate-selective electrode

Potentiometric methods are described for the determination of alkaloids, based on the formation of insoluble alkaloid picrate salts, using a picrate ion-selective indicating electrode. Micro-amounts of strychnine, papaverine, quinine and cocaine were determined by direct potentiometry and titrimetrically, using Gran plots, with average errors of about 4% and 2%, respectively. The method was successfully applied to pharmaceutical preparations. © 1981

Analytical study of new creatininium and tetramethylammonium cation selective membrane electrodes

Two new liquid membrane electrodes which respond to creatininium and tetramethylammoniwn cations are described. The creatininium cation electrode exhibits rapid and near Nernstian response to creatininium cation activity, at pH 3, in the 10-3-10-1mol/L range. The Useful concentration range extends to 10-4mol/L. The tetramethylammonium cation electrode exhibkts rapid and near Nernstian response to tetramethylammonium cation activity,at pH 2-11.5, in the 2×10-5-10-1mol/L range. Major interferences for the creatininium electrode are Na+,K+,NH+4 and creatine.The pKaof the creatininium cation was calculated. A method is described for the potentiometric precipitation titration of tetramethylammonium cation with sodium tetrapheny1boron. Amounts of tetramethylamonium in the range 20-200 wol have been determined using Gran's plots, with an average error of about 0.6%. © 1980, Taylor & Francis Group, LLC. All rights reserved

Catalytic determination of selenium with a picrate-selective electrode

A method is described for the determination of selenium, based on its catalytic effect on the picrate-sulfide reaction. The determination involves a variable-time kinetic procedure using potentiometric monitoring with a picrate-selective electrode and automatic measurement of the time required for the potential to change by a preselected amount (5.0 mV). Selenium in the range 3-30 μg was determined with an average error of about 4% and relative standard deviations of about 2%. The reaction can also be followed spectrophotometrically. © 1981

Titrimetric determination of iodide, hexacyanoferrate(ii), thiourea, cationic surfactants, and of picrate with a picrate ion-selective electrode

Methods have been developed for the semiautomatic potentiometric precipitation titration of iodide, hexacyanoferrate(II), sulfide, and sulfide-thiourea mixtures with silver nitrate, of medically important quaternary ammonium compounds with sodium picrate, and of picrate ions with tetraphenylarsonium chloride, using a picrate ion-selective electrode. Semimicro and/or microamounts of these substances were determined with relative errors and relative standard deviations of 1-2%. © 1980 Springer-Verlag

Continuous-flow determination of reducing sugars and sucrose in natural and industrial products with periodate oxidation and a periodate-sensitive flow-through electrode

A continuous-flow method for the determination of mixtures of reducing sugars and sucrose in natural and industrial products is described. The sample, before and after hydrolysis of sucrose, reacts with an excess of periodate in a flow system and the decrease in periodate activity is monitored with a periodate-sensitive flow-through electrode. The recorded peak heights are indirectly linearly related to the concentration of reducing sugars in the range 3-18 mM. The sucrose concentration is calculated by difference. The analysis is completely automated, requires no sample pre-treatment except for the hydrolysis of sucrose and samples can be analysed at a rate of 30 per hour with a relative error and a relative standard deviation of 1-3%. Comparison with Fehling's method for various natural and industrial products gave satisfactory results