Reliable method for the detection of horseradish peroxidase activity and enzyme kinetics

The Analyst ◽  
2019 ◽  
Vol 144 (4) ◽  
pp. 1442-1447 ◽  
Author(s):  
Yizhe Yu ◽  
Yinling Wang ◽  
Maoguo Li
Keyword(s):  
Horseradish Peroxidase ◽  
Enzyme Kinetics ◽  
Peroxidase Activity ◽  
Electrochemical Method ◽  
Reliable Method ◽  
Specific Activity ◽  
Kinetics Of

A new electrochemical method was proposed to detect the specific activity and study the enzyme kinetics of horseradish peroxidase.

Electrically neutral Na+-H+ exchange in endosomes obtained from rabbit renal cortex

1986 ◽  
Vol 251 (4) ◽  
pp. F702-F709 ◽  
Author(s):  
R. W. Gurich ◽  
D. G. Warnock
Keyword(s):  
Horseradish Peroxidase ◽  
Peroxidase Activity ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Renal Cortex ◽  
Specific Activity ◽  
Sucrose Density Gradient ◽  
Michaelis Constant ◽  
Fold Enrichment ◽  
Enzyme Markers

Endocytotic vesicles (i.e., endosomes) were prepared from rabbit renal cortex following the intravenous injection of horseradish peroxidase. The endosomal population was derived from a 100,000-g pellet and was found at equilibrium in the lightest area of a sucrose density gradient. This population was separate from other organelles as determined by enzyme markers and had a 2.5-fold enrichment of horseradish peroxidase specific activity compared with the homogenate specific activity corrected for soluble horseradish peroxidase activity. The endosomes contain an oligomycin-insensitive, electrogenic H+-translocating ATPase. They also contain an electroneutral Na+-H+ exchanger. This exchanger is not inhibited by amiloride, and lithium is not a substrate for the exchanger. Lithium does inhibit the Na+-H+ exchanger when added prior to the addition of sodium. The Michaelis constant for sodium of the endosomal Na+-H+ exchanger was found to be 10.0 mM. These data indicate that a population of endosomes from rabbit renal cortex contain an electrogenic H+-ATPase and an electroneutral Na+-H+ exchanger and that the exchanger is distinct from the brush-border membrane Na+-H+ antiporter.

Kinetic Aspects of the Interaction of Blood Clotting Enzymes

1968 ◽  
Vol 19 (03/04) ◽  
pp. 364-367 ◽  
Author(s):  
H. C Hemker ◽  
P. W Hemker
Keyword(s):  
Enzyme Kinetics ◽  
Blood Clotting ◽  
Competitive Inhibition ◽  
Competitive Inhibitor ◽  
Kinetics Of

SummaryThe enzyme kinetics of competitive inhibition under conditions prevailing in clotting tests are developed and a method is given to measure relative amounts of a competitive inhibitor by means of the t — D plot.

Competitive enzyme-liked immunoassay with use of soluble enzyme/antibody immune complexes for labeling. I. Measurement of human choriogonadotropin.

1976 ◽  
Vol 22 (8) ◽  
pp. 1372-1377 ◽  
Author(s):  
D E Yorde ◽  
E A Sasse ◽  
T Y Wang ◽  
R O Hussa ◽  
J C Garancis
Keyword(s):  
Horseradish Peroxidase ◽  
Peroxidase Activity ◽  
Quantitative Measurement ◽  
Original Sample ◽  
Soluble Enzyme ◽  
Soluble Complex ◽  
Human Choriogonadotropin ◽  
Preliminary Study ◽  
Free Antigen ◽  
Serum And Urine

Abstract We described the principle of a new enzyme-immunoassay, competitive enzyme-liked immunoassay (CELIA), for quantitative measurement of soluble antigens and haptens. In the assay, binding of antibody to antigen-immunosorbent is competitively inhibited by the free antigen to be measured. The amount of first antibody bound to the immunosorbent is measured by an enzymatic technique in which a heterologous bridging antibody and a soluble antibody/enzyme immune complex are applied in sequence. The soluble complex we used was rabbit antiperoxidase/horseradish peroxidase. Peroxidase activity is inversely proportional to the concentration in the original sample of the substance to be assayed. The enzyme-linked reagents are potentially widely applicable to any substance to be measured. To demonstrate the feasibility of CELIA, we report a preliminary study of its application to the measurement of human chloriogonadotropin in serum and urine. The assay described for this hormone has a working range of 1 to 50 int. units per milliliter of sample. The technique obviates the disadvantages associated with measurement and handling of radioisotopes in radioimmunoassays and the only major instrumentation required is a centrifuge and a conventional spectrophotometer.

scholarly journals Acidic horseradish peroxidase activity abolishes genotoxicity of common dyes

2017 ◽  
Vol 321 ◽  
pp. 576-585 ◽  
Author(s):  
Barbara S. Janović ◽  
Andrew R. Collins ◽  
Zoran M. Vujčić ◽  
Miroslava T. Vujčić
Keyword(s):  
Horseradish Peroxidase ◽  
Peroxidase Activity

Kinetics of the oxidation of reduced nicotinamide adenine dinucleotide by horseradish peroxidase compounds I and II

1986 ◽  
Vol 64 (4) ◽  
pp. 323-327 ◽  
Author(s):  
Mohammed A. Kashem ◽  
H. Brian Dunford
Keyword(s):  
Horseradish Peroxidase ◽  
Active Site ◽  
Nicotinamide Adenine Dinucleotide ◽  
Ph Dependence ◽  
Transient State ◽  
Nicotinamide Adenine ◽  
Compound I ◽  
Single Ionization ◽  
Reduced Nicotinamide Adenine Dinucleotide ◽  
Kinetics Of

The transient state kinetics of the oxidation of reduced nicotinamide adenine dinucleotide (NADH) by horseradish peroxidase compound I and II (HRP-I and HRP-II) was investigated as a function of pH at 25.0 °C in aqueous solutions of ionic strength 0.11 using both a stopped-flow apparatus and a conventional spectrophotometer. In agreement with studies using many other substrates, the pH dependence of the HRP-I–NADH reaction can be explained in terms of a single ionization of pKa = 4.7 ± 0.5 at the active site of HRP-I. Contrary to studies with other substrates, the pH dependence of the HRP-H–NADH reaction can be interpreted in terms of a single ionization with pKa of 4.2 ± 1.4 at the active site of HRP-II. An apparent reversibility of the HRP-II–NADH reaction was observed. Over the pH range of 4–10 the rate constant for the reaction of HRP-I with NADH varied from 2.6 × 105 to5.6 × 102 M−1 s−1 and of HRP-II with NADH varied from 4.4 × 104 to 4.1 M−1 s−1. These rate constants must be taken into consideration to explain quantitatively the oxidase reaction of horseradish peroxidase with NADH.

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