Catalase label

Ag reaction with catalase-labeled antigen. After competitive binding of free and catalase-labeled AFP, the sensor is examined for catalase activity by ampero-metric measurement after addition of H 2O2. AFP can be assayed in the rjuige 10-0.01 ng/mL. 

An electrode for the determination of vitamin H (biotin) is based on the competitive affinity between biotin and its homologue HABA for the same protein, avidin [84]. The catalase-labelled HABA-avidin complex is immobilized on a p02 electrode to produce a biotin-sensitive sensor [255]. Biotin dissociares some of the HABA-avidin complexes by its strong affinity for avidin (Figure 6.1), leading to a reduction in enzymatic activity at the pC>2 electrode which can be related to the concentration ctf biotin. The coupling between biotin and avidin occurs more from bioaffinity than from an irmnunological process. 

Catalase has also been used as an enzyme label in competitive heterogeneous enzyme immunoassays. Catalase generates oxygen from hydrogen peroxide with the oxygen determined amperometrically with an oxygen electrode. This approach has been demonstrated for a-fetoprotein theophylline and human serum albumin... 

Fig. 7.5 TEM image of microcapsules prepared theinsetcorrespondsto800nm. PLL/PGAlayers by LbL assembly of three bilayers of a PLL/PGA were assembled from a 0.05 M MES, pH 5.5 shell on catalase-loaded BMS spheres, following buffer. The MS spheres were dissolved usingHF/ removal ofthe BMS particle template (A). CLSM NH4F at pH 5. (Adapted from [82] with per-images of (PLL/PGA)3 microcapsules loaded mission of Wiley-VCH). with FITC-labeled catalase (B). The scale bar in...

Yokota, S. (1988) Effect of particle size on labeling density for catalase in protein A-Gold immunocyto-chemistry./. Histochem. Cytochem. >6, 107-109. 

CL reaction can be catalyzed by enzymes other than HRP (e.g., microperoxidase and catalase) and by other substances [hemoglobin, cytochrome c, Fe(III), and other metal complexes]. The presence of suitable molecules such as phenols (p-iodophenol), naphthols (l-bromo-2-naphthol), or amines (p-anisidine) increases the light production deriving from the HRP-catalyzed oxidation of luminol and produces glow-type kinetics [6, 7], The use of other enzymes, such as glucose-6-phosphate dehydrogenase [38-41], P-galactosidase [42], and xanthine oxidase [43-46], as CL labels has been reported. 

Fig. 13. Active site residues in a small-subunit catalase BLC (A) and a large-subunit catalase HPIl (B). The active site residues are labeled, and hydrogen bonds are shown between the serine (113 in BLC and 167 in HPll) and the essential histidine (74 in BLC and 128 in HPll). A single water is shown hydrogen bonded to the histidine. The equivalent water in BLC is located by analogy to the position of the water in HPll. The unusual covalent bond between the N of His392 and the C of Tyr415 in HPll is evident on the proximal side of the heme in B. The flipped orientations of the hemes are evident in a comparison of the two structures, as is the eis-hydroxyspirolactone structure of heme d in B.

Fig. 16. A cartoon showing the putative channels that provide access to the active site of a large-subunit catalase in A and a small-subunit catalase in B. The main or perpendicular channel is labeled P and the minor or lateral channel, which is bifurcated, is labeled L. A potential channel leading to the proximal side of the heme is shown with a dashed line.

Enzyme Decay. Moffett and Zafiriou (I) differentiated catalase- and peroxidase-mediated decay in coastal (marine) waters by using lsO-labeled H202 and 02, and by determining the labeled end products. Equation 13 shows that the products of catalase decomposition are H20 and 02. In contrast, peroxidase decomposition results in the formation of H20 without 02. From the measurement of the relative amount of labeled products it is possible to determine the contribution of both enzymes in the decay of the H202. In the coastal water, 65-80% of the decomposition was attributed to catalase and the rest to peroxidase (I). These studies are the first to use this technique. The approach should be extended to freshwater ecosystems to see if the same pattern would be found. 

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