Enzyme detection

Measured Species Enzyme Detected Species Type of sensing Reference... 

In previous papers it was shown that the enzymatic pool of Pectolyase Y23 possesses high catalytic efficiency either as free or immobilized form in solution of pectins [28, 29] and of fresh vegetable tissues [30]. According to Baldwin and Pressor [31] the following enzymatic activities were detected in the preparation PL, PG and PE. The amount of the different enzyme detected per mg of Pectolyase Y23 and the main enzyme characteristic are quoted in Table 1... 

Although this class of enzymes is involved in most electrochemical approaches, other enzymes may be investigated electrochemically indirectly. For example, the system can be arranged such that the product of the targeted nonredox enzyme serves as substrate for an appropriately selected redox enzyme. Detection then involves the redox cosubstrate of the redox enzyme. 

R. A. Evangelista, A. Poliak, and E. F. G. Templeton, Enzyme-amplified lanthanide luminescence for enzyme detection in bioanalytical assays, Anal. Biochem. 197, 213-224 (1991). 

Enzyme detection Lysosomes can also be stained via enzyme-catalyzed hydrolysis of heavily labeled and almost totally quenched substrates (124). 

Srinivasan, U., Jones, E., Weir, D. G., and Feighery, C. (1999). Lactase enzyme, detected immunohistochemically, is lost in active celiac disease, but unaffected by oats challenge. Am. J. Gastroenterol. 94, 2936-2941. 

The third subgroup of alcohol dehydrogenases consists of iron-activated enzymes. The first enzyme detected to belong to this group was the ADH II from Zymomonas mobilis [116] followed by the observation that the ADH IV from Saccharomyces cerevisiae shows more than 50% identity to this bacterial ADH [117]. No data about the secondary or tertiary structures of the enzymes in this subgroup are available currently. A prediction based upon the Chou and Fasman analysis [118] indicates that these enzymes are rich in a-helices. 

Fig. 11 Principle of FET detection with P450 wires showing the light to dark method for enzyme detection and dark to light method for inhibitor screening...

Esterases. Acetyl esterase (EC 3.1.1.6) removes acetyl esters from acetylated xylose and short-chain xylo-oligomers. It s polymeracting counterpart, acetyl xylan esterase (EC 3.1.1.72), has a similar activity, but prefers polymeric xylan.244 In addition to acetate-specific enzyme detection kits, HPLC or GC analysis of acetate release from native extracted xylan and chemically acetylated xylan, colorimetric substrates, such as p-nitrophenol acetate and /3-napthyl acetate, or the fluorometric substrate, 4-methylumbelliferyl acetate are also used to assay acetyl esterases.244,253 The third esterase, ferulic acid esterase (EC 3.1.1.73), hydrolyzes the ester bond between ferulic acid or coumaric acid and the arabinose side chain of arabinoxylan. Assays for this activity are usually carried out using starch-free wheat bran or cellulase-treated gramineous biomass as a substrate and monitoring ferulic or coumaric acid released by HPLC or TLC. When preparing enzyme-treated substrates, care must be taken to employ phenolic-acid-esterase-free cellulases.244 Other substrates include methyl and ethyl esters of the phenolic acids, as well as finely ground plant biomass.240,254,255... 

Indirect detection does require more steps, but oftentimes yields amplified signals relative to direct methods because layering of bridging molecules may increase the number of detector molecules per probe molecule. It is probably this bridging/amplification technique that has allowed current enzyme detection systems to approach the sensitivity of radiolabeled systems. The use of these indirect methods reduces steric problems that might arise from having enzyme molecules directly bound to probe molecules. 

Results are assessed either by direct visual observation or spectrophotometrically. For the sake of completeness, it should be mentioned in this section that enzyme detection systems have been described which are monitored by alternative methods. These... 

TABLE 16.1. Organ-Specific Leakage Enzymes Detectable in Scriini/l lasina... 

Enzymes, Detection of Ligand-Induced and Syncatalytic Conformatumal Changes... 

The short peptide linker ligand is specifically designed to be cleaved by the target enzyme in order to enable enzyme detection. Dissociation of the aggregates... 

Maher RC, Maier SA, Cohen LF, Koh L, Laromaine A, Dick JAG, Stevens MM (2010) Exploiting SERS hot spots for disease-specific enzyme detection. J Phys Chem C 114(16) 7231-7235... 

There are many other phenomena specifically observed in H-COj conditions. For example, H-COj cells of C. littorale exhibit higher Rubisco activity than L-COj cells, due to the increase in the amount of this enzyme detected by antibody [11]. Rubisco activase is also higher, but phosphoenolpyruvate carboxylase activity is lower in H-... 

The nucleic acid to be labeled can be divided into four types natural enzymatically amplified cloned or synthetic. The selection of an appropriate label for a given probe may depend on many factors (Table 7.1). Localization of target nucleic acid in a cell often requires high resolution. This can be obtained by low energy radioisotopes, fluorescent dyes, or enzyme detection systems that limit the spread of the generated product as much as possible. On the other hand, dot hybridization requires high detectability while resolution is not a priority. Quantification is better with radioisotope labels than with biotin systems due to a higher sensitivity, while the... 

Equilibrate the washed membrane for 2 min in the same buffer as used later for enzyme detection. 

Collins, A. R., Duthie, S. J., and Dobson, V. L. (1993). Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis 14, 1733-1735. 

The blood levels of both enzymes must be monitored. Neither enzyme concentration gives sufficient information for the duration of treatment. For example, CK is the first enzyme detected during a myocardial infarction. CK s serum concentration rises and falls so rapidly that it is of little clinical use after several days. LDH, whose serum concentration rises later, is used to monitor the later stages of heart damage. Monitoring the activity of several enzymes also prevents misdiagnosis. Recall that few enzymes are specific... 

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