Assay browning reaction

The reactivity of NO towards atoms, free radicals, and other paramagnetic species has been much studied, and the chemiluminescent reactions with atomic N and O are important in assaying atomic N (p. 414). NO reacts rapidly with molecular O2 to give brown NO2, and this gas is the normal product of reactions which produce NO if these are carried out in air. The oxidation is unusual in following third-order reaction kinetics and, indeed, is the classic... 

As another alternative, 1 mL of 1% cobalt or nickel chloride can be added to the DAB solution prior to slide incubation, and the resultant precipitate will be dark blue to black, not brown (7). This can increase the overall sensitivity of the reaction, but it is not popular because of the different color counterstain that is usually required. A 5% solution of Methyl Green used as a counterstain for 5 min provides enough contrast to the blue to be a good background for interpreting assays with a nuclear location. 

Fig. 8. Morphological changes of apoptotic eosinophils induced by dexamethasone (Z2). After eosinophils were treated (a) without or (b) with dexamethasone (2 /u,M) for 12 h, cells were harvested and detected by TUNEL assay using the In Situ Cell Death Detection Kit (Boehringer Mannheim). Briefly, cells were fixed with 4% paraformaldehyde and permeabilized by proteinase K and incubated with the TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT). After washing to remove unbound enzyme conjugated antibody, the horseradish peroxidase retained in the immune complex was visualized by a substrate reaction with diaminobenzidine. The cell nucleus was counterstained with methanol green. Apoptotic eosinophils with nuclear DNA breaks were seen to stain dark brown using a Nikon Eclipse E800 microscope (Nikon Corporation, Tokyo, Japan) in Fig. 8b.

De Leacy, E. A., Brown, N.N. Clague, A.E. (1989) Nitromethane interferes in assay of creatinine by the Jaffe reaction. Clin. Chem., 35, 1772-1774 Dellarco, VL. Prival, M.J. (1989) Mutagenicity of nitro compounds in Salmonella typhimurium in the presence of flavin mononucleotide in a preincubation assay. Environ, mol. Mutag., 13, 116-127... 

As with the Basic Protocol, this method can be used to assess latent DPO activity by addition of SDS. This simple assay system may also be used to investigate the effect of DPO inhibitors by adding suitable quantities of test compound dissolved in buffer (Ferrar and Walker, 1996, 1999). However, it must be remembered that, in this assay system, the prevention of color development (enzymic browning) may be due to chemical reactions between the inhibitor and the reaction products preventing the formation of the colored end-product, rather than the actual inhibition of the enzyme. This problem may be avoided by use of the 02-electrode assay (see Basic Protocol). 

The simplest, but least accurate, method of assaying DPO activity is to record the final color yield when the enzyme is incubated with a suitable chromogenic substrate such as catechol, DOPA, or 4-methylcatechol. DOPA is the most frequently used substrate in colorimetric assays because it yields a dark brown/black end-product. In this reaction, catecholase catalyzes the conversion of DOPA to dopaquinone and then to the red dopachrome, which subsequently polymerizes to yield dark brown melanin-type pigments. Unfortunately, this simple procedure has serious limitations, as it measures the end-product of a sequence of reactions rather than the true initial reaction rate. Furthermore, because different substrates yield different final colors, valid kinetic comparisons between substrates are not possible. Nevertheless, this simple assay technique has proved adequate for useful comparative studies of the levels of enzymic browning in different fruit varieties and similar problems (Vamos-Vigyazo, 1981 Machiex et al., 1990). 

This procedure is based on the formation of the electophile NO+, which can react with an ellagic acid residue (4.2) at two sites, either via a substitution reaction which results in 4.3, or an addition reaction that results in the nitrosyl dienone 4.4. These compounds can decay to form the quinine oxime 4.5, which under alkaline conditions forms the red product 4.6. When related compounds, such as gallic acid, phloroglucinol, hydroxycinnamic acids, and phenol are subjected to this assay, a yellow-brown product is formed, which does not interfere with the spectrophotometric detection of ellagic acid. 

Fig. 10.10. Determination of thermogenin amount in brown adipose tissue mitochondria by the enzyme-linked immunosorbent assay (ELISA) system. The amount of thermogenin was determined as elsewhere described (Cannon et al. [13] Sundin et al. [40] Hansen et al. [56]) in an assay system based on the competition between absorbed and added thermogenin for rabbit on/r-rat-thermogenin antibodies. The interaction was followed with a sheep onri-rabbit-IgG antibody conjugated to alkaline phosphatase. The reaction was linearized as indicated (abs 0 is the absorbance developed in the absence of competing thermogenin). It is seen that this assay can detect less than 0.25 fig thermogenin, i.e., the content in less than 10 fig of mitochondria. It is also seen that the thermogenin content of rat brown fat mitochondria is approximately doubled after a 24 h cold stress. (Our unpublished observations.)...

The methods are essentially those described by Brown and Cohen (BIO), with modifications to increase the sensitivity necessary for the small amounts of tissue obtained by biopsy. The simplest way of doing this was so to modify the reactions used for assaying the products of the enzyme reaction that smaller volumes and more dilute homogenates could be employed. 

---