Protein damage assay

Mechanism. Numerous studies on the mechanism of IPO toxicity have supported the view that tissue damage by the compound is due to a highly reactive, alkylating metabolite(s) (Figure 2)(12). In vitro experiments demonstrated that this metabolic activation is catalyzed by a cytochrome P-450 enzyme system which is located in the endoplasmic reticulum of target cells(lO). This metaboliteCs) forms covalent bonds with cellular macromolecules, and it causes cell death (necrosis). The amount of cellular necrosis (measured by microscopic examination of the respective tissues 24 hours after exposure to the toxin) and the extent of protein alkylation (assayed by employing or H-IPO and measuring the amount of label... 

This modification to the ADF method was designed to improve the relationship between the ADF and digestibility in ruminants. The sample drying temperature of 95°C, however, means it is unsuitable for assaying heat damage and unavailable protein (Van Soest, 1982). The method below is based on that given in MAFF/ADAS (1986, pp. 93-94), with Crown Copyright permission. See Chapter 4 for further discussion on fibre extraction procedures. 

Microbes and metazoans are exposed to a variety of toxic stresses and have evolved appropriate defenses and repair mechanisms. Some of these systems are regulated at the protein level, but others are regulated at the transcriptional level, allowing the development of reporter assays. These transcriptional responses can be used to provide an earlier marker for genotoxin exposure in a whole population of cells, rather than the detection of the endpoints discussed above, in which genotoxic stress leads to fixation of mutations or chromosomal aberrations/damage in a small subpopulation. 

Reznick A, Packer L (1994) Oxidative damage to proteins spectrophotometric method for carbonyl assay. Methods Enzymol 233 357-363... 

I are regulatory proteins involved in myocardial contractility. They are released into the plasma in response to cardiac damage. Elevated serum troponins are more predictive of adverse outcomes in unstable angina or myocardial infarction than the conventional assay of CK2. 

When ground beef is irradiated by electron beam and gamma-ray irradiation and its proteins assayed for amino acid content, the most sensitive amino acid to irradiation by either source is cystine. Approximately 50% of cystine is destroyed under the most damaging conditions used. Under the same conditions tryptophan shows about 10% destruction while arginine, phenylalanine, and tyrosine showed no destruction, and the rest of the amino acids show little destruction. From the cystine and tryptophan data, it appears that for electron beam irradiation the extent of destruction is not related primarily to total dose but to dose rate and particularly to energy level of irradiating dose. 

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