Analytical methods protein residues

Insect OBPs are secretory proteins whose only posttranslational modification is the formation of three disulfide bridges [39,45] from six cysteine residues. That six cysteine residues are well conserved in OBPs from species of the same order is a hallmark of these proteins. The disulfide links of OBPs in a few species have been determined by analytical methods, first in the OBPs from B. mori [45,46]. As part of our attempt to get better insight into the structural biology of pheromone-binding proteins, we have determined the disulfide linkages... 

Cell Culture-Derived Media-Derived Protein Impurities. Immunoassays can detect low impurity levels (<1 ppm).4 The ELISA is probably one of the most sensitive analytical methods. If bovine serum is used as a media component, then testing should include ELISAs for bovine serum albumin (BSA), bovine transferrin, bovine fetuin, and bovine IgG. Often hormones and growth factors, such as insulin or insulinlike growth factor, are used as media components. ELISAs should be used to detect and quantitate these residuals in the various production steps as well as in the final product. There are commercially available antibodies to most commonly used media components. If proprietary media components are used, then the same investment in time and effort is required for the production of specific antibodies, as described above for host cell impurities. 

This strategy is attractive because the impurities will reflect exactly those produced by the host cell during product expression and which copurify with the product through the process. The immunoabsorption procedures required for this approach, however, are difficult to perform and validate at the part-per-million level (i.e., ng of impurity per mg of protein product). All of the product, product fragments and product-impurity complexes must be removed without the loss of any of the impurities. Any residual product will produce antibodies during immunization and render the assay nonspecific. Conversely, the immunoabsorbtion step must not remove any of the impurities. An exact demonstration of these criteria at the ppm level is not possible with currently available non-antibody based analytical methods. 

The analytical method proposed by Pajot (1976) involves (a) incubating the protein of a known concentration in 6 M guanidine at pH 6.5-7 in the presence of 30 mM 2-mercaptoethanol for 30 min (if only a small amount of the protein is available, the incubation can be carried out directly in the fluorescence cuvette) and (b) measuring the fluorescence, excited at 295 nm and observed at 354 nm, yielded by the denatured protein in 6 M guanidine hydrochloride (tryptophanyl residues concentration 3-10 /xM). A linear calibration curve is obtained, thus allowing the free tryptophan fluorescence equivalents of the protein sample to be estimated by extrapolation. 

Chemical Reactions Analytical Methods. During severe heat treatments of food proteins, chemical reactions occur between the e-amino group of lysine and the carboxamide group of the glutaminyl and aspara-ginyl residues (75). 

Reactions Analytical Methods. Alkaline treatments are used in the food and feeds technology for solubilization and purification of proteins, to destroy toxic contaminants, and to obtain functional properties. These alkaline treatments induce many chemical modifications on the side chains of the amino acid residues, which have been described by many workers. 

Once incorporated, unbound lewisite is quickly hydrolyzed. Its predominant metabolite is 2-chlorovinylarsonous acid, CVAA (Figure 50.8). Analytical methods to confinn lewisite exposure have, at least in the past, focused on the detection and quantification of CVAA. However, Noort et al. (2002) also pointed out that due to the high affinity of arsenic towards sulfhydryl groups, adducts of lewisite/ CVAA and cysteine residues of proteins are formed. In an in vitro study, incubating " C-labeled lewisite with human blood samples, 90% of lewisite was found in erythrocytes, whereas 25 to 50% of arsenic was bound to globin. From these protein adducts, CVAA can be released to form an adduct with the antidote British Anti-Lewisite (BAL) (Fidder et al, 2000). The authors were also able to identify a specific protein adduct of lewisite formed with the cysteine residues 93 and 112 of P-globin. See Detection of DNA and protein adducts of vesicants, below, for analytical... 

Influent concentrations and residual concentrations of cationic surfactants, anionic surfactants, cationic polyelectrolyte, anionic polyelectrolyte, proteins, colloids, oxygen, ozone, detergents, suspended sohds, and so on, in the adsorptive bubble separation systems can be determined by the analytical methods reported in the literature (82,127-149). 

The genetic code, regardless of whether it is a product of a "frozen accident" [1] or a deterministic interaction between the nucleotides and the amino acids [2], displays an apparent correlation between the nucleotides found at particular codon positions and the physico-chemical properties of the protein amino acid residues encoded by the nucleotides [2-10]. A variety of analytical methods have been employed to quantitatively examine these relationships. SjdstrOm and Wold [5], for example, have used Principal Component Analysis (PCA) to relate twenty physical properties of the amino acids to the genetic code. They find that 58% of the variance in the data can be accounted for by considering just three factors. In order of importance, the predominant contributions are 1) hydrophobicity, 2) molar volume, and 3) electronic descriptors (e.g. pKgS and NMR chemical shifts). Fig. 1 presents a concise display of... 

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