Proteolysis modified protein

The O-ECAT reagent is a superior alternative to the use of 2,4-dinitrophenylhydrazine (DNPH Chapter 1, Section 1.1) in the study of protein oxidation. DNPH modification produces detectable complexes, but it does not provide information as to what amino acids are involved. O-ECAT modifies carbonyl end products of protein oxidation and in addition, it can provide exact information as to the amino acids that were oxidized. Mass spec analysis of modified proteins performed after proteolysis gives the exact amino acid sequences including the sites of O-ECAT reagent modification. The same antibody that is specific for the metal chelate portion of the standard ECAT reagent also can be used to capture and detect the O-ECAT... 

Preparation of Protease-Treated Proteins. The proteolysis of soy isolate was carried out by introducing 6 mL pronase E (mg/mL) to a well dispersed mixture of 12 g Mira Pro 111 in 760 mL water. Different levels of proteolysis were achieved by reaction at 50 °C for various periods of time. The reaction was stopped by heating at 100 C for 3 min, and the modified protein was then recovered by lyophilization. 

Emulsifying activity index (EAI) is a measure of the ability of protein to emulsify oil, which depends on solubility, size, charge, and surface activity of the protein molecules. The effect of proteolysis with pronase E on EAI of the modified protein was relatively insignificant (Figure 6) However, deamidation appeared to enhance EAI, especially at pH values more basic than the isoelectric point (pH 4.7). 

A concentration of 0.03M Ca2+ has been prescribed as a minimum in the formulation of imitation milk. Evidence from the study reported by Beuchat et al. (24) suggests that enzymatic hydrolysis of peanut flour modifies protein to the extent that it is highly soluble in 0.03M Ca2+ at a pH range normally associated with fluid milk. Further studies are required to assess the effect of enzyme-induced proteolysis on organoleptic properties of hydrolyzed peanut protein solutions. 

Limited digestion of globular soy proteins with rennin affords a modified protein preparation which retains a high molecular weight (47). Whipping quality, measured by foam volume and stability, was superior in comparison with native proteins. The limited rennin proteolysis of soy was identified as a key factor in functionality, since this modification conferred improved solubility. 

Fig. (1). The wound response in tomato plants. Tomato plants respond to wounding with the transcriptional activation and accumulation of Systemic Wound Response Proteins including defense proteins, proteolysis-associated proteins, signaling-associated proteins, and proteins of yet unknown function in plants defense. The change in gene expression can be monitored on SDS-PAGE gels. In comparison to control plants (I), treatment with systemin (II), or overexpression of the prosystemin cDNA (III) leads to the accumulation of SWRPs (arrowheads) and the downregulation of other, unidentified proteins (triangles). The figure was modified after [13].

Proteolytic modification has special importance for the improvement of solubility of proteins. This effect becomes significant even after very limited proteolysis. Hydrolysis of casein to DH of 2 and 6.7% with Staphylococcus aureus V8 protease increased the isoelectric solubility to 25 and 50%, respectively (Chobert et al., 1988a). However, it should be noted that the solubility profiles were not identical, due to a shift of the isoelectric point of the modified proteins. Solubility of a protein hydrolysate depends on the enzyme used (Adler-Nissen, 1986a). Protamex (a Bacillus proteinase complex) hydrolysates of sodium caseinate (DH 9 and 15%) displayed 85-90% solubility between pH 4 and 5 (Slattery and FitzGerald, 1998). 

Changes in the number of free amino residues alter the modified proteins susceptibility to proteolysis. Albumin chlorination and /V-chloramine formation decreases susceptibility to trypsin digestion. Removing of chloramine residues by treatment with thiosulfate shows that chlorination alters albumin properties by a biphasic mode the reversible chlorination and removal of chloramine moieties markedly increases albumin susceptibility to proteolysis, whereas chlorination produces the irreversible loss of amino moieties and carbonyl group formation effects decrease in albumin susceptibility to trypsin digestion. The effect is related to the number of lost amino residues. A similar relationship was observed for IgG. Fibrinogen and protamine, on the other hand, did not show dependence between chlorination and proneness to trypsin proteolysis (06). 

Gil. Giulivi, C., and Davies, K. J., Dityrosine A marker for oxidatively modified proteins and selective proteolysis. Meth. Enzymol. 233, 363—371 (1994). 

Methylation has also been found to enhance the membrane affinity of a number of different Icmt substrates. Of particular interest has been the effect of methylation on the biology of the proto-oncoprotein Ras. In an early study, K-Ras produced by in vitro translation was found to be farne-sylated, but further modification including proteolysis and methylation required incubation with intracellular membranes. Unmethylated K-Ras produced by in vitro translation in the presence of pancreatic microsomes and an inhibitor of methylation, methylthioadenosine (MTA), was found to associate less efficiently with PlOO membrane fractions from COS cells than the fully modified protein [55]. In other in vitro studies, farnesylated peptides corresponding to the C-terminus of Ras had 20-fold higher affinity for liposomes when methylated [56]. 

HPs are effective for the extraction of metabolites that function as pigments or flavorings permit the selective proteolysis of proteins that is important for the elaboration of modified milk for babies and allow the inhibition of the Maillard reaction among others (Barbosa-Canovas et al., 1998). However, it has been observed that the color of food can be affected by pressure. Within the more resistant pigments are the carotenoids, chlorophyll and anthocyanines, while myoglobin is more sensible (Cheftel, 1992). 

The functional properties of proteins depend also on their structure and interactions with the environment. The functional properties of surfactants depend on their hydrophilic-hydrophobic balance, too. Protein chains modified by proteolysis, amino acid incorporation, and transpeptidation may display different functional properties. As milk proteins possess good surface activities [131], the question of the changes in the functional properties of the enzymatically modified protein products is of especial interest. 

Proteolysis reduces the immunogenic character of the modified proteins. 

The oxidation of proteins is a natural part of aerobic life. Various free radicals, and reactive oxygen species generated as by-products of cellular metabolism, or from environmental sources, can modify the amino acids of proteins, leading to their inactivation. The majority of these modified proteins are either repaired directly, or removed by selective proteolysis. Mammalian cells contain only limited direct repair mechanisms for oxidized proteins. The two most widely studied proteolytic systems to remove oxidatively damaged proteins are the proteasome and the Lon protease. 

NCD-4 is a nonfluorescent carbodiimide derivative that forms a fluorescent adduct with the Ca -ATPase, accompanied by inhibition of ATPase activity and phos-phoenzyme formation [376-378]. Ca protected the enzyme against the inhibition by NCD-4 and reduced the extent of labeling, suggesting that the reaction may involve the Ca " " binding site. The stoichiometry of the Ca -protected labeling was i 2mole/mol ATPase. The fluorescence emission of the modified Ca -ATPase is consistent with the formation of a protein bound A-acylurea adduct in a relatively hydrophobic environment. After tryptic proteolysis of the NCD-4 labeled ATPase the fluorescence was associated with the A2 band of 24 kDa [376,379]. 

Biosynthesis of the polypeptide chain is realised by a complicated process called translation. The basic polypeptide chain is subsequently chemically modified by the so-called posttranslational modifications. During this sequence of events the peptide chain can be cleaved by directed proteolysis, some of the amino acids can be covalently modified (hydroxylated, dehydrogenated, amidated, etc.) or different so-called prosthetic groups such as haem (haemoproteins), phosphate residues (phosphoproteins), metal ions (metal-loproteins) or (oligo)saccharide chains (glycoproteins) can be attached to the molecule by covalent bonds. Naturally, one protein molecule can be modified by more means. 

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