Modifications of Proteins

Bovin trypsin has been modified with semicarbazide in the presence of a carbodiimide probably involving carboxyl groups. Also, monoamine derivatives of a, fi or y cyclodex-trins are introduced into trypsin. The thus modified trypsins are more resistant to autolysis and show some increase in esterase activity. Ribonucleases are also modified by attaching aminoethanol, taurin and 1,2-diaminoethane to approximately six to eight of the 11 available carboxylates using EDC. The modified enzymes lose activity and the cytotoxicity is increased. 

The reaction of a carbodiimide alone with a protein can lead to deactivation, but since 

An endo-1,4-j8-xylanase (xylanase A), obtained from Schizophyllum commune, is rapidly deactivated using l-(4-azonia-4,4-dimethylpentyl)-3-ethylcarbodiimide iodide, indicating that the reactive site in the enzyme is a carboxyl group. Xylanase A is of importance for the conversion of hemicellulosic biomass into fermentable products. 

Covalent immobilization of enzymes increases their stability while lowering their activity. Also, their storage stability is notably higher. The synthesis of arginine from citrulline, ATP and argenino-succinate synthetase may involve a carbodiimide intermediate.  

Membrane ATPases have also been inhibited by carbodiimides. This reaction is associated with the membrane lipoprotein. Carbodiimide binding proteins have been isolated from bacterial membranes, chloroplasts, animal liver mitochondria, bovine heart mitochondria,molds and yeasts. The site of carbodiimide attack in the protein is probably in the hydrophobic region because only lipophilic carbodiimides are effective inhibitors. The addition of methyl glycinate protects erythrocyte membrane ATPase against carbodiimide inhibition. The inhibition reaction of carbodiimides may involve an O N acyl shift in the initially formed O-acylurea. 

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While electrospray is used for molecules of all molecular masses, it has had an especially marked impact on the measurement of accurate molecular mass for proteins. Traditionally, direct measurement of molecular mass on proteins has been difficult, with the obtained values accurate to only tens or even hundreds of Daltons. The advent of electrospray means that molecular masses of 20,000 Da and more can be measured with unprecedented accuracy (Figure 40.6). This level of accuracy means that it is also possible to identify post-translational modifications of proteins (e.g., glycosylation, acetylation, methylation, hydroxylation, etc.) and to detect mass changes associated with substitution or deletion of a single amino acid. 

Hydroxylated amino acids (eg, 4-hydroxyproline, 5-hydroxylysine) and A/-methylated amino acids (eg, /V-methylhistidine) are obtained by the acid hydrolysis of proteins. y-Carboxyglutamic acid occurs as a component of some sections of protein molecules it decarboxylates spontaneously to L-glutamate at low pH. These examples are formed upon the nontranslational modification of protein and are often called secondary protein amino acids... 

Two other practical appHcations of en2yme technology used in dairy industry are the modification of proteins with proteases to reduce possible allergens in cow milk products fed to infants, and the hydrolysis of milk with Hpases for the development of Hpolytic flavors in speciaHty cheeses. 

Dialdehyde-containing nucleic acids and their components, synthesis, properties, and affine modification of proteins 99UK267. 

After their synthesis (translation), most proteins go through a maturation process, called post-translational modification that affects their activity. One common post-translational modification of proteins is phosphorylation. Two functional classes of enzymes mediate this reversible process protein kinases add phosphate groups to hydroxyl groups of serine, threonine and tyrosine in their substrate, while protein phosphatases remove phosphate groups. The phosphate-linking... 

Evolution has provided the cell with a repertoire of 20 amino acids to build proteins. The diversity of amino acid side chain properties is enormous, yet many additional functional groups have been selectively chosen to be covalently attached to side chains and this further increases the unique properties of proteins. Diese additional groups play a regulatory role allowing the cell to respond to changing cellular conditions and events. Known covalent modifications of proteins now include phosphorylation, methylation, acetylation, ubi-quitylation, hydroxylation, uridylylation and glycosyl-ation, among many others. Intense study in this field has shown the addition of a phosphate moiety to a protein... 

Vitamin K is the cofactor for the carboxylation of glutamate residues in the post-synthetic modification of proteins to form the unusual amino acid y-carboxygluta-mate (Gla), which chelates the calcium ion. Initially, vitamin K hydroquinone is oxidized to the epoxide (Figure 45-8), which activates a glutamate residue in the protein substrate to a carbanion, that reacts non-enzymically with carbon dioxide to form y-carboxyglut-amate. Vitamin K epoxide is reduced to the quinone by a warfarin-sensitive reductase, and the quinone is reduced to the active hydroquinone by either the same warfarin-sensitive reductase or a warfarin-insensitive... 

Molecular Mechanism of Oxidant Stress-induced Modifications of Protein Function... 

If cellular redox state, determined by the glutathione status of the heart, plays a role in the modulation of ion transporter activity in cardiac tissue, it is important to identify possible mechanisms by which these effects are mediated. Protein S-,thiolation is a process that was originally used to describe the formation of adducts of proteins with low molecular thiols such as glutathione (Miller etal., 1990). In view of the significant alterations of cardiac glutathione status (GSH and GSSG) and ion-transporter activity during oxidant stress, the process of S-thiolation may be responsible for modifications of protein structure and function. 

The six major proteins of milk, asl-, o s2-, and /c-casein, jS-lactoglobulin, and a-lactalbumin, contain at least one tryptophan residue [57], the fluorescence of which allows the monitoring of the structural modifications of proteins and their physicochemical environment during the coagulation processes. Emission fluorescence spectra of the protein tryptophanyl residues were recorded for the milk coagulation kinetics induced by... 

McCracken, P. G. Bolton, J. L. Thatcher, G. R. J. Covalent modification of proteins and peptides by the quinone methide from 2-ZerZ-butyl-4,6-dimethylphenol selectivity and reactivity with respect to competitive hydration, j. Org. Chem. 1997, 62, 1820-1825. 

K. Mizutani, T. Electronic and structural requirements for metabolic activation of butylated hydroxytoluene analogs to their quinone methides, intermediates responsible for lung toxicity in mice. Biol. Pharm. Bull. 1997, 20, 571-573. (c) McCracken, P. G. Bolton, J. L. Thatcher, G. R. J. Covalent modification of proteins and peptides by the quinone methide from 2-rm-butyl-4,6-dimethylphenol selectivity and reactivity with respect to competitive hydration. J. Org. Chem. 1997, 62, 1820-1825. (d) Reed, M. Thompson, D. C. Immunochemical visualization and identification of rat liver proteins adducted by 2,6-di- m-butyl-4-methylphenol (BHT). Chem. Res. Toxicol. 1997, 10, 1109-1117. (e) Lewis, M. A. Yoerg, D. G. Bolton, J. L. Thompson, J. Alkylation of 2 -deoxynucleosides and DNA by quinone methides derived from 2,6-di- m-butyl-4-methylphenol. Chem. Res. Toxicol. 1996, 9, 1368-1374. 

Originally, for preparation of such conjugates the hydroxyl groups of monomethoxy-PEG (mPEG) were activated with cyanuric chloride, and the resulting compound then coupled with proteins (10). This approach suffers from disadvantages, such as the toxicity of cyanuric chloride and its limited applicability for modification of proteins having essential cysteine or tyrosine residues, as manifested by their loss of activity. 

Formulation strategies for stabilization of proteins commonly include additives such as other proteins (e.g., serum albumin), amino acids, and surfactants to minimize adsorption to surfaces. Modification of protein structure to enhance stability by genetic engineering may also be feasible, as well as chemical modification such as formation of a conjugate with polyethylene glycol. 

Chemical modifications of proteins (enzymes) by reacting them with iV-acylimidazoles are a way of studying active sites. By this means the amino acid residues (e.g., tyrosine, lysine, histidine) essential for catalytic activity are established on the basis of acylation with the azolides and deacylation with other appropriate reagents (e.g., hydroxylamine). 

Post-translational modification of proteins plays a critical role in cellular function. For, example protein phosphorylation events control the majority of the signal transduction pathways in eukaryotic cells. Therefore, an important goal of proteomics is the identification of post-translational modifications. Proteins can undergo a wide range of post-translational modifications such as phosphorylation, glycosylation, sulphonation, palmitoylation and ADP-ribosylation. These modifications can play an essential role in the function of the protein and mass spectrometry has been used to characterize such modifications. 

Yi L, Shi J, Gao S et al (2009) Sulfonium alkylation followed by click chemistry for facile surface modification of proteins and tobacco mosaic virus. Tetrahedron Lett 50 759-762... 

Kivirikko, K.I., Myllyla, R. and Pihlajaniemi, T. (1992) Post Translational Modification of Proteins. CRC Press, Boca Raton, Florida, pp. 1-51. 

The N-Zyme Biotec business model unites three areas synergistically (i) strategic alliances in R D, (ii) a module-based services system for the manufacture and modification of proteins, and (iii) product development. Mainly oriented to the Health Sector, its capabilities could be extended to other industries. 

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