Covalent attachment essential

Plastein Formation. The ability of some proteolytic enzymes to convert soluble proteins to an insoluble aggregate was noted as early as 1947 ( 42,43) in the formation of plakalbumin from albumin by subtilisin. More recently, Fujimaki and co-workers have investigated this reaction for removal of flavor constituents from soybeans as well as a method of covalently attaching essential amino acids to the protein. 

Functionalized conducting monomers can be deposited on electrode surfaces aiming for covalent attachment or entrapment of sensor components. Electrically conductive polymers (qv), eg, polypyrrole, polyaniline [25233-30-17, and polythiophene/23 2JJ-J4-j5y, can be formed at the anode by electrochemical polymerization. For integration of bioselective compounds or redox polymers into conductive polymers, functionalization of conductive polymer films, whether before or after polymerization, is essential. In Figure 7, a schematic representation of an amperomethc biosensor where the enzyme is covalendy bound to a functionalized conductive polymer, eg, P-amino (polypyrrole) or poly[A/-(4-aminophenyl)-2,2 -dithienyl]pyrrole, is shown. Entrapment of ferrocene-modified GOD within polypyrrole is shown in Figure 7. 

Currently available proteins are all deficient to greater or lesser extent in one or more of the essential amino acids. The recently advanced plastein reaction (229) has made it possible to use protein itself as substrate and to attach amino acid esters to the protein with high efficiency. By this method, soy bean protein (which is deficient in methionine) has been improved to the extent of having covalently attached L-methionine at 11%. 

Ras is strictly localized to the inner side of the plasma membrane. A lipid anchor covalently attached to the C-terminus of Ras penetrates into the lipid bilayer. This membrane anchorage is essential for the biological activity of Ras. Hence, the inhibition of anchor attachment has become an attractive pharmacological target [ 13]. See Waldmann H, Thutewohl M,Ras-Farnesyltransferase-inhibitors as promising anti-tumor drugs, this volume. 

The electrochemistry of the heme center shows a close to ideal full width halfmaximum (Tfwhm) for the oxidation peak. The close to ideal Efwhm is good evidence that all the proteins being interrogated electrochemically are in the same environment, which essentially means the only enzymes that are wired to the electrode are those covalently attached to the molecular wires. 

The observed adjuvanticity of Bordetella pertussis is largely attributable to the presence of pertussis toxin and lipopolysaccharide (LPS). LPS, a constituent of the cell envelope of Gramnegative bacteria (Chapter 3), essentially consists of polysaccharide moieties to which lipid (lipid A) is covalently attached. 

In 1978, Miller s group and Bard s group independently showed that chemically modified electrodes could be prepared by coating electrode surfaces with polymer films [20,21]. This has since proven to be the most versatile approach for preparing chemically modified electrodes. Indeed, until the recent rebirth of chemisorption and new covalent-attachment schemes (see earlier discussion), the polymer-film method had essentially supplanted all other methods for preparing chemically modified electrodes. 

The enzymes used to generate reactive quinone methides often undergo inactivation by addition of this electrophile to essential nucleophilic amino acid side chains of the protein catalyst. This is a type of suicide enzyme inhibition.80 This was observed for the acid phosphatase and ribonuclease catalysts used to generate 43.76 79 Alkaline phosphatase has been used to remove the phosphate protecting group from a derivative of an o-difluoromethyl phenyl phosphate that was covalently attached to a solid support. Breakdown of the immobilized 4-hydroxybenzyl difluoride gives an immobilized quinone methide that, in principle, will react irreversibly with proteins and lead to their attachment to the solid support.81... 

Covalent Attachment of Essential Amino Acids to Proteins by Chemical Methods Nutritional and Functional Significance... 

Limiting essential amino acids covalently attached to proteins by using activated amino acid derivatives can improve the nutritional quality and change the functional properties of proteins. The best chemical methods for incorporating amino acids into water-soluble proteins involve using car-bodiimides, N-hydroxysuccinimide esters of acylated amino acids, or N-carboxy-a-amino acid anhydrides. The last two methods can give up to 75% incorporation of the amount of amino acid derivative used. With the anhydride method, as many as 50 residues of methionine have been linked to the 12 lysine residues of casein. The newly formed peptide and isopeptide bonds are hydrolyzed readily by intestinal aminopeptidase, making the added amino acids and the lysine from the protein available nutritionally. 

All of the above-mentioned factors affecting the biological quality of proteins should be taken into consideration when dealing with the nutritional improvement of food and feed proteins. Furthermore, the covalent attachment of essential amino acids to proteins by chemical methods must avoid damage to the biological quality of proteins if it is to have potential applications. 

The ability to covalently attach 50 (and probably more) residues of methionine (and other essential amino acids) to proteins has important, practical implications. For example, a 1 5 mixture of c-methionyl casein (50 residues of methionine) and casein (5 residues of methionine), fed at 10% weight of the diet, would meet the methionine requirements of rats (23). Therefore the cost of modification and the carry over of products from side reactions would be reduced in the feed. 

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