Alkaline solution, protein reactions

In alkaline solution, proteins are known to undergo the following types of reactions (a) denaturation, (b) hydrolysis of some peptide bonds, (c) hydrolysis of amides (asparagine and glutamine), (d) hydrolysis of arginine, (e) some destruction of amino acids, (f) e elimination and racemization, (g) formation of double bonds and (h) formation of new amino acids. 

Proteins form a complex with Cu ions in alkaline solution (Biuret reaction). The Cu ions of the complex are presumably reduced to Cu ions, which form a violet color complex with BCA. 

ImmunO lSS iy. Chemiluminescence compounds (eg, acridinium esters and sulfonamides, isoluminol), luciferases (eg, firefly, marine bacterial, Benilla and Varela luciferase), photoproteins (eg, aequorin, Benilld), and components of bioluminescence reactions have been tested as replacements for radioactive labels in both competitive and sandwich-type immunoassays. Acridinium ester labels are used extensively in routine clinical immunoassay analysis designed to detect a wide range of hormones, cancer markers, specific antibodies, specific proteins, and therapeutic dmgs. An acridinium ester label produces a flash of light when it reacts with an alkaline solution of hydrogen peroxide. The detection limit for the label is 0.5 amol. 

In general, the mechanism of heat and alkaline solution for DNA extraction may be based upon a hypothesis, previously proposed for the AR technique.32 Strong alkaline solution may denature and hydrolyze proteins, resulting in breaking cell and nuclear membranes as well as disrupting cross-linkages due to formalin fixation. It is no surprise to observe the similarity between retrieval of nucleic acid and retrieval of protein (antigen) based on a similar chemical reaction of formaldehyde with these two kinds of macromolecules (Fig. 3.1).15"19... 

Cathenamine (100) has been identified as an early intermediate in terpenoid indole alkaloid biosynthesis (cf. Vol. 8, p. 27). It has also been isolated from Guettarda eximia. Another alkaloid, 4,21-dehydrogeissoschizine (99), has now been isolated from this plant it is readily converted into (100) in alkaline solution.29 On incubation with an enzyme preparation from Catharanthus roseus cell cultures in the presence of NADPH at pH 7, (99) was converted into ajmalicine (102), 19-ep/-ajmalicine (103), and tetrahydroalstonine (104), which are the normal products with this enzyme preparation. In the absence of NADPH, cathenamine (100) accumulated.30 The reaction to give (100) proceeded linearly with time, and was dependent on the concentration of protein and substrate. No conversion occurred in the absence of enzyme. 

The significance of small amounts of metals to the Maillard reaction appears to have been first studied by Webb169 in 1935, in his work on the color of evaporated milk. The browning of lactose solutions at 49° was found to be positively catalyzed by copper and iron, but retarded by tin. Traces of cupric salts similarly accelerate the browning of amino acids or proteins with D-glucose in weakly alkaline solutions, but they have no ef-... 

Biuret Reaction. The particular capabilities of the stop-ped-flow vidicon system have been used to help explain some unusual behavior in the biuret reaction. The biuret reaction is the basis for the standard clinical chemistry technique for determining the total protein content of human blood serum (23). It involves a complexation reaction, in alkaline solution, between the cupric ion (Cu2+) and the peptide bonds of the protein. In the standard biuret technique, the reaction is allowed to go to completion and then the absorbance of the copper-protein complex is measured at 55 nm. This technique tends to be rather slow since the reaction, although very rapid over the first few seconds, does not go to completion for at least 20 to 30 minutes. 

Biuret Test. Compounds containing two or more peptide bonds (e.g., proteins) take on a characteristic purple color when treated with dilute copper sulfate in alkaline solution. The name of the test comes from the compound biuret, which gives a typically positive reaction. The color is apparently caused by the coordination complex of the copper atom and four nitrogen atoms, two from each of two peptide chains (Fig. II-5). The biuret test is fairly reproducible for any protein, but it requires relatively large amounts of protein (1 to 20 mg) for color formation. Because of its low sensitivity, the biuret assay is no longer widely used. 

Hydrolysis. Two types of hydrolytic reactions occur in proteins at alkaline pH. These are the hydrolysis of peptide and amide bonds and the hydrolysis of arginine to ornithine. Amide bonds are hydrolyzed rapidly in alkaline solution probably as shown in Equation 4 (21). 

Addition Reaction. The double bond of dehydroalanine and e-methyl dehydroalanine formed by the e-elimination reaction (Equation 6) is very reactive with nucleophiles in the solution. These may be added nucleophiles such as sulfite (44). sulfide (42), cysteine and other sulfhydryl compounds (20,47), amines such as a-N-acetyl lysine (47 ) or ammonia (48). Or the nucleophiles may be contributed by the side chains of amino acid residues, such as lysine, cysteine, histidine or tryptophan, in the protein undergoing reaction in alkaline solution. Some of these reactions are shown in Figure 1. Friedman (38) has postulated a number of additional compounds, including stereo-isomers for those shown in Figure 1, as well as those compounds formed from the reaction of B-methyldehydroalanine (from 6 elimination of threonine). He has also suggested a systematic nomenclature for these new amino acid derivatives (38). As pointed out by Friedman the stereochemistry can be complicated because of the number of asymmetric carbon atoms (two to three depending on derivative) possible. 

The reactions of proteins in alkaline solution are very important from a number of standpoints. We have already discussed several uses of alkali treatment in food processing in the introduction. When contact between the food and alkali is kept to a minimum at the lowest temperature possible with adequate control of mixing, etc. there is presently no apparent reason to discontinue its use. Low levels of lysinoalanine occur in food which has been processed in the absence of added alkali, even at pH 6 and in the dry state (20). For example, the egg white of an egg boiled three minutes contained 140 ppm of lysinoalanine while dried egg white powder contained from 160 to 1820 ppm of lysinoalanine depending on the manufacturer (20). No lysinoalanine was found in fresh egg white, 3 Elimination and addition of lysine to the double bond of dehydroalanine reduce the level of the essential amino acid lysine. This can be prevented by adding other nucleophiles such as cysteine to the reaction. Whether lysinoalanine (and other compounds formed by addition reactions) is toxic at low levels in humans is not known. 

Several s pec tropho tome trie methods are available for the determination of protein in solution. The biuret method is based on the reaction of Cu with peptides in alkaline solution to yield a purple complex that has an absorption... 

This mode of union is called peptide linkage. The presence of the peptide linkage is thought to account for the development of a violet color when a protein comes in contact with an alkaline solution of cupric ion. The color is given by biuret, formed by urea on heating NH2CONHCONH2. The reaction is known as the biuret reacUon. It is not given by any amino acid. 

Partial removal of the phosphate groups of phosvitin by -elimination in alkaline solution results in a decreased in vitro initial rate of hydrolysis by trypsin (268). The decreased rate of hydrolysis might be a result of (a) a change in conformation of the protein on removal of the phosphate groups, (b) cross-linking by the reaction of the dehydroalanine residues with lysine residues (to form lysinoalanine), or (c) racemization of some of the residues by the alkaline treatment. 

Quantitative Analysis after Chemical Reaction. A large number of fluorometric analyses have been performed after converting a nonfluorescent or weakly fluorescent compound into an intensely fluorescent species. One example, which involves only hydrolysis, is the measurement of acetylsalicylic acid as salicylic acid or the salicylate anion. Because the total concentration of these two acids in blood is very important in the treatment of rheumatic disease, acetylsalicylic acid is converted to salicylic acid by hydrolysis and the total measured as salicylate. First the serum protein is removed, then the acetylsalicylic acid is hydrolyzed under alkaline conditions to salicylic acid and acetic acid (in alkaline solution, an equilibrium mixture of the anions and acids is present). The alkaline solution is then excited at 310 nm and the fluorescence emission is measured at 410 nm. 

Because the thiol anion is the reactive species, the reactions are fastest in alkaline solution. Thiol or tributyl phosphine are usually used to break cystine residue disulfide cross-links to prepare soluble proteins from wool to be studied in solution. Especially in alkaline solution, cysteine residues in proteins are reoxidized by dissolved oxygen, and cystine residue disulfide cross-links are reformed ... 

Partially oxidized cystine residues are intermediates in the reaction. Some hydrolysis of peptide bonds occurs during oxidation with peracetic acid, less with performic acid. Methionine and tryptophan residues are also oxidized. Oxidized wool is soluble in alkaline solutions. When these solutions of dissolved, oxidized wool proteins are acidified, a low-sulfur protein, a-keratose, is precipitated. A high-sulfur protein, -y-keratose, remains in solution. (P-keratose is the residue of oxidized wool that is not soluble in alkali.)... 

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