Protein alkali treatment

Most of the common methods of isolation of heparin (described in sufficient detail in monographs128-30) are based on a procedure, developed by Charles and Scott,31 involving autolysis of the tissue (originally beef liver and beef lung), extraction with alkali, coagulation of proteins by heating, and precipitation of a heparin - protein complex by acidification. Heparin is recovered from the complex by reprecipitation with ethanol, or acetone, or both. Fats are removed by extraction with ethanol, and proteins by treatment with trypsin. Modifications of this proce-... 

Alkali treatment is used to solubilize and isolate proteins, to improve foaming and emulsifying properties, and to obtain protein solutions suitable for spinning fibers (17) ... 

In glycoproteins containing a carbohydrate-L-serine linkage, such as 50, or a carbohydrate-L-threonine linkage, the carbohydrate is released from tire protein on treatment with base. The carbohydrate released is then subjected to further degradation, until an alkali-stable structure is formed. This reaction was used by Kabat, Lloyd,... 

India ink is used to stain electroblotted proteins on blot transfer membranes. Transferred proteins (>5 ng/band) appear as black bands on a gray background. Sensitivity may be enhanced by brief alkali treatment of the membrane with 1% KOH followed by several rinses with PBS. 

The enantiomeric separation of the D- from the L-stereoisomers of amino acids is an area of growing interest. It is generally recognized that heat- and alkali-treatment of proteins can result in the racemization of L-isomers of amino acid residues to the D-analogs. Almost without exception, humans cannot utilize the D-isomers of amino acids, and some are thought to be toxic (although... 

Surfactants disrupt the cell wall by solubilizing the lipids in the wall. Sodium dodecylsulfate (SDS), sodium sulfonate, Triton X-100, and sodium taurocholate are examples of the surfactants often employed in the laboratory. Alkali treatment disrupts the cell walls in a number of ways including the saponification of lipids. Alkali treatment is inexpensive and effective, but it is so harsh that it may denature the protein products. Organic solvents such as toluene can also rupture the cell wall by penetrating the cell wall lipids, swelling the wall. When red blood cells or a number of other animal cells are dumped into pure water, the cells can swell and burst due to the osmotic flow of water into the cells. 

Since the last symposium there have been several changes in attitudes or directions. One of these has been the recognition that alkali treatment of proteins is a chemical procedure, and in particular, a chemical modification. Another has been the recognition that, although there are still difficult problems in assessing the safety aspects of chemical modifications of foods, both chemical and enzymatic treatments can be useful in laboratory experimentation and, perhaps, in eventual application to food production. Increase in nutritional value through modifications is now recognized. 

Alkali treatment of proteins is becoming more common in the food industry and may result in several undesirable reactions. When cystine is treated with calcium hydroxide, it is transformed into amino-acrylic acid, hydrogen sulfide, free sulfur, and 2-methyl thia-zoIidine-2,4-dicarboxyIic acid as follows ... 

Therefore, adding alkali to proteins may accomplish an increased solubilization of the protein while in the alkaline solution. However, upon adjustment to neutral or acid pH, the protein may be less soluble than originally because of denaturation. In texturization of proteins, this denaturation may be an advantage. However, in other cases such as alkali treatment to destroy aflatoxin or protease inhibitors it may be a disadvantage. 

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. 

The e-elimination reaction could also be used to change the solubility properties of a protein. For example, alkali treatment in the presence of sodium sulfite leads to incorporation of sulfonate groups into the protein (44,53) which would increase its water solubility and probably change its functional properties. 

Alkali Treatment. The following is a typical procedure. A 1% solution of each protein in 0.1 N NaOH (pH< 12.5) was placed in a glass-stoppered Erlenmeyer flask and incubated at the appropriate temperature in a water bath. The final pH did not differ significantly from the initial value. After three hours, the sample was dialyzed against 0.01 N acetic acid for approximately two to three days and lyophilized. Control protein samples were dialyzed and lyophilized similarly. The pH was measured with a Coming pH meter before and after treatment. 

De Groot, A. P. and Slump, P. (1969). Effects of severe alkali treatment of proteins on amino acid composition and nutritive value. J. Nutr. 98, 45-56. 

Feron, V. J., van Beek, L., Slunp, P. and Beems, R. B. (1977). Toxicological aspects of alkali treatment of food proteins. In "Biochemical Aspects of New Protein Food",... 

Alkali treatment has been used to improve the functional properties of the insoluble protein prepared by heat precipitation of an alkaline extract of broken yeast cells (63). Heating yeast protein at pH 11.8 followed by acid precipitation (pH 4.5) yielded a preparation composed of polypeptides with increased aqueous solubility. It also increased foaming capacity of the protein 20-fold. The emulsifying capacity of the modified protein was good whereas the original insoluble protein was incapable of forming an emulsion. Alkali treatment must be carefully controlled to avoid its possible deleterious effects (24,75), e.g. alkaline treatment of yeast protein resulted in a loss (60%) of cysteine (63). 

In previous papers, we have (a) reviewed elimination reactions of disulfide bonds in amino acids, peptides, and proteins under the influence of alkali (5) (b) analyzed factors that may operate during alkali-induced amino acid crosslinking and its prevention (6) (c) demonstrated inhibitory effects of certain amino acids and inorganic anions on lysinoalanine formation during alkali treatment of casein, soy protein, wheat gluten, and wool and on lanthionine formation in wool ( 7, 9) (d) demonstrated that... 

Alkali Treatments. The following procedure, illustrated with casein, was also used with the other food proteins. A solution or suspension of casein (usually 0.5 gram per 50 cc of solvent or 1% w/v) in borate buffer of appropriate pH in a glass-stoppered Erlenmeyer flask was placed in a 65°C water bath. After the indicated time, the solution was dialyzed against 0.01N acetic acid with frequent changes of water plus acetic acid for about two days and then lyophilized. 

For example, the respective values at pH 10.6 are 0.262, 0.494, and 1.04 mole per cent (ratio of about 1 2 4) at pH 11.2 the values are 0.420, 0.780, and 1.32 mole per cent and at pH 12.5 (pH of 1% protein solution in 0.IN NaOH), the respective values are 0.762, 0.780, and 2.62 mole per cent. (Note that the value of casein approaches that of gluten at this pH). The observed differences in lysinoalanine content of the three proteins at different pH values are not surprising since the amino acid composition, sequence, protein conformation, molecular weights of protein chains, initial formation of intra- versus intermolecular crosslinks may all influence the chemical reactivity of a particular protein with alkali. Therefore, it is not surprising to find differences in lysinoalanine content in different proteins treated under similar conditions. These observations could have practical benefits since, for example, the lower lysinoalanine content of casein compared to lactalbumin treated under the same conditions suggests that casein is preferable to lactalbumin in foods requiring alkali-treatment. 

Neither of the linkages formed by reactions (XXI) or (XXII) has been demonstrated in alkali-treated wool, but Patchornik and Sokolovsky (1964) and Bohak (1964) have demonstrated recently the presence of -iV-(D,L-2-amino-2 carboxymethyl)-L-lysine in hydrolyzates of proteins treated with alkali. This they attribute to the reaction of the -NH2 group of lysine residues with a-aminoacrylic acid residues formed from cystine residues by the 8-elimination reaction (Section V,A,5). Ziegler (1964) has shown that this amino acid residue is formed during the alkali treatment of wool in both the stretched and unstretched states. No assessment of the importance of these linkages in retaining set has yet been reported. 

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