Phosphotungstic acid protein precipitation

Phosphotungstic acid will precipitate many proteins, lipids and alkaloids. Many soluble basic dyestuffs will form insoluble pigments known as lakes , by complexing them with phosphomolyb-date or phosphotungstate anions. Both insolubility and improvement of light fastness are achieved (Chapter 12.8). The formation of lakes of this kind can be used as a method of detection of P (Chapter 14.1), and in staining biological specimens for electron microscopy. 

Only in a few cases has this compound been isolated from the products of hydrolysis of proteins, since its separation is extremely laborious. It can only be effected after all the other amino acids have been removed by crystallisation and by the ester method, and after the diamino acids have been precipitated by phosphotungstic acid. From the last mother-liquors it is obtained by crystallisation, and is best identified in the form of its /8-naphthalene sulpho-derivative. 

The separation and estimation of the two main groups of amino acids can be carried out in one experiment, instead of separately as described. The protein is hydrolysed by sulphuric acid, the tyrosine, cystine and diaminotrioxydodecanic acid are removed by crystallisation, and the diamino acids are precipitated by phosphotungstic acid. From this precipitate they are obtained by decomposition with baryta, and they are then separated by means of their silver compounds by Kossel, Kustcher and Patten s method. The filtrate from the phosphotungstic acid precipitate is freed from the excess of phosphotungstic acid by means of baryta, and the solution is treated by Fischer s ester method for the monoamino acids. 

Phosphotungstic acid Discriminating protein precipitant. Precipitates Milesi et al. (2007), Wilkinson et al. (1992),... 

Phosphotungstic acid finds use in the precipitation of proteins, alkaloids, and certain amino acids. The solubility of sodium phosphotungstate, contrasted with the comparative insolubility of the potassium and ammonium salts, has suggested its use as a reagent in qualitative analysis. 

Introduction. Proteins are substances produced by living matter which on enzymatic or acid hydrolysis yield amino acids. They are non-volatile and of high molecular weight, and form colloidal dispersions from which they may be precipitated by-heat, alcohol, various salts, and acids (tannic, picric, and phosphotungstic). Each protein has a minimum solubility at a characteristic pH which is called the iso-electric point. At this point the protein molecules exist as a regates and the solution has the maximum turbidity. The nature of the union of various amino acids in the protein molecule is not known. One theory assumes that the carboxyl of one amino acid unites with the amino group of another, thus ... 

III. The Ordinary Alkaloidal Reagents,—The reagents which precipitate alkaloids from their solutions react also with proteins. Among these may be mentioned phosphotungstic acid, phosphomolybdic acid, tannic acid, picric acid, potassium mercuric iodide, and potassium bismuth iodide. The alkaloidal reagents precipitate the majority of the proteins in acid solution only. 

Tannic acid, phosphomolybdic acid, and phosphotungstic acid precipitate peptones. Most of the acid aniline dyes and many of the organic color bases also precipitate proteins. 

It is also possible to titrate biochemical species. Antibodies have been titrated with antigen, and enzyme-substrate mixtures have been titrated with appropriate coenzymes. Proteins are readily titrated with acid or base, or precipitated with phosphotungstic acid, yielding very informative thermometric titration curves [7, 8]. 

The octa-, deca-, etc., peptides are immediately precipitated by phosphotungstic acid they are also thrown down by tannic acid and by concentrated ammonium sulphate solutions. They resemble, in fact, many natural proteins and would have been regarded as such if they had been found in nature. They lack only the colour reactions due to the absence of tyrosine, tryptophane, etc., in their molecules. 

Several methods are available to measure lactose content. As lactose is optically active and rotates the plane of polarized light in proportion to its concentration, polarimetry may be used to quantify it. The proteins are first removed by precipitating them with phosphotungstic acid. This method is much less used now. 

These are protein-carbohydrate compounds which contain 10-75% of carbohydrate and over 4% of hexosamines (as distinct from glycoproteins which contain from a trace up to 15% carbohydrate and less than 4% of hexosamines). Oroso-mucoid, haptoglobin and haemopexin are examples of muco-proteins. Increased serum mucoprotein levels are found in many inflammatory conditions such as acute infections and rheumatoid arthritis. Mucoproteins can be estimated by selective precipitation using phosphotungstic acid, followed by determination of the protein or carbohydrate content of the precipitate. 

Since protamine is strongly basic, it forms salt-like complexes with various inorganic and organic acids. The sulfate, hydrochloride, nitrate, phosphate, and acetate salts are soluble in water, but the picrate, flavianate, and phosphotungstate salts are almost insoluble in water. Protamines also form scarcely soluble salts with silver nitrate, copper salts, mercury salts, etc. (Kossel, 1929). When heated to 60 °C with the Mirsky histone reagent (0.34 M mercuric sulfate in 1.88 M sulfuric acid) (Daly et al., 1951), protamine and histone are not precipitated whereas many other proteins precipitate. If the mixture is cooled to 0°C, the mercury salt of protamine is precipitated but histone remains in solution. This phenomenon can be utilized to distinguish protamine from histone. 

Proteins coextracted by salt or alkaline solutions can be precipitated with the usual reagents, such as phosphotungstic acid, phosphomolybdic acid, picric acid, etc., or denatured with Sevag s reagent, followed by precipitation of the polysaccharide with alcohol. Trichloroacetic acid, phenol or acetic acid extractions have also been used with success. 

To separate the peptides from the proteins, proteins have been precipitated by means of solutions of ethanol, methanol, acetone, or acids [trichloroacetic (TCA), sulfosalicyllic (SSA), phosphotungstic (PTA), picric], followed by centrifugation or filtration. 

The history of the discovery of amino acids is closely related to advances in analytical methods. Initially, quantitative and quaHtative analysis depended exclusively upon crystallization from protein hydrolysates. The quantitative precipitation of several basic amino acids including phosphotungstates, the separation of amino acid esters by vacuum distillation, and precipitation by sulfonic acid derivatives were developed successively during the last century. 

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