Hydrolysis methods monophosphate

A method for the direct spectrophotometric determination of dinucleoside monophosphates has been developed which relies on changes in u.v. absorbance after enzymic hydrolysis. - Hydrolytic fission of the dinucleoside monophosphate with a phosphodiesterase causes a change in the u.v. absorbance of the solution allowing the 5 -nucleoside to be estimated. Addition of a phosphomonoesterase to the hydrolysate causes a further change in u.v. absorbance, allowing the 3-nucleoside to be estimated. 

Since all condensed phosphates are ultimately degraded to monophosphate in hot solution, especially at low pH, the total phosphorus(V) content of a substance may readily be determined after hydrolysis either gravimetrically or titrimetrically (109). However, as soon as it is a question of estimating the content of separate components in mixtures of condensed phosphates insuperable difficulties are encountered if methods depending on precipitation, titration, or a combination of the two are used. Even a quantitative precipitation of monophosphate is impossible if polyphosphates with chain length of n = 3 or more arc present in the solution. The precipitating cation and the compound to be precipitated by it are partly kept in solution by the polyphosphate part of the polyphosphate is also carried down by the precipitate. Both of these effects depend in their extent in different ways on the nature and quantity of the substances present and the analysis gives a correct quantitative result only in isolated instances... 

Provided only polyphosphates are present it is possible to determine titrimetrically (1) the end-group content of mixtures (2) the content of groups within the chain, after hydrolysis and by estimating the total phosphate content (3) the monophosphate content after precipitation with silver nitrate (347). These methods are likewise of no use in presence of meta- and cross-linked phosphates. 

SM2/AM1 and SM3/PM3 models were used to study the hydrolysis of pyrophosphate, which is coupled to virtually all biosynthetic reactions. However, the authors concluded that extreme care must be taken when applying semiempirical methods to compounds containing second-row atoms, since they may produce anomalously high atomic charges [98]. On the other hand, a study on syn and anti conformations of solvated cyclic 3 ,5 -adenosine monophosphate indicated that SM3/PM3 and SM2/AM1 models are inexpensive yet accurate approaches... 

Assay methods are available for most of the carbohydrates ranging from glycogen, starch, and cellulose to the monosaccharides and their monophosphates. Concentration determination of complex carbohydrates usually depends on their enzymatic hydrolysis to monosaccharides and determination of monosaccharide concentration via an enzymatic reaction. For example, starch is hydrolyzed to glucose by glucoamylase, and the... 

Enzymatic hydrolysis exposes the sulfide that undergoes episulflde formation by cyclodeesteriflcation releasing the phosphate. This method was developed for intracellular delivery of a monophosphate. This concept was also extended to the use of an 5-glucoside (GTE group) that could be activated by a glucosi-dase to release the thiol. ... 

The racemic ( )-3TC 232 and other pyrimidine and pnrine analogues were resolved by varions methods (such as separation on HPLC with a chiral column, enzyme-catalyzed hydrolysis of its 5 -0-butanoyl ester derivative. or enzy matic resolution of its monophosphate derivative with a nucleotidase derived from Crotalus atrox venom ). Resolution of racemic thia sugar precursors lias also been described. 

In another synthesis, the disilver salt of adenosine-5 -phosphate was treated directly with an excess of dibenzylphosphorochloridate, and this was followed by hydrogenolysis and hydrolysis (10.104). In a later method, ATP was obtained directly from adenosine monophosphate using ortho-phosphoric add and dicyclohexyl-carbodiimide [84,85]. 

The separation of nucleotides and deoxynucleotides, previously a formidable task involving the fractional crystallization of heavy metal and alkaloid salts 102) has been made much easier by developments in analytical techniques. Ion-exchange methods may be used for the purification, isolation, and identification of both classes of nucleotides from hydrolysis mixtures 103), Countercurrent distribution 104) and starch 106) and cellulose-column 106) as well as paper-strip chromatography 107) have also proved to be useful in separating nucleotides from natural sources. Spectro-photometric procedures based on the characteristic ultraviolet absorption spectra of the purines and pyrimidines have been the most convenient method to locate, estimate, and identify the fractions obtained in the previous separations. Since the nucleotides are acid in nature, they are often named as acids, e.g., adenylic acid, cytidylic acid. The general constitution of the purine nucleotides (and by analogy the pyrimidine nucleotides) is demonstrated by their hydrolysis by acids to a purine and ribose (or 2-deoxyribose) monophosphate and by alkalies to the nucleosides and phosphoric acid. The order of the constituents in a purine nucleotide must, therefore, be ... 

When RNA is subjected to controlled alkaline hydrolysis eight nucleotides, two isomers each of AMP, GMP, cytosine monophosphate (CMP), and uridine monophosphate (UMP), are formed. These can be separated from one another on columns of anion exchange resins ISl), by paper chromatographic methods 1S2), and by fractional crystallization (iSS). The isomers are the 2 -phosphates ( a ) (XI) and 3 -phosphates ( b ) (XII) which arise from an intermediate cyclic 2, 3 -phosphate mononucleotide ester [Eq. (44)] (1S4-1S6). 

Oxidation with periodate followed by acid hydrolysis leads to the liberation of phosphate in compounds which have at least two vicinal OH groups next to the phosphate. The method has been used for the estimation of a-glycerophosphate and of inositol monophosphate. ... 

---