Intracellular concentration of inorganic pyrophosphate in the cells of Escherichia coli: A method for its determination

A new method was developed for the separation of labelled inorganic pyrophosphate (PP₁) from the extracts of bacterial cells grown in the presence of labelled phosphate of high specific activity. First PP₁ is precipitated as manganous salt. Further purification is achieved in two chromatographic runs performed in the opposite directions on the same sheet of paper. The first solvent system consists of ethyl methyl ketone-methanol-water-36% HCl (20:40:10:1, by vol) and the second of ethyl methyl ketone-methanol-water-trichloroacetic acid-25% NH₄-EDTA (40 ml: 10 ml: 15 ml: 5 g: 0.8 ml: 50 mg). Each run takes only about 40 min of time. Thus it is possible to treat tens of samples in one working day.Using this procedure the intracellular concentration of PP₁ in the exponential phase cells of Escherichia coli growing aerobically in minimal medium at 37°C was determined to be about 1.3 mm or 5.5 nmoles/mg of dry weight.     

Determination of inorganic pyrophosphate concentration in urine

A simple method for the estimation of PPi in urine is described. The PPi and Pi may be determined simultaneously by this method.     

A rapid colorimetric method for the determination of mimosine

A rapid colorimetric method has been described for the quantitative determination of mimosine by using activated carbon as a decolorizing agent and measuring the intensity of mimosine-ferric chloride color produced.     

A simple colorimetric method for the determination of tryptophan

A simple, sensitive, and reproducible colorimetric method for the determination of tryptophan in amounts as low as 2 μg is described. It is based on the oxidation of tryptophan by sodium nitrite and the coupling of the oxidized product to the leucodye N-1-(naphthyl)ethylenediamine dihydrochloride. The purple-pink product has an absorption maximum at 550 nm. There is no interference by carbohydrates, other amino acids, neutral salts, or a number of other compounds likely to be found in tissue hydrolysates. A number of indole derivatives including indole-3-acetic acid also react to give a colored product. Dipeptides containing tryptophan are much less reactive than free tryptophan; hence proteins must be hydrolyzed completely for the method to be useful. The assay is carried out at room temperature and can be modified easily to increase or decrease its sensitivity. It has been employed to determine the tryptophan content of a number of proteins following alkaline hydrolysis. Generally, values obtained were in close agreement with values reported in the literature.     

Colorimetric determination of inorganic pyrophosphate by a manual or automated method.

A microprocedure for the colorimetric determination of inorganic pyrophosphate (PP_i) in the presence or absence of orthophosphate (P_i) has been developed. PP_i is estimated quantitatively as the amount of chromophore formed with molybdate reagent, 1-amino-2-naphthol-4-sulfonic acid in bisulfite and thiol reagent (monothioglycerol or 2-mercaptoethanol). The latter is obligatory for color formation. P_i is estimated without thiol reagent. The two chromophores differ in absorption spectra, the greatest difference being at 580 nm. For both, color develops fully by 10 min and is stable up to 1 hr. Just less than 0.4 μm PP_i can be detemined. The extinction coefficients are 2.70 × 10⁴ and 8.76 × 10³ for PP_i and P_i, respectively, both with thiol reagent present, and 2.77 × 10³ for P_i with no thiol reagent.A ten-fold excess of P_i does not interfere with the determination of PP_i and in fact can be estimated in the same mixture. A 15-fold excess, however, diminishes the accuracy of PP_i estimations. Trichloroacetic acid and sodium fluoride inhibi color formation, but this inhibition is overcome by the addition of sodium acetate buffer, pH 4.0. Nucleoside triphosphates and adenosine 3′:5′-cyclic monophosphate are stable in the reaction mixture.The method was tested in assays of Escherichia coli DNA-dependent RNA polymerase (nucleoside triphosphate: RNA nucleotidyltransferase, EC 2.7.7.6). Progress curves measured by either the rate of PP_i formation or the rate of synthesis of labeled RNA were very similar. Product PP_i formed by as little as 0.6 unit of RNA polymerase in a 225-μl incubation medium could be measured.An automated version of the method was devised which allows accurate determination of PP_i down to 1 μm (without range expander attachment) at a sampling rate of 20–40 tubes/hr.