Of the colour reactions available for the determination and identification of deoxyribonucleic acid (DNA), the reaction with diphenylamine in a mixture of acetic and sulphuric acids at 1000 (Dische, 1930) has been perhaps the most widely used. The present study arose from the observation that a more intense colour was sometimes produced if, instead of being heated at 1000 for 10 min., the reaction mixture was allowed to stand overnight at room temperature. As a result of this observation the procedure has been modified, principally by adding acetaldehyde to the reagents and by allowing the solution to stand for about 17 hr. at 30° instead of heating it at 1000. The modified method is 3-5 times as sensitive as Dische's original procedure, and several substances which interfere in the original method do not do so in the modified procedure. Some observations on the mechanism of the reaction have been made; in particular it was discovered that there is a liberation of inorganic orthophosphate from DNA during the early stages of the reaction. This finding has a bearing on the structure of DNA. The modified method has already been used in an investigation of nucleic acid metabolism during bacteriophage multiplication (Burton, 1955).

/pdf/a-study-of-the-conditions-and-mechanism-of-the-diphenylamine-3g9hz1sxlu.pdf

A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid

The Carbon Monoxide-binding Pigment of Liver Microsomes I. EVIDENCE FOR ITS HEMOPROTEIN NATURE

more acidic solutions, with pH < 7 0, a few seconds elapsed before equilibrium was attained. Taking into account the ionization of the haem-linked group on MetMb and the higher oxidation state, the variation of Kb,. with pH is shown to confirm the conclusion that 2 moles ofH+ are liberated/mole of acidic MetMb. Using 6-1 for the pK of the group in MetMb as established in other studies, the results give apK of75 for the group in the higher oxidation state at 200 and I= 0 04. 3. The variation of KRb, with temperature gives AHO = 10-0 ± 2*0 kcal./g.mol.: if the ionization of the haem-linked group is allowed for, the value 9*0 ± 1.0 kcal./g.mol. is obtained. 4. The dependence of Kob. on ionic strength is in accord with a change in charge from + 1 on MetMb to zero on the higher oxidation state. 5. The results are shown to favour the ferryl ion structure, or an isomer of this structure, for the higher oxidation state. The isomeric structures would, in general, require the presence of another ionizing group in myoglobin, but no evidence for such an ionization could be found. With other direct evidence favouring the ferryl ion structure this is to be preferred, and the higher oxidation state may provisionally be named ferrylmyoglobin.

Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat-liver tissue

Publisher Summary This chapter presents the assay of glutathione (GSH), glutathione disulfide (GSSG), and glutathione mixed disulfides (GSSR). The sum of the reduced and oxidized forms of glutathione can be determined by using a kinetic assay in which catalytic amounts of GSH or GSSG and glutathione reductase bring about the continuous reduction of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) by nicotinamide adenine dinucleotide phosphate (NADPH). The sensitivity of the assay may be enhanced by measuring NADPH fluorometrically. There are other two enzymatic assay available. In the first assay, the formation of the reduced glutathione using glyoxalase I can be monitored directly at 240 nm, or at very low levels of GSH, a dual wavelength spectrophotometer can be used at the wavelength-pair 240–270 nm with the same extinction coefficient. The second assay involves O-phthaldialdehyde, which forms a fluorescent complex with GSH and can be monitored at 350 nm–420 nm.

Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples.

https://www.jbc.org/content/246/17/5477.full.pdf

E. Racker

Papers

Crystalline alcohol dehydrogenase from bakers' yeast

The mechanism of action of glyoxalase.

Spectrophotometric measurement of hexokinase and phosphohexokinase activity.

The mechanism of oxidation of aldehydes by glyceraldehyde-3-phosphate dehydrogenase

Enzymatic synthesis and breakdown of desoxyribose phosphate