Methyl Green-Pyronin

Abstract

We have observed (1) that the selective staining of nucleic acids by methyl green and pyronin is a function of the relative states of polymerization of the nucleic acids. The purpose of this paper is to present studies on the stoichiometry of the reactions, in order to elucidate the mechanism of the selectivity and to explore the possibility of quantitative histochemical application.The studies, as applied to pyronin, met with only very limited success. Unlike methyl green, which appears to form a stable compound with polymerized DNA, pyronin-stained RNA or depolymerized DNA-precipitates lose stain continuously when washed with alcohol or aqueous buffers. The "end point" which is eventually reached, represents the retention of only a few per cent of the original dye bound (cf. Table VI and Table VIII of the preceding paper (1)). Consequently, the possibility of non-specifically adsorbed dye to the pyronin-nucleic acid complex which precipitates on the addition of pyronin to nucleic acid solutions (in high concentrations of dye) cannot be excluded, thus vitiating the significance of the stoichiometric data based upon precipitation experiments in which combined dye was determined by difference between that remaining in the supemate and the original concentration. Likewise, washing probably results in considerable dissociation of the complex so that the "end point" is also not reliable for stoichiometry. However, Table VI (preceding paper (1)) based on precipitation experiments, suggests that the low polymers of NA bind approximately one pyronin molecule for each pair of phosphoric acid groups, indicating that both amino groups are functional in the linkage.Referring again to Table VI (1), a significant difference in staining is noted between DNA and DNH. We doubt that the difference between RNA and RNP is significant, since the preparations are obtained from different sources by unrelated methods. The possibility that the slightly greater staining of RN-P is due to the staining of protein by pyronin is rendered unlikely, but by no means excluded, by the fact that heat-coagulated beef serum fails to stain with pyronin when treated in the same manner as the nucleic acid precipitates.

Methyl Green-Pyronin

“…It therefore provided an opportunity to compare the reaction with and without lanthanum. It was found that here, as with methyl green (10), lanthanum competed with the dye for the nucleic acid in that the addition of 1.5 per cent lanthanum acetate reduced the amount of stain bound by the nucleic acids by 55 per cent. DISCUSSION Whereas, we have observed that staining of DNA and presumably also R~A by methyl green and pyronln is a function of the state of polymerization of the nucleic acid substrates, we can only guess at the mechanism of the selection.…”

“…This appears particularly significant when one contrasts methyl green and crystal violet, in which the only difference lies in the methylation of one amino group of crystal violet, thus converting it into the quaternary ammonium ion of methyl green. Crystal violet stains both DNA and RNA with one dye molecule combining with two phosphoric groups (9), while methyl green stains polymerized DNA with one dye molecule per 10 phosphoric groups (10), and practically fails to stain RI~A or depolymerized DNA. This suggests that the triphenylmethane dyes are bound to nucleic acid molecules by two amino groups.…”

The Major Fraction of Deoxyribonuclease Activity from Human Urinary Proteins Purification and Properties

“…1 mU = increase of 0.001 A 260 · b) DNase activity embedded in polyacrylamide gels was determined by the methyl-green DNA decolorization test (11,23). c) Disc electrophoresis of DNase was performed as described by Ornstein (24) and Davis (25).…”

“…Furthermore, DNases were found in human body fluids, such as serum (11) urine (12), seminal plasma (13,14), and liquor cerebrpspinalis (15,16).…”

Evidence of Differences in the Desoxyribonucleoprotein Complex of Rapidly Proliferating and Non-Dividing Cells

“…Methyl green is bound quantitatively by purified highly polymerized DNA in vitro (23). The use of this stain for quantitative analysis in fixed microscopic preparations is complicated by the fact that such binding would be subject to at least two variables: one being the degree of polymerization of the substrate (25,22,23,46,18), and the other, the presence of proteins which can interfere with staining presumably by competing with the dye for the anionic groups of the DNA molecule (23,1). This suggests that the measurement of staining of the same individual cells with both the methyl green and Feulgen techniques might serve to indicate changes in the desoxyribonucleoprotein complex with regard to the degree of polymerization of the DNA moiety (22,24), and the nature of the DNA-protein bond during the cell cycle.…”