Binding Of Proflavine Research Articles

Proflavine binding to yeast rRNA and ribosomes as related to structure.

AbstractProflavine binding experiments were carried out with yeast rRNA, native and “unfolded” ribosomes; the binding constants and the number of binding sites were calculated by a spectroscopic method. The study of the intercalation complexes by fluorescence and electric dichroism shows the intercalation binding sites to involve two subtypes of sites, which could be related to different nucleotide composition and secondary structure of the rRNA regions, i.e., binding sites located in the (A + U)‐rich single strands and binding sites located in the (G + C)‐rich double‐helical strands (fluorescence quenching sites). Electric dichroism of complexed proflavine is interpreted in terms of rRNA conformation within the ribosomes. The conclusions are in agreement with the ribosomal model of Cox and Bonanou and show that, according to this model, the base planes of the nucleotides are not all parallel in the native ribsome, but rather radiate around the folding axis of the ribonucleoprotein sheet.

A quantitative analysis of proflavine binding to polyadenylic acid, polyuridylic acid, and transfer RNA.

Absorbance measurements show that the binding of proflavine to poly (A) and poly(U) can be quantitatively resolved (within ± 10% relative error) by the consideration of two bound species whose spectral properties and mechanisms of interaction are similar to those found in the DNA. proflavin systems: cooperative binding along the phosphate backbone (process I) and interaction between the aromatic rings of base and dye resulting presumably from some kind of interacalation of the ligand between consecutive bases (process II). The comparison with a simple theoretical model shows that process I and process II are not independent but coupled.The spectrophotometric method is able to resolve the binding of proflavine to a tRNA species with the same accuracy as that obtained for poly(A) and poly(U). Accordingly, it is proposed that the mode of interaction between proflavine and all classes of polynucleotides (DNA, RNA, synthetic polymers) is similar in nature. From the differences observed in the distribution of the bound dye between the two complexes, the existence of a close correlation between the secondary structure of the polymer and its ability for binding the dye in complex II is proposed.

Cyclic adenosine-3′, 5′-monophosphate and the inhibition of ribonucleic acid synthesis by proflavine

Proflavine belongs to a group of chemically synthesized drugs, the acridines, which binds nucleic acids and has been used as a biochemical tool to study the properties of DNA and RNA in vivo and in vitro [ 1 ] The binding of proflavine to the bacterial chromosome results in a rapid inhibition of the DNA dependent RNA synthesis and has therefore provided a means to study enzyme synthesis in the absence of transcription [2]. During the course of some experiments on the catabolite repression of the enzyme tryptophanase we have found that the cyclic nucleotide adenosine-3’, 5’-monophosphate (CAMP) hinders the uptake of proflavine by Escherichia coli during growth. We have also found that the overall synthesis of RNA and protein are inhibited by proflavine to a lesser degree when CAMP is present. The significance of our findings is apparent since proflavine together with CAMP has been used in earlier studies [3-51 in discerning the role of CAMP in the synthesis of catabolite repression sensitive enzymes.

Kinetic and hydrodynamic studies of the complex of proflavine with poly A-poly U.

AbstractRelaxation kinetic experiments reveal general similarity between the mechanism of binding of proflavine to poly A·poly U and DNA. There are differences in detail, however. For example, the rate constants are roughly an order of magnitude smaller for the former, and the thermodynamic parameters of the individual steps are also different. The total heat and free energy for intercalation of free dye are quite similar in the two cases. As was the case with DNA, considerable dye (up to 25% of the bound form) is attached externally to the double helix, even in the strong binding region of the isotherm. Sedimentation measurements on small, rodlike fragments of poly A·poly U reveal a length increase on binding proflavine of a magnitude similar to that found with DNA. This length increase seems to become smaller under conditions (high temperature) where the relaxation measurements indicate a higher fraction of externally bound dye.

Proflavine binding and release by sensitive and resistant Bacillus subtilis.

Proflavine binding of a sensitive strain of Bacillus subtilis and of a resistant strain derived from it was compared. Proflavine was bound very rapidly and more was bound at 0 °C than at 37 °C. Boiling increased the proflavine-binding capacity at 37 °C of sensitive but not of resistant cells. The binding capacity of sensitive and resistant cells suspended in buffer was the same; this was also true in various growth media. If cells were able to grow in the presence of proflavine their proflavine content decreased.Bound proflavine was released when cells were treated with growth media or with the salts of growth media. Sodium salts of organic acids also caused a release. This effect seemed due to their Na+ content, and was somewhat higher for resistant than for sensitive cells. The mechanism of proflavine resistance in B. subtilis is probably different from that of Escherichia coli, which is thought to depend on an energy-driven release of bound proflavine.

Binding of proflavine and ethidium bromide to two forms of T2 bacteriophage with different sedimentation coefficients

Equilibrium and kinetic studies of binding of proflavine and ethidium bromide to T2 phage indicate that (1) most of the intraphage DNA is accessible to proflavine; (2) the bulkier ethidium cation binds to a limited amount of DNA within the phage head; (3) intraphage DNA and free DNA in solutions of high ionic strength have similar equilibrium constants for binding to proflavine and ethidium; (4) on a time scale of minutes to hours, there is little difference in the permeabilities of the fast sedimenting (1000 S) and slow sedimenting (700S) forms of T2 phage as measured by proflavine binding.

Cooperative binding to linear biopolymers. 2. Thermodynamic analysis of the proflavine-poly(L-glutamic acid) system.

The binding of proflavine to poly(α‐l‐glutamic acid) at pH 7.5 has been investigated by means of absorbance measurements at 444 nm. Equilibrium properties were registered as a function of the total dye concentration and the polymer to dye ratio. The results agreed very well with the predictions of a basic model theory of binding to a linear lattice where cooperative interaction is restricted to nearest neighbor binding sites. The parameters g (number of binding sites per amino acid residue), K (cooperative binding constant), and q (factor measuring the strength of cooperativity) could be determined for various concentrations of added KCl electrolyte. While g of approx. 1.1 and q of approx. 200 were found to be little affected by increasing the ionic strength, a considerable decrease of K was observed. The latter is interpreted as the effect of competitive inhibition due to non‐cooperative binding of potassium ions (Ki= 160 M–1). Extrapolation to zero electrolyte concentration yields K→Ko= 8 × 104 M–1. It is concluded that the binding between the ligands and the polymer is of essentially electrostatic nature. Cooperative interaction, on the other hand, must be attributed to the stacking tendency of neighboring bound dye molecules.

Calorimetric investigations of the binding of inhibitors to alpha-chymotrypsin. I. The enthalpy of dilution of alpha-chymotrypsin and of proflavin, and the enthalpy of binding of indole, N-acetyl-D-tryptophan, and proflavin to alpha-chymotrypsin.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTCalorimetric investigations of the binding of inhibitors to α-chymotrypsin. I. Enthalpy of dilution of α-chymotrypsin and of proflavine, and the enthalpy of binding of indole, N-acetyl-D-tryptophan, and proflavine to α-chymotrypsinDaniel D. F. Shiao and Julian M. SturtevantCite this: Biochemistry 1969, 8, 12, 4910–4917Publication Date (Print):December 1, 1969Publication History Published online1 May 2002Published inissue 1 December 1969https://doi.org/10.1021/bi00840a039RIGHTS & PERMISSIONSArticle Views93Altmetric-Citations48LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (687 KB) Get e-Alerts Get e-Alerts

Fluorescence polarization study of DNA--proflavine complexes.

AbstractThe binding of proflavine to DNA has been studied by measuring the polarization and intensity of emission of DNA–dye complexes. Such measurements also permit the determination of the fluorescence of the bound dye as a function of the degree of binding. Techniques of emission spectroscopy permit the study of complexing at high phosphate to dye ratios, and we have examined complexes formed at up to 12,300:1 phosphates to dye. At high phosphate to dye ratios, we find that equilibrium plots of the binding data show only one type of binding. Reports in the literature of multiple binding constants are shown to be due to the incorrect assumption that the fluorescence of the bound dye is independent of the amount bound. The emission properties can be qualitatively accounted for by assuming that nearest‐neighbor interaction between bound dyes quenches the fluorescence. We report that, within experimental error, the binding constant is insensitive to the base content of the DNA. The DNA‐dye complexes show a temperature dependent depolarization, the cause of which is, as yet, unknown. Heat denaturation of the DNA–dye complex may be followed on a Perrin plot.

Viscosity and sedimentation study of sonicated DNA–proflavine complexes

AbstractThe intrinsic viscosity of sonicated calf thymus DNA (molecular weight 4–5 × 105) increases and the sedimentation constant decreases, with increasing binding of proflavine at 0. 2 ionic strength and at 25°C. The measurements correspond to a linear increase in length of the almost rodlike DNA molecules with the amount of proflavine bound; independent calculations from viscosity and sedimentation measurements yield almost identical results. Over the range of r (moles of proflavine bound per moles of nucleotides) equal to zero to r = 0.13, the length increases by about 20%. This extension is compatible with the intercalation hypothesis proposed by Lerman. Density increments at various values of r, at constant chemical potential of diffusible solutes, were determined. It was also found that, in addition to the known isosbestic point of DNA‐proflavine complexes at 455.5 mμ, an additional isosbestic point exists at 225.5 mμ; this proved extremely useful for the evaluation of binding studies.

Thermal stability of complexes of diaminoacridines with deoxyribonucleic acids of varying base content

1. 1. The dissociation of proflavine when heating its complexes with native DNA's of varying base content in the media of 1 mM and 10 mM salt concentration was followed spectrophotometrically. It was found that the dye bound more weakly to the surface (complex II) dissociated in a wide temperature interval, whereas dissociation of the dye bound more strongly (complex I) occurred in the temperature region of the helix-coil transition of the complex and was of a cooperative character. The midpoints of this “dissociation transition” T r (ref. 25) were identical with the denaturation temperatures T m for the complexes of r 25 > 0.1 (where r t is the ratio of the number of binding sites occupied by the dye to the total number of binding sites on DNA at temperature t). For the complexes of r 25 ≦ 0.1, the T r values were placed at temperatures higher than T m; in this case, however, an extension of the temperature interval of the helix-coil transition has occurred, for at a lower frequency of regions containing dye bound in complex I, which are more stable against heat denaturation than the regions not containing any dye, the heterogeneity of DNA increased. The results obtained are in agreement with the intercalation model for complex I. 2. 2. The cooperative “dissociation transition” was not found when heating proflavine complexes with denatured DNA. 3. 3. The binding of proflavine and acridine orange to DNA increases its stability against heat denaturation. The shift of T m in the complexes of a particular dye towards higher temperatures is, at constant ionic strength, a function of r 25 (complex I stabilizing DNA much more markedly than complex II) and of the base composition of DNA. If r 25 is constant, the shift of T m is proportionate to the adenine-thymine content in DNA. In the stabilization process, specific interactions between pairs of DNA bases and the dye molecules bound in complex I come into play besides the non-specific electrostatic forces between the negatively charged phosphate groups of DNA and the cations of the dyes.

The binding of proflavine to transfer ribonucleic acid: Dependence on secondary structure

Proflavine (3,6-diaminoacridine) binds strongly to transfer RNA (tRNA) from Escherichia coli. Direct evidence is obtained by ultracentrifugation, gel electrophoresis and molecular filtration of the complex. Studies on the stability of the tRNA-proflavine complex as a function of temperature show that strong binding depends on the secondary structure of the nucleic acid. Proflavine stabilizes the macromolecule against thermal denaturation. The results are comparable to those obtained with proflavine and DNA. The complexes of proflavine with tRNA and with DNA are probably of a similar nature.

Proflavine Uptake and Release in Sensitive and Resistant Escherichia coli

Both Escherichia coli B and a proflavine-resistant mutant, E. coli B/Pr, took up the same amounts of proflavine when suspended in buffer containing the dye. In growth media, however, sensitive cells took up more proflavine than did resistant cells. Adding growth media or any one of several constituents of these media, including amino acids, glycerol, pyruvic acid, and metabolizable sugars, to resistant cells that had taken up proflavine in buffer caused them to lose the dye, but had less or no effect on sensitive cells. Certian salts caused an equal release of proflavine from resistant and sensitive cells. Proflavine released from resistant cells by glucose was not changed chemically. The effects of temperature and metabolic inhibitors suggest that proflavine uptake is a passive process but that its release may be an active one, dependent on metabolism. Glucose had more effect on the proflavine binding of E. coli B grown in a minimal medium than on that of cells grown in a complex medium. E. coli B was less susceptible to proflavine when growing in a minimal medium. The effects of other synthetic media on proflavine susceptibility of E. coli B were also studied. Deoxyribonucleic acid and envelopes from sensitive and resistant cells bound the same amounts of proflavine, and no difference was seen in the site of dye binding when sensitive and resistant cells that had taken up proflavine in buffer were sonically broken and fractionated. The results suggest that sensitive and resistant cells are equally permeable to proflavine but differ in the ease with which metabolites cause them to release bound proflavine. So far, however, these differences do not account completely for the ability of resistant cells to grow in high proflavine concentrations.

Physico-Chemical Properties of Bovine Chymotrypsinogen B. A Comparative Study with Trypsinogen and Chymotrypsinogen A

1 A comparative structural study of chymotrypsinogens A and B at acidic pH has shown in both cases an isomerisation I′⇌II′ occurs due to the unmasking of an abnormal carboxylate ion. 2 Chymotrypsin Bπ does not bind the proflavine which is a competitive inhibitor for trypsin and chymotrypsin Aα. The acidic transition observed for the zymogens also exists for the corresponding enzymes (I⇌II). The unmasking of the abnormal carboxylate ions involves a dissociation of the chymotrypsin Aα or trypsin-proflavine complexes. 3 The thermal transitions of chymotrypsinogen B have been studied and compared with those of chymotrypsinogen A, trypsinogen and chymotrypsin Bπ. 4 The kinetics of denaturation by urea of chymotrypsinogen B, neochymotrypsinogen B, chymotrypsins Bπ and Bα were studied at different pH. The transformations of chymotrypsinogen B into neochymotrypsinogen and of chymotrypsin Bπ into chymotrypsin Bα are accompanied by a decreased stability in urea. The activation of chymotrypsinogen B to chymotrypsin Bπ increases the stability of the molecule. However, the rates of denaturation of states II′ and II of chymotrypsinogen and chymotrypsin Bπ are much more similar than the rates of denaturation of states I′ and I. The implications of this result are discussed. Ca2+ binds specifically to chymotrypsinogens A and B and to trypsinogen. (pKCa2+= 3.0, 3.1, and 3.2 respectively), probably on identical sites. The protection afforded by this binding against urea denaturation is of the same magnitude for trypsinogen and chymotrypsinogen A; it is much lower for chymotrypsinogen B. This observation together with the experiments concerning proflavine binding shows that chymotrypsinogen A shares with trypsinogen certain functional similarities that it does not share with chymotrypsinogen B. 5 The reactivity of phenol side-chains has been studied by titration and specific modification with acetylimidazole. The localisation of the normal and abnormal tyrosines in the sequences of chymotrypsinogens A and B is discussed. 6 The reactivity of indole side-chains was examined with 2-hydroxy-5-nitrobenzyl bromide. The four indoles of trypsinogen and the eight indoles of chymotrypsinogens A and B are masked in the native forms (I′).

Proflavine Binding to Transfer Ribonucleic Acid, Synthetic Ribonucleic Acids, and Deoxyribonucleic Acid

Abstract The ability of proflavine to directly inhibit protein synthesis has led to an investigation of complex formation between the drug and ribonucleic acid. Evidence has been obtained from absorption and fluorescence spectra, as well as heat denaturation curves, that proflavine forms a complex with transfer ribonucleic acid which is qualitatively similar to the deoxyribonucleic acid-proflavine complex. Additional studies with polyadenylate, polyuridylate, and polycytidylate emphasize the importance of nucleic acid secondary structure, the ionic environment, and the molar ratio of nucleotide to proflavine in determining the type of drug-nucleic acid complex which will be formed.

Structural studies on ribosomes: I. The binding of proflavine to Escherichia coli ribosomes

The binding of proflavine to 30-S, 50-S and 70-S ribosomes has been studied quantitatively utilising spectral changes in the dye to measure the degree of binding. All three particles produced hypochromism in the ultraviolet difference spectrum of the dye and a displacement in the visible spectrum to longer wavelengths with the formation of characteristic isobestic points. An analysis of the spectral changes revealed two distinct binding processes. A strong binding characterised by a free energy of association of 10 kcal/mole appeared to involve Van der Waals interactions between the dye and the bases on the RNA; a weak interaction was attributed to electrostatic binding of the dye to the RNA phosphates. The number of strong binding sites was 96, 70 and 108 for the 30-S, 50-S and 70-S ribosomes, respectively. Despite an increase of about 50 in the number of binding sites when 70-S ribosomes dissociate into their subunits the binding of proflavine to 30-S and 50-S ribosomes did not appear to affect the reassociation of these particles to form 70-S ribosomes. The number of strong binding sites on the 50-S ribosome increased to about 500 when the particle was unfolded in EDTA. This suggested that binding sites, which are exposed in the unfolded particle, are not available for binding dye in the intact ribosome because of the rigid tertiary folding in the native particle.

CORRECTION. Binding of Proflavine to alpha-Chymotrypsin and Trypsin and Its Displacement by Avian Ovomucoids.

CORRECTION. Binding of Proflavine to alpha-Chymotrypsin and Trypsin and Its Displacement by Avian Ovomucoids.

Binding of Proflavine to α-Chymotrypsin and Trypsin and Its Displacement by Avian Ovomucoids*

Binding of Proflavine to α-Chymotrypsin and Trypsin and Its Displacement by Avian Ovomucoids^*

Effect of proflavine on the binding of isoleucine to transfer RNA

Proflavine (3,6-diaminoacridine) reduces the amount of isoleucine which can be enzymically bound to tRNA in vitro. The activation of isoleucine by isoleucyl-sRNA synthetase ( l-isoleucine: sRNA ligase (AMP), EC 6.1.1.5), as measured by ATP-pyrophosphate exchange in the absence of sRNA, is not affected by the compound. Evidence for the strong binding of proflavine to sRNA is presented, and some characteristics of the RNA-proflavine complex are described. The binding of proflavine to tRNA may explain the reduction of isoleucyl-sRNA formation.

On the interaction of the active site of α-chymotrypsin with chromophores: Proflavin binding and enzyme conformation during catalysis

The acridine dye, proflavin, binds firmly to α-chymotrypsin. This binding alters the visible absorption spectrum of the dye. Evidence is presented that the dye binding is stoichiometric with the number of catalytically active sites. The evidence is as follows. (1) Spectrophotometric measurements of adsorption isotherms are consistent with a value of 1·0 adsorption sites/active site. (2) Competitive inhibition of specific substrate reaction occurs in the presence of dye. The enzyme-competitive inhibitor dissociation constant is the same as the dissociation constant determined (in the absence of substrate) from the adsorption isotherms. (3) Stoichiometric titration of the active site with an acylating reagent completely abolishes the dye binding site; removal of the acyl blocking group completely restores the binding site. Non-stereospecific acylating agents can react with the active site—dye complex, but the resultant acylated enzyme cannot bind dye. This finding is presented as a strong argument for a conformational change at the site during catalysis. The change in visible absorption spectrum between dye in water and dye in enzyme site is in accord with the supposed hydrophobic (non-polar) nature of the site. Due to this large change in light absorption at select wavelengths, the dye can be used as a quantitative indicator of the number of otherwise non-complexed catalytic sites, and hence as a tool for the measurement of equilibrium and kinetic (transient) interactions with inhibitors and substrates. Some examples are illustrated.