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PubMed1983

Selective inhibition of restriction endonuclease cleavage by DNA intercalators.

The preferred dye binding sites and the microenvironment of known nucleotide sequences within mitochondrial and plasmid pBR322 DNA was probed in a gross fashion with restriction endonucleases. The intercalating dyes, ethidium bromide and propidium iodide, do not inhibit a given restriction endonuclease equally at all of the restriction sites within a DNA molecule. The selective inhibition may be explained, in part, by the potential B to Z conformation transition of DNA flanking the restriction site and by preferred dye binding sites. Propidium iodide was found to be a more potent inhibitor than ethidium bromide and the inhibition is independent of the type of cut made by the enzyme.

Animals↗
PubMed1999

Time courses of mammalian cell electropermeabilization observed by millisecond imaging of membrane property changes during the pulse.

Time courses of electropermeabilization were analyzed during the electric field application using a rapid fluorescent imaging system. Exchanges of calcium ions through electropermeabilized membrane of Chinese hamster ovary cells were found to be asymmetrical. Entry of calcium ions during a millisecond pulse occurred on the anode-facing cell hemisphere. Entry through the region facing the cathode was observed only after the pulse. Leakage of intracellular calcium ions from electropermeabilized cell in low-calcium content medium was observed only from the anode-facing side. The exchanges during the pulse were mostly due to diffusion-driven processes, i.e., governed by the concentration gradient. Interaction of propidium iodide, a dye sensitive to the structural alteration of membrane, with cell membrane was asymmetrical during electropermeabilization. Localized enhancement of the dye fluorescence was observed during and after the pulsation on the cell surface. Specific staining of a limited anode-facing part of the membrane was observed as soon as the pulse was applied. The membrane fluorescence level increased during and immediately after the pulse whereas the geometry of the staining was unchanged. The membrane regions stained by propidium iodide were the same as those where calcium exchanges occurred. The fraction of the membrane on which structural alterations occurred was defined by the field strength. The density of defects was governed by the pulse duration. Electropermeabilization is a localized but asymmetrical process. The membrane defects are created unequally on the two cell sides during the pulse, implying a vectorial effect of the electric field on the membrane.

Animals↗
PubMed1994

Abnormal flow cytometry profiles in patients with interstitial cystitis.

Flow cytometry was performed on bladder cells from patients with interstitial cystitis and control patients. Cells were processed in standard fashion for flow cytometry with propidium iodide staining and analysis was restricted to samples with sufficient cells for cytokeratin gating and acceptable coefficients of variation. Of 14 interstitial cystitis patients 4 (29%) demonstrated aneuploid deoxyribonucleic acid (DNA) profiles as evidenced by a discrete peak with a DNA index of 1.2 or greater in the cytokeratin positive population. The aneuploid peak accounted for up to 54% of the cytokeratin positive population in these samples. No such aneuploid DNA profiles were evident in specimens obtained from control patients. A significant DNA tetraploid population, as evidenced by a 4C (G2) peak greater than 20%, was observed in 6 of 14 interstitial cystitis patients (43%) and 8 of 11 controls (72%). Manual counting of the per cent of binucleated cytokeratin positive cells in the cytokeratin stained population and nuclear preparations of several samples for flow analysis indicate that apparent DNA tetraploidy in the interstitial cystitis and control patients is due to an abundance of binucleated cells. Aneuploid DNA profiles on barbotage specimens from interstitial cystitis patients may reflect a real karyotypic abnormality or altered chromosome complement (true aneuploidy), abnormal chromatin structure or abnormal cytoplasmic binding of the propidium iodide stain. This finding may signal an underlying abnormality of the epithelial cell population in some patients with the clinical diagnosis of interstitial cystitis.

Adult↗
PubMed2000

Flow cytometry characterisation of Salmonella typhimurium mutants defective in proton translocating proteins and stationary-phase growth phenotype.

We have shown that the growth, starvation and population heterogeneity of Salmonella typhimurium and its isogenic nuoG and cydA mutants can be monitored by flow cytometry. Bacterial cells were analysed unstained, and after staining with rhodamine 123, propidium iodide and acridine orange. In unstained cultures it was possible to distinguish flagellated and non-flagellated cells. nuoG and cydA mutants were less stained with rhodamine confirming their defects in generating membrane potential. Increase in propidium iodide staining associated with reduced membrane integrity was seen between day 4 and 14 in all the strains. Acridine orange staining showed that there was retarded development in stationary phase in nuoG and cydA mutants. Furthermore, up to day 28, a small portion of cells showed high RNA and DNA levels. To determine whether these cells represent a sub-population better adapted for long term survival, we measured the growth of the population by both OD values and viable counts. Because the OD values increased throughout the whole study in both wild-type and mutant strains, while the viable counts gradually decreased, we propose that even in very old cultures there must be a population of cells undergoing replication.

Acridine Orange↗
PubMed1999

Flow cytometric application of the Sabin and Feldman dye test in the diagnosis of toxoplasmosis.

Although time-consuming and requiring live parasites, the Sabin and Feldman dye test (DT) is still considered the 'gold standard' among the serological tests for toxoplasmosis diagnosis. The present study was initiated to compare detection of dead parasites using optical microscopy with flow cytometry and a fluorescent nonvital dye, propidium iodide. After incubation with sera (N = 150) and a complement source, tachyzoites were washed, then stained using a fluorescein-conjugated Toxoplasma-specific antiserum. Dead tachyzoites were detected by flow cytometry after addition of propidium iodide. Intra- and inter-assay reproducibilities of percentages of dead parasites varied between 7 and 14%, and 8 and 21%, respectively. When comparing flow cytometry with the classical DT, no discrepancy was noted for positive (N = 118) and negative sera (N = 32). Correlation was good (r = 0.85) for positive sera. In conclusion, when easily available, flow cytometry is a very sensitive, specific and time-sparing method to detect specific antibodies to Toxoplasma gondii.

Animals↗
PubMed2000

Angiotensin II does not induce apoptosis but rather prevents apoptosis in cardiomyocytes.

The ability of angiotensin II (ang II) to produce apoptosis is controversial. Cardiomyocytes, isolated from 7-day embryonic chick hearts and maintained in culture for 72 h, were treated with ang II. There was no evidence of ang II-induced apoptosis consistently demonstrated by six different techniques: electrophoretic separation of fragmented DNA, staining with TUNEL, enzyme-linked immunosorbent assay specific for fragmented DNA, dual staining of cells with fluorescein diacetate and propidium iodide with analysis by flow cytometry, staining of nuclei with propidium iodide and cell microscopy. In contrast, apoptosis was readily induced by camptothecin or staurosporine or serum deprivation. The absence of ang II-induced cell death was also demonstrated in neonatal mouse cardiomyocytes in culture. We further sought to answer the question whether ang II Type 1 receptor blockade by antagonizing the potential beneficial effects mediated through this receptor and producing more ang II binding to the ang II Type 2 receptors, would lead to cardiac apoptosis. There was no evidence of ang II-induced apoptosis in the presence of the ang II Type 1 receptor antagonist losartan in embryonic chick cardiomyocytes. Rather ang II prevented serum deprivation-induced apoptosis. In summary, in these cardiomyocytes ang II does not induce but rather prevents apoptosis.

Angiotensin II↗
PubMed1998

A reliable procedure for differential staining of in vitro produced bovine blastocysts: comparison of tissue culture medium 199 and Ménézo's B2 medium.

A reliable double-dye technique has been established for counting the number of inner cell mass and trophectoderm cells of in vitro produced bovine blastocysts. The latter were first incubated in a 1:2 dilution of a rabbit antiserum raised against a mixture of recombinant bovine interferon tau and serum containing medium conditioned by in vitro produced trophoblastic vesicles for 45 min at 39 degrees C. Subsequently, the blastocysts were incubated in a 5% (v/v) solution of guinea pig complement in phosphate-buffered saline containing 50 micrograms/ml propidium iodide for 45 min at 39 degrees C. Then the blastocysts were transferred to ice-cold absolute ethanol containing 25 micrograms/ml bisbenzimide and evaluated under a fluorescence microscope. Since trophectoderm cells were permeabilised by antibody-mediated complement lysis, they were stained by propidium iodide (red or pink). Bisbenzimide can enter lysed and non-lysed cells and therefore stained also inner cell mass cells (blue) which had been protected from complement lysis by trophectoderm cells. This modified procedure proved to be very reliable for differential cell staining of bovine blastocysts produced under various culture conditions. A comparison of blastocysts produced in Ménézo's B2 vs. TCM 199 media (both supplemented with 10% serum from cows at oestrus) revealed significant (P < 0.01) differences in total cell numbers (119 +/- 24 vs. 84 +/- 10; mean +/- SD) and in the numbers of trophectoderm cells (79 +/- 19 vs. 57 +/- 8) and inner cells mass cells (40 +/- 7 vs. 26 +/- 5) between the two culture systems. The modified staining procedure presented here is a valuable tool for evaluating the quality of in vitro produced bovine blastocysts and for improving of culture conditions.

Animals↗
PubMed2002

Effects of superporous hydrogels on paracellular drug permeability and cytotoxicity studies in Caco-2 cell monolayers.

The aim of this study was to evaluate the effect of superporous hydrogel (SPH) and SPH composite (SPHC) as permeation enhancers for peptide drug delivery on Caco-2 cell monolayers. Moreover, the cytotoxic effects of these polymers were also studied using trypan blue test, MTT assay and propidium iodide staining. Transepithelial electrical resistance (TEER) studies revealed that both SPH and SPHC polymers were able to decrease TEER values to about 40% of initial values, indicating the ability of these polymers to open tight junctions. Recovery studies of TEER showed that the effects of polymers on Caco-2 cell monolayers were reversible, indicating viability of the cells after incubation with polymers. Both polymers were able to enhance the transport of the hydrophilic marker 14C-mannitol up to 2.7 and 3.8-fold in comparison to the control group. The cumulative transport of fluorescein isothiocyanate labelled dextrans with a molecular weight of 4400 Da (FD4) and 19600 Da (FD20) was enhanced by SPH and SPHC polymers by opening of tight junctions; however, this enhancement was inversely proportional to the molecular weight of marker compounds. Cytotoxicity studies confirmed that the transport enhancing properties of SPH and SPHC polymers were not caused by damage of the Caco-2 cell monolayers. The cells were able to exclude trypan blue as well as propidium iodide after incubation with SPH and SPHC polymers. MTT assay showed that the number of viable cells was higher than 95% after incubation with SPH and SPHC polymers. This indicates that the mitochondrial metabolic activities of the cells were preserved after application of the polymers.

Acrylic Resins↗
PubMed2000

Comet assay: rapid processing of multiple samples.

The present study describes modifications to the basic comet protocol that increase productivity and efficiency without sacrificing assay reliability. A simple technique is described for rapidly preparing up to 96 comet assay samples simultaneously. The sample preparation technique allows thin layers of agarose-embedded cells to be prepared in multiple wells attached to a flexible film of Gelbond, which improves the ease of manipulating and processing samples. To evaluate the effect of these modifications on assay sensitivity, dose-response curves are presented for DNA damage induced by exposure of TK6 cells to low concentrations of hydrogen peroxide (0-10 microM) and for exposure of human lymphocytes to X-irradiation (0-100 cGy). The limit of detection of DNA damage induced by hydrogen peroxide in TK6 cells was observed to be 1 uM for all parameters (tail ratio, tail moment, tail length and comet length) while the limit of detection of DNA damage in human lymphocytes was 10 cGy for tail and comet length parameters, but 50 cGy for tail ratio and tail moment parameters. These results are similar to those previously reported using the conventional alkaline comet assay. The application of SYBR Gold for detection of DNA damage was compared to that of propidium iodide. Measurements of matching samples for tail length and comet length were similar using both stains. However, comets stained with SYBR Gold persisted longer and were much brighter than those obtained with propidium iodide. SYBR Gold was found to be ideal for measuring tail length and comet length but, under present assay conditions, impractical for measuring tail ratio or tail moment due to saturation of staining in the head region of the comets.

Cell Line↗
PubMed1995

Allosteric modulation of acetylcholinesterase activity by peripheral ligands involves a conformational transition of the anionic subsite.

Replacement of residues Asp74, Trp286, and Tyr72, which are constituents of the peripheral anionic site (PAS) of human acetylcholinesterase (HuAChE), affected similarly both the binding and the inhibition constants of the PAS-specific ligand propidium, demonstrating that changes in the inhibitory activity are a direct consequence of altered binding to the PAS. In contrast, the active center HuAChE mutants W86A and Y133A show respective 350- and 25-fold increased resistance to inhibition by propidium but no change in binding affinities, demonstrating that the allosteric mechanism of PAS-mediated inhibition involves a conformational change of these Trp86 and Tyr133 residues rather than physical obstruction of substrate access by the inhibitor itself. These findings support the recent proposal that the allosteric mechanism operates via transition between active and nonactive conformations of the anionic subsite Trp86 and that replacement of Tyr133 by alanine may stabilize a nonactive Trp86 conformation that occludes the active center [Ordentlich et al. (1995) J. Biol. Chem. 270, 2082]. In further support of this mechanism and the role of Tyr133, we find that (a) the dissociation constants (Kd) for the noncovalent complexes of the irreversible inhibitors diisopropyl phosphorofluoridate or paraoxon with Y133A HuAChE are increased 20-500-fold, relative to either wild-type enzyme or its Y133F or W86A mutants; and (b) access of substrates such as 3,3-dimethylbutyl thioacetate is restored by removal of Trp86 from the Y133A enzyme (i.e., the W86A/Y133A mutant). We suggest that the conformational transition of Trp86 is coupled to the motions of the cysteine loop (Cys69-Cys96) of HuAChE and is inherent to the dynamics of the native enzyme.

Acetylcholinesterase↗
PubMed1990

Interaction of an organophosphate with a peripheral site on acetylcholinesterase.

O-Ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (MPT) is an active site directed inhibitor of acetylcholinesterase (AChE). Inhibition of the Electrophorus electricus (G4) enzyme follows classical second-order kinetics. However, inhibition of total mouse skeletal muscle AChE and inhibition of the individual molecular forms from muscle, including the monomeric species, do not proceed as simple irreversible bimolecular reactions. Similarly, complex inhibition kinetics are observed for the purified enzyme from Torpedo californica. AChE can be cross-linked with glutaraldehyde into a semisolid matrix. Under these conditions the abnormal concentration dependence for MPT inhibition is accentuated, and a range of MPT concentrations can be found where inhibition of polymerized AChE is far less than that observed at lower concentrations. Inhibition in certain concentration ranges is partially reversible after removal of all unbound ligand. Thus, there are two different modes of organophosphorus inhibition by MPT: the classical irreversible phosphorylation of the active site and a reversible interaction at a site peripheral to the active center. Propidium, a well-studied peripheral site ligand, can prevent the later interaction. Hence, the second site of MPT interaction with AChE may overlap or be linked to the peripheral anionic site of AChE characterized by the binding of propidium and other peripheral site inhibitors.

Animals↗
PubMed1998

Flow cytometric analysis of DNA content of living and fixed cells: a comparative study using various fixatives.

The majority of studies dealing with DNA analyses are made on fixed cells. In this context, the efficiency as fixatives of ethanol, methanol, acetone, Carnoy, Boehm-Sprenger and aldehydes was determined using two different DNA fluorescent probes, Hoechst 33342 and propidium iodide. The purpose of our study was to find the fixative that would provide the best results with respect to the following parameters: aggregates, cell size and granularity, and DNA staining analysis. Using murine fibroblasts, we found that 68% ethanol, 85% methanol and aldehydes did not increase aggregate formation, whereas Carnoy, acetone or Boehm-Sprenger fixatives did. The results show that aldehydes seem to alter cell size least. All fixatives induce an increase in cell granularity, which is very pronounced with alcohols, but aldehydes alter morphology less than alcohols. We observed that the fixatives giving the best resolution with Hoechst 33342 staining lead to a lower measurement variability than with propidium iodide staining. This study leads us to conclude that 68% ethanol and 85% methanol can be considered as appropriate fixatives for flow cytometry studies of DNA content.

Animals↗
PubMed2000

Evidence for apoptosis of the majority of T cells activated in vitro with Actinobacillus actinomycetemcomitans.

Our previous studies had demonstrated that nearly half of all T cells stimulated with Actinobacillus actinomycetemcomitans are activated within a few hours. However, it was not known whether all of these T cells survive. The aim of the present study was to determine whether the T cells activated in response to A. actinomycetemcomitans undergo apoptosis. To that end, peripheral blood mononuclear cells were cultured at different time points in the presence of A. actinomycetemcomitans. Flow cytometric analysis demonstrated that, following exposure to a preparation of A. actinomycetemcomitans, T cells progressively externalized their plasma membrane phosphatidylserine, as measured by annexin V binding. Approximately half of all T cells bound annexin V by 96 h. During this period, Annexin V-positive T cells also incorporated propidium iodide suggesting loss of membrane integrity. The externalization of phosphatidylserine occurred at a higher rate among activated (CD69+) T cells, where roughly two-thirds became Annexin V-positive. Flow cytometric analysis also demonstrated shrinkage of the Annexin V-positive and propidium iodide-positive T cells. The data presented here provides evidence for the induction of apoptosis among the majority of the T cells responding to A. actinomycetemcomitans.

Adult↗
PubMed2001

Genesis of progressive T-cell deficiency owing to a single missense mutation in the common gamma chain gene.

Patients with a moderate X-linked combined immunodeficiency (XCID) owing to a single missense mutation in the common gamma chain (gammac) gene (L-->Q271) were found to have a progressive T-cell deficiency. Blood T cells from four older subjects with XCIDL-->Q271 were studied to ascertain the basis of that progression. Few CD4+ T cells displayed the phenotype (CD45RA+ CD62L+) or deletion circles from T-cell receptor (TCR) Vbeta-gene rearrangements found in recent thymic emigrants. These deficiencies were more severe in older males with XCIDL-->Q271. Relative frequencies of fresh CD4+ and CD8+ T cells that bound annexin V, an early indicator of programmed cell death, or propidium iodide, an indicator of cell necrosis, were greater in XCIDL-->Q271 T cells than in normal fresh T cells. The binding of annexin V and propidium iodide to XCIDL-Q271 T cells increased marginally after stimulation with anti-CD3, but binding by fresh or stimulated XCIDL-Q271 T cells exceeded that found in normal stimulated T cells. Also, telomeres from XCIDL-->Q271 CD4+ T cells were shortened in these patients compared to normal young adults. It therefore appears that the thymus is dysfunctional and that mature T cells are not effectively rescued from apoptosis or replication senescence via gamma-mediated pathways in XCIDL-->Q271.

Adolescent↗
PubMed2001

Malnutrition-induced macrophage apoptosis.

BACKGROUND: Human and murine studies suggest protein-calorie malnutrition (PCM) results in significant host immunosuppression resulting in increased morbidity and mortality. Apoptosis has been implicated as an important mediator in the immunosuppression observed in several disease states. This study was designed to characterize macrophage apoptosis in a murine model of PCM and investigate components that regulate the apoptotic process, such as protein kinase C (PKC) and Bcl-2 activity. METHODS: Swiss-Webster mice (n = 50) were randomly assigned to receive either a control (24% protein) or a PCM diet (0% protein) for 7 days. Peritoneal macrophages were harvested and detection of apoptosis was performed by terminal deoxy-transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) and propidium iodide DNA staining under baseline and pro-apoptotic conditions. Pro-apoptotic conditions included cells treated with tumor necrosis factor-alpha (TNF-alpha) (10 ng/mL), interferon-gamma (IFN-gamma) (10 ng/mL), and a combination of both agents. In addition, levels of PKC activity and expression of Bcl-2 and p53 protein were measured. RESULTS: Peritoneal macrophages from PCM mice had a significantly greater amount of apoptosis at baseline and under stimulated conditions compared with controls. Levels of PCM apoptosis were elevated at baseline by TUNEL staining compared with macrophages from the control group (16.5% +/- 1.4%, versus 4.5% +/- 1.1%, P <.01). In addition, peritoneal macrophages from the malnourished animals were significantly more susceptible to the apoptotic effect of TNF-alpha and the effects of INF-gamma (27.3% +/- 2.1% and 31% +/- 1.4%) compared with control mice (5.5% +/- 0.7% and 7.2% +/- 0.5%, P <.01), respectively. Again, an increase in the baseline apoptosis rate was demonstrated in peritoneal macrophages from PCM mice compared with control fed mice (13.2% +/- 4.4% versus 4.3% +/- 3.1%, P <.01) as measured by propidium iodide staining. The combination of agents, TNF-alpha and INF-gamma, resulted in an additive apoptotic effect in the malnourished host compared with the control animals (43.4% +/- 4.7% versus 10.5% +/- 2.2%, P <.01), respectively. Furthermore, there was a significant decrease in the mean total PKC activity in the malnourished macrophages compared with results in controls (110,000 +/- 8000 versus 60,000 +/- 4000 cpm, P <.01). Similar changes were also observed in PKC cytosolic and membrane activity between both groups. In addition, Bcl-2 protein expression was significantly decreased in PCM animals compared with control animals. CONCLUSIONS: Thus, peritoneal macrophages from PCM mice exhibit significantly greater levels of apoptosis at baseline and when stimulated with pro-apoptotic agents compared with controls. The propensity of macrophages from PCM mice to undergo apoptosis may be attributable in part to decreased PKC activity and Bcl-2 protein expression. These findings may help to explain the associated immune dysfunction observed in malnutrition.

Animals↗
PubMed1995

Fasciculin 2 binds to the peripheral site on acetylcholinesterase and inhibits substrate hydrolysis by slowing a step involving proton transfer during enzyme acylation.

The acetylcholinesterase active site consists of a gorge 20 A deep that is lined with aromatic residues. A serine residue near the base of the gorge defines an acylation site where an acyl enzyme intermediate is formed during the hydrolysis of ester substrates. Residues near the entrance to the gorge comprise a peripheral site where inhibitors like propidium and fasciculin 2, a snake neurotoxin, bind and interfere with catalysis. We report here the association and dissociation rate constants for fasciculin 2 interaction with the human enzyme in the presence of ligands that bind to either the peripheral site or the acylation site. These kinetic data confirmed that propidium is strictly competitive with fasciculin 2 for binding to the peripheral site. In contrast, edrophonium, N-methylacridinium, and butyrylthiocholine bound to the acylation site and formed ternary complexes with the fasciculin 2-bound enzyme in which their affinities were reduced by about an order of magnitude from their affinities in the free enzyme. Steady state analysis of the inhibition of substrate hydrolysis by fasciculin 2 revealed that the ternary complexes had residual activity. For acetylthiocholine and phenyl acetate, saturating amounts of the toxin reduced the first-order rate constant kcat to 0.5-2% and the second-order rate constant kcat/Kapp to 0.2-2% of their values with the uninhibited enzyme. To address whether fasciculin 2 inhibition primarily involved steric blockade of the active site or conformational interaction with the acylation site, deuterium oxide isotope effects on these kinetic parameters were measured. The isotope effect on kcat/Kapp increased for both substrates when fasciculin 2 was bound to the enzyme, indicating that fasciculin 2 acts predominantly by altering the conformation of the active site in the ternary complex so that steps involving proton transfer during enzyme acylation are slowed.

Acetylcholinesterase↗
PubMedDate not supplied

Ocular drug targeting by liposomes and their corneal interactions.

Topical drug delivery is preferred in the eye to avoid under or over medication and undesired side effects of systemic administration. In order to maintain adequate drug levels in ocular tissues, frequent drug installation is necessary in vision threatening conditions like glaucoma, corneal ulcers due to microbial infections, etc. Only a part of the installed drug reaches the aqueous humor and the rest of it is drained by the nasolacrimal duct. Positively charged liposomes have been found to enhance the penetration of drugs into the cornea. The present study was conducted to visualize the interaction of liposomes with the cornea. Briefly, positively charged liposomes entrapped with Carboxyfluorescein (CF), Propidium iodide (PI), Horseradish peroxidase (HRP), Biotin, Hydroxy benzimide (Hoechst No: 33258), and Ethoxy benzimide (Hoechst No: 33342) were prepared by sonication. Their size and shape were analysed by laser dynamic light spectra and electron microscopy, respectively. The liposome encapsulated and free materials in buffer were instilled, in a volume of 20 microliters, into eyes of anesthetized albino rats. After 30 min and 1 h, the eyes were enucleated and quickly processed for cryosections of 3-5 microns thickness. The interaction of liposome entrapped propidium iodide and HRP was visualized on the outer epithelium of the cornea after specific processing.

Animals↗
PubMed1997

The interaction of DNA with intercalating agents probed by sodium-23 NMR relaxation rates.

We have investigated the changes of Na-23 NMR spin-lattice and spin-spin relaxation rates of Na-DNA in dilute aqueous solutions, induced by the intercalating drugs ethidium, propidium, 6,7-dihydro-pyrido[2',1':3,4]pyrazino[1,2-f]-phenanthridinediium dihydrate (dq2pyp) and by the electrostatic binder Mg2+. It has been found that the Na-23 spin-lattice relaxation is monoexponential, while the spin-spin relaxation follows a biexponential law. From the linear trends of the relaxation rates observed in the titration experiments of Na-DNA with the drugs we inferred the validity of a two-site model in the fast exchange limit. The relaxation rates of sodium in the bound state have been estimated by using the fraction of bound sodium ions per phosphate charge, predicted by the counterion condensation theory, and the number of sodium ions released per bound drug, calculated from salt dependent equilibrium binding studies. The results show that the addition of the competitors slow down the broad component of the spin-spin relaxation rate of bound sodium in the order dq2pyp approximately propidium < ethidium < Mg2+. This reduction is shown to be due mainly to the decrease of the quadrupolar coupling constant for the slow motions, thus indicating a decrease of the average electric field gradient at the sodium ions close to the DNA surface. We also show that the broad component of the spin-spin relaxation rate linearly correlates with the relative non-polyelectrolyte free energy. This result is discussed in terms of the non-polyelectrolyte interactions affecting R2bB, which can be partitioned into stacking interaction and interactions involving the molecular moieties of the intercalators exposed to the solvent in the minor groove.

Binding, Competitive↗