Influence of bovine serum albumine on the proton conductance of potato mitochondria membranes

Pulsed acid base titrations, according to the procedure of Mitchell and Moyle, have been carried out on potato mitochondria in the presence and absence of Bovine Serum Albumine (BSA). The rate of the pH decay is slower when BSA is present. The buffering capacities of the outer and inner phases, the t1/2 of the pH decay after an acid pulse and the proton conductance of the inner membrane have been measured. The results show that plant mitochondria are relatively impermeable to H⁺ and OH^–, but leakier than animal mitochondria. This may be related to the lower respiratory control ratios generally found with plant mitochondria.Abbreviations EGTA ethylene glycol bis (aminoethyl ether) NN tetraacetic acid - MERCAP sodium mercaptobenzothiazole - TRIS tris (hydroxymethyl) aminomethane - MOPS morpholinopropane sulfonic acid - BSA bovine serum albumine - RC respiratory control ratio     

The Final Frontier of pH and the Undiscovered Country Beyond

The comparison of volumes of cells and subcellular structures with the pH values reported for them leads to a conflict with the definition of the pH scale. The pH scale is based on the ionic product of water, K _w = [H⁺]×[OH⁻].We used K _w [in a reversed way] to calculate the number of undissociated H₂O molecules required by this equilibrium constant to yield at least one of its daughter ions, H⁺ or OH⁻ at a given pH. In this way we obtained a formula that relates pH to the minimal volume V_pH required to provide a physical meaning to K _w, (where N _A is Avogadro’s number). For example, at pH 7 (neutral at 25°C) V_pH = 16.6 aL. Any deviation from neutral pH results in a larger V_pH value. Our results indicate that many subcellular structures, including coated vesicles and lysosomes, are too small to contain free H⁺ ions at equilibrium, thus the definition of pH based on K _w is no longer valid. Larger subcellular structures, such as mitochondria, apparently contain only a few free H⁺ ions. These results indicate that pH fails to describe intracellular conditions, and that water appears to be dissociated too weakly to provide free H⁺ ions as a general source for biochemical reactions. Consequences of this finding are discussed.     

Effect of monovalent ion adsorption on the electric charge of phosphatidylcholine - decylamine liposomal membranes

We examined the effect of adsorbed monovalent ions on the surface charge of phosphatidylcholine (PC) – decylamine (DA) liposomal membranes. Surface charge density values were determined from electrophoretic mobility measurements of lipid vesicles performed at various pH levels. The interaction between solution ions and the PC-DA liposomal surface was described by a six component equilibrium model. The previously determined association constants of the -PO^(-) and –N⁽⁺⁾(CH₃)₃ groups of PC with H⁺, OH^-, Na⁺ and Cl^- ions (K _A1H, K _B1OH, K _A1Na, K _B1C1) were used to calculate K _B2OH, and K _B2C1, the association constants of the –N⁽⁺⁾H₃ group of DA with OH^- and Cl^- ions, providing an experimental verification for the proposed model.     

Characterization of H+/OH− currents in phospholipid vesicles

In pure phospholipid vesicles, the conductivity of H⁺/OH^– ions exceeds that for other simple inorganic ions. Protons achieve electrochemical equilibrium across egg phosphatidylcholine vesicles within tens of minutes. When pH gradients are established across vesicles, transmembrane potentials develop. Conversely, the establishment of transmembrane potentials leads to the formation of pH gradients. When the phenomenological permeability of H⁺/OH^– ions in vesicles is estimated, values are obtained that are much greater (six orders of magnitude larger) than those for Na⁺ or K⁺. A wide range in the values for this permeability has been reported; however, much of the discrepancy can be attributed to differences in the vesicle systems and experimental conditions. The H⁺/OH^– current appears to be modulated by changes in membrane dielectric constant. However, the dependence of this current on the pH gradient and on the membrane voltage argues against simple diffusion mechanisms as the source of the H⁺/OH^– current. In addition, in vesicle systems the H⁺/OH^– current shows a surprising invariance to changes in the membrane dipole potential, an observation that argues against the role of simple carriers for H⁺ and OH^– ions.     

pH Effect of the Sphingomyelin Membrane Interfacial Tension

The effect of pH on the interfacial tension of a sphingomyelin membrane in aqueous solution has been studied. Three models describing H⁺ and OH⁻ ion adsorption on the bilayer lipid surface are presented. In models I and II, the membrane surface is continuous, with uniformly distributed functional groups as centers of H⁺ and OH⁻ ion adsorption. In model III, the membrane surface is composed of lipid molecules, with and without adsorbed H⁺ and OH⁻ ions. The contribution of each individual lipid molecule to the overall interfacial tension of the bilayer was assumed to be additive in models I and II. In model III, the Gibbs isotherm was used to describe adsorption of H⁺ and OH⁻ ions at the bilayer surface. Theoretical equations are derived to describe the interfacial tension as a function of pH for all three models. Maximum interfacial tension was observed experimentally at the isoelectric point.     

Long-term effects of SO2 on plants,SO2 metabolism and regulation of intracellular pH

The impact of SO₂ on the ionic balance of plants and its implications for intracellular pH regulation was studied to find explanations for long-term effects of SO₂. When sulphur, taken up as SO₂ by the shoots of plants, is not assimilated in organic compounds, but stored as sulphate, an equivalent amount of H⁺ is produced. These H⁺ ions are not buffered chemically, but removed by metabolic processes.On the basis of knowledge on metabolic buffering mechanisms a conceptual model is proposed for the removal of shoot-generated H⁺ by (i) OH^- ions, produced in the leaves when sulphate and nitrate are assimilated in organic compounds and/or by (ii) OH^- ions produced by decarboxylation of organic anions (a biochemical pH stat mechanism). The form in which nitrogen is supplied largely determines the potential of the plant to neutralize H⁺ in the leaves during SO₂ uptake by the proposed mechanisms.In field experiments with N₂ fixing Vicia faba L. crops, the increase of sulphate in the shoots of SO₂-exposed plants was equivalent in charge to the decrease of organic anion content, calculated as the difference between inorganic cation content (C) and inorganic anion content (A), indicating that H⁺ ions produced in the leaves following SO₂ uptake were partly removed by OH^- from sulphate reduction and partly by decarboxylation of organic anions.The appearance of chronic SO₂ injury (leaf damage) in the field experiment at the end of the growing period is discussed in relation to the impact of SO₂ on the processes involved in regulation of intracellular pH. It is proposed that the metabolic buffering capacity of leaf cells is related to the rates of sulphate and nitrate reduction and the import rate of organic anions, rather than to the organic anion content in the vacuoles of the leaf cells.     

A View of Hydrogen/Hydroxide Flux Across Lipid Membranes

A topic emerging roughly 30 years ago and engendering an incompletely resolved controversy is reviewed in this article: the relatively high permeability and pH independence associated with H⁺/OH⁻ passive movements across lipid membranes. We summarize the expected characteristics of simple H⁺/OH⁻ diffusion and those of a reaction between H⁺ and OH⁻ being attracted from opposite surfaces and condensing in an interfacial zone of the membrane. An interfacial H⁺/OH⁻ reaction mechanism gives the experimentally observed behavior of an H⁺/OH⁻ flux that is independent of the pH measurement range. This mechanism assumes that H⁺ and OH⁻ within the interfacial zone become electrostatically aligned on opposite sides of the hydrophobic membrane core. Electrostatic attraction and charge delocalization among a small cluster of water molecules surrounding the ions reduce the Born energy for H⁺/OH⁻ insertion into lipid. This transmembrane condensation model predicts the magnitude of the experimentally determined H⁺/OH⁻ flux, which is significantly greater than that of other monovalent ions. The consequences of an elevated H⁺/OH⁻ permeability compared to other ions and the relative pH independence of this flux have consequences for understanding the chemical evolution of life.     

Non-enzymatic hydrolysis of creatine ethyl ester

The rate of the non-enzymatic hydrolysis of creatine ethyl ester (CEE) was studied at 37 °C over the pH range of 1.6-7.0 using ¹H NMR. The ester can be present in solution in three forms: the unprotonated form (CEE), the monoprotonated form (HCEE⁺), and the diprotonated form (H₂CEE²⁺). The values of pK_a1 and pK_a2 of H₂CEE²⁺ were found to be 2.30 and 5.25, respectively. The rate law is found to be

Rate=-dC_CEE/dt=k₊₊[H₂CEE²⁺][OH^-]+k₊[HCEE⁺][OH^-]+k₀[CEE][OH^-]     

TheChara plasmalemma at high pH. Electrical measurements show rapid specific passive uniport of H+ or OH−

Summary Above a critical external pH (about 10.5), theChara membrane acquires new propertes. In this state the membrane potential is close to the equilibrium potentials for H⁺ and OH^–, hyperpolarizing as external pH increases with a slope of –59 mV/pH unit. The membrane conductance increases by an average factor of 2.4 above the critical pH. These changes are explained by an increase in permeability to OH^– (or H⁺). The establishment of a OH^– (or H⁺ permeable membrane at high pH suggests that the large fluxes of OH^– (or H⁺ which occur in the alkaline band in photosynthesizing cells are passive.     

Effect of nickel on certain physiological and biochemical behaviors of an acid tolerantChlorella vulgaris

This study concerns the inhibitory effects of acid pH and nickel on growth, nutrient (NO₃ ^- and NH₄ ⁺) uptake, carbon fixation, O₂ evolution, electron transport chain and enzyme (nitrate reductase and ATPase) activities of acid tolerant and wild-type strains of Chlorella vulgaris. Though a general reduction in all these variables was noticed with decreasing pH, the tolerant strain was found to be metabolically more active than the wild-type. A reduced cation (NH₄ ⁺, Na⁺, K⁺ and Ca²⁺) uptake, coupled with a facilitated influx of anions (NH₄ ⁺, PO₄ ^3- and HCO₃ ^-), suggested the development of a positive membrane potential in acid tolerant Chlorella. Nevertheless, a tremendous increase in ATPase activity at decreasing pH revealed the involvement of superactive ATPase in exporting H⁺ ions and keeping the internal pH neutral. A difference in Na⁺ and K⁺ efflux of the two strains at decreasing pH suggests there is a difference in membrane permeability. The low toxicity of Ni in the acid tolerant strain may be due to the low Ni uptake brought about by a change in membrane potential as well as in permeability. Hence, the development of superactive ATPase and a change in both membrane potential and permeability not only offers protection against acidity, but also co-tolerance to metals.     

A stable Na+/H+ antiporter of thermophilic bacterium PS3

As a first step in the isolation of a stable Na⁺/H⁺ antiporter, its reaction in sonicated membrane vesicles of thermophilic bacterium PS3 has been characterized. The sonicated vesicles showed quenching of quinacrine fluorescence in either NADH oxidation or ATP hydrolysis. The quenching was reversed by the addition of Na⁺, Li⁺, Mn²⁺, Cd²⁺, and Co²⁺, but not of choline⁺ or Ca²⁺, regardless of their counter anions.²²Na⁺ was taken up into the vesicles by NADH oxidation, and the²²Na⁺ uptake was inhibited by the addition of an uncoupler. H⁺ release was observed on addition of Na⁺ to sonicated vesicles. The magnitude of the pH difference across the membrane induced by NADH oxidation was constant at pH 7.0 to 9.1, but the Na⁺/H⁺ antiport was affected by the pH of the medium (optimum pH=8.5). TheK _m 's of the antiporter for Na⁺ and Li⁺ were 2.5 and 0.1 mM, respectively, but theV _max values for the two ions were the same at pH 8.0. In the presence of Li⁺, no further decrease of fluorescence quenching was observed on addition of Na⁺ andvice versa. The Na⁺/H⁺ antiporter activity in PS3 was stable at 70°C, and the optimum temperature for activity was 55–60°C. In contrast to mesophilic cation/H⁺ antiporters, this antiporter was not inhibited by a thiol reagent.Abbreviations Tricine N-tris(hydroxymethyl)methylglycine - MOPS morpholinopropane sulfonic acid - TMAHO tetramethylammonium hydroxide - DCCD N,N-dicyclohexylcarbodiimide - FCCP carbonyl cyanidep-trifluoromethoxyphenylhydrazone - H⁺ — ATPase proton-translocating adenosine triphosphatase - electrochemical proton gradient across membrane - electrochemical Na⁺ gradient across membrane - pH pH difference across membrane     

Effect of pH on the interfacial tension of bilayer lipid membrane formed from phosphatidylcholine or phosphatidylserine

The effect of pH of an electrolyte solution on the interfacial tension of lipid membrane formed from phosphatidylcholine (PC) or phosphatidylserine (PS) was studied. The relationships were well described by an equation presented earlier based on the Gibbs isotherm but only in the proximity of the isoelectric point. Therefore, in this work models have been derived to describe the adsorption of the H⁺ and OH⁻ ions at lipid surfaces formed from PC or PS, which would reproduce changes in interfacial tension more correctly, particularly in the ranges distant from the isoelectric point. In one model, the surface is continuous with uniformly distributed functional groups constituting the centres of H⁺ and OH⁻ ion adsorption while in the other the surface is built of lipid molecules, free or with attached H⁺ and OH⁻ ions. In both models, the contributions of the individual lipid molecule forms to the interfacial tension of the bilayer were assumed to be additive.     

Amiloride-sensitive Na+/H+ exchange stimulated by cellular acidification or shrinkage in red blood cells of the frog,Rana temporaria

Simultaneous net uptake of Na⁺ and net extrusion of H⁺, both inhibited by amiloride, could be stimulated in red blood cells of the frog, Rana temporaria, either by intracellular acidification or cellular shrinkage. Net transports of Na⁺ and H⁺ were transient, dying out after 10–20 min (20°C) when stimulated by intracellular acidification but developing more slowly and proceeding for more than 60 min (20°C) when stimulated by cellular shrinkage. Evidence is presented suggesting a coupling between the transports of Na⁺ and H⁺ with an exchange ratio of 1:1 Na⁺/H⁺ exchange, stimulated by intracellular acidification, was able to readjust intracellular pH also when operating in parallel to a fully working anion exchanger in CO₂/HCO ₃ ^- -buffered media. Inhibition of anion exchange resulted in reduced cellular net uptake of Na⁺.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulphonate - DMSO dimethylsulphoxide - IU international unit - pH _e extracellular pH - pH _i intracellular pH - RBC red blood cell     

Zinc ions block H⁺/OH⁻ channels in Chara australis

Chara australis cells exposed to media of pH 10 and above exhibit high conductance, arising from the opening of H⁺/OH^‐ channels in the plasma membrane. This high conductance can be totally inhibited by 1.0 mm ZnCl₂ and restored by 0.5 mm 2‐mercaptoethanol (ME). Important for carbon fixation, H⁺/OH^‐ channels play a key role in cell pH banding. Banding was also shown to be abolished by 1.0 mm ZnCl₂ and restored in some cells by ME. The proton pump is also involved in banding, but was little affected by ZnCl₂ over the periods needed for the inhibition of H⁺/OH^‐ channels. Previously, we postulated that H⁺/OH^‐ channels open transiently at the onset of saline stress in salt‐sensitive C. australis, causing membrane potential difference (PD) noise; and remain open in latter stages of saline stress, contributing to cell deterioration. ZnCl₂ totally inhibited the saline noise and the upwardly concave I/V characteristics associated with the putative H⁺/OH^‐ currents. Again, ME reversed both these effects. We discuss the mode of action of zinc ions and ME with reference to animal voltage‐gated H⁺ channels and water channels.     

Effects of pH on the interaction of ligands with the (H+ + K+)-ATPase purified from pig gastric mucosa

The effects of K⁺, Na⁺ and ATP on the gastric (H⁺ + K⁺)-ATPase were investigated at various pH. The enzyme was phosphorylated by ATP with a pseudo-first-order rate constant of 3650 min^?1 at pH 7.4. This rate constant increased to a maximal value of about 7900 min^?1 when pH was decreased to 6.0. Alkalinization decreased the rate constant. At pH 8.0 it was 1290 min^?1. Additions of 5 mM K⁺ or Na⁺, did not change the rate constant at acidic pH, while at neutral or alkaline pH a decrease was observed. Dephosphorylation of phosphoenzyme in lyophilized vesicles was dependent on K⁺, but not on Na⁺. Alkaline pH increased the rate of dephosphorylation. K⁺ stimulated the ATPase and p-nitrophenylphosphatase activities. At high concentrations K⁺ was inhibitory. Below pH 7.0 Na⁺ had little or no effect on the ATPase and p-nitrophenylphosphatase, while at alkaline pH, Na⁺ inhibited both activities. The effect of extravesicular pH on transport of H⁺ was investigated. At pH 6.5 the apparent K_m for ATP was 2.7 μM and increased little when K⁺ was added extravesicularly. At pH 7.5, millimolar concentrations of K⁺ increased the apparent K_m for ATP. Extravesicular K⁺ and Na⁺ inhibited the transport of H⁺. The inhibition was strongest at alkaline pH and only slight at neutral or acidic pH, suggesting a competition between the alkali metal ions and hydrogen ions at a common binding site on the cytoplasmic side of the membrane. Two H⁺-producing reactions as possible candidates as physiological regulators of (H⁺ + K⁺)-ATPase were investigated. Firstly, the hydrolysis of ATP per se, and secondly, the hydration of CO₂ and the subsequent formation of H⁺ and HCO₃^?. The amount of hydrogen ions formed in the ATPase reaction was highest at alkaline pH. The H⁺/ATP ratio was about 1 at pH 8.0. When CO₂ was added to the reaction medium there was no change in the rate of hydrogen ion transport at pH 7.0, but at pH 8.0 the rate increased 4-times upon the addition of 0.4 mM CO₂. The results indicate a possible co-operation in the production of acid between the H⁺ + K⁺-ATPase and a carbonic anhydrase associated with the vesicular membrane.     

Simulation of the light-induced oscillations of the membrane potential in Potamogeton leaf cells

Summary An attempt has been made to simulate the light-induced oscillations of the membrane potential of Potamogeton lucens leaf cells in relation to the apoplastic pH changes. Previously it was demonstrated that the membrane potential of these cells can be described in terms of proton movements only. It is hypothesized that the membrane potential is determined by an electrogenic H⁺-ATPase with a variable H⁺/ATP stoichiometry. The stoichiometry shifts from a value of two in the dark to a value of one in the light. Moreover, this H⁺ pump shows the characteristics of either a pump or a passive H⁺ conductance: the mode of operation of the H⁺ translocator is considered to be regulated by the external pH. The pump conductance is assumed to be dominant at low or neutral pH, while the passive H⁺ conductance becomes more significant at alkaline pH. The pH dependence of the transport characteristic is expressed by protonation reactions in the plasma membrane. The proposed model can account for most features of the light-induced oscillations but not for the absolute level of the membrane potential.This research was supported by the Foundation of Biophysics, part of the Dutch Organization for Scientific Research (NWO) ECOTRANS publication No. 34.     

Two-substrate kinetic analysis: a novel approach linking ion and acid-base transport at the gills of freshwater trout,Oncorhynchus mykiss

Summary The novel application of a two-substrate model (Florini and Vestling 1957) from enzymology to transport kinetics at the gills of freshwater trout indicated that Na⁺/acidic equivalent and Cl^-/basic equivalent flux rates are normally limited by the availability of the internal acidic and basic counterions, as well as by external Na⁺ and Cl^- levels. Adult rainbow trout fitted with dorsal aortic and bladder catheters were chronically infused (10–16 h) with isosmotic HCl to induce a persistent metabolic acidosis. Acid-base neutral infusions of isosmotic NaCl and non-infused controls were also performed. Results were compared to previous data on metabolic alkalosis in trout induced by either isosmotic NaHCO₃ infusion or recovery from environmental hyperoxia (Goss and Wood 1990a, b). Metabolic acidosis resulted in a marked stimulation of Na⁺ influx, no change in Cl^- influx, positive Na⁺ balance, negative Cl^- balance, and net H⁺ excretion at the gills. Metabolic alkalosis caused a marked inhibition of Na⁺ influx and stimulation of Cl^- influx, negative Na⁺ balance, positive Cl^- balance, and net H⁺ uptake (=base excretion). Mean gill intracellular pH qualitatively followed extracellular pH. Classical one-substrate Michaelis-Menten analysis of kinetic data indicated that changes in Na⁺ and Cl^- transport during acid-base disturbance are achieved by large increases and decreases in J_max, and by increases in K_m. However, one-substrate analysis considers only external substrate concentration and cannot account for transport limitations by the internal substrate. The kinetic data were fitted successfully to a two-substrate model, using extracellular acid-base data as a measure of internal HCO ₃ ^- and H⁺ availability. This analysis indicated that true J_max values for Na⁺/acidic equivalent and Cl^-/basic equivalent transport are 4–5 times higher than apparent J_max values by one-substrate analysis. Flux rates are limited by the availability of the internal counterions; transport K_m values for HCO ₃ ^- and H⁺ are far above their normal internal concentrations. Therefore, small changes in acid-base status will have large effects on transport rates, and on apparent J_max values, without alterations in the number of transport sites. This system provides an automatic, negative feedback control for clearance or retention of acidic/basic equivalents when acid-base status is changing.Abbreviations Amm total ammonia in water - DMO 55-dimethyl-24-oxyzolidine-dione - J_in unidirectional inward ion movement across the gill - J_out unidirectional outward ion movement across the gill - J_net net transfer of ions (sum of J_in and J_out) across the gill - J_max maximal transport rate for ion - K_m inverse of affinity of transporter for ion - PIO₂ partial pressure of oxygen in inspired water - P_aCO₂ partial pressure of carbon dixide in arterial blood - TAlk titratable alkalinity of the water - PEG polyethylene glycol - NEN New England Nuclear     

Ion and pH gradients across the transport membrane of mitochondria following Mn ++ uptake in the presence of acetate

The electron paramagnetic resonance (EPR) spectrum of Mn⁺⁺ loaded mitochondria is affected by the presence of the permeant anion acetate (Ac^?) in the medium. The hyperfine sextet, shown earlier to have spectral characteristics like those expected of osmotically active Mn⁺⁺ in the matrix space, grows in intensity with increasing [Ac^?]. From estimates of mitochondrial water, the free internal [Mn⁺⁺] can be calculated. The gradient of free [Mn⁺⁺] across the inner mitochondrial membrane is believed to be at least 500:1 under conditions of high [Ac^?]. Since Mn⁺⁺ solubility is limited by [OH^?], it is possible to place an upper limit on the pH in the matrix space. The variation of free internal [Mn⁺⁺], as measured by EPR, with external pH indicates that the [H⁺] gradient is 1–1.5 pH units in the absence of permeant anions and considerably less in the presence of 100 mM acetate.     

Active transport,ion movements,and pH changes

The transport of substances across cell membranes may be the most fundamental activity of living things. When the substance transported is any ion there can be a change in the concentration of hydrogen ions on the two sides of the membrane. These hydrogen ion concentration changes are not caused by fluxes of hydrogen ions although fluxes of hydrogen ions may sometimes be involved. The reason for the apparent contradiction is quite simple. All aqueous systems are subject to two constraints: (1) to maintain the charge balance, the sum of the cationic charges must equal the sum of the anionic charges and (2) the product of the molar concentration of H⁺ and the molar concentration of OH⁻, established and maintained by the association and the dissociation of water, remains always at 10⁻¹⁴. As a consequence the concentrations of H⁺ and OH⁻ are determined uniquely by differences between the concentrations of the other cations and anions, with [H⁺] and [OH⁻] being dependent variables. Hydrogen ions and hydroxyl ions can be produced or consumed in local reactions whereas any strong ions such as Cl⁻, Mg²⁺, or K⁺ can be neither produced nor consumed in biological reactions. Further consequences of these truisms are outlined here in terms of the chemistry of the kinds of reactions which can lead to pH changes.     

Hydroxyl Radical-Induced Reactions in Polyadenylic Acid as Studied by Pulse Radiolysis: Part I. Transformation Reactions of Two Isomeric OH-Adducts

The absorption spectra of polyadenylic acid (polyA) radicals in N₂0 saturated aqueous solution have been measured as a function of time (up to 15 s) following an 0.4μS electron pulse. The spectra and their changes were analysed by comparison with those from monomeric adenine derivatives (nucleosides and nucleotides) which had been studied by Steenken.¹

The reaction of OH^· radicals with the adenine moiety in poly A results in the formation of two hvdroxvl adducts at the positions C-4 [polyA40H^·] and C-8 [polyA80H^·]. Each OH-adduct undergoes a unimol-ecular transformation reaction before any bimolecular or other unimolecular decay occurs. These reactions are characterized by different rate constants and _pH dependencies. The polyA40H^· adduct undergoes a dehydration reaction to yield a neutral N⁶ centered radical (rate constant K_deh= 1.4 × 10⁴s^-1 at ^pH7.3). This reaction is strongly inhibited by H⁺. In comparison with the analogous reactions in adenosine phosphates, the kinetic _pK value for its inhibition is two ^pH units higher. This shift is the result of the counter ion condensation or double-strand formation. The polyA80H^· adduct undergoes an imidazole ring opening reaction to yield an enol type of formamidopyrimidine radical with the resulting base damage (k^r.o. = 3.5 × 10⁴ s ^-1 at pH7.3). This reaction in contrast is strongly catalysed by H⁺and OH^-, similar as for adenosine but different compared to the nucleotides.