Calculating pH and salt dependence of protein-protein binding

Ionic strength- (or salt-) effects on the protein-protein binding free energy has been included in many computational studies, while comparatively fewer computational studies have looked at the corresponding effect of pH. The pH dependence can be very complex if several groups change protonation state, while the ionic strength dependence usually scales as ln(I), and the main challenge is to predict the magnitude of the correlation. However, there is now very strong indication that pH effects due to binding induced changes in protonation states make a non-negligible contribution to the binding energy of most protein-protein complexes. This observation, together with more efficient pKa prediction methods and the emergence of constant pH molecular dynamics simulations to model the protonation-dependent structural changes will spark more experimental and theoretical work in pH effects on protein-protein binding.     

pH dependence of sulpiride binding to D2 dopamine receptors in bovine brain.

In summary then these data suggest that butyrophenones such as spiperone and substituted benzamides such as sulpiride interact with different groups at the active site of the D2 dopamine receptor. The drugs bind in different modes to the receptor which must therefore contain separate but overlapping binding sites for the two classes of drug. Understanding the precise interactions involved that generate this selectivity will be important for drug design.     

The pH dependence of the tetrodotoxin-blockade of the sodium channel and implications for toxin binding

On the internally perfused, voltage-clamped squid giant axon, the effect of pH on the potency of tetrodotoxin in blocking the sodium current has been re-examined at pH 7.8 and 8.8. Confirming previous studies, tetrodotoxin is weaker at pH 8.8, when the deprotonated C-10 -OH makes the toxin molecule a zwitterion. In contrast to previous studies, our results, based on full dose--response relations, show that the relative potency at the two pH values appreciably exceeds the ratio of the abundance of the protonated C-10 form. In a medium of lowered ionic strength, tetrodotoxin is weaker, and the relative potency at pH 7.8 and 8.8 approaches the ratio of the relative abundance of the C-10 -OH. The results are believed to support hydrogen bonding at C-10 as a contributing factor in the binding of tetrodotoxin to the membrane receptor.     

Equilibrium binding of daunomycin and adriamycin to calf thymus DNA. Temperature and ionic strength dependence of thermodynamic parameters

Absorbance and fluorescence quenching monitoring of the binding of the anthracyclines adriamycin (ADM) and daunomycin (DNM) to calf thymus DNA, provides reproducible binding data only when moderate drug/DNA molar ratios are used in the assays. Under these conditions, the fraction of DNA-bound drug, in equilibrium with free anthracycline, which can be reliably detected, ranged from 40-60% to 80-95% of the total added drug, depending upon ionic strength and temperature. Use of the neighbour exclusion model adequately fits such data and predicts that (i) the affinity of ADM for binding to the DNA is always higher than that corresponding to DNM, under similar experimental conditions, (ii) the binding constant for both drugs exhibits a strong salt and temperature dependence, and (iii) the exclusion parameter, indicative of the size of the anthracycline binding sites on the DNA, equals 3.1 +/- 0.4 and 3.3 +/- 0.4 base pairs for ADM and DNM, respectively, and is independent of salt concentration. The salt and temperature dependence of the binding constant is used to estimate the thermodynamic parameters involved in the interaction of the drugs with the DNA. Binding of the drugs is an exothermic process and the binding free energy arises primarily from a large negative enthalpy which, as the entropy, strongly depends upon ionic strength, and is much larger than predicted by polyelectrolyte theory. The enthalpy and entropy changes observed, appear to compensate each other over the entire range of salt concentrations used, and may arise from a complex variety of contributions, including salt-induced changes in secondary structure of the DNA, as indicated by circular dichroism techniques.     

On the mechanism of antidepressant-like action of berberine chloride

Berberine, an alkaloid isolated from Berberis aristata Linn. has been used in the Indian system of medicines as a stomachic, bitter tonic, antiamoebic and also in the treatment of oriental sores. Evidences have demonstrated that berberine possesses central nervous system activities, particularly the ability to inhibit monoamine oxidase-A, an enzyme involved in the degradation of norepinephrine and serotonin (5-HT). With this background, the present study was carried out to elucidate the antidepressant-like effect of berberine chloride in different behavioural paradigms of despair. Berberine (5, 10, 20 mg/kg, i.p.) inhibited the immobility period in mice in both forced swim and tail-suspension test, however, the effect was not dose-dependent. Berberine (5 and 10 mg/kg, i.p.) also reversed the reserpine-induced behavioral despair. Berberine (5 mg/kg, i.p.) enhanced the anti-immobility effect of subeffective doses of various typical but not atypical antidepressant drugs in forced swim test. Berberine (5 mg/kg, i.p.) following its acute administration in mice resulted in increased levels of norepinephrine (31%), serotonin (47%) and dopamine (31%) in the whole brain. Chronic administration of berberine (5 mg/kg, i.p.) for 15 days significantly increased the levels of norepinephrine (29%), serotonin (19%) as well as dopamine (52%) but at higher dose (10 mg/kg, i.p.), there was no change in the norepinephrine (12%) levels but a significant increase in the serotonin (53%) and dopamine (31%) levels was found. The antidepressant-like effect of berberine (5 mg/kg, i.p.) in forced swim test was prevented by pretreatment with l-arginine (750 mg/kg, i.p.) or sildenafil (5 mg/kg, i.p.). On the contrary, pretreatment of mice with 7-nitroindazole (7-NI) (25 mg/kg, i.p.) or methylene blue (10 mg/kg, i.p.) potentiated the effect of berberine (2 mg/kg, i.p.) in the forced swim test. Pretreatment of mice with (+)-pentazocine (2.5 mg/kg, i.p.), a high-affinity sigma1 receptor agonist, produced synergism with subeffective dose of berberine (2 mg/kg, i.p.). Pretreatment with various sigma receptor antagonists viz. progesterone (10 mg/kg, s.c.), rimcazole (5 mg/kg, i.p.) and N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(dimethylamino)ethylamine (BD1047; 1 mg/kg, i.p.) reversed the anti-immobility effects of berberine (5 mg/kg, i.p.). Berberine at lower dose did not affect the locomotor activity and barbiturate-induced sleep time. It produced mild hypothermic action in rats and displayed analgesic effect in mice. Taken together, theses findings demonstrate that berberine exerted antidepressant-like effect in various behavioural paradigms of despair possibly by modulating brain biogenic amines (norepinephrine, serotonin and dopamine). Further, nitric oxide pathway and/or sigma receptors are involved in mediating its antidepressant-like activity in mouse forced swim test.     

Influence of pH on the binding of suramin to human serum albumin

The pH dependence of the binding of suramin to albumin has been studied by means of equilibrium dialysis and circular dichroism. Dialysis experiments have revealed that the association constants of the high and low affinity binding sites are strongly influenced by the pH. At pH 6.0 K1 = 1.4 x 10(6) M-1/n1 = 2.0 and K2 = 1.3 x 10(5) M-1/n2 = 1.0; at pH 9.2 K1 = 2.0 x 10(5) M-1/n1 = 2.0. At the high pH no low affinity sites could be demonstrated any more. The pH dependence of the induced ellipticity of the suramin-albumin complex at low molar drug-to-protein ratio r = 0.1 can be superimposed upon the neutral-to-base (N-B) transition of albumin alone. By means of the Linderstr?m-Lang equation for electrostatic interaction and a two-state model for the N-B transition of albumin, evidence is obtained of a link of the pH dependent binding behaviour of suramin to albumin and the neutral-to-base transition of albumin. The possible correlation of this link with transport processes of suramin in the body and with selective uptake of suramin in cells and parasites is discussed.     

Strong binding of ditrisarubicin B to DNA

DNA binding characteristics of ditrisarubicin B were studied by the fluorescence titration technique. Ditrisarubicin B bound to calf thymus DNA with an affinity higher than any we have ever seen among anthracyclines. The apparent association constant (Kapp) of ditrisarubicin B was 2.36 X 10(8) M-1, which is 22.7 times larger than that of doxorubicin. The apparent number of binding sites (napp) of ditrisarubicin B per nucleotide of DNA was 0.164, and this value is identical with that of doxorubicin. Betaclamycin A, which has a trisaccharide chain at C-7 but no carbohydrate at C-10 in the aglycone, interacted with DNA to give a Kapp of 5.92 X 10(6) M-1 and napp of 0.178. These results suggest to us that the high affinity of ditrisarubicin B for DNA is caused by the existence of a glycosidic chain at C-10.     

The strong binding of luzopeptin to DNA

The effect of luzopeptin on the mobility of DNA in polyacrylamide gels has been determined. Experiments on a mixture of DNA fragments of various lengths have shown that the drug does not form intermolecular cross-links. Gel analysis of complexes of the drug with short DNA fragments (15-35 base pairs) reveals a ladder of discrete bands in which each band appears to correspond to the addition of a further drug molecule. The results suggest that luzopeptin binds very strongly to DNA, occupying about four base pairs and displays little or no sequence selectivity. Luzopeptin renders certain adenine residues hyperreactive to diethylpyrocarbonate, these occur in different positions to those affected by echinomycin.     

The pH dependency of the binding of drugs to plasma proteins in man

An analysis of pH-induced changes of drug binding may contribute to the understanding of the mechanisms involved and the clinical relevance. A literature search was performed, and acceptance criteria set up, to select reported data for quantitative evaluation. The relationship between percentage of unbound drug, fu, and pH was analyzed, and the relevance of physicochemical characteristics of the ligand drugs and the importance of hydrogen ion-induced changes in plasma proteins for the pH sensitivity of the binding were evaluated. With all basic and the majority of acidic drugs, fu depended linearly on pH. Basic drugs showed a consistent behavior with fu decreasing with increasing pH. Acidic compounds behaved differently: With some, fu increased, and with others fu decreased, with pH, and with a third group of acids fu was pH independent. Large differences in the pH sensitivity of the plasma protein binding among individual compounds were found. The fu in plasma for some bases and acids increased up to 136% and 95%, respectively, at pH values seen in severe acidemia or alkemia. These changes in fu could be clinically relevant with narrow-therapeutic-range drugs. Physicochemical properties and other characteristics of the ligands affect the pH sensitivity of the interaction with plasma proteins, but there was clear evidence indicating that pH-induced changes in the plasma proteins are also involved in the observed pH-dependent interaction with ligands. It is generally accepted that the unbound, free fraction in whole blood or plasma is an important determinant of the pharmacokinetics and pharmacodynamics of drugs. pH-dependent protein binding and consequent changes in the free fraction have been reported for many drugs. From a basic science point of view, the systematic study of pH-induced perturbations of the drug-protein interaction may provide insight into the mechanism and forces involved in the binding of drugs to plasma proteins. From a clinical viewpoint it may be of interest to know the extent of pH-induced changes in the unbound fraction of drugs under extreme acidemic or alkalemic conditions. Arterial blood pH values compatible with life reportedly range between 6.7 and 8.0. pH values as low as 6.3 have been measured in survivors of drowning accidents. To the best knowledge of the authors, a review and interpretation of pH-associated changes in the protein binding of drugs has not been attempted to date. The goals of this investigation were to (1) review published results of studies that determined the impact of pH changes on the protein binding of drugs in man, (2) select representative data using predetermined criteria, (3) determine relevant factors impacting the pH sensitivity of the drug-protein interaction, and (4) attempt to interpret the results and their clinical relevance.     

pH值对盐酸小檗碱表观油水分配系数的影响

目的测定盐酸小檗碱在正辛醇-水和正辛醇-缓冲液体系中的表观油水分配系数,为其体内吸收研究提供参考。方法采用HPLC法测定盐酸小檗碱的浓度,采用经典摇瓶法测定盐酸小檗碱在正辛醇-水/缓冲液中的表观油水分配系数。结果在37.0℃时,盐酸小檗碱在水中的表观油水分配系数为0.084(lgPapp=-1.08);在pH值为1.0、2.0、11.0、12.0的缓冲液中,其油水分配系数分别为1.74、1.22、1.91、15.17;在pH值为3.0～10.0时,其油水分配系数极小,变化不大,lgPapp均<0。结论在水及pH值1.0～8.0的缓冲液中,盐酸小檗碱的lgPapp均<1.0,预测其在机体胃肠道并不易被吸收。pH值对盐酸小檗碱的表观油水分配系数有一定影响,在一定范围内,pH增加可使其分配系数增大,在pH 12.0的磷酸盐缓冲液中达到最大(Papp=15.17,lgPapp=1.18)。     

Non-covalent binding of some phenothiazine drugs to DNA

The binding of a number of phenothiazine drugs (PHs) to calf thymus DNA was studied with equilibrium dialysis. The Scatchard plots reveal two types of binding. Relatively strong binding is observed at low PH concentration. For various PHs the apparent binding constants do not differ significantly, but the binding capacity of DNA varies with the 2-substituent: 2-chloro-PHs exhibit the highest extent of strong binding. For this binding type the DNA double-helix structure is essential, suggesting that (partial) intercalation of the PH-nucleus between two adjacent DNA base-pairs plays a role. At higher PH-concentrations the apparent binding constants become very small. In this PH-concentration range the structure of the side-chain of the PH appears to be of importance for the DNA-binding.Compounds with a piperazine ring in the side-chain show cooperative binding in which next to electrostatic interactions also formation of aggregates may be important.     

pH dependence of the blocking activity of carbisocaine and its derivatives

The blocking activity and pH dependence of carbanilate local anaesthetics, carbisocaine and its homologues, were tested on isolated rat sciatic nerves at pH 6.0, 7.2 and 8.4. Carbisocaine blocked the compound action potential more strongly than the derivatives with shorter alkoxysubstituents. The blocking potency of shorter derivatives increased with the rise of external medium pH, whereas the activity of carbisocaine increased with decreasing external pH. Quaternary compounds applied in the external medium were able to block the action potential, but in higher concentrations and with a longer half-time than their tertiary analogues. The blocking potency of quaternary derivatives correlated well with the length of the alkoxysubstituent and thus also with their lipophilicity. Extracellular pH did not consistently change the inhibitory effect of quaternized derivatives. These observations support the view that the lipophilicity of local anaesthetics is one of the possible factors determining their anaesthetic activity and the pH dependence of their effect.     

The effect of pH on the binding of sodium aurothiosulphate to human serum albumin. A possible binding mechanism

The effect of pH on the binding of aurothiosulphate to human serum albumin was studied in unbuffered solutions at 37 degrees and ionic strength 0.15-0.16 M. In the investigated pH range, 6.3-8.4, the effect of pH on the high affinity association constant K1 was very different from that on the lower affinity constants K2-K4. K1 was virtually constant except for a two-fold decrease in the narrow pH range 7.5-7.9, which was explained as a H+ induced local conformation change in the environment of site 1. Contrary to this, K2-K4 decreased monotonically with increasing pH, which could be entirely accounted for by a change in electrostatic interaction. A conceivable binding mechanism consistent with the results might be: that gold binds as Au+ to the high affinity binding site by exchanging a H+ and that this site might be the free sulphydryl group in cysteine or the terminal alpha-amino group; and that gold binds as Au(S2O3)3-(2) to the lower affinity binding sites which might be the protonated basic side chain group, i.e. epsilon-amino groups.