The role of mitochondrial palmitate/Ca<Superscript>2+</Superscript>-activated pore in palmitate-induced apoptosis

The evidence of possible involvement of the mitochondrial cyclosporin A-insensitive palmitate/Ca²⁺-activated pore in palmitate-induced apoptosis is presented. It has been established that the opening of the palmitate/Ca²⁺-activated pore results in the high-amplitude swelling of mitochondria and the release of the apoptosis-inducing factor from organelles. These processes are accompanied by a short-term slight decrease of membrane potential, which recovers in 1 min. The possible role of the palmitate/Ca²⁺-activated pore in the induction of palmitate-induced apoptosis is discussed.     

On the Opening of an Insensitive Cyclosporin A Non-specific Pore by Phenylarsine Plus Mersalyl

The purpose of this work was addressed to provide new information on the effect of thiol reagents on mitochondrial non-specific pore opening, and its response to cyclosporin A (CSA). To meet this proposal phenylarsine oxide (PHA) and mersalyl were employed as tools to induce permeability transition and CSA to inhibit it. PHA-induced mitochondrial dysfunction, characterized by Ca²⁺ efflux, swelling, and membrane de-energization, was inhibited by N-ethylmaleimide and CSA. Conversely, mersalyl failed to inhibit the inducing effect of phenylarsine oxide, it rather strengthened it. In addition, the effect of mersalyl was associated with cross-linking of membrane proteins. The content of membrane thiol groups accessible to react with PHA, mersalyl, and PHA plus mersalyl was determined. In all situations, permeability transition was accompanied by a significant decrease in the whole free membrane thiol content. Interestingly, it is also shown that mersalyl hinders the protective effect of cyclosporin A on PHA-induced matrix Ca²⁺ efflux.     

On the mechanism of palmitic acid-induced apoptosis: the role of a pore induced by palmitic acid and Ca2+ in mitochondria

Palmitic acid (Pal) is known to promote apoptosis (Sparagna G et al (2000) Am J Physiol Heart Circ Physiol 279: H2124–H2132) and its amount in blood and mitochondria increases under some pathological conditions. Yet, the mechanism of the proapoptotic action of Pal has not been elucidated. We present evidence for the involvement of the mitochondrial cyclosporin A-insensitive pore induced by Pal/Ca²⁺ complexes in the apoptotic process. Opening of this pore led to a fall of the mitochondrial membrane potential and the release of the proapoptotic signal cytochrome c. The addition of cytochrome c prevented these effects and recovered membrane potential, which is in contrast to the cyclosporin A-sensitive mitochondrial permeability transition pore. Oleic and linoleic acids prevented the Pal/Ca²⁺-induced pore opening in the intact mitochondria, this directly and significantly correlating with the effect of these fatty acids on Pal-induced apoptosis in cells (Hardy S et al (2003) J Biol Chem 278: 31861–31870). The specific probe for cardiolipin, 10-N-nonyl acridine orange, inhibited formation of this pore.     

The effect of taurine on the ion transport system in mitochondria

The effect of taurine on the ATP-dependent mitochondrial swelling that characterizes the activity of mitochondrial ATP-dependent K⁺ channel and the formation of Ca²⁺-dependent pores, different in sensitivity to cyclosporin A, has been studied in rat liver mitochondria. It has been shown that taurine in micromolar concentrations (0.5–125 μM) stimulates the energy-dependent swelling of mitochondria. Taurine in physiological concentrations (0.5–20 mM) has no effect on the ATP-dependent swelling and the formation of cyclosporin A-insensitive Pal/Ca²⁺-activated pore in mitochondria. Taurine in these concentrations increased the rate of cyclosporin A-sensitive swelling of mitochondria induced by Ca²⁺ and P_i and reduced the Ca²⁺ capacity of mitochondria. The different effects of physiological taurine concentrations on the ATP-dependent transport of K⁺ and Ca²⁺ ions in mitochondrial membranes as compared with cell membranes are discussed.     

Influence of cholesterol on the formation of palmitate/Ca<Superscript>2+</Superscript>-activated pores in mitochondria and liposomes

The influence of cholesterol on the formation of a mitochondrial cyclosporin A-insensitive palmitate/Ca²⁺-activated pore has been studied. Loading of mitochondrial membranes with cholesterol increases the rate of mitochondrial swelling induced by palmitic acid (≥20 μM) and Ca²⁺ (30 μM). This effect is not related to changes in the functional activity of organelles, since cholesterol does not influence the mitochondrial respiration in different metabolic states. At the same time, palmitate/Ca²⁺-induced permeabilization of azolectin/cholesterol liposomes is more pronounced than that of azolectin liposomes. In the liposomal membrane, Ca²⁺ induces phase separation of palmitic acid into distinct membrane domains; the presence of cholesterol in membranes enhances this effect.     

Citrinin-induced mitochondrial permeability transition

The effects of mycotoxin citrinin on Ca²⁺ efflux and membrane permeabilization were studied in isolated rat liver mitochondria. The efflux rate observed when in presence of ruthenium red was higher when citrinin was added. Swelling experiments demonstrated Ca²⁺-dependent membrane permeabilization by citrinin. Catalase, butylhydroxitoluene (BHT), and dithiothreitol (DTT) did not protect swelling caused by Ca²⁺ plus citrinin. The protection conferred by ATP–Mg²⁺ and cyclosporin A in the latter experiments are strong indications of pore formation. These results suggest that citrinin can induce permeability transition by a mechanism that does not involve oxidative damage. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 291–297, 1998     

The permeability transition pore as a mitochondrial calcium release channel: A critical appraisal

Mitochondria from a variety of sources possess an inner membrane channel, the permeability transition pore. The pore is a voltage-dependent channel, activated by matrix Ca²⁺ and inhibited by matrix H⁺, which can be blocked by cyclosporin A, presumably after binding to mitochondrial cyclophilin. The physiological function of the permeability transition pore remains unknown. Here we evaluate its potential role as a fast Ca²⁺ release channel involved in mitochondrial and cellular Ca²⁺ homeostasis. We (i) discuss the theoretical and experimental reasons why mitochondria need a fast, inducible Ca²⁺ release channel; (ii) analyze the striking analogies between the mitochondrial permeability transition pore and the sarcoplasmic reticulum ryanodine receptor-Ca²⁺ release channel; (iii) argue that the permeability transition pore can act as a selective release channel for Ca²⁺ despite its apparent lack of selectivity for the transported speciesin vitro; and (iv) discuss the importance of mitochondria in cellular Ca²⁺ homeostasis, and how disruption of this function could impinge upon cell viability, particularly under conditions of oxidative stress.     

Oxidative Stress Underlies the Mechanism for Ca2+-induced Permeability Transition of Mitochondria

Recent studies demonstrated that the generation of intracellular reactive oxygen species (ROS) was enhanced prior to the onset of mitochondrial membrane permeability transition (MPT), a critical step for the induction of DNA fragmentation and apoptosis. Although Ca²⁺ induces typical MPT that involves depolarization and swelling of mitochondria and finally releases cytochrome c into cytosol, the mechanism by which ROS induce MPT remains unclear. In the presence of inorganic phosphate, Ca²⁺ increased the oxygen consumption and ROS production by isolated mitochondria as determined by a chemiluminescence (CHL) method using L-012. Ca²⁺ increased the generation of H²O² by some mechanism that was inhibited by cyclosporin A but not by superoxide dismutase (SOD) and trifluoperazine. Ca²⁺ decreased the content of free thiols in adenine nucleotide translocase (ANT) in mitochondrial membranes with concomitant increase in ROS generation. The presence of cyclosporin A, trifluoperazine, or SOD inhibited the Ca²⁺-induced increase of L-012 CHL and decrease in the free thiols of ANT. These results indicate that Ca²⁺ increases the generation of ROS which oxidize the free thiol groups in mitochondrial ANT, thereby inducing MPT to release cytochrome c.     

Permeability Transition Pore Closure Promoted by Quinine

The mitochondrial membrane permeability transition induced byCa²⁺ is inhibited by quinine in a dose-dependent fashion.Competition experiments strongly suggest that quinine displacesCa²⁺ bound to the inner membrane. This is supported byexperiments showing that quinine inhibits Ca²⁺-dependent butnot Ca²⁺-independent mitochondrial swelling induced byphenylarsine oxide. As with Ca²⁺ chelators, quinine inducespermeability transition pore closure preventing the contraction induced bypoly(ethylene glycol) 2000 in mitochondria preswollen by incubation in KSCNmedium containing Ca²⁺ and inorganic phosphate. These resultssuggest that quinine dislodges Ca²⁺ bound to the protein site,which triggers pore opening.     

Protective action of tamoxifen on carboxyatractyloside-induced mitochondrial permeability transition

AimsMitochondrial permeability transition is established after massive Ca²⁺ accumulation inside the matrix, in addition to an inducer. The closure of the pore can be accomplished by adenosine diphosphate and the immunosuppressant cyclosporin A. Recently, the estrogen antagonist, tamoxifen, has been introduced as an inhibitor of the opening of the permeability transition pore. However, the mechanism by which this drug inhibits pore opening is still under discussion. This work was performed with the purpose of establishing the membrane system involved in tamoxifen-induced pore closure. For this purpose, permeability transition was induced after the addition of carboxyatractyloside, which is a specific reagent that interacts with the adenine nucleotide translocase.Main methodsPermeability transition was assessed by analyzing matrix Ca²⁺ release, transmembrane electric gradient, and mitochondrial swelling in aged, as well as in freshly prepared mitochondria. Also, cytochrome c content was analyzed in membrane mitochondria as well as in the supernatant.Key findingsIn freshly prepared mitochondria, tamoxifen, at the concentration of 10 μM, totally inhibited nonspecific membrane permeability induced by 1 μM carboxyatractyloside. In addition, tamoxifen inhibited non-specific permeability in aged mitochondria and diminished membrane fluidity.SignificancePlausibly, the inhibitory effect of tamoxifen on nonspecific membrane permeability, as induced by carboxyatractyloside, should be ascribed to a diminution, of membrane fluidity by this drug.     

The composition of the incubation medium influences the sensitivity of mitochondrial permeability transition to cyclosporin A

The aim of this work was to study permeability transition, and the influence of the composition of the incubation medium, on the inhibitory action of cyclosporin A. It was found that cyclosporin inhibited the opening of a nonspecific pore, as induced by the uncoupler carbonyl cyanide m-chlorophenylhydrazone, provided K⁺ was present in the incubation medium, but failed to do so if mitochondria are incubated in sucrose or Na⁺-based medium. It was also found that the sensitivity of mitochondria to the uncoupler depended on the incubation mixture, being more sensitive when sucrose was the osmotic support. Matrix Ca²⁺ release, large amplitude swelling, and drop in transmembrane electric gradient revealed permeability transition. The titration of membrane thiol groups shows them to be increased in mitochondria incubated in sucrose medium, in comparison with the values found in mitochondria incubated in KCl or NaCl medium. Our proposal is that the incubation in sucrose medium propitiated a conformational change of membrane proteins in such a way that cyclosporin was unable to bind to its target site.     

Inhibition of Mitochondrial Permeability Transition by Low pH is Associated with Less Extensive Membrane Protein Thiol Oxidation

Ca²⁺ and inorganic phosphate-induced mitochondrial swelling and membrane protein thiol oxidation, which are associated with mitochondrial permeability transition, are inhibited by progressively decreasing the incubation medium pH between 7.2 and 6.0. Nevertheless, the detection of mitochondrial H₂O₂ production under these conditions is increased. Permeability transition induced by phenylarsine oxide, which promotes membrane protein thiol cross-linkage in a process independent of Ca²⁺ or reactive oxygen species, is also strongly inhibited in acidic incubation media. In addition, we observed that the decreased protein thiol reactivity with phenylarsine oxide or phenylarsine oxide-induced swelling at pH 6.0 is reversed by diethyl pyrocarbonate, in a hydroxylamine-sensitive manner. These results provide evidence that the inhibition of mitrochondrial permeability transition observed at lower incubation medium pH is mediated by a decrease in membrane protein thiol reactivity, related to the protonation of protein histidyl residues.     

Lithocholic acid induces two different calcium-dependent inner membrane permeability systems in liver mitochondria

The effect of the most hydrophobic bile acid–lithocholic–as an inducer of two different Ca²⁺-dependent inner membrane permeability systems was studied on isolated rat liver mitochondria. It is shown that the addition of lithocholic acid at a concentration of 20 μM to the Ca²⁺-loaded mitochondria leads to swelling of the organelles, rapid release of Ca²⁺ from the matrix and almost complete collapse of Δψ. Mitochondrial pore blocker cyclosporin A (CsA) eliminates mitochondrial swelling but has no effect on the process of Ca²⁺ release and Δψ collapse. In the absence of Ca²⁺ lithocholic acid causes only a transient decrease of Δψ with subsequent complete recovery. Ruthenium red, inhibitor of mitochondrial Ca²⁺ uniporter, which blocks Ca²⁺ influx into the matrix, prevents mitochondrial swelling induced by lithocholic acid. At the same time, ruthenium red, which is added before lithocholic acid to the Ca²⁺-preloaded mitochondria, does not affect the swelling of the organelles but reduces the CsA-insensitive drop in Δψ. It is concluded that lithocholic acid is able to induce two Ca²⁺-dependent energy dissipation systems in the inner membrane of liver mitochondria: CsA-sensitive mitochondrial pore and CsA-insensitive permeability, which exhibits sensitivity to ruthenium red. It is found that the effect of this bile acid as an inductor of CsA-sensitive mitochondrial pore is not associated with the modulation of Pi effects. It is assumed that CsA-insensitive action of lithocholic acid is associated with the induction of Ca²⁺ efflux from the matrix in exchange for protons. In this case, the energy-dependent Ca²⁺ transport in the opposite direction with the participation of mitochondrial calcium uniporter sensitive to ruthenium red leads to the formation of calcium cycle and thereby to energy dissipation.     

Ursodeoxycholic acid,in contrast to other bile acids,induces Ca<Superscript>2+</Superscript>-dependent cyclosporin A-insensitive permeability transition in liver mitochondria in the presence of potassium chloride

The effects of hydrophobic and hydrophilic bile acids as inducers of Ca²⁺-dependent permeability of the inner membrane were studied on isolated liver mitochondria. It is shown that in the absence of the inorganic phosphate (P_i)–a modulator of the mitochondrial pore–hydrophobic bile acids (lithocholic, deoxycholic, chenodeoxycholic) at concentrations of 20–50 μM, as well as a hydrophilic cholic acid at a concentration of 800 μM, induce swelling of liver mitochondria loaded with Ca²⁺. This effect is completely eliminated by a specific inhibitor of mitochondrial pore cyclosporin A (CsA). The effect of the bile acids as inducers of Ca²⁺-dependent CsA-sensitive mitochondrial pore is not associated with the modulation of the P_i effects. In contrast to other tested bile acids, a hydrophilic ursodeoxycholic acid (UDCA) at a concentration of 400 μM is able to induce Ca²⁺-dependent CsA-sensitive pore opening in liver mitochondria only in the presence of P_i or in the absence of potassium chloride in the incubation medium. In the presence of potassium chloride but in the absence of P_i, UDCA effects associated with the induction of the inner membrane permeability (swelling of mitochondria, drop in Δψ, and Ca²⁺ release from the matrix) are also observed in the presence of CsA. This Ca²⁺-dependent permeability of the inner membrane, in contrast to the “classical” CsA-sensitive pore, is characterized by a lower intensity of the mitochondrial swelling, a total drop in Δψ, and Ca²⁺ release from the matrix and is blocked by P_i. We suggest that the induction of the CsA-insensitive permeability in the inner mitochondrial membrane by UDCA is associated with activation of electrophoretic influx of K⁺ into the matrix and Ca²⁺ release from the matrix in exchange to H+. The effect of P_i as a blocker of such permeability is discussed.     

Long-chain α,ω-dioic acids as inducers of cyclosporin A-insensitive nonspecific permeability of the inner membrane of liver mitochondria loaded with calcium or strontium ions

Long-chain saturated monocarboxylic fatty acids can induce nonspecific permeability of the inner membrane (open pores) of liver mitochondria loaded with Ca²⁺ or Sr²⁺ by the mechanism insensitive to cyclosporin A. In this work we investigated the effect of their metabolites — α,ω-dioic (dicarboxylic) acids — as potential inducers of pore opening by a similar mechanism. It was established that the addition of α,ω-hexadecanedioic acid (HDA) at a concentration of 10–30 μM to liver mitochondria loaded with Ca²⁺ or Sr²⁺ leads to swelling of the organelles and release of these ions from the matrix. The maximum effect of HDA is observed at 50 μM Ca²⁺ concentration. Cyclosporin A at a concentration of 1 μM, previously added to the mitochondria, did not inhibit the observed processes. The calcium uniporter inhibitor ruthenium red, which blocks influx of Ca²⁺ and Sr²⁺ to the matrix of mitochondria, prevented HDA-induced swelling. The effect of HDA as inducer of swelling of mitochondria was compared with similar effects of α,ω-tetradecanedioic and α,ω-dodecanedioic acids whose acyl chains are two and four carbon atoms shorter than HDA, respectively. It was found that the efficiency of these α,ω-dioic acids decreases with reducing number of carbon atoms in their acyl chains. It was concluded that in the presence of Ca²⁺ or Sr²⁺ long-chain saturated α,ω-dioic acids can induce a cyclosporin A-insensitive permeability of the inner membrane (open pores) of liver mitochondria as well as their monocarboxylic analogs.     

Cyclosporin A binding in mitochondrial cyclophilin inhibits the permeability transition pore and protects hearts from ischaemia/reperfusion injury

When loaded with high (pathological) levels of Ca²⁺, mitochondria become swollen and uncoupled as the result of a large non-specific increase in membrane permeability. This process, known as the mitochondrial permeability transition (MPT), is exacerbated by oxidative stress and adenine nucleotide depletion. These conditions match those that a heart experiences during reperfusion following a period of ischaemia. The MPT is caused by the opening of a non-specific pore that can be prevented by sub-micromolar concentrations of cyclosporin A (CsA). A variety of conditions that increase the sensitivity of pore opening to [Ca²⁺], such as thiol modification, oxidative stress, increased matrix volume and chaotropic agents, all enhance the binding of matrix cyclophilin (CyP) to the inner mitochondrial membrane in a CsA-sensitive manner. In contrast, ADP, membrane potential and low pH decrease the sensitivity of pore opening to [Ca²⁺] without affecting CyP binding. We present a model of pore opening involving CyP binding to a membrane target protein followed by Ca²⁺-dependent triggering of a conformational change to induce channel opening. Using the ischaemic/reperfused rat heart we have shown that the mitochondrial pore does not open during ischaemia, but does do so during reperfusion. Recovery of heart during reperfusion is improved in the presence of 0.2 µM CsA, suggesting that the MPT may be critical in the transition from reversible to irreversible reperfusion injury. (Mol Cell Biochem 174: 167–172, 1997)     

Ascorbate and low concentrations of FeSO4 induce Ca2+-dependent pore in rat liver mitochondria

Oxidative stress is one of the most frequent causes of tissue and cell injury in various pathologies. The molecular mechanism of mitochondrial damage under conditions of oxidative stress induced in vitro with low concentrations of FeSO₄ and ascorbate (vitamin C) was studied. FeSO₄ (1-4 M) added to rat liver mitochondria that were incubated in the presence of 2.3 mM ascorbate induced (with a certain delay) a decrease in membrane potential and high-amplitude swelling. It also significantly decreased the ability of mitochondria to accumulate exogenous Ca²⁺. All the effects of FeSO₄ + ascorbate were essentially prevented by cyclosporin A, a specific inhibitor of the mitochondrial Ca²⁺-dependent pore (also known as the mitochondrial permeability transition). EGTA restored the membrane potential of mitochondria de-energized with FeSO₄ + ascorbate. We hypothesize that oxidative stress induced in vitro with FeSO₄ and millimolar concentrations of ascorbate damages mitochondria by inducing the cyclosporin A-sensitive Ca²⁺-dependent pore in the inner mitochondrial membrane.     

Mitochondrial Ca<Superscript>2+</Superscript> cycle mediated by the palmitate-activated cyclosporin a-insensitive pore

Earlier we found that in isolated rat liver mitochondria the reversible opening of the mitochondrial cyclosporin A-insensitive pore induced by low concentrations of palmitic acid (Pal) plus Ca²⁺ results in the brief loss of Δψ [Mironova et al., J Bioenerg Biomembr (2004), 36:171–178]. Now we report that Pal and Ca²⁺, increased to 30 and 70 nmol/mg protein respectively, induce a stable and prolonged (10 min) partial depolarization of the mitochondrial membrane, the release of Ca²⁺ and the swelling of mitochondria. Inhibitors of the Ca²⁺ uniporter, ruthenium red and La³⁺, as well as EGTA added in 10 min after the Pal/Ca²⁺-activated pore opening, prevent the release of Ca²⁺ and repolarize the membrane to initial level. Similar effects can be observed in the absence of exogeneous Pal, upon mitochondria accumulating high [Sr²⁺], which leads to the activation of phospholipase A₂ and appearance of endogenous fatty acids. The paper proposes a new model of the mitochondrial Ca²⁺ cycle, in which Ca²⁺ uptake is mediated by the Ca²⁺ uniporter and Ca²⁺ efflux occurs via a short-living Pal/Ca²⁺-activated pore.     

Mitochondria permeabilization by a novel polycation peptide BTM-P1

Bacillus thuringiensis subsp. medellin is known to produce the Cry11Bb protein of 94 kDa, which is toxic for mosquito larvae due to permeabilization of the plasma membrane of midgut epithelial cells. Earlier we found that a 2.8-kDa novel peptide BTM-P1, which was artificially synthesized taking into account the primary structure of Cry11Bb endotoxin, is active against several species of bacteria. In this work we show that BTM-P1 induces cyclosporin A-insensitive swelling of rat liver mitochondria in various salt solutions but not in the sucrose medium. Inorganic phosphate and Ca(2+) significantly increased this effect of the peptide. The uncoupling action of BTM-P1 on oxidative phosphorylation was stronger in the potassium-containing media and correlated with a decrease of the inner membrane potential of mitochondria. In isotonic KNO(3), KCl, or NH(4)NO(3) media, a complete drop of the inner membrane potential was observed at 1-2 microg/ml of the peptide. The peptide-induced swelling was increased by energization of mitochondria in the potassium-containing media, but it was inhibited in the NaNO(3), NH(4)NO(3), and Tris-NO(3) media. All mitochondrial effects of the peptide were completely prevented by adding a single N-terminal tryptophan residue to the peptide sequence. We suggest a mechanism of membrane permeabilization that includes a transmembrane- and surface potential-dependent insertion of the polycation peptide into the lipid bilayer and its oligomerization leading to formation of ion channels and also to the mitochondrial permeability transition pore opening in a cyclosporin A-insensitive manner.     

Physiological modulators of cyclosporin A-insensitive nonspecific permeability of the inner membrane of liver mitochondria induced by calcium ions and α,ω-hexadecanedioic acid

Long-chain saturated α,ω-dioic acids can induce nonspecific permeability of the inner membrane (pore opening) of liver mitochondria loaded with Ca²⁺ or Sr²⁺ by the mechanism insensitive to cyclosporin A (CsA). In this work we found that 200 μM Ca²⁺ and 20 μM α,ω-hexadecanedioic acid (HDA) in the presence of 1 μM CsA induced high-amplitude swelling of liver mitochondria (pore opening) only in the presence of succinate as oxidation substrate. Under these conditions protonophore uncoupler of oxidative phosphorylation 2,4-dinitrophenol at the concentration of 75 μM, which is optimal for its uncoupling activity, inhibited mitochondrial swelling induced by Ca²⁺ and HDA, despite the presence of succinate in the incubation medium. Natural uncouplers of oxidative phosphorylation, oleic and linoleic acids, produced a similar effect. These data suggest that energization of organelles, which promotes Ca²⁺ transport into the matrix, is one of the basic requirements of pore opening in liver mitochondria induced by Ca²⁺ and HDA. It is shown that ATP at the physiological concentration of 2 mM inhibits HDA-induced high-amplitude swelling of mitochondria by reducing free Ca²⁺ concentration in the medium. ADP at the same concentration had a similar effect. This modulating effect of nucleotides apparently is attributable to their ability to chelate calcium ions. Polycation spermine, which is known as an inhibitor of the classical CsA-sensitive pore, at the physiological concentration of 1 mM inhibited CsA-insensitive swelling of liver mitochondria induced by sequential addition of Ca²⁺ and HDA. It is assumed that such action of spermine is due to its ability to shield the negative surface charges on the inner membrane of mitochondria. Bovine serum albumin (BSA), which is able to bind free fatty acids and thus prevent the induction of Ca²⁺-dependent pore, inhibited HDA-induced swelling of mitochondria. However, at the same BSA/fatty acid molar ratio inhibitory effect of BSA was much less pronounced if HDA was used as the pore inducer instead of palmitic acid. Apparently, this can be accounted by the fact that BSA binds α,ω-dioic acids weaker than their monocarboxylic analogues.