Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Aliphatic alcohols are among the most widespread compounds found to occur naturally in plants and foods. At higher concentrations, they are known to cause a variety of biochemical responses. The fact that aliphatic alcohols are relatively stable and nonreactive has led to their use as models for the study of drug-target mechanisms, especially for modeling amphiphilic, head-tail type compounds. They have repeatedly been used in studies of the mechanisms of anesthesia (Pringle et al., 1981; Franks and Lieb, 1986; Alifimoff et al., 1989; Miller et al., 1989; Chiou et al., 1990) because they fit the trends described by the Meyer-Overton rule, which correlates anesthetic potency with lipid solubility (Meyer and Hemmi, 1935). At concentrations higher than those needed to induce anesthesia, alkanols can cause death and we reported their potential for use as a means of pest control against larval mosquitoes (Hammond and Kubo, 1999). In mosquitoes, potency increased with chain length until leveling off at undecanol and eventually reaching a cutoff after pentadecanol, with the activity of tetradecanol and pentadecanol occurring only after a lag-phase of several hours. The variety of biological functions affected by alkanols leaves some doubt, however, as to the actual mechanisms responsible for death, and whether these mechanisms overlap at all with those of general anesthesia. In this article, we have examined the effects of alkanols on respiration of intact mitochondria to see whether their activity occurs at relevant concentrations, whether it follows the same trends regarding lipid solubility as those observed in vivo, and whether they have the same characteristics of cutoff seen in other test systems. Butanol, pentanol, and hexanol were previously reported to inhibit localized enzymes of the electron transport chain, albeit at relatively high concentrations for most sites (Chazotte and Vanderkooi, 1981). A kinetic study examined alkanol inhibition of cytochrome c oxidase alone, finding activity up to tetradecanol, but not for hexadecanol (Hasinoff and Davey, 1989). An analysis of the uncoupling properties of n-hexane, hexanol, and 1-hexanethiol resulted in the conclusion that they uncouple mitochondrial respiration by a nonprotonophoric mechanism (Canton et al., 1996). Measurement of alkanols' effects on intact mitochondria is an essential intermediate step to linking their action in enzyme assays to their action in living organisms. Our review of the literature indicates that this is the first study to measure the effects of medium- or long-chain alcohols and herein establish the point of cutoff in intact mitochondria. It is also the first attempt to measure the effects of alkanols on a mitochondrial substrate carrier.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Animals. Male Sprague-Dawley mice were purchased at 2 to 3 months of age (200-300 g) from Simonsen Laboratories (Gilroy, CA) and fed on a normal diet. They were fasted for approximately 14 h before removal of the liver.

Materials. Alkanols were purchased from Aldrich (Milwaukee, WI), were the highest grade available, were checked for purity by thin-layer chromatography, and were used without further purification: octanol, >99%; decanol, undecanol, and pentadecanol, 99%; hexanol and dodecanol, 98%; and tridecanol and tetradecanol, 97%. Substrates, inhibitors, and buffer constituents were all obtained from Sigma Chemical Co. (St. Louis, MO). Cytochrome c from horse heart (Sigma catalog no. C2506) was 95% pure and the solution was prepared fresh for each day's experiments.

Isolation of Mitochondria. Liver mitochondria were isolated from fasted rats by the established methods. The rat was briefly anesthetized with isoflurane ("iso-thesia"; Abbott Laboratories, North Chicago, IL) and then exsanguinated by laceration of the jugular vein. The liver was quickly excised and placed in ice-cold homogenization buffer (210 mM mannitol, 70 mM sucrose, 5 mM HEPES, and 1 mM EDTA, and adjusted to pH 7.4). All subsequent isolation work was performed at 0-4°C. The liver was minced with surgical scissors and homogenized with a Potter-Elvehjem tissue grinder and Teflon pestle (Kontes, Vineland, NJ) connected to a Heidolph RZR-1 stirrer (Pennsauken, NJ) operated at a duty cycle of 3.5, approximately 600 rpm. This homogenate was centrifuged for 10 min at approximately 700g; the supernatant was poured off to another tube, the pellet was resuspended, and both were centrifuged a second time under the same conditions to precipitate nuclear and cellular debris. The mitochondria in the resulting supernatant were isolated by centrifugation for 10 min at approximately 8000g. The supernatant was poured off, the pellet was gently resuspended in 40 ml of buffer, and centrifuged one more time for 10 min at 8000g. The mitochondrial pellet was gently loosened, removed, and stored on ice without dilution (typical yield approximately 1.2 ml) for experiments the same day. Mitochondrial protein concentration was determined by the biuret method with BSA as standard.

Polarographic Measurements of Oxygen Consumption. Oxygen consumption measurements were performed with a Clark style electrode and an oxygraph equipped with a water-jacketed chamber maintained at 20°C (all from Yellow Springs Instrument Co., Yellow Springs, OH). The assay buffer solution consisted of 154 mM KCl, 1 mM EGTA, 5 mM K₂HPO₄, and 3 mM MgCl₂, adjusted to pH 7.4. Alkanols were added to the buffer from a dimethyl sulfoxide (DMSO) solution; sonication was used to facilitate solubilization of high concentrations and longer chain alcohols. Mitochondria (1 mg of mitochondrial protein/ml of assay buffer) were preincubated with continuous stirring for 15 min, when rotenone (1 µM) was added to block respiration through complex I and the reaction was started by addition of succinate (5 mM) as substrate. The total assay volume was 3 ml. Controls were treated with the highest dose of DMSO used in each set of experiments, which never exceeded 0.4% and had no discernible effect on respiration. After a slow rate of respiration had stabilized in the initial resting state (State 2), State 3 respiration was initiated by addition of 200 µM ADP. When all ADP has been consumed, respiration returns to a resting rate (State 4). Functional integrity of the mitochondria was measured in terms of respiratory control ratio (RCR), defined as the ratio of the rate of State 3/State 4. RCR values averaged 4.5. Uncoupled respiration was measured after addition of the classic uncoupler of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP; 0.15 µM).

Oxygen Consumption by Freeze-Thawed Mitochondria. Experiments concerning the effect of alkanols on intact versus freeze-thawed mitochondria were performed in the manner described above for intact, coupled mitochondria with the exception of a few modifications. The preincubation period was 5 to 10 min, with preincubation and all respiration measurements performed at 30°C. Respiration was measured with 0.2 mg of mitochondrial protein/ml of assay buffer except where noted. Because breaking the mitochondrial membranes allows endogenous cytochrome c, which freely floats along the intermembrane surface of the inner membrane rather than being membrane-bound, to be washed away, the assay buffer for experiments with freeze-thawed mitochondria included a final concentration of 60 µM cytochrome c added as 10 µl of an aqueous solution. Freeze-thawed mitochondria are inherently uncoupled by the act of breaking the membranes and only the uncoupled state of respiration was measured; this rate was compared with uncoupled respiration in intact mitochondria effected by addition of the least amount of CCCP that would maximally stimulate respiration rates without causing any subsequent inhibition (0.1-0.15 µM). The functional integrity of intact mitochondria was verified before uncoupling by at least two assays yielding an RCR of at least 4.5. 

View this table: [in this window] [in a new window]   TABLE 1 Alkanol concentrations (micromolar ± S.E.) causing 50% inhibition (IC50) in the rate of State 3 respiration, uncoupled respiration, and in the RCR of intact mitochondria isolated from rat liver Data for State 4 respiration refers to the concentration causing 50% stimulation (EC50), and gaps in this column signify that 50% stimulation was not reached (tetradecanol and pentadecanol) or the RCR was decreased to approximately 1 before State 4 reached 50% stimulation (undecanol). Data for mosquito refers to the in vivo LD50 for first instars of C. tarsalis (Hammond and Kubo, 1999).

Data Analysis for Intact Mitochondria. To calibrate for the gradual respiration changes that occur within hours of isolating intact mitochondria, control assays were conducted throughout each day's experiments. The respiration rates of these controls (minimum of five) were plotted and used as the benchmark for comparison with alkanol treatments conducted that day. Each compound-concentration pair was tested at least in triplicate. The IC₅₀ and EC₅₀ values presented in Table 1 were derived by fitting a nonlinear regression curve (SigmaPlot 4.0) to the inhibition data and solving for y = 50. The best curve fit was obtained with the cubic polynomial equation y = y₀ + ax + bx² + cx³.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Change in State 3 respiration after treatment with various alkanols. Alkanols inhibit State 3 respiration in a dose-dependent manner. Potency peaks at undecanol, then tapers off until a cutoff, such that pentadecanol was completely inactive. Average control value was approximately 72 nmol of oxygen/min/mg of protein.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Primary aliphatic alcohols of 6, 8, and 10 to 15 carbons were assayed against succinate-supported mitochondrial respiration up to the concentration of each alkanol that completely abolished respiratory control (RCR 1.1) from that preparation's average control value. There were multiple effects, simultaneously influencing all respiration states measured, as outlined below. Undecanol was the most potent compound in each category (Table 1).

Effects of Alkanols on State 3 Respiration. Alkanols inhibited succinate-supported State 3 respiration in a dose-dependent manner. Potency increased with increasing chain length up to undecanol, then tapered off such that dodecanol was similar in potency to decanol, and potency of tridecanol was between that of octanol and decanol (Table 1). Tetradecanol appeared to have some inhibitory effect on State 3, but the data obtained was not consistently linear or dose dependent (see Discussion for possible explanation). Pentadecanol caused no measurable inhibition up to the maximum dose tested, which is well beyond its solubility limit (Fig. 1).

Effects of Alkanols on State 4 Respiration. All compounds up to 13 carbons caused uncoupling (stimulation) of State 4 respiration at low doses, potency increasing with chain length. However, the effects of higher doses (i.e., concentrations greater than needed to abolish respiratory control) were not the same throughout the alkanol series. Hexanol stimulated State 4 respiration at low concentrations and inhibited it at higher concentrations, whereas for longer alkanols, State 4 respiration was stimulated only; progressively higher doses of longer chain homologs exerted no inhibitory effect on State 4 respiration (Fig. 2). No effect of tetradecanol or pentadecanol on State 4 respiration was detected.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Change in State 4 respiration after treatment with various alkanols. Octanol and longer chain alkanols act as uncouplers. Hexanol uncouples at low concentrations and inhibits State 4 respiration at higher concentrations. Average control value was approximately 16 nmol of oxygen/min/mg of protein.

Effects of Alkanols on Uncoupled Respiration. Respiration uncoupled by CCCP, which normally results in a rate comparable with that of State 3 respiration, was inhibited by alkanols in a dose-response fashion similar to that of State 3 (Fig. 3). Inhibitory potency increased with chain length, peaked at undecanol, then tapered off to tridecanol. Neither tetradecanol nor pentadecanol exhibited any effect on respiration uncoupled by CCCP. However, just as for State 4 respiration, hexanol affected uncoupled respiration differently than long-chain alcohols. When applied at concentrations that lowered RCR to approximately 1, hexanol inhibited uncoupled respiration by >80%, whereas octanol, decanol, undecanol, dodecanol, and tridecanol lowered uncoupled rates of respiration by approximately 67, 70, 67, 68, and 55%, respectively. Whereas several experiments with high concentrations of hexanol produced a rate of uncoupled respiration even lower than the State 4 rate of controls, the longer chain alkanols reduced uncoupled respiration only to a rate similar to that of State 4, but no lower (Fig. 4), even when the dose was increased severalfold.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effect of various alkanols on the rate of uncoupled respiration. Almost identical with State 3, uncoupled respiration is inhibited in a dose-dependent manner, with potency peaking at undecanol and pentadecanol completely inactive. Average control value was approximately 81 nmol of oxygen/min/mg of protein.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Effects of decanol (top) and hexanol (bottom) on succinate-supported mitochondrial respiration. Each trace depicts a representative experiment, in which the reaction was started by adding succinate (leftmost arrow of each trace), State 3 was initiated with ADP (second arrow of each trace), and uncoupled respiration with CCCP (third arrow). Assay conditions and concentrations are given in Experimental Procedures. Note that in the highest concentration experiments, where respiratory control had already been abolished, additional doses of hexanol (5 mM each arrow) decreased the rate of uncoupled respiration below the State 4 rate, whereas higher doses of decanol (400 µM each arrow) did not.

Effects of Alkanols on ADP/Oxygen Ratio. Measurement of ADP/oxygen ratio inherently requires some degree of coupling, i.e., some noticeable difference between State 3 and State 4 respiration rates. The RCR for all alkanol treatments was reduced to 1 (where State 3 rate = State 4 rate) before the inhibition of ADP/oxygen reached 50%, so we were unable to establish IC₅₀ values for alkanol effects on ADP/oxygen. Nevertheless, some decrease in ADP/oxygen was evident for treatments with hexanol, octanol, and decanol through tridecanol; at the concentration of these alkanols that, respectively, produced 50% inhibition of RCR, we observed approximately 10% inhibition of ADP/oxygen, i.e., relative to an average control value of 1.75 nmol of ADP/nmol of molecular oxygen. At alkanol concentrations causing near total loss of RCR (RCR approaching 1.1), decline in ADP/oxygen averaged approximately 25 to 40%. Tetradecanol and pentadecanol did not affect ADP/oxygen ratios.

Inhibition of Respiration in Intact Versus Broken Mitochondria. In subsequent assays with independent mitochondrial preparations, a medium- and a long-chain alkanol (hexanol and decanol) were each tested for their differential effects on the uncoupled respiration of isolated rat liver mitochondria that was 1) intact and used immediately on isolation, or 2) subjected to two freeze-thaw cycles so as to physically break the inner and outer mitochondrial membranes. Consistent with the earlier data, both hexanol and decanol inhibited respiration of intact mitochondria in a dose-dependent manner, but notably, each compound had far less effect on the freeze-thawed mitochondrial preparation.

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Effects of hexanol on the respiration rate of intact (uncoupled) and freeze-thawed mitochondria isolated from rat liver.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Effects of decanol on the respiration rate of intact (uncoupled) and freeze-thawed mitochondria isolated from rat liver.

Effect of Mitochondrial Protein Concentration on Inhibition. To gain further insight into the molecular nature of the inhibition, several experiments were performed under identical conditions but with half as much mitochondrial protein to initiate respiration. The results show that for hexanol, there is a similar degree of inhibition, even when the protein concentration is halved. The level of inhibition seems to depend on the inhibitor concentration, regardless of how much mitochondrial protein is present (Table 2).

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The inhibitory data we report herein for alkanols of 11 carbons and longer reflect concentrations that substantially exceed the published aqueous solubility limits for alkanols (Bell, 1973; Barton, 1991). Similar findings have been reported elsewhere, for both alcohols and acids (Huhtanen, 1980; Takeuchi et al., 1991; Kubo et al., 1995; Gustavson et al., 1998; Matsuki et al., 1999). Mitochondrial membranes are composed largely of lipids, so if an aqueous assay system is designed to create a supersaturated suspension, even temporarily, then high doses of amphiphilic compounds may partition into the lipid environment and thereby be delivered to the target site. A supersaturated solution is facilitated by adding the alkanols from a solution having detergent properties (such as DMSO), or by adding the solution while simultaneously applying sonication. Moreover, long-chain alcohols are known to have a marked propensity for forming supersaturated solutions without forming micelles (Tanford, 1991).

The concentrations found to inhibit mitochondrial respiration are comparable with those causing death in mosquitoes and other organisms (Fig. 7), prompting the question whether decreased mitochondrial functions might in some way contribute to mosquito death. If mosquito larvae were prevented from making ATP, approximately 90% of which is produced in mitochondria, then certainly they would appear paralyzed and, ultimately, would die. Although more work is required to substantiate the apparent correlation, in a single preparation of mosquito larvae mitochondria that we succeeded in isolating, a decanol concentration of 100 µM caused approximately 30% inhibition of uncoupled respiration, which is roughly comparable with the effects seen in rat liver mitochondria. Differences in the chain length of cutoff from one organism to another may be related to differences in the composition of their respective cell membranes because alkanols have been shown to bind more tightly to those phospholipid vesicles having longer acyl chains (Kamaya et al., 1981), and increasing the cholesterol content of lipid membranes has been shown to suppress anesthetic potency (Rehberg et al., 1995).

View larger version (27K): [in this window] [in a new window]   Fig. 7.   The biological activity of alkanols against various organisms in vivo compared with their inhibition of RCR in isolated rat mitochondria. Cutoff in activity occurs at different chain lengths in different organisms. For isolated mitochondria, tetradecanol may show some activity if applied for longer preincubation times. , tadpole (Rana pipiens), loss of righting reflex, EC50 (Alifimoff et al., 1989); , minnow (Pimephales promelas), 96 h LC50 (Veith et al., 1983); , protozoan (Tetrahymena pyriformis), inhibitory growth concentration, EC50 (Schultz et al., 1990); , Staphylococcus aureus, minimum bactericidal concentration, EC50 (Kubo et al., 1995); , isolated rat mitochondria, respiratory control ratio, IC50; ×, mosquito (Culex tarsalis), 24 h LD50; Tween used to maximize solubility of alkanols 13 carbons in length (Hammond and Kubo, 1999); , Propionibacteruim acnes, minimum bactericidal concentration, EC50 (Kubo et al., 1995); and +, Clostridium botulinum, minimum inhibitory concentration (Huhtanen, 1980).

Independent evidence of a role for mitochondria in anesthetic action has indeed been found. By mutating a gene of the nematode Caenorhabditis elegans that has been determined to control expression of a mitochondrial protein located within NADH dehydrogenase of complex I, an increased sensitivity to volatile anesthetics was conferred (Kayser et al., 1999). Although the precise function of the gene's mitochondrial protein product has still not been identified, Kayser et al. (1999) found that the mutation in complex I increases the animal's sensitivity to anesthetics, either by a direct anesthetic effect on a mitochondrial protein or by indirect effects resulting in mitochondrial dysfunction. Such malfunctions could result from lipid peroxidation and membrane damage caused by depletion of the antioxidant ubiquinol pool, or simply from lack of electrons to fuel synthesis of ATP, the regulator of innumerable other cellular processes (Kayser et al., 1999).

Numerous studies have reported certain biological activities for short-chain alcohols, and different, or even opposite, activities for longer chain homologs. For example, patch-clamp studies on neuroblastoma cells showed that butanol and hexanol potentiated responses to the activator 5-hydroxytryptamine at low concentrations, and inhibited them at high concentrations; in contrast, higher alcohols produced no potentiation, but only inhibition, at all alcohol concentrations (Jenkins et al., 1996). In studies of the effects of alkanols on calcium transport in brain mitochondria, it was found that short-chain alcohols inhibit whereas long-chain alcohols activate the cyclosporin-sensitive Ca²⁺ efflux (Rottenberg and Marbach, 1992). There is convincing evidence that short-chain and long-chain alkanols act at distinct sites on the nicotinic acetylcholine receptor channels of electric eels, thus accounting for their dissimilar effects on ion flux (Wood et al., 1991, 1993).

The finding that hexanol stimulates State 4 respiration at low concentration, yet inhibits it at high concentrations (Fig. 2) corroborates the results of a previous study (Canton et al., 1996). In intact rat mitochondria tested at 20°C, decanol inhibits uncoupled respiration at a relatively low concentration, but hexanol inhibits it more completely (Fig. 4). This difference between the degree of maximal effects on mitochondria displayed by decanol and hexanol may conceivably be a consequence of the latter acting via a wider array of mechanisms. Nevertheless, lack of sufficient solubility probably plays a part in explaining the limits to decanol's inhibitory effect. The data comparing freeze-thawed and intact mitochondrial preparations (Figs. 5 and 6), where similar assays were performed at 30°C, shows decanol inhibiting uncoupled respiration (77%) more thoroughly than hexanol (63%), so it is possible that the additional doses of decanol depicted in Fig. 4 produced no additional inhibitory effect simply because they could not enter solution. Hexanol, in contrast, is easily soluble up to concentrations more than three times greater than any dose tested herein (Bell, 1973).

The fact that alkanols inhibit State 3 and uncoupled respiration while stimulating State 4 causes the oxygen electrode trace to tend toward a straight line at high concentrations (Fig. 4). Determining the slope of each respiratory state under these conditions becomes somewhat imprecise and contributes to the relatively large standard errors in the figures.

In earlier in vivo studies of 1-alkanols as mosquito larvicidal agents, we found that the action of chain lengths >11 carbons was clearly time dependent, with dodecanol immobilizing mosquito larvae after 12 min, tridecanol after 45 min, tetradecanol after 3 h, and pentadecanol after 12 h (Hammond and Kubo, 1999). Nonlinear data for tetradecanol against State 3 respiration therefore leads us to speculate that although it may indeed possess inhibitory activity, its action is time dependent, requiring more than the 15-min preincubation period used herein.

Where inhibition of State 3 respiration is greater than that of uncoupled respiration at equivalent doses, one can reasonably infer some inhibitory effect on ATP synthase (complex V) (Gudz et al., 1997). But the fact that inhibition by alkanols, regardless of chain length, is nearly identical for both State 3 and uncoupled respiration (Fig. 8) implies that complex V is not directly affected by alkanols at the concentrations used herein.

View larger version (14K): [in this window] [in a new window]   Fig. 8.   Effects of octanol on RCR (), State 3 (), and uncoupled respiration (), expressed as percentage of inhibition from control. Other alkanols displayed similar patterns, such that State 3 and uncoupled respiration were each inhibited to a similar degree. Curve fitting is described in Experimental Procedures.

It appears then that the inhibition occurs either at the level of the electron transport chain and/or on the dicarboxylate substrate carrier enzyme itself, which is needed to shuttle succinate across the inner membrane for its introduction into the respiratory chain at complex II (Tyler, 1992). Our preliminary assays with duroquinol as substrate, which feeds into complex III, suggest that there is no inhibition of the respiratory chain by alkanols at complexes III or IV up to the maximum doses tested herein (data not shown).

Previous work has shown that long-chain fatty acids affect mitochondrial respiration by uncoupling State 4 and inhibiting State 3 (Takeuchi et al., 1991), and by directly inhibiting activity of the dicarboxylate carrier (Wieckowski and Wojtczak, 1997). Fatty acids and fatty alcohols, both head-tail type compounds, may share common mechanisms for inhibiting mitochondrial respiration. Interestingly, on several occasions we found that at a dose approximately five times the concentration of alkanol needed to lower the RCR to 1, the mitochondrial test solution turned from milky to clear, inhibition was released, and respiration increased. Presumably, this occurs because on reaching the critical micelle concentration of the alkanol, its detergent properties serve to solubilize the mitochondrial membranes. A finding that succinate-supported, uncoupled respiration increases after permeabilizing the mitochondrial membrane is consistent with the possibility that the actual site of inhibition may be the dicarboxylate substrate carrier, and not, or not only, complex II of the electron transport chain itself.

Effects of a compound on mitochondrial substrate carriers such as the dicarboxylate carrier, which transports succinate, can be distinguished from effects on the electron transport chain by the use of freeze-thawed mitochondria (Modica-Napolitano et al., 1990; Krahenbuhl et al., 1991; Modica-Napolitano, 1993; Krahenbuhl et al., 1994; Gudz et al., 1997). Because freezing to 20 or 80°C and subsequently thawing has the consequence of breaking the inner and outer mitochondrial membranes, substrates that would normally need to be transported across the membrane before being fed into the respiratory chain can instead freely access complex II. The data presented in Figs. 5 and 6, where hexanol and decanol are both shown to inhibit uncoupled respiration in intact mitochondria but that such inhibition is released in freeze-thawed mitochondria, indicates that the inhibitory effects can therefore be inferred to occur at the level of trans-membrane substrate transport rather than on the electron transport chain itself.

Researchers describing the effects of butanol, pentanol, and hexanol on submitochondrial particles (Chazotte and Vanderkooi, 1981) concluded that inhibition occurs at various enzyme sites along the electron transport chain, but at 5- to 10-fold the concentrations reported herein. Although there is undoubtedly an effect of alkanols on electron transport, our data show that inhibition occurs on the substrate carrier first, and at lower concentration.

It would be interesting to see whether further experiments would corroborate the preliminary findings of Table 2, where decanol inhibits respiration more thoroughly in a solution containing 0.1 mg of mitochondrial protein/ml than in one containing 0.2 mg/ml, but hexanol inhibits them both equally. The pattern suggested is consistent with a scenario whereby decanol acts in a specific manner, binding molecule per molecule to a finite number of protein sites, whereas hexanol acts as a nonspecific agent of conformational or structural changes, probably perturbing the lipid-protein interface, but not acting via a "lock-and-key" type molecular interaction.