Methane-induced hemolysis of human erythrocytes

Human erythrocytes were exposed to high concentrations of methane and nitrogen through the application of elevated partial pressures of these gas molecules. Cell leakage (hemolysis) was measured for cells exposed to these gases under a wide range of experimental conditions. Application of methane produces hemolysis at pressures far below the hydrostatic pressures known to disrupt membrane or protein structure. The effects of changes in buffer, temperature, diffusion rate, and detergents were studied. Methane acts cooperatively with detergents to produce hemolysis at much lower detergent concentration than is required in the absence of methane or in the presence of nitrogen. At sufficiently high concentrations of methane, all cells are hemolyzed. Increased temperature enhances the effect. Methane produces 50% hemolysis at a concentration of about 0.33 M compared to about 7.5 M methanol required for the same degree of hemolysis.

INTRODUCTION

Small gaseous hydrocarbons exhibit properties potentially advantageous for the manipulation of membranes, membrane components and detergents. At relatively low concentrations these molecules exhibit anesthetic behavior, presumably mediated through their interaction with membrane-associated binding sites [1,2]. At higher concentrations they disrupt the membrane structure and function. The potential for using this disruption for the preparation and processing of membrane components is largely unexplored. Although only sparingly soluble at atmospheric pressure (1 standard atmosphere ~ 0.1 megapascal (MPa)), small hydrophobic molecules such as methane can be forced into aqueous solution at higher concentrations by application at high partial pressures. For instance, methane has a solubility of about 1.5 mM at atmospheric pressure in water, but over 0.5 M at 100 MPa [3]. Since most proteins do not denature at hydrostatic pressures less than 150-200 MPa [4-5], it is possible to expose biological molecules in their native conformation to relatively high concentrations of small hydrophobic gas molecules. The partitioning of gaseous hydrophobic molecules between aqueous solutions and the hydrophobic interiors of membrane systems is likely to result in much higher effective concentrations of the gas molecules within the membranes. For instance, the partition coefficient of methane between n-octonol and water is about 100:1, depending on the pressure and temperature of the system [3,6]. This makes it possible to produce very high local concentrations of methane within membrane systems at moderate pressures. It is also, presumably, one reason for the broad anesthetic properties of most hydrophobic gases.

In principle, the effect of small gaseous hydrocarbons on membrane systems may be mediated through several mechanisms. First, they partition into hydrophobic compartments, such as lipid bilayers [7], potentially altering their structure and affecting their stability. Second, they compete for water of hydration with other hydrophobic solutes, such as detergents, and force these molecules out of solution, moving them into either micelles or membranes. Third, to achieve moderate concentrations of small gaseous hydrocarbons in biological system requires the application of high partial pressures of the gas and the system will be affected by the pressure changes. The consequent effect of this may not solely be due to the hydrostatic pressure. For example, Dodson et al. have found that the physiological effects of high hydrostatic pressure on tadpoles Rana pipiens, were different from the effects produced by high pressure helium or nitrogen, for the same pressure [8]. Similar effects have been observed by Nelson and co-workers on the growth of microbial organisms and they have used the term hydrostatic and hyperbaric to distinguish these effects [9].

The effect of hydrostatic pressure on biological systems has been characterized in detail [4,10,11]. For instance, hydrostatic pressures of several hundred atmospheres have been shown to affect cell function and properties such as ion transport [12], release of membrane proteins [13], receptor-protein dissociation [14], and lipid phase transitions [15]. Mild pressure in the range of a few atmospheres, does not usually affect cell function, even though it has been shown that application of pressures of the order of 0.2-0.5 MPa increases the ability of intact erythrocytes to withstand lytic actions of snake venom phospholipase A2 [16]. In contrast to the detailed study of the hydrostatic effects, there have only been limited studies on the effects of hyperbaric pressure effects on biological systems. The majority of this work has been by those who study the hyperbaric effect of gases like O2, N2 and He and high pressure nervous syndrome or high pressure narcosis on humans who dive under water [17].

The interactions of moderate concentrations of small gaseous hydrocarbons with membranes are assessed here both in the presence and absence of detergents, by measuring the level of hemolysis induced by moderate concentrations of methane, and comparing this to the activity of nitrogen and of methanol. Hemolysis is a complex process that has been used as an assay for the membrane- targeted action of many compounds [18,19]. The extent of hemolysis is readily measured and quantified, but the results are difficult to interpret in terms of a specific molecular mechanism. However, under experimental conditions where other probes are difficult to apply, hemolysis can be used to measure the relative activity of two substances, thereby providing qualitative information not accessible by other techniques.

MATERIALS AND METHODS

Materials

N-octyl b-D-glucopyranoside (minimum purity 98%) was obtained from Sigma (St. Louis, MO); Triton X-100 (Polyethylene glycol tert-octylphenyl ether, electrophoresis grade) was obtained from Bio-Rad (Richmond, CA). High-pressure cell was purchased from Ruska (Houston, TX). Methane and nitrogen were procured from Airco Gases Inc., (Murray Hill, NJ) in high-pressure cylinders (~6000 psi). The purity of methane was 99%. Typical impurities present in methane are 0.6% N2, 0.2% O2, 0.2% CO and CO2 and 0.1% ethane. Nitrogen was 99.998% pure and the impurities are 0.0005% O2 and 0.0001% total hydrocarbon.

High-pressure instrumentation

Commercially available [Model # 2239-800, Ruska, Houston, TX] high-pressure gas cell has been modified in our lab and is shown schematically in Figure 1 a & b. The cell can be pressurized up to ~ 70 MPa (10,000 psi) at ambient temperatures. The cell is made of 450 series steel and has 4 inlet/outlet ports. There are two rectangular optical windows and two lids perpendicular to the windows. The lids and windows are secured with screws tightened against gaskets to a preset torque to ensure uniform and leak-free closure. Opening and closing of only one lid is required for all the operations described below. The center of the cell has a cylindrical cavity with a volume of 100 ml. In order to reduce the risks of working with large amounts of gas under pressure, a sample holder was designed to fill the 100 ml working volume of the pressure cell, reducing the volume of the gas within the cell to ~ 25 ml. The stainless steel sample holder used in this study (Figure 1 c) has two rows of six equally spaced holes (1.25 cm dia. and 1.5 cm deep) designed to hold appropriately cut Pyrex glass tubes containing the samples. The high-pressure cell is mounted on a steel support and the support itself is fitted to the base plate of a safety cabinet made of plywood and steel. The high-pressure cell is enclosed inside the safety cabinet. The cell is connected to a gas-booster [Hogan Fluid Power, Houston, TX] and the gas booster in turn is connected to high-pressure cylinder through high-pressure lines containing several high-pressure valves [Swagelok, Solon, OH]. Gas from a high-pressure cylinder is pressurized using the gas-booster to gas pressures in the range 0.1-70 MPa (15-10,000 psi) to an accuracy of 0.25 MPa. Once the desired pressure has been achieved, the cell can be isolated and the pressure maintained for the course of the experiment by closing the high-pressure valve. After introducing the sample, the lids and the safety cabinet were securely closed. The cell was first flushed out by pumping the desired gas to about 0.5-1 MPa, maintaining this range for ~ 2 min. and then slowly leaking the gas to a vented hood. This procedure was repeated at least three times to remove all traces of air and moisture from the interior of the cell. After flushing, the cell was pressurized in steps of 5 MPa until the desired pressure was reached. A few minutes were allowed to lapse after each pressure step, to avoid any increase in temperature due to the compression of the gas and to minimize the stress on the high-pressure cell. At the end of the experiment, pressure was reduced gradually by equalizing the pressure inside the booster with that inside the cell and then reopening the isolation valve. Subsequently, the pressure in the cell was brought down to atmospheric over a period of 1-1.5 hours by releasing the gas to the vented hood. Characterization and further details of the high-pressure cell are described elsewhere [ T. Somasundaram and L. Makowski, Rev. Sci. Instrum. 66(5), 3311-3316 (1995) ].

Figure 1

Schematics of the high-pressure cell. a) Front view of the high-pressure cell. b) Side view of the cell without the lid and the sample holder. c) Top view of sample and the sample holder. A: Lid; B: Cell body; C: Holding screws; D: Inlet/outlet port; E: Rectangular opening; F: Optical window; G: Sample; H: Cylindrical interior cavity; I: Pressure gasket; J: Stainless Steel Sample holder; and K: Pyrex tubes containing sample. Sample holder (J) containing sample tubes (K) is inserted into the cylindrical interior cavity (H)and the lid (A) is tightened to a preset torque against the gasket (I) to ensure a leak-free operation. The entire cell is enclosed inside a safety cabinet. Inlet/outlet ports (D) are used for pressurization and depressurization.

Isolation of human erythrocytes

Whole blood (peripheral) was drawn and collected in Vacutainers (Beckton-Dickinson, Rutherford, NJ) containing lithium heparin as anticoagulant. Erythrocytes were isolated by standard techniques [20]: Blood was centrifuged at 1000 x g for 5 min. in a clinical centrifuge and supernatant plasma was removed by Pasteur pipette. Pelleted cells were resuspended in pre-filtered (0.2 mm filter) phosphate buffered saline pH 7.4 (PBS) and centrifuged as above. Buffy coat and supernatant were removed and the process repeated five times to remove white blood cells and platelets. Erythrocytes were finally resuspended in PBS, refrigerated at 4_ C and used until 15 days old. For experiments a 5 ml aliquot of erythrocyte suspension was drawn from the stock, centrifuged at 1000 x g for 3 min. and 0.5 ml of the pellet was removed and diluted with 49.5 ml of PBS, to give a 1% erythrocyte suspension. This 1% erythrocyte suspension has approximately 1.2 x 108 cells/ml. Erythrocyte suspensions were also prepared by a similar process in pre-filtered (0.2 mm filter) Alsever's buffer, pH 6.4 (0.42% (w/v) NaCl, 0.8% (w/v) tri sodium citrate dihydrate, 2.05% (w/v) glucose and pH adjusted with citric acid).

Application of high-pressure to erythrocytes

Aliquots of a 1% erythrocyte suspension were placed at room temperature in glass tubes that were then put in the sample holder and subsequently transferred to the high-pressure cell. The high-pressure cell was then sealed. Erythrocytes were incubated against high-pressure gas as described above. A similar set of erythrocyte suspensions was kept in air at room temperature, one atmosphere pressure, as a control. After depressurizing, all samples (control and pressurized) were centrifuged in Eppendorf tubes at 1000 x g in an Eppendorf Micro 5415 Centrifuge (Brinkmann, Westbury, NY) for 5 min. Supernatant was removed and the erythrocyte pellet was lysed by adding distilled water to each sample to a 1 ml final volume. The supernatant was also diluted to 1 ml. The lysed erythrocytes (ghosts) were pelleted by centrifuging in Eppendorf tubes at 15, 000 x g for 30 min. Each sample was then diluted 10-fold.

Determination of standard conditions

UV spectra of hemoglobin released from erythrocytes after exposure to 64 MPa of methane or nitrogen for 24 hours were identical to control. This indicated that these exposures did not irreversibly alter hemoglobin structure. No information about the structure of hemoglobin at high gas pressures was obtained in these studies. However, this result demonstrated that absorbance at 414 nm could be used as a direct measure of hemoglobin concentration. Hemoglobin released from erythrocytes was measured to quantitate percent cell survival. Early experiments were designed to determine under what conditions the effect of methane and nitrogen could best be studied. The effect of sample volume was measured to determine the effect of diffusion time of gases through the aqueous solutions in the absence of a facility to stir the specimens in the pressure cell. Sample temperature, buffer and incubation time were also varied to determine appropriate conditions for subsequent experiments.

Percent erythrocyte survival (or percent hemolysis) was calculated for each 1 ml sample by measurement of absorbance at 414 nm using a Hewlett Packard Diode Array Spectrophotometer HP 8451A (Hewlett Packard, Corvallis, OR). Percent hemolysis and percent erythrocyte survival were calculated using the following formula for each sample. Percent Hemolysis = 100 S/(S+P) and percent erythrocyte survival = 100 - percent hemolysis ; where S is total supernatant absorption and P is total pellet absorption. To measure the effect of the diffusion of gas through the aqueous samples, a wide range of specimen volumes was studied. Erythrocyte suspensions (1%) in PBS and Alsever's buffer were used to make aliquots of increasing volume (0.05, 0.1-1.0 ml in 0.1 ml step). Experiments were done with increasing erythrocyte suspension volume for various incubation periods (5-25 h). The effect of high-pressure gas on detergent-induced hemolysis was studied by adding detergent to 1% erythrocyte suspensions and subjecting them to high gas pressures. N-octyl _-D- glucopyranoside was used over a concentration range of 0-0.45% (w/v) and Triton X-100 over a range of 0-0.008% (w/v). Experiments on these samples were performed at gas pressures of 20, 41, 50 and 64 MPa of methane or nitrogen. Appropriate control specimens with identical detergent concentrations were left at one atmosphere pressure.

RESULTS AND DISCUSSION

Effect of methane and nitrogen

The effect of methane and nitrogen on erythrocyte survival was measured as a function of the pressure of the applied gas. Cell suspensions (0.3 ml in PBS) were incubated for 24 hours in the presence of 20, 41, 50 and 64 MPa methane and in the presence of identical pressures of nitrogen. Following the incubation time the solutions were brought to atmospheric pressure and extent of hemolysis determined as described in Materials and Methods. Both the nitrogen and methane-incubated cell suspensions contained large amount of gas bubbles in the solution (see below). Figure 2 is a plot of percent hemolysis as a function of pressure for nitrogen and methane. Methane pressure of 64 MPa for 24 hours hemolyses 90% of cells. Nitrogen-induced hemolysis on the other hand is constant at all pressures at a low value of 7%. This implies that with increasing pressure of methane, increased methane-induced hemolysis occurs. Nitrogen has little effect on hemolysis as a function of pressure. The solubility of methane in aqueous solvent is 2.1 times that of nitrogen. Therefore at a given pressure the aqueous concentration of methane is more than twice that of nitrogen. This difference does not, however, explain the great difference seen in the hemolysis induced by the two gases.

Figure 2

Effect of methane and nitrogen on the degree of hemolysis of erythrocyte suspension. Aliquots of 0.3 ml of erythrocyte suspension in PBS buffer were exposed to various methane or nitrogen pressures at 23deg.C for 24 h and percent erythrocyte survival estimated as described in Materials and Methods section.

Another possibility is that the cells saturated with the gaseous solutes collapsed during the decompression due to the formation of intracellular bubbles leading to the leakage of hemoglobin. Experiments carried out by Hemmingsen et al. [23] rule out this possibility. They have shown that intact human erythrocytes and ghosts (loaded with fluorescent markers) incubated either with nitrogen or argon pressures of 30 MPa and decompressed rapidly (~1 s) are not hemolysed despite the presence of profuse bubbles in the medium. They have attributed this ability of the erythrocytes to withstand the gas supersaturations to the absence of intracellular bubble formation, in contrast to Tetrahymena pyroformis cells (containing food vacuoles) which were ruptured due to the intracellular bubble formation [24]. Even though we have carried out experiments at higher pressures than Hemmingsen et al.[23], our decompression times are at least three orders of magnitude longer and our nitrogen results are in agreement with experiments of Hemmingsen et al.

Earlier work of Gerth and Hemmingsen had shown that for N2 and CH4 the gas supersaturation tension (in atmospheres) i.e., the difference between the gas equilibration pressure and pressure at which bubbles appear at the glass-water interface is not significantly different for gas equilibration pressures 30-57 MPa [25]. Therefore, the hemolysis seen for methane-incubated solutions are not likely due to the bubble formation in the external medium.

Effect of sample volume and buffer

The effect of diffusion time was studied by measuring erythrocyte survival at 64 MPa of methane and of nitrogen for different volumes of cell suspension in order to confirm the results shown in Figure 2. Results of these experiments carried out in PBS as well as Alsever's buffer are shown in Figure 3. Except for the smallest sample volume, less than 3% of the control erythrocytes (in PBS) hemolysed at one atmosphere, in the 10 hours of this experiment. The one atmosphere, 0.050 ml sample exhibited 7% hemolysis, presumably due to disruption of erythrocytes at the air-water interface or to the errors involved in handling small volumes. In Alsever's buffer, pH 6.4, erythrocyte survival at one atmosphere was similar to the behavior shown by PBS (data not shown). At low sample volumes (0.1 to 0.4 ml) erythrocyte survival for 10 hours at 64 MPa methane in PBS is about 20% and in Alsever's buffer about 60%. Cell survival increases rapidly with increasing volume for both the buffers. In the case of 64 MPa nitrogen pressure even for the smallest volume, erythro- cyte survival was more than 90%.

Figure 3

Effect of sample volume on erythrocyte survival in PBS and Alsever's buffers in the presence of 64 MPa of methane or 64 MPa of nitrogen for 10 hours at 23_C. The diffusion of gases in unstirred samples with volumes greater than 0.4 ml is sufficiently slow to lower the degree of hemolysis observed for experiments of 10 h duration. The diffusion time for the transport of methane through a height h (in cm) is given by h2= ½ Dt, where, t = time (in s), and D = diffusion constant. Since the volume of the sample is V=hA where A is the cross sectional area and is essentially a constant in these experiments, V2should be proportional to Dt for a diffusion limited process. The inset shows a plot of time to achieve 50% hemolysis vs. the square of the volume, which is proportional to the square of the height of the solution (since area is held constant). This relationship is linear, supporting the proposition that methane-induced hemolysis is a diffusion limited process.

The diffusion constant D of methane in water is 1.904 x 10-5 cm 2/sec [21] making it unlikely that methane diffused completely to the sedimented erythrocytes at the bottom of the sample tubes, over the course of these experiments. For example, given this value of D and using the expression for diffusion in one dimension over a height h as, ½ Dt = h2, we have t ~ 8 h, for h = 0.5 cm. To further substantiate that the hemolysis is diffusion limited in the absence of stirring, the time required to produce 50% hemolysis (PBS buffer) was plotted as a function of the square of the volume since the volume V = h A. For diffusion-limited process, this should produce a straight line, as seen in the inset of Figure 3. These experiments validated the use of sample volumes of 0.3 ml and incubation time of >12 hours. Decrease of this time led to incomplete diffusion of the applied gas into the specimen. Increased temperature also enhanced the effect of methane on hemolysis (data not shown). However, due to problems in maintaining the pressure cell at a constant, uniform temperature, most experiments were carried out at room temperature.

Comparison with methanol-induced hemolysis

Figure 4 shows the percentage of hemolysis induced by methane and methanol as a function of their molar concentrations. Methane is over 20 times as efficient as methanol in hemolysing erythrocytes. Small aliphatic alcohols are considered to be potent hemolysing agents and believed to destabilize membrane by pore formation [26]. Methane acts much more effectively to hemolyse erythrocytes but whether it acts through the same mechanism or a different one is not known. The partition coefficient of methane between octonol and an aqueous solution is ~ 70 times higher than for methanol for the same interface [22], the difference is much higher if the partition coefficients are considered for hexadecane and an aqueous solution [22] and this may contribute to the increased potency of methane for hemolysis. Chi et al., for example, have found that the correlation between the observed hemolysis and concentration of small chain alcohols become better when they have used the membrane concentration (calculated using the partition coefficient) rather than the aqueous concentration [27]. A related reason may be due to the region of the membrane where the n-alcohols and n-alkanes get partitioned (see below).

Figure 4 Comparison of hemolysis induced by methane and methanol as a function of concentrations of these molecules. The horizontal axis is logarithm of concentration to allow plotting the substantially different activities of methane and methanol on the same curve. Note that at these high pressures of methane, Henry's law is not well obeyed [3].

Effect of gases on detergent-induced hemolysis

Erythrocyte suspensions having increasing concentrations of n-octyl b- D -gluco-pyranoside (0.0 to 0.45%) were incubated under methane pressures of 20, 41, 50, and 64 MPa for 12 hours, at 22_C. Similar experiments were performed in the presence of Triton X-100 at detergent concentrations of 0.0-0.01%. The results of representative experiments are shown in Fig. 5. The curves in Figures 5a and 5b correspond to experiments with n-octyl b-D-glucopyranoside and Triton X-100, respectively. These results indicate that methane acts to promote the destabilization of membranes by detergents. Nitrogen does not significantly increase hemolysis as a function of detergent concentration. For example, erythrocytes incubated for 12 h at 22_C under 64 MPa nitrogen in presence of 0.225%(w/v) n-octyl b-D-glucopyranoside, show 50% hemolysis compared to 48.4% hemolysis exhibited by the unpressurized control sample. Detergent-induced hemolysis occurs close to the critical micellar concentration of the detergents, especially for the non-ionic detergents [28,29]. However, there is not always a simple correlation between the critical micellar concentration and hemolysis [29]. There have, however, been suggestions that the detergent induced hemolysis may involve intrabilayer non-bilayer phases [30,31].

Figure 5

Effect of methane on detergent induced hemolysis of erythrocytes in PBS buffer. a) Effect of methane on hemolysis induced by n-octyl b-D-glucopyranoside, and b) effect of methane on hemolysis induced by Triton X-100.

The mechanism by which methane enhances the detergent-induced hemolysis is not immediately clear. By competing with detergent molecules for the water of hydration, methane will drive detergent molecules out of solution, either into micelles or into the membrane. This enhances the effect of any given concentration of a detergent on the membrane by increasing the number of the detergent molecules that are interacting with the membrane. The natural aggregate for a detergent is to form a micelle; detergents have too large an area-to-volume ratio to form a bilayer [32]. When they interact with a membrane, the lipid structure will be disrupted in a way that will be consistent with an increase in the area-to-volume ratio of the hydrophobic phase. Small hydrophobic molecules such as methane would be expected to produce the opposite effect on the area-to-volume ratio, presumably increasing the volume of the hydrophobic phase with little or no effect on its area. Consequently, these two opposing effects might be expected to cancel one another, however, such a cancellation is not observed and therefore other effects must be contributing to the methane induced hemolysis.

King et al. have studied the interaction of n-hexane with dioleoylphophatidylcholine (DOPC) bilayer and reported that the partial molar volume of hexane in bilayer is close to zero [33]. According to them the thickness of the hydrocarbon region (Dh) in DOPC does not change even when one molecule of hexane is added per lipid molecule. This observation is in contrast to earlier reports and widely held belief that added hexane should increase the bilayer thickness. Whether the very small volume change seen for n-hexane in pure DOPC bilayer is universal and applicable to small gaseous alkanes in heterogeneous membrane lipids is not known. If the near-zero volume change seen for n-hexane is true for methane also, then it can partly explain the co-operative effect seen in the case of methane-induced hemolysis by detergents. White et al. have also shown that partitioned hexane is predominantly present in the hydrocarbon core (HCR) of the bilayer (the middle) [34]. From the structure of fluid DOPC bilayer determined by the combined use of x-ray and neutron diffraction by White and co-workers [34,35], one can infer the possible partitioning of n-alcohols in the interfacial region (IFR) of the bilayer as opposed to the predominant partition at the HCR for the n-alkanes. This can partly explain the difference between methane and methanol that is likely to be present mostly in the interfacial region and hence require much higher concentration in order to get partitioned further into the membrane to produce hemolysis. However, detailed structural information is required for the further understanding of methane induced hemolysis both in the presence and absence of detergents.

Although the molecular mechanism of methane-induced hemolysis is unclear, these results suggest that the strong interactions of methane with membranes may have clear advantages for a number of biochemical procedures. First, methane may make possible the use of smaller detergents for the solubilization of membrane components. This could be advantageous for the isolation, purification and crystallization of membrane proteins. Second, methane could be used to improve the efficiency of current isolations, or to solubilize detergent-resistant components under relatively mild conditions. Finally, methane can be removed completely from any biological specimen through the release of pressure at the end of an experiment, making it a completely clean reagent for processing of products for human usage.

The results presented here demonstrate that methane is highly interactive with membranes, and its application can result in the complete hemolysis of erythrocytes in suspension at hydrostatic pressures that have little effect on protein structure. Methane is much more effective than methanol at producing these effects whereas nitrogen has little effect. Methane also enhances the destabilization of the erythrocyte membrane by the action of detergents.

ACKNOWLEDGMENTS

We would like to thank Dr. B. Chasan for many useful discussions and Dr. N. P. Franks for comments on an early draft of the paper. We would also like to thank the financial support provided by General Electric Corporation and Community Technology Foundation.

ABBREVIATIONS USED

MPa, megapascal (0.1 MPa ~ 1 standard atmosphere); psi, pounds per square inch (14.69 psi = 1 standard atmosphere); PBS, phosphate buffered saline, pH 7.4; DOPC, dioleoyl-phosphatidylcholine; HCR, hydrocarbon core region; IFR, interfacial region

REFERENCES

1. Franks, N. P. and Lieb, W. R. (1994) Nature (London) 367, 607-614.

2. Evers, A. S., Berkowitz, B. A. and d'Avignon, D. A. (1987) Nature (London) 328, 157- 160.

3. Clever, H. L. and Young, C. L. (1987) Methane. Solubility data series, Vol. 27/28,

Pergamon Press, New York.

4. Heremans, K. (1982) Ann. Rev. Biophys. Bioeng. 11, 1-21.

5. Kundrot, C. E. and Richards, F. M. (1987) J. Mol. Biol. 193, 157-170.

6. Wilcock, R. J., Battino, R., Danforth, W. F. and Wilhelm, E. (1978) J. Chem. Thermodyn. 10, 817-822.

7. Miller, K. W., Hammond, L. and Porter, E. G. (1977) Chem. Phys. Lipids 20, 229-241.

8. Dodson, B. A., Furmaniuk, Jr., Z. W. and Miller, K. W. (1985) J. Physiol. 362, 233- 244.

9. Nelson, C. M., Schuppenhauer, M. R. and Clark, D. S. (1992) Appl. Environ. Microbiol. 58, 1789-1993.

10. Jannasch, H. W., Marquis, R. E. and Zimmerman, A. M. (1987) Current perspectives in high-pressure biology, Academic Press, London, UK.

11. Weber, G. and Drickamer, H. G. (1983) Q. Rev. Biophys. 16, 89-112.

12. Hall, A. C., Ellory, J. C. and Klein, R. A. (1982) J. Membr. Biol. 68, 47-56.

13. Deckmann, M., Haimovitz, R. and Shinitzky, M. (1985) Biochim. Biophys. Acta 821, 334-340.

14. Royer, C. A., Weber, G., Daly, T. J. and Matthews, K. S. (1986) Biochemistry 25, 8308-8315.

15. Braganza, L. F. and Worcester, D. L. (1986) Biochemistry 25, 2591-2596.

16. Halle, D. and Yedgar, S. (1988) Biophys. J. 54, 393-396.

17. Bennett, P. B. and Marquis, R. E. (1994) Basic and applied high pressure biology, University of Rochester Press, Rochester, NY, USA

18. Portlock, S. H., Clague, M. J. and Cherry, R. J. (1990) Biochim. Biophys. Acta 1030,

1-10.

19. Lieber, M. R., Lange, Y., Weinstein, R. S. and Steck, T. L. (1984) J. Biol. Chem. 259, 9225-9234.

20. Dodge, J. T., Mitchell, C. D. and Hanahan, D. J. (1963) Arch. Biochim. Biophys. 100, 119-130.

21. Hille, B. (1984) Ionic channels of excitable membranes, p. 172, Sinauer, Sunderland, MA, USA.

22. Stein, W. D. and Lieb, W. R. (1986) Transport and diffusion across cell membranes,

pp. 89-90, Academic Press, New York.

23. Hemmingsen, B. A., Steinberg, N. A. and Hemmingsen, E. A. (1985) Biophys. J. 47, 491- 496.

24. Hemmingsen, B. A. and Hemmingsen, E. A. (1983) J. Protozool. 30, 608-612.

25. Gerth, W. A. and Hemmingsen, E. A. (1976) Z. Naturforsch. 31a, 1711-1716.

26. Chi, L. -M. and Wu, W. (1991) Biochim. Biophys. Acta 1062, 46-50.

27. Chi, L. -M., Wu, W., Sung, K. -L. P. and Chien, S. (1990) Biochim. Biophys. Acta 1027, 163-171.

28. Tr‰gner, D. and Csordas, A. (1987) Biochem. J. 244, 605-609.

29. Zaslavsky, B. Y., Ossipov, N. N., Krivich, V. S., Baholdina, L. P. and Rogozhin, S. V. (1978) Biochim. Biophys. Acta 507 1-7.

30. Isomaa, B., H‰gerstrand, H., Paatero, G. and, Engblom, A. C. (1986) Biochim. Biophys. Acta 860, 510-524.

31. Isomaa, B., H‰gerstrand, H. and Paatero, G. (1987) Biochim. Biophys. Acta 899, 93-103.

32. Tanford, C. (1980) The hydrophobic effect: formation of micelles and biological membranes, John Wiley, New York.

33. King, G. I., Jacobs, R. E. and White, S. H. (1985) Biochemistry, 24, 4637-4645.

34. White, S. H., King, G. I. and Cain, J. E. (1981) Nature 290, 161-163.

35. White, S. H. and Wimley, W. C. (1994) Curr. Opin. Struc. Biol. 4, 79-86.

http://www.sb.fsu.edu/~soma/Publications/bcjnew.html.