Langmuir 1991, 7,81-88
81
General Anesthetic Agents and the Conformation of Proteins. 4. Strengthening of Hydrophobic Interaction in Bovine and Human Serum Albumins, in Human Transferrin, and in Lysozyme by 1-Alkanols and the Cutoff Effect in Anesthetic Potency Mohammad Abu-Hamdiyyahf Chemistry Department, University of Kuwait, Safat, 13060 Kuwait Received March 27, 1990.In Final Form: June 15,1990 By use of the ability of a nonionic amphiphile to depress the levorotation of a protein in the visible spectrum as a measure of the ability to strengthen hydrophobic interaction (HI) (Langmuir 1986,2,310), the action of homologous 1-alkanols(ethanol (Cz),propanol (C3),butanol (C4),hexanol (Ce), octanol (C8), dodecanol (C~Z), tridecanol (CIS),tetradecanol (C14),pentadecanol ( C d , and hexadecanol (C16)) on bovine and human serum albumin (BSA and HSA, respectively), human transferrin (HT), and lysozyme (LYS) has been investigated. The results obtained parallel those already found in P-lactoglobulin (PLG) (Langmuir 1989, 5, 808); namely, strengthening of HI increases with decreasing protein concentration and increases with increasing alcohol chain length, showing two distinctive regions of interaction. The first region is for alcohols below Clz tending to show a saturation effect in strengthening of HI as alcohol chain length increases, and the second region is for dodecanol and higher alcohols where strengthening of HI increases linearly with alcohol chain length. Experiments on BSA and PLG show that strengthening of HI in the second region is not observed when the protein molecule is unfolded. The interaction in the first region is interpreted as due to strengthening of HI at the protein-water interface involving sites composed of amino acid side chains exposed or accessible for the solvent. Unlike PLG, where a definite site of interaction is indicated in the lower region which is drastically altered when the PLG is unfolded, the proteins BSA, HSA, HT, and LYS do not seem to show a definite single site in the lower region of interaction. The results point to more than one site that is more exposed to water than the site in PLG, and this site does not seem to be much changed when the protein (BSA) molecule is unfolded. The region for strengthening of HI for dodecanol and higher alcohols appears to be common to all the proteins examined so far, including PLG, and involves the folded segments of the protein molecule. Implications of these results to the phenomenon of the cutoff effect in anesthetic potency are discussed.
Introduction In a recent review' on water structure and hydrophobic bonding tendency, it was pointed out that the action of anesthetic agents in nerve cell membranes at clinical concentrations appears to be akin to the action of nonpolar or of amphiphilic substances at low concentrations in surfactant or protein solutions. It was suggested that anesthetic potency, In l/EDm, is related to the ability to strengthen hydrophobic interaction in aqueous solutions. These ideas were later2 applied, by using published experimental EDm values obtained on tadpoles with reflex responses as the end point,w4to the action of anesthetic agents in aqueous solutions of sodium dodecyl sulfate and in BLG solutions. The ability to depress the critical micelle concentration (cmc), In (-d(cmc)/dC&,,,+, represents the ability to strengthen HI in the surfactant solution, and the ability to depress the levorotation of the protein in the visible spectrum, In (da/dC,)C,+, represents the ability to strengthen HI in the protein solution. The correlation obtained was encouraging, although some of the experimental data were obtained at concentrations that exceed ED50 values. Recently, the action of homologous 1-alkanols on the levorotation of PLG at 546 nm was studied: and the t Current address: 1OEdgecombe Rd., Aylesbury HP219UG, Buckinghamshire, England. (1) Abu-Hamdiyyah, M. In Solution Behavior of Surfactants;Mittal, K. L., Fendler, E. J., Eds.; Plenum: New York, 1982; Vol. 1, pp 697-712. (2) Abu-Hamdiyyah, M. Langgmoir 1986,2, 310. (3) Brink, F.; Posternak, J. M. J . Cell. Physiol. 1948, 32, 211. (4) Pringle, M. J.; Brown, K. B.; Miller, K. W. Mol. Pharmacol. 1981,
19, 49.
relationship between anesthetic potency and the ability to strengthen HI in a protein solution at concentrations that do not exceed ED50 values was verified. It was also found that the ability to strengthen HI increases with decreasing protein concentration and increases also with alcohol chain length, showing two distinctive regions of interaction. The first region is for alcohols below C12, which tends to show a saturation effect suggesting the size of the hydrophobic site of interaction to be about CS.The second region is for dodecanol and higher alcohols, which shows a linear increase in strengthening of HI with alcohol chain length. The interaction in the first region was interpreted as due to strengthening of HI at a site at the proteinwater interface while that in the second region was probably due to HI bridging between two sites (regions) at the surface of the protein. The objective of this study is to find out whether other globular proteins behave in a manner similar to that in PLG toward strengtheningof HI by homologous 1-alkanols. In this study, the ability of 1-alkanols to strengthen HI in BSA, HSA, HT, and LYS is reported. It will be shown that the general trends observed in PLG are also obtained in these protein solutions, especially the increasing ability to strengthen HI with decreasing protein concentration and also the existence of two characteristic regions for strengthening of HI as a function of alcohol chain length. Furthermore, experimental evidence will be reported which indicates that the two regions of interaction mentioned above are distinct and separate. The implications of the results will be discussed, especially with respect to the cutoff effect in anesthetic potency on ascending a homologous series of additives.
0743-7463/91/2407-0081$02.50/0 0 1991 American Chemical Society
Abu-Hamdiyyah
82 Langmuir, Vol. 7, No. 1, 1991
Experimental Section Materials. Bovine serum albumin (Sigma A0281) essentially fatty acid free (less than 0.005%) and lysozyme from chicken egg white, 3 times crystallized, lyophilized, and dialyzed, were from Sigma Chemical Co. Human serum albumin and transferrin were pure quality from Behring. Some experiments were also done on BSA samples (pure quality) from Behring. 2-Mercaptoethanol was from Sigma Chemical Co. All were used as received. The alcohols, 6-lactoglobulin, and urea were described earliere5 Procedure. All solutions were made volumetrically and prepared as described previously.5 Long-chain alcohols from dodecanol to hexadecanol were prepared from saturated solutions made by shaking the solid alcohols in the relevant buffer solution for protein measurements. Solutions of short-chain alcohols were prepared by weighing. Saturated alcohol solutions in buffer were filtered through Millipore type HA filter paper. The numerical values of the alcohol concentrations of the saturated solutions were taken from the literatures by using the equation used for saturated water solutions. BSA stock solutions were prepared in a phosphate buffer, pH 5.5, and 0.15 M NaCl. Some experiments were carried out using BSA a t pH 2.0 and 0.15 M NaCl. HSA, LYS, and H T were prepared in aphosphate buffer, p H 7.1, and0.15 M NaC1. Some experiments were carried out on 0.5 % @LGin a phosphate buffer of pH 6.7, 0.15 M NaC1, and 10 M urea. For measurements in 0.5 76 BSA (both p H 5.5 and pH 2.0 were used), 10 M urea solution in presence of 2-mercaptoethanol, 5 mL of 1% protein in 10 M urea, pH 5.5 phosphate buffer, 0.15 M NaCl, and 0.09 M mercaptoethanol were mixed with 5 mL of alcohol solution in the same buffer solution containing 0.09 M mercaptoethanol, and the resulting solutions were used for measurements. The optical rotation measurements were carried out, as previously described, at 25 "C using a Perkin-Elmer 241MC polarimeter, 546-nm monochromatic light, and a cell 10 cm long of 1-ml volume. The instrument was calibrated for each rotation measurement with the respective buffer solution alone for protein solution in absence of alcohol or the buffer solution containing the same concentration of alcohol for measuring the optical rotation of the protein in presence of alcohol.
Results cy readings in absence or presence of alcoholswere steady and could be read to a third decimal with a maximum spread of 0.002O. Generally, different stock solutions of a given protein gave somewhat different values of cy resulting from small variation in the protein concentration. For a 95% confidence level, the fluctuation in concen) the mean. The values of cy in the tration is ( f l % of presence or absence of alcohols on standing overnight kept at 2 "C varied within the above spread (0.002O). In both situations, the values for cy for a given protein solution in different alcohol concentrations are shifted in the same direction, leaving the shape of CU-cad plots essentially unchanged. The levorotation of all the proteins in this study measured at 546 nm in the absence of alcohols increased linearly with increasing protein concentration in the range 0.1-3.0 g/ 100mL of solution, with the lines passing through the origin. The slopes of these lines are 0.772,0.745,0.744, 0.565, and 0.519 for HSA, BSA (Behring), BSA (Sigma), LYS, and HT, respectively. For 0.5% BSA (pH 2.0), the optical rotation cy (-0.377O) is more levorotatory than cy for the same concentration of protein at pH5.5 (-0.360O). Similarly, in the presence of urea cy becomes more levorotatory. For example, a (0.5% BSA pH 2.0,0.15 M NaC1) is -0.377O and -0.655' in 0.5 % BSA (pH 2.0,0.15 M NaC1,
( 5 ) Abu-Hamdiyyah, M.;
Kumari, K. Langmuir 1989, 5, 808.
Tetradacanol 2 I6
2.08
I
20
40
60
nm
/I
I Pontadocanol
Tridacanol
4 2 06
I
1
0
I
5
10
mM
I5
0 I
0 3
0,s
I
mM
Figure 1. Levorotation of 3% BSA (pH 5.5,0.15 M NaCl) at 546 nm as a function of alcohol concentration.
10 M urea). These changes in a reflect structural changes in the protein molecule and are well documented in the literature.&" Effect of Alcohols on the Levorotation of BSA, HSA, HT, and LYS. The levorotation of each of the above proteins decreased linearly with alcohol concentration at low concentrations, with a tendency to reach a minimum, and then the levorotation increased at higher alcohol concentrations, as was noted in the case of ,t7LG.5 This trend was noted in each of the protein concentrations used in this study covering the range 0.1-3%. Figures 1 and 2 illustrate these trends for 3% BSA (Sigma) (phosphate buffer pH 5.5, 0.15 M NaC1) in the presence of ethanol, propanol, butanol, hexanol, octanol, dodecanol, tridecanol, tetradecanol, pentadecanol, and hexadecanol. The minimum is not noted in butanol. Similar trends were obtained for BSA at pH 2.0 for all the alcohols examined. BSAsamples from Behring gave results similar to those obtained from BSA (Sigma); therefore, only the results obtained from the latter samples are reported to avoid confusion. The linear decrease in levorotation with alcohol concentration has also been obtained in 0.5% BSA (pH 5.5 and 2.0) in 10M ureasolutions for all the alcohols examined in the range C2-Cls. This pattern of behavior was also noted in 0.5 % BSA (pH5.5 and 2.0) in 10M urea containing 0.09 M 2-mercaptoethanol,for the alcohols Cz-Ce, as dodecanol and higher alcohols lost the ability to depress the levorotation of the protein (see later). Ability of an Alcohol To Depress the Levorotation of a Protein as a Function of Alcohol Chain Length. (6) Umes, P.; Doty, P. Adu. Prot. Chem. 1961, 16, 401. (7) Tanford, C.Ado. Prot. Chem. 1969, 23, 121. (8)Warren, J. R.; Gorden, J. A. J. Biol. Chem. 1970,245, 4097. (9) McKenzie, H. A.; Raleton, G. B. Experientia 1971, 27, 617. (IO)Greene, R. F.; Pace, C. N. J. Eiol. Chem. 1974,249, 5338. (11)Peters, T.,Jr. Adu. Prot. Chem. 1985, 37, 161.
Langmuir, Vol. 7, No. I, 1991 83
General Anesthetic Agents and Protein Conformation
2
1 -1
1 - O
A
BLG
0
ESA
x
HSA
A
HT
0
LYS
1'
4
6
'
' -
k Average
2
n
Butanol
1
20
0
60
40
Octanol
I1
0
4
IrM
n
I
8
IrM
Figure 2. Levorotation of 3% BSA (pH 5.5,0.15 M NaCl) at 546 nm as a function of alcohol concentration.
BBA
Average n
Figure 4. Rate of change of the ability to depress the levorotation per CH2obtaine by dividing the difference in the values of In da/dC for two successive alcohols by the difference in the number of carbon atoms in the alcohol chain length plotted against the average number of carbon atoms in the two respective alcohols in the range c2-C~.
0
I
v
-
0
10-
v
Y \
U
E C
A
5-
I 7' d
0
I
I
I
1
1
I
I
1
I
The ability of an alcohol to depress the levorotation of a protein is taken as the logarithm of the initial rate of decrease of levorotation with alcohol concentration, In (do/ dC)c+. Figure 3 shows the variation of In (da/dC)c+ with alcohol chain length in BSA (pH 5.5). The figure displays the characteristic variations of the ability to depress the levorotation of a protein as a function of alcohol chain length previously obtained5for PLG, namely, the existence of two distinctive regions of interactions. The first region is for alcohols below dodecanol ( C I ~ )where , the ability to depress the levorotation increases with alcohol chain length, and then the effect tends to level off for higher alcohols (below C12). The other region is for dodecanol and higher alcohols,where the ability to depress the levorotation increases linearly with alcohol chain length. The variation of In (da/dC)c+ as a function of alcohol chain length in HSA, HT, and LYS shows similar behavior. Figure 3 shows the variation of In (da/dC)c+ as a function of alcohol chain length for two protein concentrations 0.5 % and 3.0%. The trend obtained is essentially similar in both concentrations. In fact, this trend has also been obtained in the lowest protein concentration used in this study, namely, 0.1%, for BSA, HSA, and LYS.
However, there are differences in the details of the interaction in the first region in BSA, HSA, HT, and LYS and in pLG.5 In PLG, the initial linear increase in In (da/ dC)c+ with alcohol chain length continues up to Cg, tending to level off thereafter. The proteins BSA, HSA, HT, and LYS as illustrated in Figure 3 for BSA do not show a strict linear increase in In (da/dC)c+ with alcohol chain length in the lower region of interaction, pointing to multiplicity of sites with sizes not exceeding C4-Cb. Moreover, the values of In (da/dC)c+ obtained in BSA, HSA, HT, and LYS are larger than the corresponding values in PLG for short- and medium-chain alcohols (see later). Figure 4 shows the rate of change of In (da/dC)c+ per CH2 (A In (da/dC)c+/An) obtained by dividing the difference in the values of In (da/dC)c+,for two successive alcohols by the difference in the number of carbon atoms in the alcohol chain lengths plotted against the average number of carbon atoms in the two respective alcohols (in the range c2-C~)in 0.5% BSA, HSA, HT, and LYS. It shows that the interaction per CH2 increases with chain length, reaching a maximum and then rapidly decreasing after C4. In PLG, the corresponding rates fall within 1.25 f 0.25.
Effect of Unfolding Conditions on the Ability To Depress the Levorotation of a Protein. Experiments were carried out in 0.5 % BSA (pH 2.0) where the protein molecule exists in the so-called "E" or expanded state." The variation of In (da/dC)c+ as a function of chain length obtained gives a plot that is similar to that in Figure 3 for BSA (pH 5.5) where the protein molecule exists in the "N" or native state except that the absolute values of In (da/ dC)c+ for dodecanol and higher alcohols are consistently lower than the corresponding values obtained at pH 5.5. The values of In (da/dC)c+ in 0.5% BSA (pH 5.5) and
Abu- Hamdiyyah
84 Langmuir, Vol. 7, No. 1, 1991 0.5% B S A
0.5% BSA i n i 0 M U r e a
in 1 0 M U r e a + O . O S M 2 - M E
o PH 2.0 x pH 5 . 5 0 0.5% B S A , A
0 . 6 % BLG
5.5 N o Urea 1 0 M Urea
pH
o/o -
12
n in
C,
00
2
n i n C.H2,+1
16
H p n + ,OH
Figure 5. Ability to depress the levorotation of 0.5% BSA in 10 M urea at pH 2.0 and 5.5 as a function of alcohol chain length.
in 0.5% BSA (pH 2.0) for (212, c13, c14, c14, c15, and CIS are 12.2, 14.0, 15.1, 16.8, 17.8 and 11.6, 13.1, 14.13, 15.7, 17.2, respectively. Experiments were also carried out on BSA (pH 5.5 and 2.0) in 10 M urea solutions. The results are illustrated in Figure 5. The characteristic regions of interaction are still present in BSA at pH 5.5 and 2.0 under these conditions of unfolding. However, when 2-mercaptoethanol is added to BSA (pH 5.5 or 2.0) in 10 M urea so that the S-S bridges are broken, it was found that dodecanol and the higher alcohols loose the ability to depress the levorotation of the protein, and only interaction in the first region occurs. It is of interest to report here also the results of the ability to strengthen HI in PLG under unfolding conditions. I t is found that PLG (phosphate buffer pH 6.7) in 10 M urea behaves like BSA in 10 M urea in the presence of 0.09 M 2-mercaptoethanol. That is, dodecanol and higher alcohols did not depress the levorotation of PLG in 10M urea, and the characteristic hydrophobic site in the lower region of PLG (below C12) is drastically altered to become similar to that of BSA. Figure 6 illustrates these points. Table I lists the readings of the levorotations of BSA (pH 5.5 and 2.0) in 10 M urea and 0.09 M 2-mercaptoethanol and also for PLG (pH 6.7) in 10 M urea for dodecan01and higher alcohols. No reduction in the levorotation occurs in these proteins under these conditions. Effect of Protein Concentration. It is obvious from Figure 3 that the ability of a given alcohol to depress the levorotation of a protein increases with decreasing protein concentration. Figure 7 illustrates this trend for HSA. This trend was found for all the alcohols investigated so far in BSA, HSA, HT, and LYS as well as in PLG.5 The variations of In (da/dC)c+ with protein concentration (g/lOO mL of solution) for the alcohols examined have been fitted by a second-degree polynomial and the limiting values of In (da/dC)c+ at infinite dilution of the protein, as well as the initial slopes, were obtained by the leastsquares method and are shown in Table 11. The limiting values of In (da/dC)c+ as [PI 0 for BSA and LYS (for which sufficient data are available) when plotted against alcohol chain length give the two characteristic regions of interactions already noted for
-
6
4
8
IO
OH
Figure 6. Ability to depress the levorotation of 0.5% BSA in 10 M urea + 0.09 M 2-mercaptoethanolat pH 2.0 and 5.5; 0.5% BSA, pH 5.5, no urea; 0.5% PLG, pH 6.7, 10 M urea; and 0.5% PLG, pH 6.7, no urea as a function of alcohol chain length. Table I. Effect of 1-Alkanols on the Levorotation of BSA and of @LG under Complete Unfolding Conditions 0.5% BLG, pH 6.7, 0.15 M NaCl, 0.5% BSA, 0.15 M NaCl, 10 M urea 10 M urea + 0.09 M 2-ME" 1-alkanol, M pH 2.0 pH 5.5 set 1 set 2 0.626 0.616 0.616 dodecanolb 0.655 0.626 0.615 0.616 0.64 X lo-' 0.655 0.629 0.616 0.618 0.86 X loT7 0.668 0.630 0.618 0.667 0.614 1.12 x tridecanol 1.52 X 2.28 X lo-@ 3.04 X tetradecanol 0.40 x 0.53 X 0.80 X pentadecanol 1.05 X 1.40 X lo4 2.11 X hexadecanol 0.26 X 0.39 X 0.52 X a
0.660 0.669 0.669
0.615 0.615 0.618
0.618 0.622 0.622
0.625 0.626 0.626
0.662 0.669 0.669
0.615 0.615 0.616
0.616 0.616 0.620
0.626 0.625 0.630
0.670 0.675 0.680
0.615 0.615 0.614
0.617 0.619 0.620
0.628 0.628 0.629
0.660 0.664 0.672
0.616 0.616 0.615
0.618 0.619 0.619
0.628 0.628 0.631
2-ME = 2-mercaptoethanol. 0 M.
finite protein concentrations. The other proteins with data only for four alcohols give a skeleton of the general trend. Anesthetic Potency and the Ability To Depress the Levorotation of BSA, HSA, HT, and LYS. Figure 8 shows the logarithm of the negative of the relative ability to depress the levorotation of LYS and of HSA, In -11 [a(da/dC)c+], for the anesthetically active alcohols plotted against anesthetic potency (the logarithm of the reciprocal of ED50 in mo1.L-l). The anesthetic data were taken from the l i t e r a t ~ r e . ~Both ! ~ sides of the plot have the same units. It is clear that the correlation is not strictly linear, as was found in the case of /3LGV5Similar plots were obtained by using the data for BSA or HT. The same pattern was obtained by using different protein concentrations. The deviations from linearity start after c4.
General Anesthetic Agents and Protein Conformation
Langmuir, Vol. 7, No. 1, 1991 85
of the additive with the protein and therefore to (-AGOm/
RT),where AGO, is the standard free energy of associ-
r-
e
1-Octanol
l-Butanol
l
3;
I
I 2
e
I 3
Protein Cone. g l l 0 0 ml Solution
Figure 7. Ability of an alcohol to depress the levorotation of HSA as a function of protein concentration in g/100 mL of solution.
Discussion In a protein solution under normal conditions of pressure and temperature and in the absence of unfolding agents in solution, the protein molecule is in the native (or quasinative) state7where due to hydrophobic interactions the molecule is folded into a globular structure to minimize the contact of nonpolar amino acid side chains with ~ a t e r . ' ~ J 5In this state, there is equilibrium between the buried amino acid side chains (due to folding) and free amino acid side chains protruding in water, i.e., side chains exposed or accessible wholly or partially to the solvent, as complete removal of hydrophobic side chains from contact with water is generally not possible.12 The accessibility of such hydrophobic groups to the solvent provides the source for hydrophobic interaction with nonionic amphiphiles or nonpolar additives. I t was suggested2that nonionic amphiphiles when added at low concentrations to a protein solution tend to strengthen HI in a manner similar to that which occurs in surfactant solutions, where the nonionic amphiphiles aggregate with free surfactant molecules (ions) in solution to form mixed micelles resulting, amongst other things, in the reduction of the cmc. In a protein solution however, because all side chains are permanently bonded to the polypeptide backbone, free translation of side chains is not possible, and the coaggregation process results in a small perturbation of the conformation of protein molecule (toward more folding) which is manifested by a small decrease in the levorotation of the protein in the visible spectrum. The logarithm of the rate of initial decrease of levorotation with alcohol concentration, which is taken to represent the ability to strengthen HI in the protein solution, has been found to depend on the alcohol chain length, the state of the protein molecule in solution (Le., extent of unfolding), protein concentration, and the nature or type of protein. In analogy with work on strengthening of HI by nonionic amphiphiles in surfactant solutions,16it will be assumed that the ability to strengthen HI in protein solutions is proportional to the logarithm of the association constant (12) Tanford, C. The Hydrophobic Effect,2nd ed.;Wiley-Interscience: New York, 1980. (13) Creighton, T. E. J. Phys. Chem. 1985,89, 2452. (14) Dill, K. A. Biochemistry 1988, 24, 1501. (15) Kauzmann, W. Nature 1987,325, 763. (16) Abu-Hamdiyyah, M. J. Phys. Chem. 1986,90, 1345.
ation. Effect of Alcohol Chain Length. The finding that two regions of interactions exist in all the proteins examined (BSA, HSA, HT, and LYS) and depend in a similar manner on the alcohol chain length, one for alcohols below C12 the other for C12 and higher alcohols, is significant. The same trend was obtained earlier in BLG.6 It was then arrived at on the basis of the shape of the plot of ability to strengthen HI versus alcohol chain length. This has now been verified experimentally in the present study for both BLG and BSA. The other proteins most likely behave similarly. This sort of behavior, where an amphiphilic additive is able to distinguish different regions of the protein molecule by using the (additives) chain length as a probe, does not appear to have been reported earlier. Several authors studied the interaction of homologous amphiphiles with p r ~ t e i n s , l however ~ - ~ ~ under somewhat different conditions and using different methods (equilibrium dialysis and partitioning). Reynolds et al.23 measured the binding constants of octanol, decanol, and dodecanol in 0.1 9% BSA, and when In K is plotted against the alcohol chain length the increment per CH2 decreases with increasing alcohol chain length, showing the same general trend obtained in our work in the lower region of interaction. No studies are available for higher alcohols. However, Ashbrook et al. studiedl8Jg the binding of homologous carboxyates ((35, C7, CS, C11, c13, c15, and C17COO-) in 0.7% HSA (pH 7.4) at 37 "C and analyzed their results by multiple stepwise equilibria. When the logarithm of any of the binding constants is plotted against the number of carbon atoms in the hydrocarbon chain, two regions of interaction are apparent, as seen in Figure 2 of ref 19, one for dodecanoate and higher members and the other region for the shorter homologues. The resulting shape, however, was attributed to an artifact due to differences in the methods used to obtain the binding constants of the short (C5-C9) and the higher homologues. According to our results, it looks as if the two regions of interaction are common features of folded proteins in water and probably of folded proteins that are only partly in contact with water (see later). The region for dodecanol and higher alcohols involves the folded segments of the protein structure that disappears on unfolding. The region for the shorter alcohol homologues involves sites at the protein-water interface that are slightly affected (BSA and probably HSA, HT, and LYS) or modified (6LG) but not destroyed on unfolding. The region involving the folded structures in BSA, HSA, HT, LYS, and in BLG appears to be the same in all. The values of the intrinsic ability to strengthen HI, In (da/dC)c+ as [PI 0, which is independent of protein concentration, for dodecanol and hexadecanol (for which data are available for all these proteins) are the same in all these proteins, being 13.4, 13.3, 13.4, 13.2, 13.4 and 18.4, 18.8, 18.7, 18.0, 18.4 respectively, as obtained by extrapolation. This is further seen in the values of the slopes of In (da/dC)c+ versus alcohol chain length, which reflect values of -AGo(CH2)/ R T in each of these proteins. These values in 0.5% BLG,
-
(17) Goodman, D. J. Am. Chem. SOC.1958, BO, 3892. (18) Ashbrook,J. D.;Spector, A. A.;Fletcher,J. E. J. Biol. Chem. 1972, 247, 7038. (19) Ashbrook, J. D.; Spector, A. A.; Santos, E. C.; Fletcher, J. E. J. Biol. Chem. 1976, 250, 2333. (20) Spector, A. A.; Fletcher, J. E. J. Lipid Res. 1969, 10, 56. (21) Spector, A. A.; Fletcher, J. E. Lipids 1970,5, 403. (22) Karush, F.; Sonenberg, M. J. Am. Chem. SOC.1949, 71, 1369. (23) Reynolds, J. A.; Herbert, S.;Steinhardt, J. Biochemistry 1968,7, 1357.
Abu - Hamdiyyah
86 Langmuir, Vol. 7,No. 1, 1991
+
Table 11. Coefficients of the Equation In (da/dC), = ao al[P] -t az[PI2 where [PI is the Protein Concentration. As Obtained by the Least-Squares Method Together with the Standard Deviation a0 -a1 x 10-2 a2 x 10-4 BSA HSA LYS HT BSA HSA LYS HT BSA HSA LYS HT 4.8 4.45 2.63 1.82 0.43 0.17 ethanol f 0.3 f 0.48 f 0.46 f 0.82 f 0.14 f 0.25 0.992 0.973 R propano1 1.86 3.44 5.91 6.46 0.29 0.76 f 0.14 f 0.30 f 0.24 f 0.51 f 0.07 f 0.15 0.996 0.988 R 9.63 8.61 butanol 9.38 10.9 2.67 2.09 2.26 3.42 0.53 0.44 0.51 0.62 f 0.25 f 0.18 f 0.38 f 0.7 f 0.43 f 0.30 f 0.67 f 2.23 f 0.13 f 0.09 f 0.21 f 1.35 R 0.989 0.959 0.989 0.985 hexanol 11.15 11.3 2.56 2.83 0.53 0.60 f 0.38 f 0.26 f 0.64 f 0.15 f 0.08 f 0.19 0.995 R 0.975 12.2 11.77 11.47 2.87 1.36 2.55 octanol 13.3 4.60 0.64 0.12 0.56 1.85 f 0.4 f 0.4 f 0.04 f 0.19 f 0.67 f 0.07 f 0.34 f 1.20 f 0.20 f 0.02 f 0.11 f 0.73 0.969 0.992 R 0.9997 0.992 12.8 1.47 dodecanol 13.3 1.81 1.86 3.40 0.26 13.97 12.9 0.30 0.38 0.74 f 1.75 f 0.08 f 0.4 f 0.20 f 0.56 f 0.27 f 0.65 f 0.41 f 0.2 f 0.20 f 0.13 f 1.05 0.966 0.989 R 0.963 0.982 2.29 tetradecanol 16.54 2.54 0.47 15.67 0.55 f 0.49 f 0.40 f 0.68 f 0.83 f 0.20 f 0.25 R 0.962 0.947 18.8 19.2 17.66 hexadecanol 18.45 1.34 2.05 1.52 3.00 0.26 0.38 0.30 0.58 fO.ll f0.15 f0.28 f 0.1 f 0.19 f 0.25 f 0.49 0.40 f 0.06 f 0.07 f 0.15 f 0.25 R 0.992 0.995 0.965 0.993
*
a
a/100m L of solution. 14-
12
-
10
-
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Figure 8. Negative relative ability of an alcohol to depress the levorotation of a protein as a function of its anesthetic potency.
BSA, HSA, and HT fall in the range 1.70-1.10 and in the range 1.50-1.02 for LYS assuming a 95 % confidence level and a degree of freedom of 3 (n - 2). The region of interaction of higher alcohols occurs also in BSA at pH 2.0, where the protein molecule has been described" to be fully uncoiled within the limits of its disulfide bonds but with the values of In (da/dC)c+ being less than that for pH 5.5. This behavior is also observed for BSA of pH 5.5 and 2.0 in 10 M urea. This points to
the conclusion that the S-S bridges in BSA not only stabilize the tertiary structures but also confer stability to the folded segments of the proteinsz4 However, dodecanol and higher alcohols loose the ability to strengthen HI in PLG in the presence of 10 M urea; i.e., the region of interaction disappears. In BSA, this happens in the presence of 10M ureaand 0.09 M 2-mercaptoethanol where the S-S bridges are broken and the protein molecule becomes unfolded with no internal restraints. The difference observed in the behavior of PLG and BSA toward 10M urea is in line with established trends in the literature. It has been reported7 that PLG in 10 M urea undergoes complete transition to the unfolded state. The complete transition does not occur in BSA at room temperature under these conditions (in the presence of urea alone). The question now is whether the site of interaction in the region involving the higher alcohols is between the folded segments or across them. The site of interaction could be composite; that is, the alcohol molecule bridges two sites on the folded structure on the surface of the globular protein which are separated from each other by a distance that has to be spanned by the alcohol molecule. Or it could be a single site between the folded segments of the protein such that only molecules of certain sizes but not smaller are able to interact with it. No distinction could be made at present between these two alternatives. The region of interaction for alcohols below dodecanol is interpreted to be due to interactions at the proteinwater interface which persist even in the unfolded state. However,there are differences between the proteins BSA, HSA, HT, LYS, and PLG. The interactions are stronger and the binding sites smaller in the former proteins than in PLG. This is clearly seen in Figure 6, where the ability to strengthen HI in 0.5 5% PLG in the absence of 10 M urea is compared with the corresponding ability in the presence of 10 M urea. It is clear that not only is the region of interaction for dodecanol and higher alcohols destroyed on unfolding, but also the region of interaction for the lower alcohols (below CIZ)is drastically altered, with the (24)Takeda, K.; Sasa, K.; Kawamoto, K.; Wada, A.; Aoki, K. J. Colloid Interface Sci. 1988, 124, 284.
Langmuir, Vol. 7, No. 1, 1991 87
General Anesthetic Agents and Protein Conformation I 500
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Figure 9. Negative initial slope of the ability to depress the levorotation of protein with respect to protein concentration plotted against alcohol chain length for BSA and LYS.
interaction becomingstronger and resembling that in BSA. The figure also shows the ability to strengthen HI in 0.5% BSA (pH 5.5) in the absence of urea, in the presence of 10 M urea, and in the unfolded conditions in the presence of 10 M urea plus 0.09 M 2-mercaptoethanol. There appears no significant difference in the interaction under these different conditions. The binding sites in the folded molecule of BSA in the lower region appear to be more exposed to water than the binding site in the folded PLG, and there is no significant change in the ability to strengthen HI in going from native to completely unfolded state as in the case of PLG. It is obvious that in PLG in the folded state the binding site is discrete with size about C8,which appears to be destroyed on unfolding. Effect of Protein Concentration. In all the proteins examined, a given alcohol shows an increasing ability to strengthen HI with decreasing protein concentration. The concentration range examined covered 0.1-3%. The rate of decrease in the ability to strengthen HI with protein concentration is slow between 3 % and 1% and then rises steeply between 0.5% and 0.1 5%. This trend is found for all the alcohols and all the proteins examined. The data for each alcohol have been fitted to a polynomial of degree 2, and Table I1 lists the coefficients of the equations obtained together with the standard deviations and correlation coefficients. The extrapolated values of In (da/ dC)c+as [PI 0 obtained through the regression analysis agree reasonably with those obtained graphically. It was already pointed out earlier that dodecanol and higher alcohols have essentially the same intrinsic values for the ability to strengthen HI in all the proteins examined. The table also shows that the intrinsic ability to strengthen HI in BSA, HSA,and LYS for each alcohol in the lower region falls in the same range. The values for butanol and octanol in H T appear to be higher than the corresponding values in BSA, HSA, and LYS. Whether this deviation is real or due to an experimental error remains to be seen. The inset in Figure 4 shows - A G 0 c ~ J R Tfor BSA, LYS, and PLG for different alcohols obtained from the graphical differentiation of the intrinsic ability to strenghen HI as a function of alcohol chain length in the lower region. It
-.
shows the essential constancy of interaction per CH2 in PLG up to c8 and the variation of this quantity in BSA and LYS showing a maximum value about Cq. The rate of change of In (da/dC)c+ with protein concentration in the limit of infinite dilution of the protein (i,e,, the initial slope) is given by a1 in Table 11. The negative values of the initial slopes for the various alcohols in LYS and in BSA have been plotted against the alcohol chain length in Figure 9, with the spread in the individual values representing twice the standard deviation. The least-squares values of the coefficients of the lines y = PO + PIX for BSA and LYS in Figure 9 are 2.74 f 0.34, -0.065 f 0.036 and 2.78 f 0.41, -0.05 f 0.043, respectively. For BSA, the slope of the plot in Figure 9 falls in the range 0.05 to -0.17 for a 95% confidence level and 6 degrees of freedom (n - 2). For LYS, the corresponding slope falls in the range 0.26 to -0.34. Thus it appears that, within the confidence limits imposed, the values of the ordinates in Figure 9 are independent of the alcohol chain length. This result serves as a consistency test for the experimental data obtained since the slope of the plot in Figure 9 (for BSA or LYS) is
which is equal to
The value of the last expression should be zero as the quantity being differentiated with respect to [PI is independent of protein concentration. Moreover, similar analysis for PO values for BSA and LYS assuming a 95 7% confidence level gives the ranges 2.78-2.70 and 2.81-2.75, respectively. Thus, the variation in the ability to strengthen HI as a function of protein concentration for all the alcohols c2-cl6 appears to be the same and of common origin. This variation was interpreted earlier5 as being due to an effective increase in the concentration of nonpolar side chains exposed to water, with the protein molecules interacting together through the side chains at higher protein concentration. I t must be stressed, however, that this interaction between protein molecules through the side chains does not involve detectable conformational changes, as the levorotation in each of these proteins increases linearly with protein concentration passing through the origin. The effective increase in the concentration of side chains as the protein concentration decreases reflects the increased exposure of side chains to water. Thus, the increased interaction of the protein with a given alcohol as the protein concentration decreases is simply a consequence of an increased hydration of the side chains. In hydrophobic interaction, when a nonpolar moiety approaches a hydrophobic side chain on the protein, some water is eliminated from the hydration shell. The free energy change accompanying this interaction depends, amongt other things, on the amount of water being eliminated.25v26 Implications for the Cutoff Effect in Anesthetic Potency. It is now generally recognized that anesthetic action involves transmembrane proteins forming channels that are utilized in nerve impulse conduction and trans(25) Ooi, T.;Oobatake, M.; Nemethy, G.; Scheraga, H. A. Proc. Natl. Acad. Sei. U.S.A. 1987, 84, 3086. ( 2 6 ) Ooi, T.; Oobatake, M. J . Biol. Chem. 1988, 103, 114.
88 Langmuir, Vol. 7, No. 1, 1991
mission. Recent studies indicate27t28 that these channels are formed by bundles of folded a-helical segments spanning the width of the membrane enclosing a central pore with the most hydrophobic surface of these segments bonded to the lipid layer. The inner surface of the pore or channel provides the protein-water interface. There are two more interfaces, the so called annular (proteinbilayer) and the protein-protein (involving the folded segments) interfaces. Recent work by Lee and co-workers29 on the binding of homologous alkyl derivatives (C14-C~o) to a reconstituted membrane protein in the lipid bilayer showed that binding a t the protein-protein interface increased with increasing alkyl chain length, a trend opposite that found for the binding at the annular interface. The trend obtained for the binding at the protein-protein interface is similar to that found in this work for dodecanol and higher alcohols. Thus it appears that the binding of alkyl derivatives at the protein-protein interface involves the folded segments of the membrane proteins. Assuming that the interaction of the alcohols in the channels, i.e., at the protein-water interface, is represented by the interaction of the alcohol at the protein-water interface in the first region (for alcohols below dodecanol) in globular proteins (PLG and BSA), then a cutoff effect is readily predicted. However, according to our results in BSA and in PLG, dodecanol belongs to the alcohols that interact with the folded segments and thus should be anesthetically inactive. Whether this discrepancy is due to a difference in the binding properties of the folded structures (segments) of membrane proteins involved in anesthetic action and those in the globular proteins BSA and PLG such that dodecanol is unable to interact with the folded segments of the membrane protein or due to an artifact in our experimental measurements remains to be established.
A bu- Hamdiyyah results essentially parallel those already found in PLG, namely, strengthening of HI increases with decreasing protein concentration and increaseswith increasingalcohol chain length showing two distinctive regions of interactions. The first region is for alcohols below C12 and tends to show a leveling off effect after c4-C~;the other region is for dodecanol and higher alcohols, where strengthening of HI increases linearly with alcohol chain length. The region for higher alcohols was found to involve folded segments of the protein. The region for lower alcohols is assumed to involve sites at the protein-water interface which are only modified for @LGbut not destroyed, unlike that in BSA, where no significant change was observed on unfolding. Assuming the similarity of the binding of alcohols at the protein-water interface in globular proteins (BSA and PLG) and at the protein-water interface in the membrane channel and between the binding at the folded segments in globular protein (BSA and PLG) and in membrane proteins, the cutoff effect in anesthetic potency on ascending a homologous series of additives is readily predicted. According to our results, dodecanol is predicted to be anesthetically inactive. This discrepancy remains to be resolved.
Acknowledgment. This research was supported by the University of Kuwait project no. SC042.
(27)Furois-Corbin,S.;Pullman, A. In Transport through membranes: Carriers, Channels a n d h m p s ; Pullman,A. et al.,Eds.; Kluwer Academic,
Note Added in Proof: Since submitting the above work, we have obtained results that seem to support the notion that the anesthetic site is not connected with a helices but with strand structures. Experiments were carried out on a-chymotrypsinogen A, chymotrypsin, and concanavalin A, all @-proteinswith essentially no a helices (Levitt,M.; Chothia, C. Nature 1976,261,552)which show that the lower alcohols below dodecanol are active in each case when in the native or pseudo native state and give plots of In (da/dC) vs C, that are similar to the corresponding plot for PLG. Dodecanol and higher alcohols on the other hand were unable to depress the levorotation of any of these proteins. In the presence of 6 M guanidinium hydrochloride all the alcohols investigated were inactive including the lower alcohols below dodecanol.
1988; pp 337-357. (28)Smith,E. V.M. Elements ofhfolecularNeurobiology, Wiley: New York, 1989. (29)Froud, R. J.; East, J. M.; Rooney, E. K.; Lee, A. G. Biochemistry 1986,25, 7535.
Registry No. Ethanol, 64-17-5; propanol, 71-23-8; butanol, 71-36-3; hexanol, 111-27-3; octanol, 111-87-5; dodecanol, 11253-8;tetradecanol, 112-72-1;hexadecanol, 36653-82-4;tridecanol, 112-70-9; pentadecanol, 629-76-5; lysozyme, 9001-63-2.
Summary and Conclusion We have investigated the ability of l-alkanols to strengthen HI in BSA, HSA, HT, and LYS solutions. The
