808
Langmuir 1989,5, 808-816
is determined by the solvent polarity.16 In spite of this, the data of Tables I1 and I11 show that the quenching rate constanta in reverse micelles follow a similar pattern to that obtained employing fumaronitrile as quencher: (kQ)ht values are smaller than those obtained in water or ethanol and increase when R increases. For acrylonitrile, this increase must then reflect a more favorable spatial distribution and/or a more polar environment when R increases. Quenching of PTS by Fumaronitrile. Quenching of PTS by fumaronitrile in homogeneous solvents is slower than that obtained for the other pyrene derivatives. Furthermore, and due to ita high negative charge, PTS will be incorporated to the micellar water p00l.9.~ The values of (kQ)min AOT solutions show a different pattern than those observed for the other pyrene derivatives, increasing by a factor of nearly 2 when R increases. However, at R = 6, when it can be considered that a significant "pool" is not present,16 the value of (kQ)ht/ (kQ)EaHis similar to that obtained for other pyrene derivatives whose chromophores can be considered to remain in the interfacial region (see Table IV). At larger R values, PTS must be displaced toward the water pools? Under these conditions, (15) Encinae, M.V.;LiSei, E. A. J. Photochem. 1986,29, 386. (16) (a) Wong,M.; Thomas, J. K.; Now&, T. J. Am. Chem. SOC.1977, 99,4730. (b) Baedez, E.;Monnier, E.;Valeur, B. J. Phys. Chem. 1986, 89, 5031.
the fluorescence decay of PTS must be represented by T-l
=
(To)-'
+ (k~)~i[Ql,i
(2)
where [Q] is the average quencher concentration in the water pooYThese values can be evaluated from the reported values of the quencher partition constant between the n-heptane pseudophase and the poolg and the size of the pool," which can be approximately evaluated from the amount of water associated to it. The values show a small increase with R, and absolute values are less than a factor of 2 smaller than those in bulk water. It is important to note that, since PTS must be concentrated toward the center of the pool3 and since fumaronitrile, due to its lack of charge, can be considered as nearly equally distributed over all the pool, the obtained rate constant can be considered as a representative measure of the characteristics of the center of the core of the water pool of the reverse micelle as a reaction media.
Acknowledgment. This work was carried out as part of a project supported by CONICET (Argentina) and CONICYT (Chile), with financial support from FONDECYT (Grant No. 1433). Redstry NO.AOT, 577-11-7; NEA,3309-13-5; NAA (sodium Salt), 61-31-4; MP, 2381-21-7; PSA, 38886-82-7; PUTMA, 103692-03-1;PMTMA, 72185-47-8;PTS (sodium salt), 10829262-2; fumaronitrile, 764-42-1; acrylonitrile, 107-13-1;heptane, 142-82-5.
General Anesthetic Agents and the Conformation of Proteins. 2. Strengthening of Hydrophobic Interaction in @-Lactoglobulinby 1-Alkanols and the "Cutoff"Effect in Anesthetic Potency Mohammad Abu-Hamdimah* and Kamlesh Kumari Chemistry Department, University of Kuwait, Safat, 13060,Kuwait Received June 16,1988. I n Final Form: January 12,1989 By use of the ability of a nonionic amphiphile to depress the levorotation of a protein at 546 nm as a measure of the ability to strengthen hydrophobic interaction (HI) in the protein solution, the effect of homologous 1-alkanols (ethanol, propanol, butanol, hexanol, octanol, decanol, dodecanol, tridecanol, tetradecanol, pentadecanol, and hexadecanol) on the optical rotation of &lactoglobulin (j3LG) has been determined at 25 "C. For the anesthetically active alcohols, we have investigated the effect at concentrations that do not exceed the concentration necessary to anesthetize 50% of subjecta (EDSO)and verified the previous conclusion that anesthetic potency is linearly related to the ability to strengthen HI in the protein solution. The ability to strengthen HI depends on the protein concentration and the alcohol chain length. HI increases with decreasing protein concentration. The effect of chain length shows two distinctive regions, the first region from C2to C14showing an initial linear increase up to Cswith the ability thereafter tending to level off at dodecanol. In the second region, the ability to strengthen HI increases linearly from C13 to Cia. The onset of curvature in the ability to strengthen HI after C8 suggests the size of hydrophobic region to be about Cs. HI in the second region is interpreted as probably due to intramolecular bridging of two hydrophobic regions on the surface of the protein. The corresponding strengthening of HI by 1-alkanols in solutions of sodium dodecyl sulfate (SDS)was also determined. The ability to strengthen HI as a function of chain length shows no diecontinuity or significant curvature. It is argued that the hydrophobic interaction of anesthetic agents with membrane proteins, forming ionic or hydrophilic channels, is represented by the interaction observed in the first region (C2-C12)in BLG, thus rationalizing the "cutoff" effect in anesthetic potency in homologous 1-alkanols.
Introduction Studies on the effect of amphiphiles on proteins in aqueous solutions indicate that there are two main effects observed.'-" The first, which has been extensively in(1) Duggan, E.L.; Luck, J. M. J. B i d . Chem. 1948,172, 205. (2) Tanford, C.; De,P. K.; Taggart J.Am. Chem. SOC.1960,82,6028.
vestigated, occurs at relatively high concentration of the additive resulting usually in denaturation of the protein (3) Tanford, C.; De, P. K. J. Biol. Chem. 1961,236, 1711. (4) Roaenberg, R. N.; Rogers, D. W.; Haebig, J. E.; Steck, T.L. Arch. Biochem. Biophys. 1962,97,433. ( 5 ) Herskovitz, T. T.; Mescanti, L. J. Biol. Chem. 1966, 240, 639.
0743-1463/89/2405-0808$01.50/00 1989 American Chemical Society
General Anesthetic Agents and Protein Conformation and is characterized by an increase in the viscosity and in the levorotation of the protein solution. The second effect, which occurs at low concentration of the additive, is relatively a minor one usually observed as a small decrease in the levorotation (in the visible spectrum) of the protein2s3vgand with no significant change in the viscosity of the protein solution. Nevertheless, the presence of the additive in the low concentration range tends to "stabilize" and protect the protein against denaturation by urea, heat, or low pH." In essence, the effect produced at low concentration is opposite to that produced at high concentration of the amphiphilic additive. There are few detailed investigations of the low concentration effect. Markus and Karushg reported the effect of sodium octyl, decyl, and dodecyl sulfates on the optical rotation of human serum albumin (HSA). Reynolds et al.la reported the effect of SDS on the optical rotation of bovine serum albumin (BSA). However, the phenomenon of the depression of the levorotation of protein by nonionic amphiphiles at low concentrations was not investigated in a systematic manner as a characteristic parameter for the study of changes in the hydrophobic effect in protein solutions and was not given a definite interpretation on the molecular level." For example, in one studyI2the effect of several nonionic amphiphiles on the optical rotation of BSA was measured at only one concentration M) of the additive, and the percent change in 'YD(where D signifies the sodium line) was correlated with the additive's water-octanol partition coefficient. This was the first attempt to correlate the effect of an amphiphile on the optical rotation of the protein with the additive's hydrophobic character, with the latter parameter represented by the water-octanol partition coefficient. However, the kind of conformational perturbation involved in the protein molecule was not given. In another study,l3 the effect of anesthetic agents on the levorotation of P-lactoglobulin (PLG) and of BSA was also measured at one concentration only in an attempt to find a relationship between the decrease in the levorotation (at 546 nm) and anesthetic potency. In general, studies on the ability of nonionic amphiphiles at low concentrations to decrease the levorotation of proteins (in the visible spectrum) are sketchy and incomplete, being indicative only, and in many cases are only appendages in the denaturation curves. In a recent study,16 it was concluded that the ability of a nonionic amphiphile to depress the levorotation of a protein in the visible spectrum may be taken as a measure of the ability to strengthen HI in the protein solution. Using this principle and utilizing the limited data in the above-mentionedstudy13on the effect of anesthetic agents on the optical rotation of proteins, we have shown that anesthetic potency is linearly related to the ability to strengthen HI in the protein solution. The objectives of this study are as follows: (1)to examine the effect of alcohols on the optical rotation of PLG (6) Herskovitz, T. T.; Gadegbeku, B.; Jaillet, H. J. Biol. Chem. 1970, 245,2588. (7) Herskovitz, T. T.;Jaillet, H.; Gadegbeeku, B. J.Biol. Chem. 1970, 245,4544. (8) (a) Herskovitz, T. T.;Jaillet, H.; Desena, A. T. J . Biol. Chem. 1970, 245,6511. (b) Tanford, C. Adu. Prot. Chem. 1969,23,121; 1970,24, 2. (9) Markus, G.; Karush, F. J. Am. Chem. SOC.1967, 79, 3264. (10) Reynold, J. A.; Herbert, S.;Polet, H.; Steinhardt, J. Biochemistry 1967, 3, 937. (11) Markus, G.; Love, R. L.; Wider, F. C. J.Biol. Chem. 1964,239, 3687. (12) Helmer, F.; Kiehs, K.; Hansch, C. Biochemistry 1968, 8, 285. (13) Balaaubramanian, D.; Wetlaufer, D. B. Proc. Nutl. Acud. Sci. U.S.A. 1966,55,762.
Langmuir, Vol. 5, No. 3, 1989 809 in the visible spectrum at concentrations that do no exceed ED, values, (2) to examine the effect at relatively higher concentrations of the alcohol to establish the general characteristic of the optical rotation-alcohol concentration (a- Cad) curve, (3) to see how the ability of an alcohol to strengthen HI in PLG solutions varies with alcohol chain length, and (4) to compare the ability to strengthen HI in PLG and in SDS solutions with anesthetic potency and discuss the bearing of our results on the phenomenon of the "cutoff" effect in anesthetic potency. Finally, we shall report on the ability of an alcohol to strengthen HI as a function of protein concentration and how this ability is modified by the addition of urea at a given protein concentration. Experimental Section Materials. &Lactoglobulin3 times crystallizedand lyophilized
powder (IAl30) were purchased from Sigma Chemical Co. Sodium dodecyl sulfate (SDS)was described previ0us1y.l~ Ethanol was obtained from Merck, 1-propanol from British Drug Houses, 1-butanol, 1-hexanol, 1-tetradecanol, and 1-pentadecanolfrom Fluka, and 1-octanol, 1-decanol, 1-dodecanol, 1-tridecanol,and 1-hexadecanol from Sigma Chemical Co. They were all used as received. Urea was obtained from Riedel De Haen. Procedure. All solutions were made volumetrically. Longchain alcohols from dodecanolto hexadecanolwere prepared from saturated solutions made by shaking the solid alcohols in water for SDS and in the relevant buffer solution for protein measurements, respectively. Solutions of short-chain alcohols were prepared by weighing. Saturated alcohol solutionsin buffer were filtered through millipore type HA filter paper. The numerical values of the alcohol concentrations of the saturated solutions were taken from the literatureI5 by using the equation used for saturated water solutions. @-Lactoglobulinstock solutions were prepared in a phosphate buffer, pH 6.7 and 0.15 M NaCl. The protein solution was centrifuged for about 20 min at 15000 rpm and then filtered with type HA millipore filter paper (0.45 pm). Then 5-mL portions of the stock solution were mixed with 5-mL portions of the alcohol solutions in the same buffer, and the resulting solutions were used for measurement. For the measurements in 0.5% BLG in buffered 6 M urea, 5 mL of l % BLG in 6 M urea, pH 6.7 phosphate buffer, and 0.15 M NaCl were mixed with 5 mL of alcohol solution in 6 M urea, phosphate buffer (pH 6.7), and 0.15 M NaCl. On dissolving solid urea in the buffered solution, it was necessary to add a few drops of HCl to keep the pH value constant at 6.7. The concentration of the protein was determined8 by UV absorption at 278 nm by using ti,", = 9.6. At least 0.5 h elapsed before the polarimetric measurements were taken. The optical rotation measurements were carried out at 25 "C by using a Perkin-Elmer 241MC polarimeter, 546-nm monochromatic light, and a cell 10 cm long of 1-mL volume. The instrument was calibratedfor each rotation measurementby using the respective buffer solution alone for protein solutionin 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. Conductancemeasurementswere carried out as described previously." Results In the absence of alcohol, the levorotation of PLG increased linearly with increasing protein concentration in the concentration range 0.03%-3% with the line passing through the origin when all the protein solutions were made from the same stock solution. For example, the values of levorotations (in degrees) for the protein concentrations 0.03%, 0.5%, 1.0%, 1.5%, 2.0%, and 3.0% are (14) Abu-Hamdiyyah, M.; Ftahman, I. A. J. Phys. Chem. 1985, 89, 2377. (15) Franks, N. P.; Lieb, W. R. Proc. Nutl. Acad. Sci. U.S.A. 1986,83, 5116.
Abu- Hamdiyyah and Kumari
810 Langmuir, Vol. 5, No. 3, 1989 112 EDsO ?j
1.32
t!
Rtbrral
1
I
I
I
I
I
I
1
1.301
L
I
0
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1.33
I
I
I
I
I
I
I 4.0
1.31
I
I
0,008M
I
2.0
0
(
16’M 1
Figure 1. Effect of alcohol on the optical rotation of 3% @-lactoglobulin (pH 6.7, phosphate buffer, 0.15 M NaCl) in the low-concentration range of the additive. 1-Dodrcanol
1.
29 0
0.2
0.4
0 . 6 mM
0
1
2
3bM 5
1.34r
l-Ocknol
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1.30
0
8 1.32
1-Docanol
10
20
I
30 A M
‘
u
0
0.5
-
1.0
0
1.5 (10 M I
Figure 2. Effect of alcohol on the optical rotation of 3% &lactoglobulin (pH 6.7, phosphate buffer, 0.15 M NaC1) in the low-concentration range of the additive.
0.014,0.231,0.464,0.684,0.923,and 1.366,respectively. The specific rotation a!$’& in this range is constant. All measurements were taken immediately after mixing. The readings were stable, and a could be read to a third decimal (maximum spread of 0.002). We also checked the variation of a values with time, keeping the mixed solutions for 24 or 48 h at 2-5 OC. It was found that a values were slightly different; however, the differences between these a readings and those readings obtained immediately after mixing were constant within the experimental error. That is to say, the general shape of the .-C, plot remained unchanged. This was also found to be the case for the variation of a with different protein stock solutions or with different protein lot preparations. Several lots of protein preparations and many stock solutions were used. In contrast to the above, the readings of a for urea solutions showed fluctuations during measurements.
wCd Plots at Low Alcohol Concentration. We have measured the variation of optical rotation of PLG at very low concentrations of alcohols in order to determine unambiguously the initial slopes. Table I gives a sample of the values of a for one set of measurements for all the alcohols examined for their effect on the levorotation of 3% PLG. Figures 1 and 2 illustrate the effects of these alcohols on a values at low concentrations. We have indicated on the diagrams for the anesthetically active alcohols the ED, values with arrows. It is clear that the effect is linear with alcohol concentration. Similar plots were obtained for lower protein concentrations. General Shape of o - C , ~Plots. The variation of optical rotation of the protein solution at the low and relatively high alcohol concentrations has been obtained for all the alcohols examined (except ethanol) in 0.5% BLG. Figures 3 and 4 illustrate these variations for some of the
Langmuir, Vol. 5, No. 3, 1989 811
General Anesthetic Agents and Protein Conformation Table I. Optical Rotation8 of 3% &Lactoglobulin at 25 ' C alcohol alcohol alcohol concn -a,deg alcohol concn -a,deg 1.334 1.327 1.325 1-hexanol 0.20 mM ethanol 0.04 M 1.321 0.35 mM 1.317 0.06 M 1.311 0.50 mM 0.08 M 1.311 1.326 1.326 l-octanol 5.0 pM 1-propanol 0.005 M 1.318 15.0 pM 1.319 0.015 M 1.310 30.0 pM 1.314 0.025 M 1.319 5.0 pM 1.325 1-decanol 1-butanol 0.0025 M 1.310 6.5 pM 1.317 0.005 M 1.307 8.0 pM 1.309 0.008 M 1.329 0.685 pM 1.327 l-dodeca1-tridecanol 0.181 pM 1.325 1.37 pM 1.323 no1 0.271 pM 1.315 2.74 pM 0.3625 pM 1.315 1.329 1.329 l-tetradec- 0.04 pM 1-pentadec- 0.128 nM 1.323 0.094 pM 1.324 anol anol 0.187 nM 1.320 1.319 0.187 pM 0.250 nM 1.331 1-hexadec- 0.0312 nM 1.327 anol 0.0469 nM 1.323 0.0625 mM
1-Octanol
114 8aturat.d
94
0
1-Hexadecanol
42 1
I
I
I
5
10
15
J
20 nM
Alcohol concentration
Figure 4. Effect of alcohols on the optical rotation of 0.5% &lactoglobulin (pH 6.7, phosphate buffer, 0.15 M NaCl). -2r
.4s
0
0,005
0.010
(MI
* 1-Dodrcanol
44
-22 1
3
5
7
9
(1
13
15
47
n inC,H2,+,0H
Figure 3. Effect of alcohols on the optical rotation of 0.5% &lactoglobulin (pH 6.7,phosphate buffer, 0.15 M NaCl).
Figure 6. Logarithm of the alcohol concentration that cause8 the maximum decrease in the levorotation of @LG as a function of alcohol chain length.
alcohols (this was not done at higher protein concentrations in order to save protein). The levorotation of the protein decreases with addition of alcohol, reaching a minimum, then increasing at higher concentrations, and tending to reach a plateau, as shown in Figures 3 and 4. For propanol and butanol, however, the levorotation increases at higher concentrations to values much higher than the values in the absence of the alcohol (not shown in the figures). The values for the concentrations corresponding to the minimum in a!-C curves, C-, have been plotted as In Cagainst the alcohol chain length in Figure 5. C- depends on the alcohol chain length as well as on the protein concentration. For a given alcohol, C- decreases with decreasing protein concentration. Cmh also decreases with increasing alcohol chain length. This decrease in In Cmh increases linearly with increasing chain length up to octanol for both protein concentrations, with the rate of the decrease of In Cminwith chain length decreasing until do-
decanol. Thereafter, In Cmh decreases linearly with increasing chain length. Initial Slopes of a-CadPlots. As shown in Figures 1 and 2, the determination of the initial slope in 3% protein solution is unambigous, as the variation of a! with alcohol concentration is linear. At least three alcohol concentrations are involved. However, as Cmh decreases with decreasing protein concentration, sometimesonly two alcohol concentrations were obtained in the linear range, below Cmh, which were used with the zero concentration value to obtain the initial slope for 0.5% BLG or lower protein concentrations. The initial slope depends on the protein concentration as well as on the alcohol chain length. The effect of protein concentration on the initial slope is illustrated in Figure 6 for dodecanol. Figure 7 shows the logarithm of the initial slopes of the various lines in Figure 6. We shall from now on use the quantity In (da/dC& to represent the ability of the alcohol to depress t h e c vorotation of the protein in solution. This ability, as seen
Langmuir, Vol. 5, No. 3, 1989
Abu-Hamdiyyah and Kumari
4 5 r
0.5% in BY urea
Dodrcanol Concentration kY
Figure 6. Effect of 1-dodecanolin the low-concentrationrange on the levorotation of 8-lactoglobulinas a function of the protein
concentration.
A
-21
$, 2
4
I
I
1
I
I
I
6
8
10
12
(4
16
n in
C, HP,+I OH
Figure 8. Ability of an alcohol to decrease the levorotation of @-lactoglobulinas a function of alcohol chain length in phosphate buffer (pH 6.7 and 0.15 M NaC1).
OL 0
I I 1 2 Protein concentration (%)
I
3
Figure 7. Ability of an alcohol to decrease the levorotation as a function of @-lactoglobulinconcentration.
in Figure 7, increases only slightly between 3% and 1.5% with the rate of change of In (da/dCd) with protein concentration becoming more significant between 1.5% and 0.1% PLG. Ability To Depress the Levorotation as a Function of Alcohol Chain Length. This is illustrated in Figure 8 for 0.5% and 3% PLG and also for 0.5% PLG in the presence of 6 M urea. Each point on the curves for 3% and 0.5% PLG is the average of three and two values, respectively, of In (da/dC,d). The curves have two characterstic regions. In the first region, a linear increase in the ability to depress the levorotation with alcohol chain length followed by a decrease in the rate of increase in ability with alcohol chain length until dodecanol is reached. The second region, starting with tridecanol, shows essentially a linear increase in the ability to depress the levorotation with alcohol chain length. The slopes of the linear portions (C2-C8and CI2-Cl6)in Figure 8 are essentially the same and equal to about 1.38. Whether the slight variation in curvature observed in 0.5% PLG compared to that in 3% PLG is an artifact due to experimental error or a real concentration effect remains to be seen. The observation
that In C- is linear with alcohol chain length in 0.5% PLG (Figure 5) up to octanol seems to support the first suggestion. However, the transition between the two regions is distinct in 3% BLG and shows some curvature in 0.5% PLG, suggesting the onset of a concentration effect. The absolute value of In da/dC,d in the higher region (c13-Cl6) for each of these alcohols is less than the value one would obtain by extending the linear range (C2-C,) to the respective higher alcohol coordinates. Finally, we have investigated the effect of alcohols on the optical rotation of 0.5% @LGin the presence of 6 M urea. The results are shown in Figure 8. The ability to depress the levorotation of 0.5% PLG increases in the presence of urea for all the alcohols examined. However, the general shape of the curve is essentially unchanged, except that the transition between the two regions now appears at decanol. The figure shows that the increase in the ability of an alcohol to depress the levorotation of PLG in the presence of urea is in the same direction as obtained on decreasing the protein concentration. However, the specific rotation of the protein increases in the presence of urea (becomes more levorotatory), whereas the specific rotation value does not change by decreasing the protein concentration. Ability of Alkanols To Depress the cmc in SDS Solution. We have determined the critical micelle concentration (cmc) of SDS in the presence of all the alcohols examined with BLG in this study and obtained the initial slopes of cmeCadplots. Figure 9 shows the results plotted as In -(d(cmc)/dCad)cd against the alcohol chain length. The ability to depress the cmc increases linearly with alcohol chain length, exhibiting no discontinuities or significant curvature in the plot.
Discussion In a recent study,16 the decrease in the levorotation of protein in the visible spectrum in the presence of am-
Langmuir, Vol. 5, No. 3, 1989 813
General Anesthetic Agents and Protein Conformation
I
I
I
8 (0 n In C n H Z n + , OH
6
I
I
I
12
(4
(6
Figure 9. Ability of an alkanol to strengthen the hydrophobic effect in sodium dodecyl sulfate solutions as a function of alcohol chain length.
phiphilic and nonpolar substances was interpreted as due to strengthening of HI in the protein solution in a manner similar to that which occurs when amphiphiles are added a t low concentration to surfactant solutions. This strengthening of the hydrophobic effect involves the coaggregation of the amphiphilic additive with free monomers in surfactant solution or free unaggregated nonpolar side chains of proteins (i.e., exposed to the aqueous phase) in protein solutions. In surfactant solutions, the strengthening of HI is manifested by the depression of the cmc. In protein solution, however, because the side chains are permanently bonded to the polypeptide backbone and thus free translation of the side chains is not possible, the coaggregation process results in a small perturbation of the protein conformation which is manifested by the small decrease in the levorotation of the protein molecule in solution.18 As alcohol concentration is increased eventually an opposing effect seta in, which increases with concentration. At the minimum, Cmh, the two effects are equal. After C&, any addition of alcohol increases the levorotation of the protein. For soluble amphiphiles, the levorotation continues to increase, and the protein may eventually d e n a t ~ r e . 2However, ~ for sparingly soluble amphiphiles, the levorotation does not increase much above the value in the absence of the amphiphilic additive, as seen in Figures 3 and 4. The ability of an amphiphilic additive to strengthen HI in a surfactant solution is taken as the initial slope of the cmc-Cnd curve. In protein solutions, it is the initial slope of the .-Cad curve. We take the logarithm of the initial slope to represent the ability to strengthen the hydrophobic effect, Le., In (-d(cmc)/ dC,&, in surfactant and In (da/dC,d)c +, in protein solutions, respectively. Our resulta show tgat the ability of an alcohol to strengthen HI in protein solutions depends ~____
(16) Abu-Hamdiyyah, M. Langmuir 1986,2,310. This is regarded BS part 1.
on the protein concentration and alcohol chain length. Effect of Alcohol Chain Length. Figure 8 shows that the ability to increase hydrophobic interaction in ,8LG falls into two regions. In the first region, a kind of saturation effect in the hydrophobic interaction tends to appear. Strengthening of the hdyrophobic effect increases linearly with alcohol chain length up to octanol; thereafter, only part of the alcohol chain seems to contribute fully to the interaction, the rest of the molecule in excess of C8 contributing only weakly. This region ends with dodecanol. The fact that the value for the ability to increase HI for the tridecanol does not fall on the continuation for C2-C12 alcohols but rises rather sharply suggests the onset of another region of interaction which is size dependent. The second region could be a single one at a protein-protein interface such that only molecules of certain sizes, but not smaller, are able to interact with it. Or the second region could be composite: made of two regions on the surface of the globular protein (i.e., protein-water interface) such as the first region and another adjacent region separated from the first one by a distance that has to be spanned by the alcohol molecule. According to this, a molecule such as C13 increases HI at two regions, i.e., the alcohol molecule effecting intramolecular HI at the surface of the protein molecule. The s u m of the interactions in the two bridged regions (spanned by c1&6 alcohols) represents the measured value, which is still less than the value expected from full HI of the whole molecule, i.e., less than the value obtained by extending the line C2-CB. The proposed interaction at the protein-protein interface would have to be much weaker than in the first region to explain the absolute values of In (da/dC,d) for c13-c16 alcohols. However, in view of the fact that the slopes for C2-C8 and c13-c16 are essentially the same, the second explanation becomes less favorable. It has been shown14that -d(cmc)/dCnd X l/cmcO= OK, where cmco is the value of the cmc in the absence of the additive, 8 is a constant for a given surfactant, and K is the distribution coefficient, which may be regarded as a binding constant. Thus In (-d(cmc)/dCnd)is proportional to In K. In an analogous manner, In (l/ao)(da/dC,d) = O'K', with aothe levorotation in absence of the additive, 8' a constant for a given protein, and K' the binding or association constant, so that ln (da/dC,d) is proportional to In K' and hence to -AGO, the standard free energy of association of the additive with the protein. Assuming AGO = AGO,, nAGocH2,then the slope of the In (da/ dC& +, vs n in Figure 8 between C2and C8 and between (212 an8 c16 gives -AGo,,,/RT, which is equal to 1.38. This value is comparableto the standard free energy change per CH2for the transfer of alcohols (n = 4-10) from aqueous solution to liquid alcohols or the transfer of long-chain acids (n = 8-16) from aqueous solution to hydrocarbon solutions.2o To get information on the sue of the second hydrophobic region, it would be essential to investigate alcohols higher than hexadecanol. However, assuming that In (da/ dC&,+, is proportional to In K where K is the binding constant, our results would read as follows. In K increases with increasing alcohol chain length, tending to level off at C12;from tridecanol upward, In K increases linearly with chain length. The only binding data that exist for @LG are those of Spector and Fletcher18 for long-chain fatty
+
(17)Ray, A.; Reynolds, J. A.; Polet, H.; Steinhkdt, J. Biochemistry 1966,5, 2606. (18)Spector, A. A.; Fletcher, J. E. Lipids 1970,5,403. (19)Reynolds, J.; Herbert, S.; Steinhardt, J. Biochemistry 1968, 7, 1357. (20) Lin, I. J.; Somasundaran,P. J. Colloid Interface Sci. 1971,37,731.
814 Langmuir, Vol. 5, No. 3, 1989
Abu-Hamdiyyah and Kurnari
-2L\ 0
/
12
*\
I
t I
O
I 0
-(2 -14
cw o C a n (thir work) A Experimontal C a n (Brink and Partarnak)
\ \
A \
tc
A /
V
I
2
I
4
1
6
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8
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-16 1
2
Figure 10. Experimental anesthetic potency data (A,Brink and Pasternak; 0, Pringle et al.) plotted against the relative ability of an alcohol to decrease the levorotation of 0.5% @-lactoglobulin.
acids which show that palmitic acid binds more strongly than stearic acid, with lauric acid showing the weakest affinity. The difference, however, between palmitic and stearic acids is quite small, and it is hard to judge whether this observed reversal signals a size limitation. Reynolds et al.19 determined the binding constants for octanol, decanol, and dodecanol with BSA, and their data show increasing binding strength with increasing chain length. Anesthetic Potency and the Ability To Strengthen HI in BLG Solution. Taking the ability of an amphiphilic (or nonpolar) additive to strengthen HI in protein or surfactant solution to represent a measure of its anesthetic potency and using previously reported experimental data, we have shownl6that anesthetic agents affect micellization equilibria as well as optical rotation of proteins in the direction of increasing HI. In the above study,16the experimental data on the effect of anesthetic agents on the optical rotation of PLG which were used covered only one concentration (pressure) of the anesthetic agent.13 Even then, the concentration used for a given anesthetic agent was greater than its reported EDmvalue. These limitations were a major drawback. This has now been rectified in this study as seen in Figures 1 and 2, where we have measured the effect at sufficiently low concentrations that do not exceed EDm values. Using our new results, we have plotted in Figure 10 the logarithm of the relative ability of an alcohol to depress the levorotation of 0.5% PLG (In [(l/aro)(da/dC,d)l) against the correspondingexperimental anesthetic potency value (In (l/EDso)) taken from the literature,21i22where EDm is in mo1.L-l. Both plotted quantities are equivalent dimensionally. A linear relation is also obtained for 3% PLG having the same slope except that the line is shifted to the right of the origin.
(21) Brink, F.;Postern&, J. M. J. Cell Physiol. 1948, 32, 211. (22) Pringle, M. J.; Brown, K.B.; Miller, K. W.Mol. Phorrnacol. 1981, 19,49.
4
I
I
I
6 8 io ninCnHpn+f OH
Figure 11. Anesthetic concentration (C,) (C,)
1
I
19
14
(6
and water solubility
of alkanols as a function of alcohol chain length, n.
Implications for the "Cutoff"'Effect in Anesthetic Potency. The anesthetic potency increases with increasing alcohol chain length, reaching a maximum at dodecanol with the higher alcohols being nonactive. This is the socalled "cutoff" effect in anesthetic potency in ascending a homologous series of additives. Two main hypotheses are currently used to explain the action of anesthetic agents, one postulating the lipid bilayer as the primary site, the other implicating directly the protein in nerve cell membrane.15 Taking the surfactant SDS to represent a lipid, the results shown in Figure 9 display no "cutoff" tendency in the ability to strengthen HI. The effect increases linearly from the lowest membrane examined, ethanol, to the highest, hexadecanol. This is in line with the results of Frank and Liebls using lipid bilayers instead of micelles. However, the ability to strengthen HI in /3LG shows two distinctive patterns depending on the alcohol chain length. In the first, the ability to strengthen HI increases with alcohol chain length, reaching a maximum value a t dodecanol. This would have been a straightforward demonstration of the "cutoff" effect in the soluble protein PLG if the higher alcohols c13-c16 did not interact with the protein. We have suggested that the hydrophobic interaction of the higher alcohols with PLG as being due either to intramolecular bridging between two adjacent but separated regions on the surface of the globular protein (protein-water interface) or to interaction with the protein-protein interface, i.e., between the a-helixes or domains. Now the question must be asked whether the results on the ability to strengthen HI in the globular protein PLG can be taken as representative for HI in membrane proteins. The proteins involved in nerve impulse conduction and transmission are transmembrane proteins which are hydrophobically bonded to the lipid bilayer on all sides forming a sort of cylindrical structure with ionic or hydrophilic channels in the middle, respectively. The only protein-water interface that exists in such a structure is that in the center along the length of the channel that
General Anesthetic Agents and Protein Conformation
Langmuir, Vol. 5, No. 3, 1989 815
in the protein exposed to water or an increase in extends across the lipid bilayer. Recent s t ~ d i e son ~ ~ * ~groups ~ the concentration of the hydrophobic groups in the protein binding of long-chain alkyl derivatives (Cl4-C20) to a exposed to water. An increase in size of nonpolar side membrane protein reconstituted into a lipid bilayer inchains is not possible, and so the observed trend is due to dicate that binding occurs at the annular (protein-lipid) an increase in the concentration of nonpolar side chains interface and at the protein-protein (nonannular) interexposed to water. At high protein concentrations, the face. protein molecules interact together through the side chains It was found that the binding affinity at the proteinon the surface of the protein molecules. On dilution of the protein interface increased with increasing chain length, protein solution, the side chains become available for inunlike that at the annular interface, which increased with teraction with the amphiphilic additive. The interaction decreasing chain length. Assuming that the interaction between protein molecules through the nonpolar side of alcohol in the protein-water interface in the channel is chains does not involve detectable conformational changes similar to that at the protein-water interface in PLG, then in the protein molecule, as the specific rotation [a]does the ability to increase HI in the channel would increase not change on dilution. Apparently, the protein molecule with alcohol chain length until the chain length becomes becomes more labile with decreasing protein concentration sufficiently long to interact preferentially at the proteintoward hydrophobic interaction with the alcohol molecule. protein interface, thus displaying the "cutoff" effect at that This lability can be also noted from the variation of Cmin point. According to the above, anesthetic action occurs with protein concentration. Figure 5 shows that the cononly when an additive increases HI in the ionic or hycentration of alcohol that causes the maximum decrease drophilic channel (i.e., protein-water interface). in levorotation decreases with decreasing protein concenThe idea that the hydrophobic site in PLG (for shorttration. chain amphiphilic or nonpolar substances) has a limited Ray et al." found that the binding of dodecanol (but not size was first arrived at by Wishnia and PinderZ6using octanol) to BSA is also concentration dependent, with the butane, pentane, and iodobutane in binding studies. Frank number of moles of alcohol bound per mole of protein (Y) and Lieb studying the effect of anesthetic agents on the increasing with decreasing protein concentration. Their activity of luciferase came to the conclusion" that the site v-protein concentration plot resembles our results in of action in the enzyme is of limited size, comparable to Figure 7. However, we found that the concentration effect that found in PLG.% In another s t u d F b using homologous as seen in Figure 7 occurs for short- as well as for longalkanols and alkanes, they found two "leveling-off" effects chain alcohols (C4, Cg, C12, and C16) in PLG and HSA in the inhibition of the enzyme activity as a function of (unpublished results). the chain length. Two "leveling-off" effects for alkanols Effect of Urea. Urea is known to weaken hydrophobic were shown to occur: one at hexanol and the other just interaction in protein solutions, tending to expose nonpolar after dodecanol. However, the "cutoff" in anesthetic poside chains which were aggregated or buried to the aqueous tency of the higher members was attributed26bto the inphase, with [a]becoming more levorotatory. Thus, in the ability of that member to achieve enough concentration presence of urea the concentration of nonpolar side chains in the aqueous phase in order to halve the enzyme activity. exposed to the solvent increases, and hence the ability of According to the data in Figure 2 of ref 26b, the solubility a given alcohol to increase HI would also increase.16 This line intersects the ED, curve such that only the solubility is borne out by the experimental results shown in Figure of hexadecanol occurs below its corresponding ED, value, 8. Similar strengthening of HI occurs in SDS solutions and therefore it is the only alcohol which would be ancontaining urea.27 esthetically inactive. Applying this concept to our data The curve for 0.5% PLG in 6 M urea shows that the on SDS shown in Figure 9 and using the previously obability to increase HI is greater in the presence of urea than tained relationle between the ability of an anesthetic agent in its absence for all the alcohols examined. The shape to depress the cmc of sodium dodecyl sulfate and its anof the curve is not affected by urea except that the tranesthetic potency, namely, In 1/C, = In - d(cmc)SDS/dC, sition between the two regions appears now at decanol 8.54, where C , is equivalent to EDm above, which is in instead of dodecanol. If this observation is true, it means mol-L-'; we obtained the corresponding In C,. These the resulting exposure of nonpolar side chains due to the values have been plotted together with In Cw (the water presence of urea in the solution shortens the distance solubility in mo1.L-') and In (C,)ex,, (the experimental between two neighboring hydrophobic regions so that a anesthetic concentration2'Vn) against the numbe of carbon dodecanol molecule can now span the distance between atoms in the alcohol molecule. The solubility line interthe two regions on the surface of the protein. Although sects the estimated anesthetic concentration line just beboth the initial slope (da/dCad)and the levorotation value fore pentadecanol. The point to be made here is that we of aoin the absence of the additive increase with increasing are not dealing with a protein solution but with a surfacurea concentration, the prediction of the value of the tant solution which does not show any leveling-off effect binding constant K cannot be made without determining in its ability to strengthen HI as a function of alcohol chain the value of the constant t9 for the given protein as a length. However, we obtained a "cutoff" effect. function of urea concentration. For K to decrease in the Effect of Protein Concentration. Figure 7 shows that presence of urea, 0 must increase faster than the increase the ability to strengthen the hydrophobic effect increases in the ratio of initial slope to ao (keeping in mind the with decreasing protein concentration. The increase is assumption -da/dCad x 1/ao= O K ) . slight between 3% and 1.5% and becomes greater between 1.5% and 0.5% (0.1% for dodecanol). An increase in hydrophobic interaction in the system as shown in Figure Summary 6 implies either an increase in the size of the hydrophobic Studying the effect of alcohols at concentrations that do not exceed EDm values, we verified the previous con(23) Froud, R. J.; East, J. M.; Rooney, E. K.; Lee, A. G. Biochemistry clusion that anesthetic potency is linearly related to the 1986,25, 7535. (24) Froud, R. J.; East, J. M.; Jones, 0. T.; Lee, A. G . Biochemistry ability to strengthen the hydrophobic effect in PLG solu-
+
1986,25, 7544.
( 2 5 ) Wishnia, A.; Pinder, W. Biochemistry 1966, 5, 1534. (26) Frank, N. P.; Lieb, W. R. (a) Nature 1984,310,599; (b) 1986,316,
349.
(27) Abu-Hamdiyyah, M.; Kumari, K., submitted to J. Phys. Chem.
Langmuir 1989,5, 816-818
816
tion. The ability to strengthen the hydrophobic effect depends on the protein concentration and alcohol chain length. The effect increases for a given alcohol with decreasing protein concentration. For a given protein concentration, two characteristic regions appear in the ability to strengthen HI as a function of alcohol chain length. In the first region, the effect increases linearly with increasing chain length up to octanol, with the ability tending to level off at dodecanol. In the second region, the effect increases sharply and linearly from tridecanol to hexadecanol. No such discontinuity or curvature was observed in the corresponding effect in a surfactant solution as a function of alcohol chain length.
Taking the effect in the first region to be representative of the effect anesthetic agents have on HI in the ionic or hydrophilic channels in membrane proteins, the "cutoff" effect in anesthetic potency in homologous alkanols is rationalized.
Acknowledgment. This research was supported by the University of Kuwait Grant no. SC027. Registry NO.Ethanol, 644-17-5;l-prOpanOl, 71-23-8; 1-butanol, 71-36-3; 1-hexanol, 111-27-3; 1-tetradecanol, 112-72-1;l-pentadecanol, 629-76-5; 1-octanol, 111-87-5; 1-decanol, 112-30-1; 1dodecanol, 112-53-8; 1-tridecanol, 112-70-9; 1-hexadecanol, 36653-82-4;urea, 57-13-6.
Particle Sizes and Electrophoretic Mobilities of Poly(N-isopropylacrylamide) Latex R. H. Pelton,*it H. M. Pelton,? A. Morphesis,* and R. L. Rowells McMaster University, Department of Chemical Engineering, Hamilton, Ontario, Canada, L8S 4L7,and Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003 Received September 12,1988. In Final Form: January 25, 1989 Average particle diameters and electrophoretic mobilites of poly(N-isopropylacrylamide) latex were measured as a function of temperature. Diameters decreased from 788 nm at 10 "C to 380 nm at 50 "C in 0.001 M KC1; the corresponding electrophoreticmobilities increased from -0.193 X lo4 (18 "C) to -3.06 x lo4 m2V-' s-' (47 OC). The most dramatic changes with temperature occurred around 31 "C, the lower critical solution temperature of poly(N4sopropylacrylamide) in water. The increased electrophoreticmobility with temperature reflected increasing charge density when the particle diameter decreased. Charge density increased with decreasing particle diameter because the number of charged groups per particle was constant.
Introduction We have been interested in the development of sterically stabilized aqueous latexes which can be flocculated by heating. As a first step, latexes based on cross-linked poly(N-isopropylacrylamide)(polyNIPAM) were prepared by surfactant-free emulsion polymerization.' The term latex is perhaps misleading for a t room temperature polyNIPAM is water soluble and the polyNIPAM-based particles appear to be water-swollen colloidal gel particles. In the absence of added electrolyte, the latex was colloidally stable over an extended temperature range, whereas in the presence of 0.1 M CaCl,, the latex coagulated when the temperature was raised above 31 "C, thus demonstrating the desired temperature-sensitive colloidal stability. The temperature sensitivity of polyNIPAM latex properties is a reflection of the lower critical solution temperature (LCST) of 31 "C for polyNIPAM in water. From the coagulation behavior, it was concluded that below the LCST the latex particles were colloidally stabilized both by steric stabilization and electrostatic stabilization whereas above the LCST only electrostatic stabilization was operative. On the basis of imprecise electron micrographs and turbidity measurements, it was postulated that the polyNIPAM latexes underwent significant reduction in particle size as the temperature rose above the LCST. In this work, particle size and electrophoretic mobilities of polyNIPAM latex are reported as a function of temperature.
'McMaster University.
* University of Massachusetts. 0743-7463/89/2405-0816$01.60/0
Table I. PolvNIPAM Latex Reciw' NIPAM 14 g L-' methylenebis(acry1amide) 1.4 g L-' potassium persulfate 0.83 g L-' distilled water 720 mL at 70 O
C
Experimental Section All measurements were made by using a latex prepared by the surfactant-free emulsion polymerization of N-isopropylacrylamide-see Table I. More details of monomer purification and latex polymerization along with electron micrographs of this latex are given in ref 1. Latex dispersions were cleaned by successive ultracentrifugation, decantation, and dispersion in doubly distilled
water. Particle size measurements were made with a h4alvern M2OOO photon correlation spectrometer (PCS) and a Spectra Physics 124B, 15-mW helium-neon laser. All data were collected at a scattering angle of 90° and at one latex concentration. Electrophoretic mobilities were measured by the Pen Kem System 3000 electrokinetic analyzer. Care was taken to ensure that temperature equilibrium was reached before recording measurements.
Results and Discussion Particles sizes are shown in Figure 1 as a function of temperature. The diameters of particles dispersed in 0.001 and 0.01 M KC1 gradually decreased from 788 nm at 10 "C to 604 nm at 32 OC, after which the particle size dropped to 492 nm over a range of 3 "C. Above 40 OC, the diameter decreased very slowly with increasing temperature. There was little distinction between the two KC1 concentrations. (1)Pelton, R. H. Colloids Surf. 1986,20, 247.
0 1989 American Chemical Society
