Enthalpy of Interaction Between Some Cationic Polypeptides and n-Alkyl Sulphates in Aqueous Solution B Y MARIA I. PAZ-ANDUDE,? MALCOLM N. JONES AND HENRY A. SKINNER* Department of Biochemistry and Chemistry, University of Manchester, Manchester M13 9PL Received 1 5 th May, 1978 The enthalpies of interaction of a homologous series of n-alkyl sulphates with poly(L4ysine)- hydrobromide, poly(L-arginine)hydrochloride and poly(L-histidine)hydrochloride have been measured at 25°C. A linear relationship between the enthalpy of interaction and carbon chain length has been found for alkyl chain lengths above C8. The data support a model based on a stoichiometric interaction between the anionic head group of the n-alkyl sulphates and the cationic side chains of the polypeptides. The results are discussed in relation to the interaction between surfactants and proteins, and lead to the view that a major contribution to the enthalpy of interaction arises from the surfactant-cationic residue interactions, but that there remains an additional contribution from surfactant-apolar aminoacid residue interactions.The interactions between surfactants and globular proteins have been extensively studied inaiiily because surfactants at low concentrations unfold proteins and also because of the general use of surfactants as solubilizing agents.l’ The enthaipy of interaction between a globular protein and a surfactant is generally a complex function of surfactant concentrati~n.~-~ There are at least two contributions to the net enthalpy of interaction arising from (1) the binding of surfactant ions, (generally an exothermic process) and (2) the unfolding of the native conformation (an endothermic process).It is believed that the initial stage in the interaction involves the binding of the surfactant ion to oppositely charged sites on the surface of the native protein molecule, although the ionic interaction is modulated by hydrophobic effects3 The complexity of protein-surfactant interactions has prompted us to investigate simpler systems consisting of cationic polypeptide plus anionic surfactants. Micro- calorimetric measurements are reported here of the enthalpies of interaction of three synthetic polypeptides, [poly(L-lysine)hydrobromide, poly(L-arginine)hydrochloride and poly(~-histidine)] with a homologous series of n-alkyl sulphates in aqueous solution.Previous work on such systems has been limited mainly to spectroscopic and binding studies on poly(L-lysine) plus surfactants. 6* EXPERIMENTAL MATERIALS Poly(L-1ysine)hydrobroniide (lot LY 190A, mol. wt . 40 300), poly(L-arginine)hydro- chloride (lot AR 55, mol. wt. 13 900) and poly(L-histidine) (lot HS 36, mol. wt. 11 100) were purchased from Miles-Yeda, Israel. The materials were used as supplied. The molecular weights were determined by the manufacturers by ultracentrifugation. or supplied by t Present address : Departmento de Fisica, Universidad de Santiago, Santiago de Compostela, Sodium n-dodecyl sulphate was prepared as previously described Spain. 29232924 INTERACTION BETWEEN POLYPEPTIDES AND N-ALKYL SULPHATES Cambrian Chemicals, Croyden.No difference was found between results obtained with the two samples. All the other n-alkyl sulphates were supplied by Cambrian Chemicals. All solutions were made up in doubly distilled water. MICROCALORIMETRY The Beckman 19OB twin-cell conduction calorimeter has been described in detail else- where ’ and was calibrated electrically.1° Matched drop well annular glass cells were used. The procedure adopted was to charge one cell with 0.30(+0.01)x kg of polypeptide solution in the drop well and lO.O(+O.l)x kg of surfactant solution of known con- centration in the annular space. The reference twin cell was charged similarly with the same amount of polypeptide solution and lO.O(+O.l)x kg of water. On mixing, the dilution of polypeptide is identical in both cells so that the dilution enthalpies cancel.The enthalpies of surfactant dilution are negligible under these conditions. The majority of the measurements were made with surfactant concentrations below their respective critical micelle concentrations in water at 25°C. The initial polypeptide concentrations were : poly(L-1ysine)HBr (1.5 % w/v), poly(L-arginine)HCl (1.4 % w/v) and poly(L-histidine) (0.9 % w/v). On dilution the final amino acid residue concentrations were approximately 2.1,2.1 and 2.0 mmol dm-3 calculated on the basis of molecular weights of 209.09, 192.65 and 137.14, respectively. Because poly(L-1ysine)HBr is very hygroscopic the solutions were made up in a glove box purged with dry nitrogen. Poly(L-histidine) required acidification with hydrochloric acid to facilitate solution.The enthalpy data presented below are expressed in kJ mol-1 of poly- peptide residues [kJ (mol res.)-l] and were calculated from the equation. AH = 99’7.1($) where Q is the measured enthalpy in kJ, w is the weight of polypeptide solution of residue concentration c mol dm-3. The equation assumes a negligible difference in density between polypeptide solution and water (density 0.9971 g ~ m - ~ ) at 25°C. RESULTS Fig. 1 shows the enthalpy of interaction of poly(L-lysine) as a function of surfactant concentration for sodium n-alkyl sulphates covering carbon chain lengths from C to Cq. In every case the enthalpies come to limiting values at a surfactant concentration indicative of saturation of the polypeptide chain with surfactant ions.The data for n-hexyl sulphate are slightly anomalous at the higher surfactant concentrations. The most exothermic interaction is found with the longest carbon chain (C,,), and it is noteworthy that in this case the two linear portions of the curve extrapolate to intersect at a concentration of approximately 2.4mn1oldrn-~, which is close to the lysine residue concentration of 2.1 mmol dm-3. This conforms with the view of Satake and Yang that there is a stoichiometric (1 : 1) interaction between the cationic amino acid side chain and the anionic n-dodecyl sulphate ion. For the other surfactants saturation occurs in the region of 2-4 mmol dm-3. Although interaction of the surfactant with the poly(L-lysine) hydrobromide resulted in a turbid solution or a flocculant precipitate for the Cl0, CI1 and CI2 alkyl sulphates at concentrations > 1 mmol dm-3, all the other systems remained visually clear.In the former cases there may be a thermal contribution from precipitate forma- tion, but the consistency between the results for the same system above and below the precipitation threshold, and between these systems which gave precipitates and those that did not, makes it unlikely that precipitation contributes significantly to the interaction enthalpies.M. I. PAZ-ANDRADE, M . N. JONES AND H. A. SKINNER 2925 The enthalpy data for poly(L-arginine)hydrochloride and poly(L-histidine)hydro- chloride are plotted in fig. 2 and 3 respwtively. These plots show similar patterns to that given by poly(L-lysine)hydrochloride, and likewise show that for n-dodecyl sulphate, saturation occurs at surfactant concentrations close to the aminoacid residue concentrations of 2.1 and 2.0 mmol dm-3 for poly(L-arginine) and poly(L- histidine), respectively.mmol dm-3 -10 -20 5 " - O--C 12 - 4 3 2 1 0 - 1 -2 -3 - 4 -5 -6 -7 -8 I 1 FIG. 1 .-Enthalpy change [AN/kJ (mol amino acid residue)-l] on interaction of sodium n-alkyl sulphates (chain length Ca with poly(L-1ysine)hydrobromide in water at 25°C. mmol dm-3 4 2 d o g - 4 - - 6 & - 2 3 -10 w 8 -8 -12 4 -11 - 16 t , o - o , , , , , 0-c 4, 6 7 8 9 1 0 1 1 8-CB O ClO r FIG. 2.-Enthalpy change [AH/kJ (mol amino acid residue)-l] on interaction of sodium n-alkyl sulphates (chain length C,) with poly(L-arginine)hydrochloride in water at 25°C.For the three polypeptides the limiting enthalpy values for each surfactant are plotted as a function of the number of carbon atoms in fig. 4. These plots are linear for surfactants with carbon chain lengths in the range C8 to CI2 but markedly deviate from linearity below C8. The equations for the linear regions based on a regression analysis of all the values of the limiting enthalpies are as follows (+ represents the standard deviation of slope and intercept) poly(L-1ysine)HBr AH/kJ (mo! res.)-l = -2.374(+0.036)n,+22.61(+0.12)2926 INTERACTION BETWEEN POLYPEPTIDES AND N-ALKYL SULPHATES pol y (L-arginine)HCl pol y( L-histidi ne) HCl AH/kJ (mol res.)-l = - 1.445(+0.120)n,+0.180($_0.380) AH,,kJ (mol res.)-l = - 1.863( +0.063)n,+9.190(+0.201). (3) (4) In water, the side chains of all three polypeptides should be fully ionised.The pH of the solutions were - 5, except for the poly(L-histidine) solutions which were acidified to pH N 3. Some measurements were carried out on the system poly(L-1ysine)f sodium n-dodecyl sulphate in glycine buffers of ionic strength 0.0167. At both pH 2.95 (glycine-HC1) and pH 8.50 (glycine-NaOH) the limiting enthalpies did not differ significantly from those measured with pure water. mmol dm-3 FIG. 3.-Enthalpy change [AH/kJ (mol amino acid residue)-'] on interaction of sodium n-alkyl sulphates (chain length C,) with poly(L-histidine)hydrochloride in water at 25°C. nC 6 4 2 M O -2 2 - 4 Q - 6 g -e 2 -10 $ -12 - --- Q -11, -1 6 -1 8 -29 -22 FIG. 4.-Enthalpy change [AH/kJ (mol amino acid residue)-'] on interaction of sodium n-alkyl sulphates as a function of carbon chain lengths in water at 25°C 0, poly(L-1ysine)hydrobromide : A, poly(L-histidine)hydrochloride ; 0, poly(L-arginine)hydrochloride.M. I .PAZ-ANDRADE, M . N. JONES AND H. A. SKINNER 2927 DISCUSSION The linear relationships between the enthalpy of interaction and the carbon chain lengths of the surfactants from c8 to C12 as represented by eqn (2)-(4) correspond to methylene increments of -2.37, - 1.45 and - 1.86 kJ (mol res.)-' for poly(L-lysine), poly(L-arginine) and poly(L-histidine), respectively. In comparison with methylene increments calculated for other processes these values are very large, e.g., the enthalpies of micellization of the Clo and C12 alkyl sulphates at 25°C give a methylene increment of -0.42 kJ mol-1 ; the enthalpies of transfer of normal aliphatic hydro- carbons (C,C,) from aqueous solution to the liquid state (data quoted by Nemethy and Scheraga)12 give -0.82 kJ mol-', whereas the extensive assessment of hydro- phobic interactions in n-alkanes (C2-C10) reported by Gill and Wadso l3 gives -0.318 kJ mol-1 for the latter process.A higher value of the methylene increment is obtained for the transfer of aliphatic alcohols from water to the liquid state, e.g., for n-butanol and n-pentanol l4 we calculate - 1.59 kJ mol-'. The present study is of the interaction of surfactant with a polyelectrolyte of random conformation, in which it seems probable that the surfactant head group interacts ionically with the cationic charge on an amino acid side chain.The main question is that of the resultant orientation of the surfactant hydrocarbon chain in the polyelectrolyte + surfactant complex. Two extremes can be envisaged ; (a) the surfactant chains pack intimately into the polypeptide to maximise contact with the side chain methylene groups and possibly with part of the polypeptide backbone, or (b), the surfactant chains orient themselves away from the polypeptide chain, into the aqueous phase, possibly then to interact with similarly bound surfactant on adjacent amino acid side chains, leading to incipient formation of micelle~.~~ To form roughly spherical micelles in this way would require the polypeptide chain to adopt a coiled conformation. Of these two extremes, the first seems to be more likely in that, (1) the methylene increments are much larger than for micellization in aqueous solution at 25"C, (2) the methylene increments are specific for a given polypeptide and (3) circular dichroism spectroscopy has indicated that sodium n-octyl sulphate in neutral solution induces a partially helical conformation with poly(L-lysine), whereas the higher homologues (Clo-C1 6 ) induce a P-pleated sheet structure, the percentage of p-structure increasing with surfactant chain lengtha6 Such rigidity precludes globular micellar structures of the type postulated for the complex formed between polyethylene oxide and sodium-n-dodecyl sulphate, in which the polymer is wrapped around a surfactant micelle.'6 On the other hand, favouring the alternative possibility, is the fact that n-alkyl sulphates with carbon chain lengths below c8 do not form micellar s ~ h t i o n s , ' ~ and our results (fig.4) show that the breakdown of linearity occurs below c8. We consider now to what extent the present results relate to the measured enthalpies of interaction of surfactants with globular p r ~ t e i n s . ~ ' ~ Native proteins have relatively rigid structures, so that for comparison with a simple polypeptide in solution the unfolded protein is a more strict starting point. The final conformation of a protein + surfactant complex will resemble that of a polypeptide + surfactant complex, apart from conformational restriction imposed by disulphide bonds, and a less uniform distribution of binding sites. At saturation, globular proteins bind 2 1.4 g of sodium n-dodecyl sulphate per gram of protein,19-21 which, for an average molecular weight of -130 per amino acid residue, corresponds to the binding of 0.6 surfactant molecules per residue, i.e., rougly half that in a polypeptide + surfactant complex.Table 1 records thermochemical data for several protein-surfactant systems.2928 INTERACTION BETWEEN POLYPEPTIDES AND N-ALKYL SULPHATES The numbers of ionised lysine, arginine and histidine residues in the proteins 2 2 are shown together with the experimentally measured enthalpies of interaction (AHexp) at a surfactant concentration of 4 mmol dm-3. This concentration is below that required for saturation, but high enough to have induced denaturation, (except in case of the lysozyme + Clo surfactant system).If we now assume ; (i) that surfactant molecules interact preferentially with cationic sites, the enthalpy of interaction, AHc, TABLE 1 .-CONTRIBUTIONS TO ENTHALPIES OF INTERACTION OF GLOBULAR PROTEINS WITH SURFACTANTS IN AQUEOUS SOLUTION AT 25°C no. aminoacid system residues (ionised) AHb Lys Arg His Y O /kJmol-l /kJmol-1 /kJmol-l /kJmol-I SDS ref. AH& AHc AHa AHb /kJmol-1 lysozyme + CI2Hz50SO;Na+, pH 3.6,IO.W 6 11 1 25 lysozyme + el 2H2 50SO;Na+, pH 9, Z 0.009 5.9 11 0 25 lysozyme + CloH210SO;Na+, pH 3.6,10.004 6 11 1 17 Iysozyme + CloHz 10SO;Na+, pH 9,10.009 5.9 11 0 10 ribonuclease A + C1 2H250SO;Na, pH 7,I0.005 10 4 0.4 68 trypsin+ C1 2H2 5osOiNa+, pH 3.5, Z 0.01 14 2 3 38 trypsin + C1 2H2 ,OSO;Na+, pH 5.5,IO.Ol 14 2 2.3 33 /3-hctoglobulin + C1 2H250SO;Na+, Cl2HZ50SO;Na+, pH 5.5,IO.Ol 2 3 1.6 46 ovalbumin + C1 2H250SO;Na+, pH 7,10.005 20 15 0.7 97 bovine serum albumin+ C12H250SO;Na+, pH 7.9, 10.005 58 23 1.7 198 pH 3.5,10.01 2 3 2 48 ~-1~toglobulin + - 168.8 - 118.7 - 88.7 - 30.0 - 17.1 - 193.4 -116.5 - 600 - 150 - 220 - 660 -237 22223 -223 22223 -159 22223 -64 2za3 -133 25023 -156 -280b -147 ~ 2 8 0 b -89.6 190 24 -84.3 19024 -384 ~ 5 3 0 b -758 ~ 7 9 0 b - 154 - 118 - - - 134 - 317 - 250 - 700 - 256 - 366 - 692 - 22 - 15 I - - 2.5 - 17 - 17 - 17 - 6.5 - 6.0 - 6.0 18 18 18 18 3 5 5 4 4 3 3 a At total surfactant concentration of 4 mmol ~Irn-’~; estimated on the basis of 12 J g-l.being calculated from the number of such interactions, and eqn (2)-(4) above, (ii) that the proteins are completely unfolded, involving a denaturation enthalpy term AHd, and (iii) that following saturation of the cationic sites, the remaining bound surfactant ions interact with apolar aminoacid residues in the protein with an enthalpy AH,,, then AHexp = AHC +AH, 4- AH,.( 5 ) The enthalpies of unfolding have been taken from the literature 2 3 9 24 or were estimated from the work of Privalov and Knechinashivili 23 on the basis of 12 J 8-lM. I . PAZ-ANDRADE, M. N . JONES AND H . A. SKINNER 2929 protein. Values of AHb were then calculated from eqn (5). Assuming that surfactant ions not bound to cationic sites bind to (V-number of cationic residues) apolar sites, AH, can be expressed in terms of the enthalpy per mole of bound surfactant, values for which are given in column 10.The lysozyme + Clo surfactant system is a special case, in that the bound surfactant is insufficient to cover the total number of cationic sites ; hence, in calculating AHc, it was assumed that some of the arginine sites remain unoccupied, as the lysine residues are known to be exposed on the protein surface. Furthermore, the net enthalpy of interaction is less than that arising from interaction with the cationic sites, so that in this particular case the protein is not fully unfolded. For the other systems it is noteworthy that AHb is substantially larger than the enthalpy of a micellization process,25 (e.g., for sodium n-dodecyl sulphate in a medium of ionic strength 0.023, AH(micellization, = -0.64 kJ mol-1 at 25°C). This implies a stronger interaction between surfactant molecules and protein chains than between surfactant molecules in micelles, consistent with the fact that complex formation with proteins occurs below the critical micelle concentration of the surfactant concerned.Despite the approximate nature of the present analysis, it seems clear that the surfactant non-cationic residue interactions can make a significant contribution to the overall enthalpies of interaction of sodium dodecylsulphate with proteins, but there is no obvious correlation with the number and nature of apolar aminoacid residues in the protein. We thank Mr. A. Butler and Miss M. Griffin for experimental assistance and One of us (M. I. P.-A.) Dr. G. Pilcher for calibration of the microcalorimeter. expresses her thanks to the Spanish authorities for sabbatical leave.M . N. Jones, Biological Interfaces (Elsevier, Amsterdam, 1973, chap. 5. C. Tanford and J. A. Reynolds, Biochim. Biophys. Actu, 1976,457,133. E. Tipping, M. N. Jones and H. A. Skinner, J.C.S. Faruduy I, 1974, 70, 1306. M. N. Jones and A. E. Wilkinson, Biochem. J., 1976,153,713. M. N. Jones, Biochim. Biophys. Actu, 1977,491,121. I. Satake and J. T. Yang, Biochem. Biophys. Res. Comm., 1973,54,930. ' I. Satake and J. T. Yang, Biopolymers, 1975, 14, 1841 ; 1975, 15,2263. ' M. N. Jones, H. A. Skinner, E. Tipping and A. Wilkinson, Biochem. J., 1973, 135,231. H. A. Skinner in Biochemical Microculorimetry, ed. H. D. Brown (Academic Press, New York, 1969). lo M. N. Jones, G. Agg and G. Pikher, b, Chem. ThermodyMmics, 1971, 3,801, l1 E. D. Goddard and G. C. Benson, Csnud. J. Chem., 1957,35,986. l2 G. Nemethy and H. A. Scheraga, J. Chem. Phys., 1%2,36, 3401. l3 S. J. Gill and I. Wadso, Proc. Nat. Acad. Sci., 1976, 73, 2955. l4 E. M. Arnett, W. B. Kouer and J, V. Carter, J. Amer. Chem. SOC., 1963,91,4028. l6 B. Cabane, J. Phys. Chem., 1977,81,1639. l8 M. N. Jones and P. Manly, unpublished results. l9 J. A. Reynolds and C. Tanford, Proc. Nut. Acud. Sci., 1970,66, 1002. 'O R. Pitt-Rivers and F. S. A. Impiombato, Biochem. J., 1968,109, 825. 21 T. Takagi, K. Tsiyii and K. Shirahama, J. Biochern., 1975,77,939. 22 Analytical Methods ofprotein Chemistry, ed. P. Alexander and H. P. Lundgren (1966), vol. 4, 23 P. L. Privalov and N. N. Knechinashvili, J. Mol. Biol., 1974, 86, 665. 24 S. Lapanje, M. Launder and J. Skerjanc, Proc. 4th Int. Con5 Chem. Thermodynamics (Nouvelle 2 5 M. N. Jones, G. Pilcher and L. Espada, J. Chem. Thermodynamics, 1970,2,333. I. Satake and J. T. Yang, Biopolymers, 1976, 15,2263. P. Mukerjee and K. J. Mysels, National Standard Reference Data System, N.B.S. 36, 1971. chap. 5. Berthier, Arles, 1975), sect. 5, no. 16, 92. (PAPER 8/897)