Potentiometric titration and enthalpy evaluation of horseradish peroxidase in the presence of n-dodecyl trimethylammonium bromide

Colloids and Surfaces B: Biointerfaces 18 (2000) 63 – 70 www.elsevier.nl/locate/colsurfb

Potentiometric titration and enthalpy evaluation of horseradish peroxidase in the presence of n-dodecyl trimethylammonium bromide K. Nazari a,*, A.A. Moosavi-Movahedi b a

Chemistry Department, Science and Research Campus, Islamic Azad Uni6ersity, P.O. Box 19395 -1775, Tehran, Iran b Institute of Biochemistry and Biophysics, Uni6ersity of Tehran, Tehran, Iran Received 25 May 1998; accepted 31 August 1999

Abstract The reversible proton dissociation equilibria of horseradish peroxidase (HRP) is investigated in 100 mM NaCl solution at 25°C and over a pH range of 3.0 – 11.0 in the absence and presence of n-dodecyl trimethylammonium bromide (DTAB). Intrinsic dissociation constants for the carboxyl groups of acidic residues and also for the imidazole side chains of histidine residues in HRP are estimated using the Tanford expression. From the obtained titration curve the intrinsic pKa (pKa,int) of titratable groups, the electrostatic free energy (Wel), the enthalpy of unfolding (DHu) and especially the enthalpy of ionisation (DHion) for the enzyme molecule are estimated. Also the state of ionisation is discussed in terms of ionisation enthalpy changes. Results indicate that 79% of the total carboxyl groups are present in the form of buried or masked groups, which are not exposed to titration. While in the presence of DTAB, 65% of these groups remain buried and the isoelectric pH varies from 8.8 (in the absence of DTAB) to 5.5 (in the presence of DTAB). In the absence of surfactant no conformational changes are observed in the whole pH region of the titration experiment. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Horseradish peroxidase; n-Dodecyl trimethylammonium bromide; Enthalpy of ionisation; Titratable groups; Electrostatic free energy

1. Introduction Horseradish peroxidase (HRP, E.C. 1.11.1.7, donor H2O2 oxidoreductase) catalyzes oxidation of a wide variety of aromatic compounds by hydrogen peroxide[1 – 3]. HRP (isozyme C) is a haem glycoprotein containing 308 residues in its * Corresponding author.

primary structure and is the most abundant member of the peroxidase family [4,5]. It folds to an alpha-helical structure consisting of eight helices while the haem group (protoporphyrin IX) is sandwiched between two helices [6]. Aromatic substrates bind easily near the haem group at a specific site including Arg38, Tyr185, and 8-CH3 group of the pyrol(IV) [7–9]. Charged groups are thought to be important in the structure, function,

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and characterisation of globular proteins [10]. Accurate measurements of the acid-base titration curves of proteins are capable of providing information about the nature and number of ionising residues on the molecule, give estimates of the intrinsic dissociation constants of the ionising groups, the electrostatic free energy of the protein molecule, and, in certain cases, detect conformational changes as a function of pH [10,11]. Protein denaturation studies are capable of yeilding information about the tertiary structure and solubilization of the polar, non-polar and peptide groups. Solubilization of non-polar side chains of HRP in urea and guanidine hydrochloride solutions is attributed to the initial favourable interaction between these denaturants and some of the polar groups of HRP [12]. Ionic surfactants are one of the most potent denaturants, which denature the proteins in the milimolar range [13 – 16] and their interactions with biological macromolecules involve both electrostatic and hydrophobic effects [17,18]. Previously we reported some structural information for HRP that is inferred from its interaction with chemical denaturants (urea and guanidine hydrochloride) and surfactants [18,19]. Also spectrophotometric measurements for the interaction of HRP with the competitive inhibitor, N-phenyl benzhydroxamic acid, indicated that a histidine residue has an important role in the catalytic action of the enzyme [20]. A pKa =6.4 was found for this residue, so that below or above this pH value, the catalytic role is restricted and the enzyme loses its activity in acidic and basic regions [21]. In the present paper, the electroststic free energy and the enthalpy of ionisation as well as acid – base titration properties of HRP-surfactant interactions are reported.

2. Experimental

2.1. Materials Horseradish Peroxidase type II with a purity index of R.Z = 2.30 and n-dodecyl trimethylammonium bromide were obtained from Sigma. All

other chemicals were analytical grade and solutions were prepared in CO2-free doubly distilled water.

2.2. Methods Standard buffers with pH values of 4.0, 7.0, and 9.0 were used for initial scale adjustment of the pH-meter. An automatic and thermostat-controlled microtitrator, model Titroline alpha was used for performing the experiment. The instrument was computer controlled and the data were processed by an AT&T Globalyst 550 P.C. 486. It is necessary to carry out a blank experiment on a solution without dissolved HRP for blanking the acid–base equilibria of the solvent. For this purpose standardisation was acheived as follows. A 4 ml sample of 0.10 M NaCl solution was added to the titration vessel and equilibrated at 25°C. In order to remove carbon dioxide from the mixture, during the experiment nitrogen gas is passed through the stirring solution. The pH was then adjusted to 7.0 with a standard sodium hydroxide solution, and the resultant solution was titrated from pH 7.0 to 2.0 and from pH 7.0 to 12.5 separately. For estimating the heat of ionisation, it is possible to perform rapid transitions of temperature at a fixed addition of acid or alkali using two thermostat controlled water baths at temperatures 25 and 50°C simultaneousely. The critical micelle concentration (CMC) for DTAB at pH 6.4 was 11 mM using a conductometric method [22]. Such standardisation was also used after each reversible titration of the protein sample solution. Thus to 4 ml of an unbuffered solution of 0.5% w/v HRP in the absence or presence of DTAB at isoionic pH, known amounts of acid or alkali were added followed by potentiometric measurment of the pH of the solution at 25°C. Addition of acid or alkali is continued until the solution reached pH 2.0 and 12.5, respectively. Protein concentrations were measured at pH 7.0 on a Shimadzu spectrophotometer model 2101 PC on samples withdrawn from the titration vessel using an extinction coefficient of 1.02× 105 cm − 1 M − 1 at 403 nm. In all calculations a molecular weight of 42 500 was used for HRP [23].

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3. Results and discussion An electrometric or direct titration curve as an electrometric hydrogen ion titration curve represents clearly the relation between pH and the number of moles of protons bound by, or removed from, one mole of protein in the reaction (I): PHi + rH+ l PHi + r

or

PHi −rH+ lPHi − r (I)

where PHi represents a protein species at the begining of the titration, and r is the number of hydrogen ions bound by, or removed from, the protein. Experimental r values are obtained as the difference between the number of moles of strong acid or base added to a solution containing one mole of protein to bring it from its initial pH to the final value, and the number of moles of strong acid or base added to the solvent to give the same pH change with other conditions (ionic strength, temperature, volume, etc.) being the same as for the protein solution [11,24].

Fig. 1. Titration curves for HRP (0.5% w/v) in 100 mM sodium chloride at 25°C. , in the absence of DTAB; , in the presence of DTAB(2 mM).

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Hydrogen ion activities are determined with high accuracy by pH measurements [25], with the aid of the assumption of single ion activity [26– 28]. It means that we can identify the experimental pH with the − log aH + which in turn can be considered the product of hydrogen ion concentration, cH + , and an apparent activity coefficient, gH + . Thus we have: pH=paH + = pcH + + pgH +

(1)

where ‘p’ in each symbol stands for the negative logarithm. The idea of single ion activity may be extended to hydroxyl ions, by: pOH=pKw − pH= pcOH − + pgOH −

(2)

where Kw is the dissociation constant of water. The apparent activity coefficients remain the same, at constant ionic strength, in the presence and absence of protein in the solution. The values of pgH + and pgOH − for NaCl solution at 25°C and ionic strength of 0.10 M are 0.076 and 0.116, respectively [29]. Fig. 1 shows the number of hydrogen ions dissociated from one mole of HRP, in the acidic form, calculated with the aid of such activity coefficients. The curves are the average of five titration measurements on samples of the enzyme and each has been shown to be reversible within the pH range of 3.0–11.0. The average reproducibility between one experiment and another was equivalent to 9 0.4 groups per mole of HRP. Starting from the point of maximum net charge (zero point on the r scale), the first groups which lose their protons are carboxyl groups with the lowest pKa, so that the net proton charge on the molecule decreases as a result of the negative charges introduced thereby. These are the side chain carboxyl groups of glutamate and aspartate residues, and ionise over the pH range of 3.0–5.5. Over the pH range of 5.5–8.5, the more basic groups, among which the imidazole groups of histidine residues, will lose their hydrogen ions and their positive charges. There will be a pH at which the protein molecule carries zero net proton charge. This is the zero point on the Z scale, which shows the number of protons bound by (positive Z values) or removed from (negative Z values), one mole of HRP originally at the point

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of zero net proton charge or ‘the isoelectric point’ [11]. For HRP (isozyme C) the isoelectric pH is 8.8–9.0 [30,31] which corresponds to the pH at Z= 0 for the curve in the absence of DTAB indicated in Fig. 1. The number of ions dissociating from the protein over the entire pH range gives the number of groups of this type in the molecule exposed to the solvent. Since all groups with the same pKa are titrated simultaneously, therefore we may treat a titration curve as a combination of a small number of titration equilibria instead of having as many equilibria as there are titratable groups. The nature of the interaction between groups is considered to be solely electrostatic and interpreted as based on the Linderstorm – Lang theory [32,33]. For dissociation of titratable groups of type ‘i’, the equilibrium constant, Ka,i, is given by: Ka,i = aH + [a/(1 – a)]

or

pKa,i = pH − log[a/(1 −a)]

(3)

where a is the average degree of dissociation that may be obtained by dividing r by the total number of ionisable groups of a particular type in the molecule and aH + is the thermodynamic hydrogen ion activity. Because of the attraction of counterions for water molecules (with their high dipole moments) a large energy is required to remove a charged protein group from the solution. Therefore most of the charged groups tend to be found at the protein surface. The interaction encountered by a hydrogen ion which is being added to the protein molecule is dependent on the net charge only, and can be calculated as the electrostatic work that must be done to increase the net charge by one unit, against the opposing force of the charges already present. The mode of dependence is found by Tanford as below [34,35]: Ka,i = Ka,i(int)e 2vZ

or

pKa,i = pKa,i(int) −0.868vZ =pKa,i +(dWel/dZ)

(4)

where Ka,i(int) is the intrinsic dissociation constant of titratable groups of type ‘i’ in the absence of interaction, at Z =0. The work function, v, in Eq. (4) is given by: v = e 2/2DkBT[(1/b) −k/(1 + ka)]

(5)

where ‘e’ is the charge of electron, ‘D’ is the dielectric constant of the medium, kB is the Boltzmann constant, ‘T’ is the absolute temperature, ‘b’ is the radius of the spherical protein molecule, ‘a’ is the distance of closest approach of ions to the center of the sphere i.e. the sum of the radii of the sphere and the average radii of the small ions which make up the ionic strength and ‘k’ is the Debye–Huckel parameter expressed by: k= (8pNe 2I/103 DkBT)1/2

(6)

where N is the Avogadro number and ‘I’ is the ionic strength. The value of ‘v’ can be calculated from Eq. (5) with reasonable values for ‘b’ and ‘a’, which are calculable either from molecular weight and assumed hydration, or from hydrodynamic data [10]. For HRP with a molecular weight of 42 kD, a partial specific volume of ¯ 2 = 0.73 cm3 g − 1 and assuming 20% hydration, V b= 24.2 A° and a= 6.5 A°, a value of 0.058 is reported for ‘v’ in KCl solution of 150 mM [30]. It is noteworthy that ‘v’ which depends on the ionic strength, has the same value for all titratable groups, and is constant as long as the protein molecule does not lose its size and shape. Combining Eqs. (3) and (4) we obtain: pH− log[(a /1− a)]=pKa,i(int) − 0.868vZ

(7)

where ‘v’ is related to the electrostatic free energy of the molecule by the Eq. (8): Wel = RTZ 2 v

(8)

Eq. (7) provides a way to evaluate pKa,i(int) (Y-intercept), v (-slope/0.868), and Wel (from Eq. (8)), experimentally by plotting the left hand side of the Eq. (7) against Z. Such plots are shown in Figs. 2 and 3 for carboxyl groups and imidazole groups(inset). In the absence of DTAB pKa,i(int) values are estimated as 4.4690.02 and 6.34 9 0.05 and values of the work function (v) obtained are 0.069 9 0.004 and 0.313 9 0.024 for the acidic residues and imidazol groups, respectively.The difference between ‘v’ values obtained by Eqs. (5) and (7) is related to the possible deviations of the actual shape from a sphere [36]. The above parameters are also calculated for the protein in the presence of DTAB (2 mM) and are recorded in Table 1. It is clear from Table 1 that denatured

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Fig. 2. Logarithmic plots of carboxyl titrations of native HRP at 25°C. Inset: logarithmic plots of imidazole titrations of native HRP at 25°C.

Fig. 3. Logarithmic plots of carboxyl titrations of denatured HRP at 25°C. Inset: logarithmic plots of imidazole titrations of denatured HRP at 25°C.

HRP because of having more acidic binding sites for the cationic surfactant, has a greater v value

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than the titrated native form of HRP. Electrostatic free energies are estimated according to Eq. (8) over the Z range observed from Fig. 2 and the value obtained at 25°C are given in Table 1. During the titration process the net charge on the protein molecule and the electrostatic energy decrease. HRP has about 69 titratable carboxyl groups (50 Asp.+ 19 Glu.) [4,5], but Fig. 1 shows that in the absence of DTAB only 15 groups of this type are titrated during the titration process. This means that 79% of total carboxyl groups are located inside the native HRP molecule, and hence these are protected against titration. While in the presence of DTAB (2 mM) about 24 carboxyl groups are titrated up to pH 9. The transition concentration for denaturation of HRP with DTAB at pH 6.4 is equivalent to 2 mM, as reported previously [19]. Thus the same concentration of the surfactant is selected for exposing the buried charged groups to the solvent. Binding of DTAB to the negatively polar sites on HRP decreases the pI value by about 4 units of pH as illustrated in Fig. 1. Figs. 2 and 3 enable the average values of pKa,i(int) for carboxyl groups of acidic residues and imidazole groups of histidine residues(insets) to be obtained from the Y-intercept of the graphs in the absence and the presence of detergent, respectively. Because of low sensitivity at 11 B pHB2, the reference point of calculations was chosen to be pH 3 and estimation of a value for pKa,i(int) of the basic residues was not examined. Results obtained from thermal denaturation [19] and isothermal chemical denaturation of HRP [18] indicated that the enzyme has a relatively hydrophobic nature in its native form, so that its conformational Gibbs free energy(intrinsic stability) at neutral pH and 25°C is about 26 kJ mol − 1. Probably it may be introduced as the source of resistance of the enzyme against pH-denaturation. From three histidine residues in the primary structure of HRP, His170 is covalently bound to the iron atom and hence is not titratable [30]. Over the pH range of 5.5–8.5 two histidine residues are titrated, from those His 42 is of paramount importance [20]. Binding studies on the interaction of HRP with competitive inhibitor, N-phenyl benzhydroxamic acid and hydrogen donors indicated that this residue with a pKa =

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Table 1 Acid–base titration parameters for titratable groups of native and denatured states of HRP at 25°C Carboxylic residues 6 6.42

Z Wel (kJ.mol−1)

10 17.84

Histidine residues 14 34.97

18 57.80

1 2 0.776 3.102

v

Native HRP Denatured HRP

0.0699 0.004 0.1059 0.011

0.313 9 0.024 0.254 9 0.015

pKa(int)

Native HRP Denatured HRP

4.64 90.02 4.819 0.03

6.34 9 0.05 6.25 90.04

6.4 has an important catalytic role in the binding of donors to the HRP molecule [20,21]. This pKa value is in good agreement with pKa 6.34 obtained for histidine residues according to the Yintercept of the Fig. 3 (inset). The optimum pH for maximum activity of the enzyme observed at pH 7.1 and the enzyme activity decreases sharply below or above the optimum pH. Fig. 4 shows the apparent enthalpies of ionisation of the various groups titrated in HRP. These values are obtained from the temperature dependence of pH for each solution at a fixed amount of added acid or alkali, using the following relation [30]: DHion =2.303R [d pH/d (1/T)]

3 6.979

4 12.408

in terms of ionisation enthalpy change (DHion). Z values are extracted at the corresponding pH values from the lower curve of Fig. 1. Fig. 4 (inset) shows the enthalpy of ionisation at pH range between 4 and 10, has a strong dependency on the net charge of HRP molecule so that it decreases from + 2 kJ mol − 1 at Z= − 6, pH 10 to −2.2 kJ mol − 1 at Z= 2, pH 4, while in the pH region

(9)

where R is the universal gas constant. Eq. (9) is a modified form of the van’t Hoff relation and could be obtained from the temperature dependence of Ka,i(int) in Eq. (3). The higher limit of temperature (50°C) is so selected that it would be below the thermal transition temperature (52°C at pH 6.4) for HRP [19]. DHion may be estimated in the presence or in the absence of the surfactant. The lower curve in the Fig. 4 indicates variation of DHion with pH of the solution in the presence of DTAB (2 mM), while the upper curve shows such a variation in the absence of DTAB. Less positive values of DHion in the presence of DTAB (2 mM) may be attributed to titration of the newly exposed titratable groups on the denatured state of HRP molecule. As it is shown in Fig. 1 the state of ionisation can be illustrated as the relationship between pH of the protein solution and the net charge on the protein molecule (Z). Fig. 4 (inset) characterises the state of ionisation

Fig. 4. Ionisation enthalpy for titratable groups of HRP as a function of pH in 100 mM sodium chloride solution. Enthalpy values are calculated from titration curves at 25 and 50°C using Eq. (9). , in the absence of DTAB; , in the presence of DTAB (2 mM). Inset: variation of DHion versus the net charge (Z) on HRP molecule during the titration experiment in the presence of DTAB (2 mM).

K. Nazari, A.A. Moosa6i-Mo6ahedi / Colloids and Surfaces B: Biointerfaces 18 (2000) 63–70 Table 2 Enthalpy changes (kJ mol−1) associated with denaturation of HRP by DTAB at pH6.4 and 25°C DHcala DHbb DHionb DHua DHuc

−20.090.3 −44.091.1 −0.2090.02 +24.090.7 +24.290.7

a

Directly taken from [37] without considering ionisation enthalpy. b Taken from the lower curve of Fig. 4 at pH 6.4. c Obtained from Eq. (10).

of 3–4 DHion has no significant pH-dependency. For the pH region of 4 – 10 an average charge ionisation enthalpy equal to 0.42 (kJ mol − 1/ charge) could be obtained for titration process (an enthalpy change equal to 4.2 kJ mol − 1/10 charges produced) (see inset of Fig. 4). The total enthalpy change associated with the denaturation of HRP with DTAB could be estimated by the isothermal titration calorimetry. The denaturation process includes, (a) binding of the surfactant to the protein molecule, (b) unfolding of the protein molecule, and (c) titration of the titratable groups exposed to the solvent during unfolding of the enzyme by the surfactant. Thus for the interaction of HRP with DTAB the calorimetric enthalpy (DHcal) consists of contributions of enthalpy of binding (DHb), enthalpy of unfolding (DHu) and enthalpy of ionisation (DHion). Hence Eq. (10) could be written as follows: DHcal = DHb +DHu +DHion

(10)

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4 and is recorded in Table 2. Putting the values of DHcal., DHb and DHion (see Table 2) into Eq. (10), it is possible to determine a reliable value for enthalpy of unfolding (DHu). Thus a value of DHu = 24.29 0.7 kJ mol − 1 is obtained for the unfolding of HRP by DTAB (2 mM) at pH 6.4. This positive enthalpy change compensates the large negative value of DHb in Eq. (10). It is generally accepted that ionic surfactant unfolds globular proteins by means of the electrostatic interaction of its polar head group and subsequent hydrophobic interaction of its nonpolar hydrocarbon tail. During unfolding of HRP the newly exposed groups could be titrated. The smaller value of DHion corresponds to the lower number of such titratable groups, as we saw in the case of unfolding of HRP by DTAB. On the other hand, the larger extent of unfolding corresponds to a higher value of DHu. Although the resulting enthalpy change (DHion) has a relatively small value but it is influenced considerably by electrostatic properties (including: work function (v), electrostatic free energy (Wel) and the isoelectric pH) (see Table 1). Binding of the cationic surfactant generates a positive state of charge on the surface of the enzyme molecule. Here, repulsion between these positive charges is the main source for begining of the unfolding process. After initial electrostatic binding,the process proceeds with extensive hydrophobic binding of the surfactant, unfolding of the enzyme and exposing buried titratable groups of the produced denatured state to the solvent.

or DHu = DHcal −(DHb +DHion)

Acknowledgements

as previously reported for estimating DHu (DHu =DHcal −DHb) [38]. Here, we suggest a reliable definition (Eq. (10)) for DHu estimation. Table 2 shows enthalpy values of the interaction of HRP with DTAB (2 mM) at pH 6.4 and a temperature of 25°C. As previously reported, DHcal and DHion (in above conditions) are obtained from calorimetric measurements and the equilibrium dialysis technique, respectively [37]. DHion in the presence of DTAB (2 mM) and pH 6.4 could be obtained from the lower curve of Fig.

The financial support provided by the Research Council of the Science and Research Campus and the Complex Laboratory of the Islamic Azad University and the University of Tehran is gratefully acknowledged.

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