Hydrogen ion titration of lysozyme in alcohol-water solutions

Biochimica et Biophysica Acta, 405 (1975) 82-88

© Elsevier Scientific Publishing Company, Amsterdam- Printed in The Netherlands BBA 37149 HYDROGEN SOLUTIONS

ION T I T R A T I O N OF LYSOZYME IN A L C O H O L - W A T E R

M. TRIBOUT et J. LI~ONIS Chimie Gdndrale I, Facultd des Sciences, Univerisitd Libre de Bruxelles, Bruxelles (Belgium)

(Received January 20th, 1975) (Revised manuscript received June 9th, 1975)

SUMMARY H + titration curves of hen egg-white lysozyme were obtained at 0.15 1 in the presence of small amounts (less than 15 ~o) of methanol, ethanol and n-propanol. The acidity constants of two groups (whose pK values in water are, respectively, 4.2 and 3.5) are increased in water-alcohol mixtures in comparison to water. From the evaluation of these constants as a function of alcohol concentration and hydrocarbon chain length, it is suggested that these alcohols interact specifically with lysozyme. As pK values of 4.2 and 3.5 in water are generally assigned to Asp-101 and Asp52 respectively, it seems that interaction occurs within the active site of the enzyme.

INTRODUCTION Many studies of the effects of alcohols on protein conformation have been carried out [1-6] which lead to the conclusion that the effectiveness of the alcohols as protein denaturants increases with increasing hydrocarbon chain length, and is explained in terms of a hydrophobic mechanism of alcohol-protein, side-chain interactions. However, Hamaguchi and his colleagues [7, 8] have found that the nearultra violet, circular dichroism and absorbance spectra of lysozyme depend nonlinearly on the concentration of certain alcohols (particularly methanol, ethanol and npropanol); saturation effects are observed at a concentration below that required to unfold the protein. X-ray diffraction studies on alcohol-treated lysozyme crystals (less than 3 ~o alcohol) [3], suggested that ethanol and n-propanol bind specifically at a limited number of sites in the protein. The purpose of the present work is to decide whether any specific interaction of lysozyme with methanol, ethanol and n-propanol molecules can be detected from their effect on titratable groups. Proton titration curves of the enzyme were thus obtained from solutions in which the alcohol content did not exceed 15 ~ by weight, so that no conformation change should be detected.

83 EXPERIMENTAL PROCEDURE Hen egg-white lysozyme was a three times recrystallized, dialyzed and lyophilized preparation obtained from Sigma Chemical Co, and the alcohols used were spectroscopic grade (methanol and ethanol) or analytical grade (n-propanol) reagents. All the solutions, prepared on the day of the experiments, were 0.15 M in KC1 and contained about 4 mg/ml protein. The pH measurements were made in a thermostated (25 4- 0.2 °C) titration vessel under a nitrogen atmosphere with a combined calomelglass electrode (Ingold, HA405), and the pH meter used was a Radiometer Model P H M 4b. HCI or NaC1 solutions (Titrisol standardized solution, Merck) were added by means of an Agla micrometer syringe. Standard aqueous buffers of p H 4.6 and 8 (Merck) were used to standardize the pH meter. Measured pH values should be corrected for the solvent effect on the liquid junction potential. However, as this correction does not exceed 0.01 pH unit for the alcohol-water solutions used [9], we did not consider it in the analysis of our results (two experimental tests are reproducible within 0.02 pH units). RESULTS AND DISCUSSION

Construction and method of analysis of difference titration curves For each solution of protein two separate titrations were performed from pH 5-6 to the acid end point, and the other from p H 4-5 to alkaline pH. A resultant curve was then obtained empirically by superimposing the separate curves in the overlap region. We located the origin of the curves at a pH between 6 and 7 where the groups with acid pK should begin to titrate (eight carboxylic groups are titrated in aqueous solution). The precise location of this origin in the titration curves is not very critical because the number of titrating functions varies only slightly in that pH region, and because resultant difference titration curves are nearly constant at a more alkaline pH than 6. Only difference titration curves were be analyzed; indeed, results presented by Tanford and Roxby [10] show clearly that accurate assigning of individual pH values to each titratable group is still impossible. Difference titration curves constructed for the protein in various solutions, (the aqueous medium was used as reference) are shown in Figs 1-3. Because the difference function d~n approaches a zero value at a p H higher than 6.5, the alkaline part of the curves is not shown, although measurements were extended to very high pH values. It is well established [10] that lysozyme titration behaviour differs greatly from that predicted by the Linderstrom-Lang theory, particularly in the acid region. As we are concerned with difference curve analysis, we did not take electrostatic interactions between the titratable groups into account; independent titration of the different groups is thus assumed. Consequently, all the acidity constants are apparent values. The equation applicable to the difference (d~n) between the number of protons bound in alcohol-water solution and in water, as a function of the proton concentration (Cn), is A~n

C.,F,~Nj i

I

Kj -F C n

1

K; q- CH)

84 ANH .5

-.5

3

4

5

6

7

pH

Fig. 1. Difference titration curves in methanol-water solutions. The methanol c,~ncentrations (in wt 70) are, respectively, 2.1870 (1), 4.2470 (2), 6.4970 (3), 8.8270 (4), 11.32~ (5).

/W H .5 5-

-5

3

Z,

5

6

7

pH

Fig. 2. Difference titration curves in ethanol-water solutions. The ethanol concentrations (in wt %) are, respectively, 2.1% (1), 4.24% (2), 6.47% (3), 8.80% (4), 11.2870 (5).

AVH

3

4

5

6

7

pH

Fig. 3. Difference titration curves in n-propanol-water solutions. The n-propanol concentrations (in wt 70) are, respectively, 2.157o (l), 4.3070 (2), 6.6370 (3), 9.01 70 (4).

85 Nj represents the number of groups with ionization constant changing from K~ in water to Kj in a water-alcohol mixture. So an equation that provides a complete description of dvn in terms of the properties of the individual titratable groups will be exceedingly complex and will contain many more terms than can conceivably be extracted from the experimental data. The customary procedure used to arrive at a realistic representation of d r . is to assume that the difference in the number of bound protons is predominantly determined by differences in the titration behaviour of a very small number of groups.

Discussion of difference titration curve analysis The sharp rise in A v H at the most acid pH (Figs 1-3) results from an increase of pK in alcohol-water solution. To analyze this region of the curves, measurements should be extended to pH values lower than 2, where some denaturation of the enzyme is already observed in water. However, data from several sources [11, 12, 13] indicate the presence of 1-3 carboxyl groups with extremely low pK values (lower than 2) in native lysozyme. In the denaturated state, these groups ionize normally [14], so the fact that a small increase of these pK values is already detectable in the presence of small amounts of alcohol seems very plausible. Interference due to the slight maximum observed about pH 5 was considered while analyzing the negative part of the difference titration curves. This maximum can be ascribed to one or two groups whose pK value of about 4.7 in water is slightly increased by the addition of alcohol. This is the expected effect on the ionization constant of weak acids when the dielectric constant of the solvent is lowered. The negative valt~es of Av. in the difference titration curves are attributed to the increased acidity in alcohol-water solution of two titratable groups which are characterized by pK values of, respectively, 4.2 and 3.5 in water. Values estimated in the different alcohol-water mixtures are shown in Table I. TABLE I Alcohol concentration

pK~

pK2

4.2

3.5

3.7 3.7 3.5 3.4 3.7

2.6 2.9 3.0 3.1 3.5

3.5 3.8 3.7 4.0 4.2

3.1 3.5 3.5 3.5 3.5

4.0 3.7 3.8 4.1

3.0 3.2 3.3 3.3

(wt %) 0.00 Methanol 2.18 4.24 6.49 8.82 11.32 Ethanol 2.10 4.24 6.47 8.80 11.28 n-Propanol 2.15 4.30 6.63 9.01

86 /kpK 1

-5

-.E

|

I

1

2

3 CRO H (M)

Fig. 4. Difference between pK, values in alcohol-water mixtures and in water as a function of alcohol concentration. O, methanol; D, ethanol; A, n-propanol. The values shown must evidently be considered with caution owing to the simplifications we have shown in the preceeding heading, and should be considered solely as indicative of a trend and its order of magnitude. It is seen in Figs 4 and 5 that the negative ApK values tend to vanish at the highest alcohol concentrations. Since ApK must be zero in water, the curves must go through a minimum which is observed in Fig. 4, but not in Fig. 5, where it apparently occurs at even lower concentrations (dotted part of the curves).

/kpK 2

l ,

-.6

1 T

J.--'--r

\

~

'

~

1 1

~

J, J /

"

-.8

1

2

3 CROH (M)

Fig. 5. Difference between pK~ values in alcohol-water mixtures and in water as a function of alcohol concentration. O, methanol; D, ethanol; A, n-propanol. Such a development indicates the occurrence of at least two types of interaction with opposite effects on pK values. The first one, predominant at the lowest alcohol concentrations, and leading to decreased pK values, is progressively compensated by a second one responsible for the rise of the curves in Figs 4 and 5, at higher concentrations. This second interaction could correspond to the denaturation of the protein. In fact, the acidity constants of all the titratable groups become normal

87 on denaturation [14], and in particular, the abnormally low pK values of some residues are increased (as was already detected in the most acid part of the titration curves). Furthermore, the lower dpK values reached in methanol solutions, together with the abscissa of the minimum in these curves can at least be partly accounted for by the lower effectiveness of methanol as a denaturant, in comparison to ethanol and n-propanol. The most striking feature of our results is the modified ionization constant of two carboxylic groups, resulting from the first type of interaction. A pK value of 4.2 is generally attributed to Asp-101 [15], a residue which is located in the cleft of the native enzyme. According to certain authors [16, 17], a pK of 3.5 lies in the range of the values ascribed to Asp-52. However, there is a disagreement with other authors [18, 19, 20] who propose a pK of 4.5 for this same residue, and this conflict has not yet been resolved. Interaction of alcohols with lysozyme in the substrate binding site has already been pointed out by some authors. In analyzing absorbance and circular dichroism spectra, Hamaguchi et al. [7, 8] suggest a two-step interaction with the tryptophan residues in the cleft of the enzyme (Trp-62, -63, and -108): one occurs between an alcohol molecule and a tryptophan other than Trp-62 in the substrate binding site, the other is the interaction with Trp-62 or with a charged group near Trp-62. On the other hand, the spectral properties of tryptophan residues (the dominant chromophores in lysozyme) are sensitive to the ionization of neighbouring groups; for example, Trp-108 is in van der Waal's contact with Glu-35, a group with an abnormally high pK, also involved in the enzymatic catalysis. According to Lehrer and Fasman [21, 22], Asp-101 and Asp-52 could affect the properties of Trp-63, and likewise, the ionization of Asp-52 could affect Trp-108 which is located at a distance of approx. 9-10 A [15]. The modification of the behaviour of two carboxylic groups with pK values generally ascribed to residues located in the cleft of the enzyme, leads to the conclusion that alcohol molecules interact with lysozymein the substrate binding site. The occurrence of a particular mode of interaction at very low alcohol concentrations has also been detected in fluorescence spectra of lysozyme solutions and in X-ray diffraction studies on crystals [3, 23]. Several authors have discovered a modification of pK values for lysozymesaccharide complexes. Many studies (summarized in ref. 15) have shown that the association of the enzyme with tri-N-acetylglucosamine perturbs Asp-101 ; its pK value is decreased by about 0.6 unit, owing to the formation of two hydrogen bonds between the carboxyl group and the trisaccharide; Banerjee et al. [16] have pointed out a slightly decreased pK value for Asp-52 (residue involved in the enzymatic catalysis) when N-acetyl-glucosamine is bound to the protein. According to these authors, the environment of Asp-52 in the monosaccharide-lysozyme complex is similar to that of Asp-101 in the trisaccharide-lysozyme complex. Thus, the results presented here would suggest that, owing to an interaction between lysozyme and alcohol molecules, the environment of two carboxylic residues becomes more polar. The lower hydrophobicity of methanol can therefore account for the greater effects observed with the latter in comparison with ethanol and n-propanol. However, if the action of these alcohols was directly related to their relative hydrophobicity, ethanol should affect the ionization constants more strongly

88 t h a n n - p r o p a n o l . T h e o p p o s i t e sequence is observed in Figs 4 a n d 5, but a possible e x p l a n a t i o n o f this feature is t h a t the presence o f a l c o h o l molecules in the cleft o f the enzyme is essentially a consequence o f interactions between a h y d r o p h o b i c site and the h y d r o c a r b o n c h a i n o f the a l c o h o l ; the h y d r o x y l i c function o f the b o u n d alcohol could then interact with a n e i g h b o u r i n g c a r b o x y l i c residue. To conclude, the interaction o f alcohol molecules with lysozyme leads to increased i o n i z a t i o n c o n s t a n t s for two residues located in the s u b s t r a t e b i n d i n g site o f the enzyme. A l t h o u g h the m e c h a n i s m o f this process is not yet obvious, the fixing o f alcohol molecules in the cleft o f the enzyme c o u l d originate f r o m h y d r o p h o b i c interactions. REFERENCES 1 Kurono, A. and Hamaguchi, K. (1964) J. Biochem. Tokyo 56, 432 A.A.O 2 Hamaguchi, K. and Kurofio, A. (1963) J. Biochem. Tokyo 54, 497-503 3 Vincentelli, J. B. and IAonis, J. (1973) in Protides of The Biological Fluids (Peeters, H., ed.), pp. 493-497, Pergamon Press, Oxford 4 Herskovits, T., Gadegbeku, B. and Jaillet, H. (1970) J. Biol. Chem. 245, 2588-2598 5 Parodi, A., Bianchi, E. and Cifferi, A. (1973) J. Biol. Chem. 248, 4047-4050 6 Gerlsma, S. and Stuur, E. (1972) Int. J. Peptide Protein Res. 4, 377-383 7 Ikeda, K. and Hamaguchi, K. (1970) J. Biochem. Tokyo 68, 785-794 8 Shimaki, N., Ikeda, K. and Hamaguchi, K. (1970) J. Biochem. Tokyo 68, 795-803 9 Bates, R. G., Paabo, M. and Robinson, R. A. (1963) J. Phys. Chem. 67, 1833-1838 10 Tanford, C. and Roxby, R. (1972) Biochemistry 11, 2192-2198 1l Donovan, J. W., Laskowski, M. and Scheraga, H. A. (1960) J. Am. Chem. Soc. 82, 2154-2163 12 Sophianopoulos, A. J. and Weiss, B. J. (1964) Biochemistry 3, 1920-1928 13 Aune, K. C. and Tanford, C. (1969) Biochemistry 8, 4579-4585 14 Roxby, R. and Tanford, C. (1971) Biochemistry 10, 3348-3352 15 Imoto, T., Johnson, L. N., North, A. C. T., Phillips, D. C. and Rupley, J. A. (1972) in The Enzymes (Boyer, P. D., ed.), 3rd edn, Vol. 7, pp. 665-868 Academic Press, New York 16 Banerjee, S. K., Vandenhoff, G. E. and Rupley, J. A. (1974) J. Biol. Chem. 249, 1539-1444 17 Banerjee, S. K., Kregar, I., Turk, V. and Rupley, J. A. (1973) J. Biol. Chem. 248, 4786-4792 18 Studebaker, J. F., Sykes, B. D. and Wien, R. (1971) J. Am. Chem. Soc. 93, 4579-4585 19 Parsons, S. M. and Raftery, M. A. (1972) Biochemistry 11, 1623-1638 20 Kowalski, C. J. and Schimmel, P. R. (1969) J. Biol. Chem. 244, 3643-3646 21 Lehrer, S. S. and Fasman, G. D. (1966) Biochem. Biophys. Res. Commun. 23, 133-138 22 Lehrer, S. S. and Fasman, G. D. (1967) J. Biol. Chem. 242, 4644--4651 23 Vincentelli, J. (1971) Th6se de doctorat, Universit6 Libre de Bruxelles