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Dielectric studies of the binding of water to lysozyme
J. Mol. Biol. (1982) 157, 571-575
LETTER TO THE EDITOR
Dielectric Studies of the Binding of Water to Lysozyme Dielectric dispersion measurements as a function of hydration are reported for lysozyme powder. The dispersion that occurs in the frequency range 10 kHz to 10 GHz can be analysed in terms of bound water molecules that form single or multiple hydrogen bonds, and the numbers found in these two categories agree well with recent X-ray data for lysozyme crystals. The dielectric data also indicate that at 20% (w/w) hydration the bound water acts as a plasticizer to increase the vibrational freedom of the protein structure, and that this may be of relevance to the fact that the onset of enzymatic activity occurs at this hydration level. Also, a sudden transition in the polarizability of the protein-water system is found to occur at 7% (w/w) hydration.
Knowledge of the ways in which water molecules bind to proteins is of relevance to studies of the dynamics of protein conformation and enzyme activity. As a consequence of each water molecule possessing a relatively large electric dipole moment the nature of the protein-water interaction, in terms of the rotational freedom of the bound water, can be investigated by measuring the dielectric polarizability of a hydrated protein. Such measurements are reported here for lysozyme. Lysozyme is a particularly stable protein (Imoto et al., 1972). There is evidence to show that its conformation in a dry powder preparation is similar to that when in solution (Careri et al., 1979; Yang & Rupley, 1979) and when partially dehydrated it continues to exhibit enzymatic activity (Yang & Rupley, 1979). Dielectric measurements on Iysozyme powders of low hydration should, therefore, be of biological relevance, and in working with powder preparations rather than with protein solutions there is the added experimental advantage of avoiding effects associated with protein molecule vibrations and counter-ion relaxation effects (Pethig, 1979). Estimates are presented here of the extent to which water molecules are either rotationally or tightly bound to the protein structure, and the results are in good agreement with the recent X-ray data of P. J. Artymiuk, C. C. F. Blake & W. C. A. Pulford (unpublished results) for lysozyme crystals, where the bound water was categorized as forming either single or multiple hydrogen bonds. The dielectric measurements also support the conclusions of Careri et al. (1980) regarding the redistribution of water at 7% (w/w) hydration and the associated changes that occur in the vibrational freedom of the protein-water system. Hen
dialysed
egg-white
lysozyme,
obtained
against water, freeze-dried
1-3x 10M4 m2, thickness
of the order
from
Sigma
Chemicals,
was thoroughly
and compressed into discs of surface area 15x
10T3 m and density
890+20kg
rne3.
Dielectric measurements in the frequency range low4 Hz to 1 MHz, and at. 9.95 GHz, were made as a function of hydration using techniques and methods of 551 Om2-283ti/82/!50589%05
003.00/O
0
1982 Academic
Press Inc.
(London)
Ltd.
S. BOSE
572
AXI)
It. I’E’I’HI(:
analysis described elsewhere (Eden et al., 1980; HOW it ~1.. 1977) for work on IX)\-incs serum albumin. A dielectric dispersion. extending over t,he frequency range IO kHz to 10 GHz and centred at’ around 10 MHz. was observed for the lysozyme samples and had essentially the same charact,eristics as t,hosr that havfb been described for the dispersion observed for bovine serum albumin (Gascoyne & Pethig, 1981). The magnitude de of this dispersion is determined as:
Ar = El -<*.
(11
where c1 and c2 are the values of the relativcl permittivity determined at 10 kHz and 9.95 GHz, respectively. The value for AC varied with hydration as shown in Figure 1. The hydration content was det,ermined using a sensitive quart)z-crystal microbalance technique (Gascoyne & Pethig, 1977) and t,he zero hydration levt4 corresponded to the situation where the lysozyme samples were in equilibrium wit,h a vacuum of the order of 6 x IO-’ N m -* at 293 K. On heating to 377 Ii for three hours the “dry” lysozyme samples reversibly lost about, 05O/,, of t,heir weighty, of Pigure I each lysozymr molecult suggesting that,, at the zero hydration contained approximately four water molecules tightly bound into its structure This number can be compared to the three water molrc*ulrs that appear to 1~ trapped within the polypeptide structure of lysozymr (lmoto d al., 1972). ‘I%P AC = I .35 that exists for dry lSvsozymt, can. as underlying dispersion of magnitude described for the case of bovine serum albumin (Ciascoyne et 01.. 19X1 ), be explained in terms of vibrations of the constituent dipolar peptide units and associated sidrchains of the protein structure. The increase of AC with h.vdrat’ion r&e&s the increase in polarizability of the protein samples. dur to the increasing content of
0
5
IO h(% (w/w)
I5 H,O)
FIG. 1. Variation of de. the diffiirenw in permittivity at 10 kHz anti 995 GHz. its a function hydration for 3 different samples of hen egg-white Iysozymr.
of’
LETTER
TO
THE
573
EDITOR
dipolar water molecules, and also to any changes that might occur in the vibrational freedom of the protein structure. For dielectric materials such as proteins of low hydration, the Onsager correction for the local electric field can be considered to be relevant, so that the effective moment VEof the dipoles responsible for the dielectric dispersion is given as (see e.g. Davies, 1965) : (%-%)P%+~,) %(Ecc +v2
_ ,v?ii2
(2)
9q-,kT ’
where N is the number density of dipoles, c0 is the permittivity of free space, kT is the Koltzmann energy and for our purposes l s and Ed refer to the relative permittivity values at 10 kHz and 9.95 GHz, respectively. (In the work of Gascoyne & Pethig (1981), a typographical error occurred in this equation.) The number density, N, of bound water molecules in the lysozyme sample is given by:
where X, is Avogadro’s number, h is the percentage (w/w) hydration and d is the density of the sample. In Figure 2 the left-hand side of equation (2) is plotted against A’ of equation (3). Up to a hydration level corresponding to, on average, 32 water molecules being added to each lysozyme molecule, the values of AC andf(E) (1.h.s. of eqn (2)) of Figures 1 and 2 are relatively insensitive to the degree of hydration, which suggests that each of these 32 water molecules is bound to the protein structure by two or more hydrogen bonds in such a way as to prevent them
I 50
I 0
I I00
No. of H,O molecules/lysozyme FIG. 1. Variation off(~), samples of Fig. 1. The water molecule.
the left-hand concentration.
I 150 molecule
side of eqn (2), aa a function of hydration for the lysozyme A’. of eqn (3) has ken reealculatcd as molecules per lysozyme
,574
S. BONE ANI) R. PE’THI(:
from rotating. These water molecules, together with the four that remain tightly bound after our dehydration procedure, can be envisaged to be tightly incorporated into the vibrating protein structure, with relaxation times equivalent to those of the protein. For the hydration region corresponding to the situation when there arc between 32 and 101 water molecules associated with each lysozyme molecule. the gradient of Figure 2 corresponds to the effective dipole moment ~fi for each water molecule having a value of 5.79 x 10S3’ Cm (1.74 Dehye). This moment, can 1~ compared to the value 6.14 x lo- 3o Cm (I.84 Debve) for normal bulk water (Eisenberg & Kauzmann, 1969) and suggests that each water molecule in this group (about 69 in number) has one hydrogen bond link either to the protein or to another water molecule and is able to rotate in a relatively unhindered manner. At a hydration of 7% (w/w) corresponding to 57 water molecules being added t,o thrl enzyme, there is a sudden discontinuity in the plots of AE andf(E). This coincides with discontinuities observed in the infrared absorption and specific heat by (‘arcri et al. (1980), which were interpreted by them as a redistribution of protons betnecAn the carboxylic acid and basic groups. For hydration levels above, about I%..‘iU,, (w/w) the superlinear increase of AC and!(r) indicates that correlation effects are developing between absorbed water molecules, or that there is an increase in motional freedom of the bound water molecules and of the protein structure. All of these effects probably occur and it is of interest to note that the region where thrsc effects become significant at 200?, (w/w) hydration coincides with the onset of enzymatic activity, as observed by Yang & Rupley (1979). Recent X-ray studies by Artymiuk et al. (unpublished results) have investigated the nature of the binding of water in crystals of tortoise egg-white lysozymr and human lysozyme. A total of 111 water molecules was detected for tortoise egg-\+++ lysozyme with 31 of them making two or more hydrogen-bonds to the protein. For human lysozyme of the 143 waters detected, 34 were found t,o be bound in this way. Our estimate of 36 tightly bound waters for hen egg-white lysozvme compares well with these X-ray data. Artymiuk et al. (unpublished results) have proposed t,hat, the hydration can be categorized in terms of water that forms multiple or single hydrogen bonds and our dielectric studies fully support this viewpoint. Our results also suggest that after the addition of 100 water molecules to dry lysozymr flIrther hydration acts as a plasticizer to “loosen” the protein st,ructure. and that this must occur before enzymatic activity can be realized. This work was supported by the National Foundation for (:ancrr Research. We ark grateful to members of the Oxford Enzyme Group and, in particular to Sir David Phillips, Drs 1’. .I. Artymiuk, and C. C. F. Blake, and W. C. A. Pulford for valuable discussions and for access to their X-ray data before publication. School of Electronic Engineering Science University College of North Wales Dean Street, Bangor, Gwynedd, LLS7 IUT, Wales
s. H01w R. Pethig
REFERENCES Bone, S., Gaacoyne, P. R. C. & Pethig, R. (1977) J. Chem. Sot. Faraday Trans. 1611. Careri, G., Giansanti, A & Gratton, E. (1979). Biopotymers. 18, 1187-1203.
I, 73, lciO&
LETTER
TO THE
EDITOR
b75
Careri, G., Gratton, E., Yang, P-H. & Rupley, J. A. (1980). %ture (London), 284, 572-573. Davies. M. (1965). In Some Elrctrienl and Optirx~l ./1spects of Molecular Behaviour. p. 73. Pergamon Press, Oxford. Eden. J., Gascoyne, P. R. C. & Pethig, R. (1980). J. Chem. Sot. Faraday Trans. I, 76, 426.434. Eisenberg, D. bz Kauzmann, W. (1969). In The Structure and Properties of Il.&r. p. 12. Clarendon Press. Oxford. Gascoyne, P. R. C. &, Pethig, R. (1977). J. Chem. Sot. Faraday Trans. I, 73, 171-180. Gascoyne, P. R. C. & Pethig, R. (1981). J. Chem. Sot. Faraday Trans. I., 77, 1733-1735. (iascoyne, P. R. C., Pethig, R. & Szent-GyiSrgyi, A. (1981). Proc. 2Vat. Acox!. Sci., U.S.A. 78, 261-26.5. Tmoto, T., Johnson, L. N., North, A. C. T., Phillips, D. C. & Rupley, J. A. (1972). In The Enzymes (Boyer, P. D., ed.), pp. 665-868, Academic Press, New York. Pet,hig, R. (1979). Lkeleetrie and Electronic Properties of Biobgicat Materiab. J. Wiley and Sons, Chichester. Yang, P.-H. & Rupley, J. A. (1979). Biochemistry, 18, 2654-2661. Edited
by R. Huber
LETTER TO THE EDITOR
Dielectric Studies of the Binding of Water to Lysozyme Dielectric dispersion measurements as a function of hydration are reported for lysozyme powder. The dispersion that occurs in the frequency range 10 kHz to 10 GHz can be analysed in terms of bound water molecules that form single or multiple hydrogen bonds, and the numbers found in these two categories agree well with recent X-ray data for lysozyme crystals. The dielectric data also indicate that at 20% (w/w) hydration the bound water acts as a plasticizer to increase the vibrational freedom of the protein structure, and that this may be of relevance to the fact that the onset of enzymatic activity occurs at this hydration level. Also, a sudden transition in the polarizability of the protein-water system is found to occur at 7% (w/w) hydration.
Knowledge of the ways in which water molecules bind to proteins is of relevance to studies of the dynamics of protein conformation and enzyme activity. As a consequence of each water molecule possessing a relatively large electric dipole moment the nature of the protein-water interaction, in terms of the rotational freedom of the bound water, can be investigated by measuring the dielectric polarizability of a hydrated protein. Such measurements are reported here for lysozyme. Lysozyme is a particularly stable protein (Imoto et al., 1972). There is evidence to show that its conformation in a dry powder preparation is similar to that when in solution (Careri et al., 1979; Yang & Rupley, 1979) and when partially dehydrated it continues to exhibit enzymatic activity (Yang & Rupley, 1979). Dielectric measurements on Iysozyme powders of low hydration should, therefore, be of biological relevance, and in working with powder preparations rather than with protein solutions there is the added experimental advantage of avoiding effects associated with protein molecule vibrations and counter-ion relaxation effects (Pethig, 1979). Estimates are presented here of the extent to which water molecules are either rotationally or tightly bound to the protein structure, and the results are in good agreement with the recent X-ray data of P. J. Artymiuk, C. C. F. Blake & W. C. A. Pulford (unpublished results) for lysozyme crystals, where the bound water was categorized as forming either single or multiple hydrogen bonds. The dielectric measurements also support the conclusions of Careri et al. (1980) regarding the redistribution of water at 7% (w/w) hydration and the associated changes that occur in the vibrational freedom of the protein-water system. Hen
dialysed
egg-white
lysozyme,
obtained
against water, freeze-dried
1-3x 10M4 m2, thickness
of the order
from
Sigma
Chemicals,
was thoroughly
and compressed into discs of surface area 15x
10T3 m and density
890+20kg
rne3.
Dielectric measurements in the frequency range low4 Hz to 1 MHz, and at. 9.95 GHz, were made as a function of hydration using techniques and methods of 551 Om2-283ti/82/!50589%05
003.00/O
0
1982 Academic
Press Inc.
(London)
Ltd.
S. BOSE
572
AXI)
It. I’E’I’HI(:
analysis described elsewhere (Eden et al., 1980; HOW it ~1.. 1977) for work on IX)\-incs serum albumin. A dielectric dispersion. extending over t,he frequency range IO kHz to 10 GHz and centred at’ around 10 MHz. was observed for the lysozyme samples and had essentially the same charact,eristics as t,hosr that havfb been described for the dispersion observed for bovine serum albumin (Gascoyne & Pethig, 1981). The magnitude de of this dispersion is determined as:
Ar = El -<*.
(11
where c1 and c2 are the values of the relativcl permittivity determined at 10 kHz and 9.95 GHz, respectively. The value for AC varied with hydration as shown in Figure 1. The hydration content was det,ermined using a sensitive quart)z-crystal microbalance technique (Gascoyne & Pethig, 1977) and t,he zero hydration levt4 corresponded to the situation where the lysozyme samples were in equilibrium wit,h a vacuum of the order of 6 x IO-’ N m -* at 293 K. On heating to 377 Ii for three hours the “dry” lysozyme samples reversibly lost about, 05O/,, of t,heir weighty, of Pigure I each lysozymr molecult suggesting that,, at the zero hydration contained approximately four water molecules tightly bound into its structure This number can be compared to the three water molrc*ulrs that appear to 1~ trapped within the polypeptide structure of lysozymr (lmoto d al., 1972). ‘I%P AC = I .35 that exists for dry lSvsozymt, can. as underlying dispersion of magnitude described for the case of bovine serum albumin (Ciascoyne et 01.. 19X1 ), be explained in terms of vibrations of the constituent dipolar peptide units and associated sidrchains of the protein structure. The increase of AC with h.vdrat’ion r&e&s the increase in polarizability of the protein samples. dur to the increasing content of
0
5
IO h(% (w/w)
I5 H,O)
FIG. 1. Variation of de. the diffiirenw in permittivity at 10 kHz anti 995 GHz. its a function hydration for 3 different samples of hen egg-white Iysozymr.
of’
LETTER
TO
THE
573
EDITOR
dipolar water molecules, and also to any changes that might occur in the vibrational freedom of the protein structure. For dielectric materials such as proteins of low hydration, the Onsager correction for the local electric field can be considered to be relevant, so that the effective moment VEof the dipoles responsible for the dielectric dispersion is given as (see e.g. Davies, 1965) : (%-%)P%+~,) %(Ecc +v2
_ ,v?ii2
(2)
9q-,kT ’
where N is the number density of dipoles, c0 is the permittivity of free space, kT is the Koltzmann energy and for our purposes l s and Ed refer to the relative permittivity values at 10 kHz and 9.95 GHz, respectively. (In the work of Gascoyne & Pethig (1981), a typographical error occurred in this equation.) The number density, N, of bound water molecules in the lysozyme sample is given by:
where X, is Avogadro’s number, h is the percentage (w/w) hydration and d is the density of the sample. In Figure 2 the left-hand side of equation (2) is plotted against A’ of equation (3). Up to a hydration level corresponding to, on average, 32 water molecules being added to each lysozyme molecule, the values of AC andf(E) (1.h.s. of eqn (2)) of Figures 1 and 2 are relatively insensitive to the degree of hydration, which suggests that each of these 32 water molecules is bound to the protein structure by two or more hydrogen bonds in such a way as to prevent them
I 50
I 0
I I00
No. of H,O molecules/lysozyme FIG. 1. Variation off(~), samples of Fig. 1. The water molecule.
the left-hand concentration.
I 150 molecule
side of eqn (2), aa a function of hydration for the lysozyme A’. of eqn (3) has ken reealculatcd as molecules per lysozyme
,574
S. BONE ANI) R. PE’THI(:
from rotating. These water molecules, together with the four that remain tightly bound after our dehydration procedure, can be envisaged to be tightly incorporated into the vibrating protein structure, with relaxation times equivalent to those of the protein. For the hydration region corresponding to the situation when there arc between 32 and 101 water molecules associated with each lysozyme molecule. the gradient of Figure 2 corresponds to the effective dipole moment ~fi for each water molecule having a value of 5.79 x 10S3’ Cm (1.74 Dehye). This moment, can 1~ compared to the value 6.14 x lo- 3o Cm (I.84 Debve) for normal bulk water (Eisenberg & Kauzmann, 1969) and suggests that each water molecule in this group (about 69 in number) has one hydrogen bond link either to the protein or to another water molecule and is able to rotate in a relatively unhindered manner. At a hydration of 7% (w/w) corresponding to 57 water molecules being added t,o thrl enzyme, there is a sudden discontinuity in the plots of AE andf(E). This coincides with discontinuities observed in the infrared absorption and specific heat by (‘arcri et al. (1980), which were interpreted by them as a redistribution of protons betnecAn the carboxylic acid and basic groups. For hydration levels above, about I%..‘iU,, (w/w) the superlinear increase of AC and!(r) indicates that correlation effects are developing between absorbed water molecules, or that there is an increase in motional freedom of the bound water molecules and of the protein structure. All of these effects probably occur and it is of interest to note that the region where thrsc effects become significant at 200?, (w/w) hydration coincides with the onset of enzymatic activity, as observed by Yang & Rupley (1979). Recent X-ray studies by Artymiuk et al. (unpublished results) have investigated the nature of the binding of water in crystals of tortoise egg-white lysozymr and human lysozyme. A total of 111 water molecules was detected for tortoise egg-\+++ lysozyme with 31 of them making two or more hydrogen-bonds to the protein. For human lysozyme of the 143 waters detected, 34 were found t,o be bound in this way. Our estimate of 36 tightly bound waters for hen egg-white lysozvme compares well with these X-ray data. Artymiuk et al. (unpublished results) have proposed t,hat, the hydration can be categorized in terms of water that forms multiple or single hydrogen bonds and our dielectric studies fully support this viewpoint. Our results also suggest that after the addition of 100 water molecules to dry lysozymr flIrther hydration acts as a plasticizer to “loosen” the protein st,ructure. and that this must occur before enzymatic activity can be realized. This work was supported by the National Foundation for (:ancrr Research. We ark grateful to members of the Oxford Enzyme Group and, in particular to Sir David Phillips, Drs 1’. .I. Artymiuk, and C. C. F. Blake, and W. C. A. Pulford for valuable discussions and for access to their X-ray data before publication. School of Electronic Engineering Science University College of North Wales Dean Street, Bangor, Gwynedd, LLS7 IUT, Wales
s. H01w R. Pethig
REFERENCES Bone, S., Gaacoyne, P. R. C. & Pethig, R. (1977) J. Chem. Sot. Faraday Trans. 1611. Careri, G., Giansanti, A & Gratton, E. (1979). Biopotymers. 18, 1187-1203.
I, 73, lciO&
LETTER
TO THE
EDITOR
b75
Careri, G., Gratton, E., Yang, P-H. & Rupley, J. A. (1980). %ture (London), 284, 572-573. Davies. M. (1965). In Some Elrctrienl and Optirx~l ./1spects of Molecular Behaviour. p. 73. Pergamon Press, Oxford. Eden. J., Gascoyne, P. R. C. & Pethig, R. (1980). J. Chem. Sot. Faraday Trans. I, 76, 426.434. Eisenberg, D. bz Kauzmann, W. (1969). In The Structure and Properties of Il.&r. p. 12. Clarendon Press. Oxford. Gascoyne, P. R. C. &, Pethig, R. (1977). J. Chem. Sot. Faraday Trans. I, 73, 171-180. Gascoyne, P. R. C. & Pethig, R. (1981). J. Chem. Sot. Faraday Trans. I., 77, 1733-1735. (iascoyne, P. R. C., Pethig, R. & Szent-GyiSrgyi, A. (1981). Proc. 2Vat. Acox!. Sci., U.S.A. 78, 261-26.5. Tmoto, T., Johnson, L. N., North, A. C. T., Phillips, D. C. & Rupley, J. A. (1972). In The Enzymes (Boyer, P. D., ed.), pp. 665-868, Academic Press, New York. Pet,hig, R. (1979). Lkeleetrie and Electronic Properties of Biobgicat Materiab. J. Wiley and Sons, Chichester. Yang, P.-H. & Rupley, J. A. (1979). Biochemistry, 18, 2654-2661. Edited
by R. Huber