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A new experimental method for the direct determination of the water content of protein crystals
J. Mol. Bid. (1972) 71, 369-813
A New Experimental Method for the Direct Determination of the Water Content of Protein Crystals A new experimental method for measuring directly the water content of protein crystals is described iu which a microbalance with sensitivity of 1 pg is used to record continuously the weight variations of a protein crystal as a function of time under drying agents or on heating. This procedure leads to precise aud reproducible results either on large or small crystals. The method was verii%d on several types of lysozyme crystals. In a study of the crystal structure of a protein by X-ray diffraction, it is very important to be able to determine directly the water content of the crystals. Important facts which must be known at the beginning of such a study are the number of molecules in the unit-cell and, eventually, the number of subunits in the molecule. The only way to obtain a precise value of the protein content of the unit-cell is to determine previously the water content of the crystal. The weight of protein in the unit-cell has often been computed indirectly using the volume of the unit-cell, the molecular weight determined by biochemical methods and the measured density of the crystal. These data must be corrected by using an empirical coefficient, which is sometimes the apparent volume in A3 of one unit of molecular weight (1 dalton) of protein (Matthews, 196&z) in the crystal saturated by the mother liquor and sometimes the “partial specific volume” of the protein, i.e. the real volume of 1 g of protein in the crystal, the liquor which surrounds it not being included (Poljak & Dir&is, 1966; Magdoff-Fairchild, Lodl & LOW, 1969; Matthews, 19683). But these empirical ooefllcients which are obtained from previously determined protein structures may vary within rather broad limits when the nature of the protein changes and the molecular weight determinations by biochemical methods are sometimes largely in error. In consequence of these two facts, the indirect methods may, iu certain circumstances, lead to ambiguous results, as regards the number of molecules contained in the unit-cell or the number of subunits in the molecule. The direct measurements of the water content of protein crystals by desiccation should give a more precise determination of the protein content of the unit-cell. If the density of the crystal has been measured, the weight of the content of the unitcell is known. We can then obtain the protein content by subtracting from the total weight (a) the weight of water measured by desiccation, and (b) the weight of salt contained in the mother liquor. This can be estimated according to the method described by North (1969), for example. Mention may be made here that the evaluation of the salt content raises some diillculties due to the fact that a monomolecular layer of water bound to the surface of the protein molecule seems to be impermeable to salt ions (Peru@ 1946). This direct method has been used by many authors (North, 1969; Swan, 1970). It gives rise to difficulties with very small crystals which, when taken out of their mother liquor, lose water very quickly, so that it is not possible to distinguish between the 809
810
J. BERTHOU,
F. CESBRON
AND
A. LAURENT
evaporation of the mother liquor on the surface of the crystal and that enclosed in the crystal. There results a lack of precision in the initial weight, which is more important when the crystal is small (i.e. the surface/volume ratio is large). The method described here is precise and can also be applied to small monocrystals (from O-5 mg to 10 mg) or even to masses of very small crystals (of the order of 0.1 mg each). The error is, however, greater in the second case due to the small quantity of mother liquor which is retained between the crystals by capillarity. This method consists essentially in the continuous recording of the weight variations of a monocrystal by means of a Cahn R. G. microbalance, the sensitivity of which is approximately 1 pg. Our early experiments showed us that if the crystal separated from its mother liquor dehydrates very rapidly, it can also rehydrate very easily. We, thus, defined the following procedure: the crystal was carefully dried on filter paper and placed in the platinum crucible suspended from the beam of the balance by a silica fibre, within a closed Pyrex vessel (Fig. 1). During these manipulations the dehydration has already
C
G
F
FIG. 1. Experimental 8ppCAdUS: A, Beam of the balance; B, Pyrex vessel; C, Pyrex tube with ground-glass joint and 0 ring; D, platinum crucible (38 mg); E, W8ter, mother liquor or drying agent; F, rubber bung; G, silica fibre (Q 50 p).
begun (part AB of the theoretical curve) but it is possible to weigh the crystal at any moment after a previous calibration of the balance. A glass tube filled with distilled water was placed under the platinum crucible; a very fast rehydration was then observed (BC), the partial pressure of water being very high. When the increase of weight is sufficient (a few per cent of the starting weight) the water was replaced by the mother liquor. We then observed either an increase of the weight, slower if the initial
LETTERS
TO THE
EDITOR
811
loss has been important, or a loss of weight if the rehydration in the saturated atmosphere has been oarried too far. In both cases the system tends towards an equilibrium (Fig. 2, CD) corresponding to the maximum rehydration of the crystal at a given temperature and concentration of the mother liquor. The crystal in equilibrium with its mother liquor was then carefully weighed. The balance being of zero type, the weighing consists in measuring the electric current which maintains the beam horizontal. We observed that this equilibrium was very sensitive to room temperature variations. In many cases, we observed a variation of 1% of the weight per degree between 20 and 24°C. This observation points out the importance of having a constant room temperature, or, better, the whole apparatus in a thermostat.
Time
FIG. 2. Example of dehydration curve obtained.
The starting weight of the crystal having been carefully measured, the dehydration can be carried out in many ways: (1) isothermally, at room temperature in air or in the presence of a drying agent (P,O,; Mg(ClO&J. With P,O, the partial pressure is roughly 10Ts mm of Hg (e.g. Fig. 2, DE); (2) on heating, with a programmed furnace around the tube, a thermocouple being placed under the crucible. To avoid the decomposition of the protein the maximum temperature is f&d at 110°C. Many experiments showed that with a powerful drying agent such as P,Os the dehydration was very fast and almost complete within a few hours. When the crystals were large the equilibrium was reached more slowly and it was necessary to heat to achieve the dehydration. In this case, we observed a supplementary loss of 1% of weight (Fig. 2, EF). The theoretical curve (Fig. 2) clearly shows the successive stages of the dehydration, In the following only the total weight losses are given, The time elapsed for each experiment varied from about 6 hours when heating was carried out throughout, to two days for isothermal dehydration of a large crystal at, say, 22°C. Table 1 summarizes results of measurements on singk? cr@&h!8 of lysozyme, both hen egg white lysozyme HCl in either the tetragonal (A in this note) or the orthorhombic (B) forms and, duck egg white lpozyme, type II, which is monoclinic. We studied this class of enzymes tist because we had a large amount of this material and, secondly, the tetragonal form having been well studied by Blake et al. (19&j), it was possible to verify and compare our results. Furthermore, it was of value to know if polymorphic crystals had large or small differences in their respective water content. 62
812
J. BERTHOU,
F. CESBRON
AND
A. LAURENT
TABUU 1 water coratcnt of 8onM lysozy?ne cy6tal8 Nature
of CrySt8h
Starting weight (h mf3)
:P
Loss of weight
Meen lose in y0
32.5
I II III
0.681 I.499 1.171
32.2 32.6 32.6
I II III Iv Duckegg white I lyeozyme II II
Q-030 7.868 6.203 5.077 1.706 l-010
36.3 36.5 35.6 36.9 27.9 28-4
Hen egg white 1ysozymeA Hen egg white lyeozyme B
36.1
28.1
Crystals are of hen egg white lyeozyme HCl, either tetragonal P4,2,2 (A form (a = 79-l; P212,21 (B form) a = 56.3; b = 73.8; c = 30.4 or of duck egg white c = 37.9 A) or orthorhombic lysozyme type II monoclinic P21 (a = 28.6; b = 66.2; c = 32-l; /I = 113’C). All experiments were carried out at 22’C.
The single cry&& weighed between 0.6 mg and 10 mg and their length reached 5 mm. The study of the B crystals, ofwhich we had large amounts, showed that crystals of this weight and size gave reproducible results regardless of the method used (dehydration under P,Os or on heating), i.e. whether the dehydration was slow or fa.st. The experience gained enabled us to estimate the water content of duck egg white lysozyme from only two measurements. The water content of the A crystals is 32*5%, a value very similar to the estimation of Blake et al. (1966). The B crystals obtained at higher temperature (up to 66’%) (Jolles & Berthou, 1972) have a greater water content (36% against 32.6%). It does not seem to be a general rule that a higher temperature of crystal growth is associated with a higher water content, for duok egg white crystals which can be obtained at 4”C, 20°C and 37°C have a water content of 28%. The respective values of measured densities of these three types of crystals are 1.26, 1.24, 1.27 g crnm3 in complete agreement with their water content, and the crystallographic data. One of the interesting phenomena observed on dehydration curves is the part EF. It represents 1 y. of the total loss of weight and appears when one heats the crystal up to llO”C, after dehydration under P,O,, for example. Does it represent another type of water, more strongly bonded to the surface of the molecule? It would be of value to know its exact nature. We intend to carry out a series of isothermal dehydrations at different temperatures in order to clear up this problem, bearing in mind that the mother solution may contain salts unstable even at 110”(rammonium salts, for example-which must be taken into account in the estimation of the loss of weight. A second series of experiments dealt with &ycyti. Very often, indeed, protein crystals are rather small (< O-6 mm), so we measured the loss of weight of masses of miorocrystals, the mass of which lay between 3 and 5 mg. In the csse of the A form, for example, the value of the water content reaohes 36%, instead of 32.6%. This overestimation is obviously due to the fact that a oertain quantity of mother liquor remains between the orystals by oapillarity. We think that it is possible to reduce this diver-
LETTERS
TO THE
EDITOR
313
gence if the individual drying of each crystal is possible and to reduce the total weight to about 1 mg. In conclusion, the results obtained on this type of crystal show that this very simple new direct experimental method, using a commercial apparatus, can be applied to measure the water content of any protein crystal. It gives direct, reproducible and precise results. The method is fast and in theory two measurements may be suflicient. It has the advantage that the crystal can be observed at any moment and its macroscopic properties (geometry, transparence, oolour) followed during the experiment. Laboratoire de Min&alogie Crietallographie de l’Universit6 de Paris Tour 16, 9 quai St Bernard Paris V”, France
J. BERTEOTJ F. CESBRON A. LAURENT
Received 29 June 1972 REFERENCES Blake, C. C. F., Koenig, D. F., Muir, G. A., North, A. C. T., Phillips, D. C. & Sarma, V. R. (1966). Nature, 206, 757. Jolles, P. & Berthou, J. (1972). FEBS Lettere, 23, 21. Magdoff-Fairchild, B., Love& F. M. t Low, B. W. (1969). J. Bid. Chews. 244, 3497. Matthews, B. W. (190&). J. Mol. Bid. 33, 491. Matthews, B. W. (19686). J. Mol. Bid. 33, 499. 12, 612. North, A. C. T. (1959). Acta c9y8t. Perutz, M. F. (1946). Disc. Faraday Sot. B,42, 187. Poljak, R. L. & Dintzis, H. M. (1966). J. Mol. Bid 7, 646. Swan, I. D. A. (1970). Ph.D. Tbeais, Oxford University.
A New Experimental Method for the Direct Determination of the Water Content of Protein Crystals A new experimental method for measuring directly the water content of protein crystals is described iu which a microbalance with sensitivity of 1 pg is used to record continuously the weight variations of a protein crystal as a function of time under drying agents or on heating. This procedure leads to precise aud reproducible results either on large or small crystals. The method was verii%d on several types of lysozyme crystals. In a study of the crystal structure of a protein by X-ray diffraction, it is very important to be able to determine directly the water content of the crystals. Important facts which must be known at the beginning of such a study are the number of molecules in the unit-cell and, eventually, the number of subunits in the molecule. The only way to obtain a precise value of the protein content of the unit-cell is to determine previously the water content of the crystal. The weight of protein in the unit-cell has often been computed indirectly using the volume of the unit-cell, the molecular weight determined by biochemical methods and the measured density of the crystal. These data must be corrected by using an empirical coefficient, which is sometimes the apparent volume in A3 of one unit of molecular weight (1 dalton) of protein (Matthews, 196&z) in the crystal saturated by the mother liquor and sometimes the “partial specific volume” of the protein, i.e. the real volume of 1 g of protein in the crystal, the liquor which surrounds it not being included (Poljak & Dir&is, 1966; Magdoff-Fairchild, Lodl & LOW, 1969; Matthews, 19683). But these empirical ooefllcients which are obtained from previously determined protein structures may vary within rather broad limits when the nature of the protein changes and the molecular weight determinations by biochemical methods are sometimes largely in error. In consequence of these two facts, the indirect methods may, iu certain circumstances, lead to ambiguous results, as regards the number of molecules contained in the unit-cell or the number of subunits in the molecule. The direct measurements of the water content of protein crystals by desiccation should give a more precise determination of the protein content of the unit-cell. If the density of the crystal has been measured, the weight of the content of the unitcell is known. We can then obtain the protein content by subtracting from the total weight (a) the weight of water measured by desiccation, and (b) the weight of salt contained in the mother liquor. This can be estimated according to the method described by North (1969), for example. Mention may be made here that the evaluation of the salt content raises some diillculties due to the fact that a monomolecular layer of water bound to the surface of the protein molecule seems to be impermeable to salt ions (Peru@ 1946). This direct method has been used by many authors (North, 1969; Swan, 1970). It gives rise to difficulties with very small crystals which, when taken out of their mother liquor, lose water very quickly, so that it is not possible to distinguish between the 809
810
J. BERTHOU,
F. CESBRON
AND
A. LAURENT
evaporation of the mother liquor on the surface of the crystal and that enclosed in the crystal. There results a lack of precision in the initial weight, which is more important when the crystal is small (i.e. the surface/volume ratio is large). The method described here is precise and can also be applied to small monocrystals (from O-5 mg to 10 mg) or even to masses of very small crystals (of the order of 0.1 mg each). The error is, however, greater in the second case due to the small quantity of mother liquor which is retained between the crystals by capillarity. This method consists essentially in the continuous recording of the weight variations of a monocrystal by means of a Cahn R. G. microbalance, the sensitivity of which is approximately 1 pg. Our early experiments showed us that if the crystal separated from its mother liquor dehydrates very rapidly, it can also rehydrate very easily. We, thus, defined the following procedure: the crystal was carefully dried on filter paper and placed in the platinum crucible suspended from the beam of the balance by a silica fibre, within a closed Pyrex vessel (Fig. 1). During these manipulations the dehydration has already
C
G
F
FIG. 1. Experimental 8ppCAdUS: A, Beam of the balance; B, Pyrex vessel; C, Pyrex tube with ground-glass joint and 0 ring; D, platinum crucible (38 mg); E, W8ter, mother liquor or drying agent; F, rubber bung; G, silica fibre (Q 50 p).
begun (part AB of the theoretical curve) but it is possible to weigh the crystal at any moment after a previous calibration of the balance. A glass tube filled with distilled water was placed under the platinum crucible; a very fast rehydration was then observed (BC), the partial pressure of water being very high. When the increase of weight is sufficient (a few per cent of the starting weight) the water was replaced by the mother liquor. We then observed either an increase of the weight, slower if the initial
LETTERS
TO THE
EDITOR
811
loss has been important, or a loss of weight if the rehydration in the saturated atmosphere has been oarried too far. In both cases the system tends towards an equilibrium (Fig. 2, CD) corresponding to the maximum rehydration of the crystal at a given temperature and concentration of the mother liquor. The crystal in equilibrium with its mother liquor was then carefully weighed. The balance being of zero type, the weighing consists in measuring the electric current which maintains the beam horizontal. We observed that this equilibrium was very sensitive to room temperature variations. In many cases, we observed a variation of 1% of the weight per degree between 20 and 24°C. This observation points out the importance of having a constant room temperature, or, better, the whole apparatus in a thermostat.
Time
FIG. 2. Example of dehydration curve obtained.
The starting weight of the crystal having been carefully measured, the dehydration can be carried out in many ways: (1) isothermally, at room temperature in air or in the presence of a drying agent (P,O,; Mg(ClO&J. With P,O, the partial pressure is roughly 10Ts mm of Hg (e.g. Fig. 2, DE); (2) on heating, with a programmed furnace around the tube, a thermocouple being placed under the crucible. To avoid the decomposition of the protein the maximum temperature is f&d at 110°C. Many experiments showed that with a powerful drying agent such as P,Os the dehydration was very fast and almost complete within a few hours. When the crystals were large the equilibrium was reached more slowly and it was necessary to heat to achieve the dehydration. In this case, we observed a supplementary loss of 1% of weight (Fig. 2, EF). The theoretical curve (Fig. 2) clearly shows the successive stages of the dehydration, In the following only the total weight losses are given, The time elapsed for each experiment varied from about 6 hours when heating was carried out throughout, to two days for isothermal dehydration of a large crystal at, say, 22°C. Table 1 summarizes results of measurements on singk? cr@&h!8 of lysozyme, both hen egg white lysozyme HCl in either the tetragonal (A in this note) or the orthorhombic (B) forms and, duck egg white lpozyme, type II, which is monoclinic. We studied this class of enzymes tist because we had a large amount of this material and, secondly, the tetragonal form having been well studied by Blake et al. (19&j), it was possible to verify and compare our results. Furthermore, it was of value to know if polymorphic crystals had large or small differences in their respective water content. 62
812
J. BERTHOU,
F. CESBRON
AND
A. LAURENT
TABUU 1 water coratcnt of 8onM lysozy?ne cy6tal8 Nature
of CrySt8h
Starting weight (h mf3)
:P
Loss of weight
Meen lose in y0
32.5
I II III
0.681 I.499 1.171
32.2 32.6 32.6
I II III Iv Duckegg white I lyeozyme II II
Q-030 7.868 6.203 5.077 1.706 l-010
36.3 36.5 35.6 36.9 27.9 28-4
Hen egg white 1ysozymeA Hen egg white lyeozyme B
36.1
28.1
Crystals are of hen egg white lyeozyme HCl, either tetragonal P4,2,2 (A form (a = 79-l; P212,21 (B form) a = 56.3; b = 73.8; c = 30.4 or of duck egg white c = 37.9 A) or orthorhombic lysozyme type II monoclinic P21 (a = 28.6; b = 66.2; c = 32-l; /I = 113’C). All experiments were carried out at 22’C.
The single cry&& weighed between 0.6 mg and 10 mg and their length reached 5 mm. The study of the B crystals, ofwhich we had large amounts, showed that crystals of this weight and size gave reproducible results regardless of the method used (dehydration under P,Os or on heating), i.e. whether the dehydration was slow or fa.st. The experience gained enabled us to estimate the water content of duck egg white lysozyme from only two measurements. The water content of the A crystals is 32*5%, a value very similar to the estimation of Blake et al. (1966). The B crystals obtained at higher temperature (up to 66’%) (Jolles & Berthou, 1972) have a greater water content (36% against 32.6%). It does not seem to be a general rule that a higher temperature of crystal growth is associated with a higher water content, for duok egg white crystals which can be obtained at 4”C, 20°C and 37°C have a water content of 28%. The respective values of measured densities of these three types of crystals are 1.26, 1.24, 1.27 g crnm3 in complete agreement with their water content, and the crystallographic data. One of the interesting phenomena observed on dehydration curves is the part EF. It represents 1 y. of the total loss of weight and appears when one heats the crystal up to llO”C, after dehydration under P,O,, for example. Does it represent another type of water, more strongly bonded to the surface of the molecule? It would be of value to know its exact nature. We intend to carry out a series of isothermal dehydrations at different temperatures in order to clear up this problem, bearing in mind that the mother solution may contain salts unstable even at 110”(rammonium salts, for example-which must be taken into account in the estimation of the loss of weight. A second series of experiments dealt with &ycyti. Very often, indeed, protein crystals are rather small (< O-6 mm), so we measured the loss of weight of masses of miorocrystals, the mass of which lay between 3 and 5 mg. In the csse of the A form, for example, the value of the water content reaohes 36%, instead of 32.6%. This overestimation is obviously due to the fact that a oertain quantity of mother liquor remains between the orystals by oapillarity. We think that it is possible to reduce this diver-
LETTERS
TO THE
EDITOR
313
gence if the individual drying of each crystal is possible and to reduce the total weight to about 1 mg. In conclusion, the results obtained on this type of crystal show that this very simple new direct experimental method, using a commercial apparatus, can be applied to measure the water content of any protein crystal. It gives direct, reproducible and precise results. The method is fast and in theory two measurements may be suflicient. It has the advantage that the crystal can be observed at any moment and its macroscopic properties (geometry, transparence, oolour) followed during the experiment. Laboratoire de Min&alogie Crietallographie de l’Universit6 de Paris Tour 16, 9 quai St Bernard Paris V”, France
J. BERTEOTJ F. CESBRON A. LAURENT
Received 29 June 1972 REFERENCES Blake, C. C. F., Koenig, D. F., Muir, G. A., North, A. C. T., Phillips, D. C. & Sarma, V. R. (1966). Nature, 206, 757. Jolles, P. & Berthou, J. (1972). FEBS Lettere, 23, 21. Magdoff-Fairchild, B., Love& F. M. t Low, B. W. (1969). J. Bid. Chews. 244, 3497. Matthews, B. W. (190&). J. Mol. Bid. 33, 491. Matthews, B. W. (19686). J. Mol. Bid. 33, 499. 12, 612. North, A. C. T. (1959). Acta c9y8t. Perutz, M. F. (1946). Disc. Faraday Sot. B,42, 187. Poljak, R. L. & Dintzis, H. M. (1966). J. Mol. Bid 7, 646. Swan, I. D. A. (1970). Ph.D. Tbeais, Oxford University.