Eur. J. Biochem. 77, 107-111 (1977)
Dimerization of Papain Induced by Mercuric Chloride and a Bifunctional Organic Mercurial Lamoraal A. AE. SLUYTERMAN, Jacob WIJDENES, and Gysbertus VOORN Philips Research Laboratories, Eindhoven (Received February 22,1977)
The bifunctional mercurial meso-l,4-bis(acetatomercuri)-2,3-diethoxybutane and mercuric chloride are capable of dimerizing papain, by the attachment of the thiol group of two molecules of papain to each molecule of reagent. This is evident from the titration data, gel filtration and sedimentation equilibrium. The conformational change of papain necessary for this reaction is discussed. It has been shown recently [l] that one proton is released if one zinc ion or one methylmercuric ion is attached to the single essential thiol group of one papain molecule at pH 6. As will be reported in this paper, these experiments were repeated with mercuric chloride, a divalent metal which binds very strongly to thiol groups. The proton release proved to be different, in that two protons are released from two papain molecules on addition of one mercuric ion. It will be shown that this effect is due to the formation of a papain dimer. EXPERIMENTAL PROCEDURE Active papain, free from activators, was prepared in the following manner. A solution of 1 % mercuric papain, purified on the agarose-mercurial column [2], containing 10 mM phosphate, 20 mM dithiothreitol and 1 mM tetraethylenepentamine (Merck), pH 7.5, was stored for 10 min and concentrated to 10% of its initial volume, under nitrogen pressure, in a ‘Diaflo’ cell provided with a type-PM10 membrane (Aminco Corp.). The solution was readjusted to about the initial volume by addition of oxygen-free water containing 0.5 mM tetraethylenepentamine and 10 mM acetate pH 5.5 and concentrated again to 10% of the initial volume. Finally water was added containing 30 mM KCI and 0.1 mM acetate pH 5.5 and dialysis was carried out with the same solution in the ‘Diaflo’ Note. A preliminary report was presented at the 10th Meeting of the Federation of European Biochemical Societies, Paris, July 1975. Abbreviations. Reagent I, meso-l,4-bis(acetatomercuri)-2,3-diethoxybutane ; ClHgBzSO3, p-chloromercuribenzene sulphonate. Enzyme. Papain (EC 3.4.22.2).
cell. The emerging solution was checked for thiol compounds by adding 1 ml of 10 mM 5,5‘-dithio-bis(2nitrobenzoic acid) in 0.1 M phosphate pH 8.0 to 3 ml eluent and measuring the absorbance at 440 nm [ 3 ] . When the absorbance was < 0.05, corresponding to a thiol concentration in the eluent of < 8 pM, the papain solution was subjected to one more cycle of tenfold dilution with the KCl/acetate buffer and reconcentration. The final solution contained 1-2% (0.40.8 mM) papain. Titrations were carried out in a quartz vessel. The pH was read on a Philips digital pH meter. The calomel electrode was separated from the vessel by an agarose-containing salt bridge. The solution was gently stirred with a magnetic stirrer. The mercurials were added in increments of 0.1 - 1.O ml. All solutions were made in distilled deionized water and deaerated in vacuo. The organic mercurials were p-chloromercuribenzene monosodium sulphonate (ClHgBzS03) purchased from Sigma Chem. Corp. (St Louis) and meso1,4-bis(acetatomercuri)-2,3-diethoxybutane (reagent I) provided by Dr J. Drenth. The gel filtrations presented in Fig.2 and 3 were carried out in a type-K15/30 column of Pharmacia (Sweden). The height of the bed of Sephadex G-75 superfine (Pharmacia) was about 15 cm. Aliquots of 0.25 ml protein solution, to which 4 % sucrose has been added, were layered on the top of the bed under the supernatant elution buffer. The elution was effected with 0.1 M acetate pH 6.0. The solvent flow of 12 ml/h was adjusted with a peristaltic pump type ‘varioperpex’ of L.K.B. (Sweden). The transmittance at 280 nm of the effluent in a 3-mm cell was recorded with a Uvicord I1 of L.K.B. The transmittance data were read from the record, converted into absorbance
108
Dimerization of' Papain
and plotted (Fig.2). Since the optical pathway was 3 mm, the absorbance per cm path length was 3.3 times the measured absorbance. Sedimentation equilibria were measured in the Spinco model E ultracentrifuge of Beckman (U.S.A.) in an eight-channel short-column equilibrium center piece, in accordance with the method of Yphantis [4]. The partial specific volume of papain was taken as 0.723 ml/g. When this method is used, only a small difference between top and bottom concentrations occurs. I
A 1
RESULTS When a heavy metal compound combines with a non-ionized thiol group a proton is liberated : RSH
+ M + e RSM + H + .
This is evident in the reaction of glutathione with the monovalent reagent C1HgBzS03. Small aliquots of ClHgBzS03 were added to a solution of glutathione. The pH dropped after each addition and was readjusted to pH 5.9. The resulting titration curve is shown
I
I
I
RSH = glutathione
RSH=papain
.o
I
I
E
I
cn
[L
+-
I
0 1
.o
0
0
1
.o
0
1
.o
Mercurial/RSH (rnol/rnol)
Fig. 1. The liberation ofprorons on the addition ofmercuriuls to glutathione andpupain. Solvent for glutathione and reagents = 0.1 M KCI, 0.1 mM acetate. (A) 20.0 pmol glutathione in 15 ml solvent of pH 5.9 with 1.31 mM CIHgBzS03 pH 5.7. (B) 20.0 pmol glutathione in 15 ml solvent of pH 5.9 with 10.6 mM reagent I. (C) 19.5 pmol glutathione in 15 ml solvent of pH 5.5 with 6.67 mM HgC12. Solvent for papain and reagents 0.01 M KCI, 0.1 mM acetate. (D) 15.5 pmol papain in 25 ml solvent of pH 5.9 with 11.2 mM ClHgBzS03. (E) 15.5 pmol papain in 30 ml solvent of pH 6.1 with 6.0 mM reagent I. (F) 10.2 pmol papain in 30 ml solvent of pH 6.0 with 7.3 mM HgClz. The HgClz in excess of one mole of HgClz to one mole of papain (indicated by the arrow) gave a slow time-dependent liberation of protons. The same phenomenon was observed on addition of HgClz to papain with blocked thiol group. Readings were made after 30 s
109
L. A. AE. Sluyterman, J. Wijdenes, and G. Voorn
in Fig.1A. At the equivalence point one proton is liberated on addition of one molecule of ClHgBzS03 per molecule of glutathione. The results with papain and ClHgBzS03 were similar (Fig.1D). (For the present argument it is irrelevant whether the proton originates from the thiol group itself or from the neighbouring imidazole group 111.) On addition of one molecule of reagent I : AcHg - CH2 - CH - CH - CH2 - HgAc
I
I
CZHsO OCzH5 in which the mercury atoms can be about 0.6 nm apart, two protons are released from two molecules of glutathione (Fig.lB), and from two molecules of papain (Fig. 1 E). Similar results were obtained with HgC12 : two protons are liberated from two molecules of glutathione (Fig. 1C) and from two molecules of papain (Fig. 1F) on addition of one molecule of HgC12. All of these reactions are virtually instantaneous, i.e. they are certainly completed within 30 s, the time taken for the measurement of each titration point. Amperometric titration of small thiol compounds has indicated the following equilibria [ 5 ]: RSH Hg2+ S RSHg+ H + (1)
+ + RSH + RSHg' S RSHgSR + H + RSHgSR + Hg2+ 2 RSHg' .
(2) (3)
If reaction (2) occurs, one mercuric ion is capable of liberating two protons from two molecules of thiol compound, producing a dimer of the latter. When more HgC12 or reagent I is added after dimerization is complete, the reversion of dimer to monomer according to reaction (3) liberates no more protons. Since papain also carries a single thiol group the results of the titrations indicate dimerization. This was confirmed by the results of gel filtration and of ultracentrifugation. Solutions were made of HgCI2 and papain in ratios R of 0, 0.1, 0.5, 0.7 and 1.0. Aliquots of these solutions were put on a column of Sephadex G-75 and subjected to gel filtration. The results are shown in Fig.2. As R increases, a new peak of high elution rate appears, reaching a maximum when R = 0.5 and disappearing again when R = 1.O. Similar results were obtained with reagent I. These elution diagrams confirm the occurrence of equilibria (2) and (3): when the Hg: papain ratio is 0.5, equilibrium (2) predominates (Fig. 2 C) and when the ratio is 1.0, equilibrium (3) predominates. Complete dimerization will not be observed with this method owing to dilution during the run. Dilution will tend to shift equilibria (1) and (2) to the left and to remove the small fraction of free HgC12 by the dialysis action of such a column, displacing the equilibria
al c n m L
0
n Q
0: 0.; 0'
c
5 10 15 Elution vohme (mi)
20
Fig. 2. Elution profiles of papain with various concentrations of' HgC12. R = HgCIZ:papain
further to the left. This mechanism produces most of the monomer peak of curve C from the dimer peak during the run. This in its turn explains why the monomer peaks of curves C and D apparently travel at a slightly higher rate than the monomer peaks of curves A, B and C. A molecular weight determination of the compound of the first peak was attempted by the wellknown procedure of comparing its elution volume with that of other proteins [6]. The determination was complicated by the observation that standard papain exhibits a larger elution volume than expected from its molecular weight of 23400 (Fig. 3 and [7,8]). The same phenomenon is exhibited by ficin 171 and by Chinese gooseberry proteinase, as is apparent on comparison of the reported molecular weights [9,10]. Using the graphical procedure depicted in Fig. 3, the molecular weight of the dimer was estimated as 52000. In view of the uncertainties involved, this is reasonably near the expected value of 47000.
110
Dimerization of Papair
15
4.0
I
I
I
I
I
I
I
I
42
4.4
46
4.8
1
50
log Mr
Fig. 3. Elution volume of various proteins versus the logarithm of’ molecular weight. Proteins were: (1) cytochrorne c, (2) soybean trypsin inhibitor, (3) pepsin, (4) ovalbumin, ( 5 ) bovine serum albumin. (6) papain monomer, (7) papain dimer
Table 1. Sedimentation equilibrium of’ papain preparations in 0.1 M acetate p H 6.0, at 25 “C 0.5 Hg refers to a preparation of papain with an Hg: papain ratio of 0.5 Molecular weight
M , ratio, 0.5 Hg/ standard
Papain
Concentration
Standard 0.5 Hg
0.30 0.26
27000 53000
Standard 0.5 Hg
0.21 0.18
23000 46000
4000
2.00
Standard 0.5 Hg
0.10 0.10
21000 k 3000 43000 +_ 7000
2.05
x t
2000 1000
1.96
k 1000
Further confirmation was obtained from sedimentation equilibrium experiments in very short columns according to the procedure of Yphantis [4].Both standard papain and a preparation with an Hg:papain ratio of 0.5 were measured at a few protein concentrations in 0.1 M acetate pH 6.0. Although the results presented in Table 1 indicate some concentration dependence, similar to the data of Pandit and Narasingd Rao [ll], it is quite evident that mercuri-papain has double the molecular weight of standard papain. Both the mercury monomer and the mercury dimer of papain regain full activity on the addition of activators. DISCUSSION It can be concluded from the results of the titrations, the gel filtration and the sedimentation that mercuric ions are capable of dimerizing papain, when there is one H$ ion per two molecules of active papain. In 1954 Kimmel and Smith [12] proposed the existence of a papain dimer on the evidence that preparations of papain of potential activity c1 = 1.1 +
contained 0.47% Hg, i.e. about 0.5 Hg per molecule of papain. This claim was not supported by ultracentrifugation, reported in their next paper [13], which revealed a monomer at pH 4 and general aggregation at pH 8 (i.e. near the isoelectric point of 8.6 [13]) as ordinary papain does. Furthermore Finkle and Smith demonstrated in 1958 [14] that papain, prepared by the same method, is a mixture of active and nonactivatable papain and that the active species, containing one thiol group, must have an activity of CI = 2.2. The mercury papain of c1 = 1.1 of 1954, therefore, contained about 0.5 - SH group and therefore one Hg atom per molecule of active papain. The proposal made by Kimmel and Smith is therefore inconsistent with their later data. It is also contradictory with our results, because Kimmel and Smith [12] used an excess of HgClz in preparing mercury papain, which leads to monomers (cf. Fig.2). A decrease in Hg content from 1.10- 1.25 atom per molecule of M , 23400 to 0.60 atom per molecule on crystallisation is no doubt due to removal of traces of mercury mercaptide of thioglycolic acid, originating from the ion exchanger highly charged with this compound. The papain solution with its few counter ions was passed through this column as the last step before Hg analysis. In the X-ray analysis of papain [15] an excess of HgC12 was present, which meant that no dimer could have been formed, even if the crystal packing had allowed it. It is known from X-ray analysis of crystals [15] that the thiol group is located at the bottom of the cleft of the active centre, which is about 0.8 nm deep. A single mercuric ion cannot bridge a distance of 1.6 nm, even a molecule of reagent I is too short. Two explanations are possible : either the spatial structure of papain in crystals is not the same as in solution, or the structure of the enzyme is flexible enough locally to allow a conformational change to bring the thiol group to the surface. Papain inside crystals suspended in substrate solution was found to be as active towards two substrates as dissolved papain [16], indicating the same conformation of the active site in both phases. Hence a change of conformation is required to bring the thiol group of cysteine-25 to the surface. In this case, therefore, the occurrence of dimerization is a direct demonstration of a conformational change and of a certain flexibility of the active site. Examination of the model shows that little more than one turn of the central helix needs be unwound to bring the thiol group outside, with a number of changes in the preceding part of the main chain. It is not to be expected that such a small change in conformation will cause a significant change in circular dichroism. In fact, no such change was observed. Other methods of detecting conformational changes
311
L. A. AE. Sluyterman, J. Wijdenes, and G. Voorn
are not likely to distinguish between the effects of conformational change and these of dimerization. Only X-ray analysis can reveal the nature of the changes involved in the dimerization.
REFERENCES 1. Sluyterman, L. A. AE. & Wijdenes, J. (1976) Eur. J . Biochem. 71,383 - 391, 2. Sluyterman, L. A. AE. & Wijdenes, J. (1970) Biochim. Biophys. Acta, 200, 593 - 595. 3. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77. 4. Yphantis, D. A. (1960) Ann. N . Y . Acad. Sci. 88, 586-601. 5. Kolthoff, J. M., Stricks, W. & Morren, L. (1954) Anal. Chem. 26,366 - 372. 6. Andrews, P. (1970) Methods Biochem. Anal. 18, 1-53.
7. Fossum, K. & Whitaker, J. R. (1968) Arch. Biochem. Biophys. 125, 367 - 375. 8. Maggio, E. T. & Shafer, J. A. (1973) Anal. Biochem. 54, 616618. 9. McDowall, M. A. (1973) Biochim. Biophys. Acta, 293, 226231. 10. McDowall, M. A. (1970) Eur. J . Biochem. 14,214-221. 11. Pandit, M. W. & Narasinga Rao, M. S. (1974) Biochim. Biophys. Acta, 371, 211 -218. 12. Kimmel, J. R. & Smith, E. L. (1954) J . Biol. Chem. 207, 515531. 13. Smith, E. L., Kimmel, J. R. & Brown, D. M. (1954) J . B i d . Chem. 207,533- 549. 14. Finkle, B. J. & Smith, E. L. (1958) J . Bid. Chern. 230, 669690. 15. Drenth, J., Jansonius, J. N., Koekoek, R. & Wolthers, B. G. (1971) Adv. Protein Chem. 25, 79-115. 16. Sluyterman, L. A. AE. & de Graaf, M. J. M. (1968) Biochim. Biophys. Acta, 171, 277-287.
L. A. AE. Sluyterman, J. Wijdenes, and G. Voorn, Natuurkundig Laboratorium, N.V. Philips Gloeilampenfabrieken, Eindhoven, The Netherlands
Dimerization of Papain Induced by Mercuric Chloride and a Bifunctional Organic Mercurial Lamoraal A. AE. SLUYTERMAN, Jacob WIJDENES, and Gysbertus VOORN Philips Research Laboratories, Eindhoven (Received February 22,1977)
The bifunctional mercurial meso-l,4-bis(acetatomercuri)-2,3-diethoxybutane and mercuric chloride are capable of dimerizing papain, by the attachment of the thiol group of two molecules of papain to each molecule of reagent. This is evident from the titration data, gel filtration and sedimentation equilibrium. The conformational change of papain necessary for this reaction is discussed. It has been shown recently [l] that one proton is released if one zinc ion or one methylmercuric ion is attached to the single essential thiol group of one papain molecule at pH 6. As will be reported in this paper, these experiments were repeated with mercuric chloride, a divalent metal which binds very strongly to thiol groups. The proton release proved to be different, in that two protons are released from two papain molecules on addition of one mercuric ion. It will be shown that this effect is due to the formation of a papain dimer. EXPERIMENTAL PROCEDURE Active papain, free from activators, was prepared in the following manner. A solution of 1 % mercuric papain, purified on the agarose-mercurial column [2], containing 10 mM phosphate, 20 mM dithiothreitol and 1 mM tetraethylenepentamine (Merck), pH 7.5, was stored for 10 min and concentrated to 10% of its initial volume, under nitrogen pressure, in a ‘Diaflo’ cell provided with a type-PM10 membrane (Aminco Corp.). The solution was readjusted to about the initial volume by addition of oxygen-free water containing 0.5 mM tetraethylenepentamine and 10 mM acetate pH 5.5 and concentrated again to 10% of the initial volume. Finally water was added containing 30 mM KCI and 0.1 mM acetate pH 5.5 and dialysis was carried out with the same solution in the ‘Diaflo’ Note. A preliminary report was presented at the 10th Meeting of the Federation of European Biochemical Societies, Paris, July 1975. Abbreviations. Reagent I, meso-l,4-bis(acetatomercuri)-2,3-diethoxybutane ; ClHgBzSO3, p-chloromercuribenzene sulphonate. Enzyme. Papain (EC 3.4.22.2).
cell. The emerging solution was checked for thiol compounds by adding 1 ml of 10 mM 5,5‘-dithio-bis(2nitrobenzoic acid) in 0.1 M phosphate pH 8.0 to 3 ml eluent and measuring the absorbance at 440 nm [ 3 ] . When the absorbance was < 0.05, corresponding to a thiol concentration in the eluent of < 8 pM, the papain solution was subjected to one more cycle of tenfold dilution with the KCl/acetate buffer and reconcentration. The final solution contained 1-2% (0.40.8 mM) papain. Titrations were carried out in a quartz vessel. The pH was read on a Philips digital pH meter. The calomel electrode was separated from the vessel by an agarose-containing salt bridge. The solution was gently stirred with a magnetic stirrer. The mercurials were added in increments of 0.1 - 1.O ml. All solutions were made in distilled deionized water and deaerated in vacuo. The organic mercurials were p-chloromercuribenzene monosodium sulphonate (ClHgBzS03) purchased from Sigma Chem. Corp. (St Louis) and meso1,4-bis(acetatomercuri)-2,3-diethoxybutane (reagent I) provided by Dr J. Drenth. The gel filtrations presented in Fig.2 and 3 were carried out in a type-K15/30 column of Pharmacia (Sweden). The height of the bed of Sephadex G-75 superfine (Pharmacia) was about 15 cm. Aliquots of 0.25 ml protein solution, to which 4 % sucrose has been added, were layered on the top of the bed under the supernatant elution buffer. The elution was effected with 0.1 M acetate pH 6.0. The solvent flow of 12 ml/h was adjusted with a peristaltic pump type ‘varioperpex’ of L.K.B. (Sweden). The transmittance at 280 nm of the effluent in a 3-mm cell was recorded with a Uvicord I1 of L.K.B. The transmittance data were read from the record, converted into absorbance
108
Dimerization of' Papain
and plotted (Fig.2). Since the optical pathway was 3 mm, the absorbance per cm path length was 3.3 times the measured absorbance. Sedimentation equilibria were measured in the Spinco model E ultracentrifuge of Beckman (U.S.A.) in an eight-channel short-column equilibrium center piece, in accordance with the method of Yphantis [4]. The partial specific volume of papain was taken as 0.723 ml/g. When this method is used, only a small difference between top and bottom concentrations occurs. I
A 1
RESULTS When a heavy metal compound combines with a non-ionized thiol group a proton is liberated : RSH
+ M + e RSM + H + .
This is evident in the reaction of glutathione with the monovalent reagent C1HgBzS03. Small aliquots of ClHgBzS03 were added to a solution of glutathione. The pH dropped after each addition and was readjusted to pH 5.9. The resulting titration curve is shown
I
I
I
RSH = glutathione
RSH=papain
.o
I
I
E
I
cn
[L
+-
I
0 1
.o
0
0
1
.o
0
1
.o
Mercurial/RSH (rnol/rnol)
Fig. 1. The liberation ofprorons on the addition ofmercuriuls to glutathione andpupain. Solvent for glutathione and reagents = 0.1 M KCI, 0.1 mM acetate. (A) 20.0 pmol glutathione in 15 ml solvent of pH 5.9 with 1.31 mM CIHgBzS03 pH 5.7. (B) 20.0 pmol glutathione in 15 ml solvent of pH 5.9 with 10.6 mM reagent I. (C) 19.5 pmol glutathione in 15 ml solvent of pH 5.5 with 6.67 mM HgC12. Solvent for papain and reagents 0.01 M KCI, 0.1 mM acetate. (D) 15.5 pmol papain in 25 ml solvent of pH 5.9 with 11.2 mM ClHgBzS03. (E) 15.5 pmol papain in 30 ml solvent of pH 6.1 with 6.0 mM reagent I. (F) 10.2 pmol papain in 30 ml solvent of pH 6.0 with 7.3 mM HgClz. The HgClz in excess of one mole of HgClz to one mole of papain (indicated by the arrow) gave a slow time-dependent liberation of protons. The same phenomenon was observed on addition of HgClz to papain with blocked thiol group. Readings were made after 30 s
109
L. A. AE. Sluyterman, J. Wijdenes, and G. Voorn
in Fig.1A. At the equivalence point one proton is liberated on addition of one molecule of ClHgBzS03 per molecule of glutathione. The results with papain and ClHgBzS03 were similar (Fig.1D). (For the present argument it is irrelevant whether the proton originates from the thiol group itself or from the neighbouring imidazole group 111.) On addition of one molecule of reagent I : AcHg - CH2 - CH - CH - CH2 - HgAc
I
I
CZHsO OCzH5 in which the mercury atoms can be about 0.6 nm apart, two protons are released from two molecules of glutathione (Fig.lB), and from two molecules of papain (Fig. 1 E). Similar results were obtained with HgC12 : two protons are liberated from two molecules of glutathione (Fig. 1C) and from two molecules of papain (Fig. 1F) on addition of one molecule of HgC12. All of these reactions are virtually instantaneous, i.e. they are certainly completed within 30 s, the time taken for the measurement of each titration point. Amperometric titration of small thiol compounds has indicated the following equilibria [ 5 ]: RSH Hg2+ S RSHg+ H + (1)
+ + RSH + RSHg' S RSHgSR + H + RSHgSR + Hg2+ 2 RSHg' .
(2) (3)
If reaction (2) occurs, one mercuric ion is capable of liberating two protons from two molecules of thiol compound, producing a dimer of the latter. When more HgC12 or reagent I is added after dimerization is complete, the reversion of dimer to monomer according to reaction (3) liberates no more protons. Since papain also carries a single thiol group the results of the titrations indicate dimerization. This was confirmed by the results of gel filtration and of ultracentrifugation. Solutions were made of HgCI2 and papain in ratios R of 0, 0.1, 0.5, 0.7 and 1.0. Aliquots of these solutions were put on a column of Sephadex G-75 and subjected to gel filtration. The results are shown in Fig.2. As R increases, a new peak of high elution rate appears, reaching a maximum when R = 0.5 and disappearing again when R = 1.O. Similar results were obtained with reagent I. These elution diagrams confirm the occurrence of equilibria (2) and (3): when the Hg: papain ratio is 0.5, equilibrium (2) predominates (Fig. 2 C) and when the ratio is 1.0, equilibrium (3) predominates. Complete dimerization will not be observed with this method owing to dilution during the run. Dilution will tend to shift equilibria (1) and (2) to the left and to remove the small fraction of free HgC12 by the dialysis action of such a column, displacing the equilibria
al c n m L
0
n Q
0: 0.; 0'
c
5 10 15 Elution vohme (mi)
20
Fig. 2. Elution profiles of papain with various concentrations of' HgC12. R = HgCIZ:papain
further to the left. This mechanism produces most of the monomer peak of curve C from the dimer peak during the run. This in its turn explains why the monomer peaks of curves C and D apparently travel at a slightly higher rate than the monomer peaks of curves A, B and C. A molecular weight determination of the compound of the first peak was attempted by the wellknown procedure of comparing its elution volume with that of other proteins [6]. The determination was complicated by the observation that standard papain exhibits a larger elution volume than expected from its molecular weight of 23400 (Fig. 3 and [7,8]). The same phenomenon is exhibited by ficin 171 and by Chinese gooseberry proteinase, as is apparent on comparison of the reported molecular weights [9,10]. Using the graphical procedure depicted in Fig. 3, the molecular weight of the dimer was estimated as 52000. In view of the uncertainties involved, this is reasonably near the expected value of 47000.
110
Dimerization of Papair
15
4.0
I
I
I
I
I
I
I
I
42
4.4
46
4.8
1
50
log Mr
Fig. 3. Elution volume of various proteins versus the logarithm of’ molecular weight. Proteins were: (1) cytochrorne c, (2) soybean trypsin inhibitor, (3) pepsin, (4) ovalbumin, ( 5 ) bovine serum albumin. (6) papain monomer, (7) papain dimer
Table 1. Sedimentation equilibrium of’ papain preparations in 0.1 M acetate p H 6.0, at 25 “C 0.5 Hg refers to a preparation of papain with an Hg: papain ratio of 0.5 Molecular weight
M , ratio, 0.5 Hg/ standard
Papain
Concentration
Standard 0.5 Hg
0.30 0.26
27000 53000
Standard 0.5 Hg
0.21 0.18
23000 46000
4000
2.00
Standard 0.5 Hg
0.10 0.10
21000 k 3000 43000 +_ 7000
2.05
x t
2000 1000
1.96
k 1000
Further confirmation was obtained from sedimentation equilibrium experiments in very short columns according to the procedure of Yphantis [4].Both standard papain and a preparation with an Hg:papain ratio of 0.5 were measured at a few protein concentrations in 0.1 M acetate pH 6.0. Although the results presented in Table 1 indicate some concentration dependence, similar to the data of Pandit and Narasingd Rao [ll], it is quite evident that mercuri-papain has double the molecular weight of standard papain. Both the mercury monomer and the mercury dimer of papain regain full activity on the addition of activators. DISCUSSION It can be concluded from the results of the titrations, the gel filtration and the sedimentation that mercuric ions are capable of dimerizing papain, when there is one H$ ion per two molecules of active papain. In 1954 Kimmel and Smith [12] proposed the existence of a papain dimer on the evidence that preparations of papain of potential activity c1 = 1.1 +
contained 0.47% Hg, i.e. about 0.5 Hg per molecule of papain. This claim was not supported by ultracentrifugation, reported in their next paper [13], which revealed a monomer at pH 4 and general aggregation at pH 8 (i.e. near the isoelectric point of 8.6 [13]) as ordinary papain does. Furthermore Finkle and Smith demonstrated in 1958 [14] that papain, prepared by the same method, is a mixture of active and nonactivatable papain and that the active species, containing one thiol group, must have an activity of CI = 2.2. The mercury papain of c1 = 1.1 of 1954, therefore, contained about 0.5 - SH group and therefore one Hg atom per molecule of active papain. The proposal made by Kimmel and Smith is therefore inconsistent with their later data. It is also contradictory with our results, because Kimmel and Smith [12] used an excess of HgClz in preparing mercury papain, which leads to monomers (cf. Fig.2). A decrease in Hg content from 1.10- 1.25 atom per molecule of M , 23400 to 0.60 atom per molecule on crystallisation is no doubt due to removal of traces of mercury mercaptide of thioglycolic acid, originating from the ion exchanger highly charged with this compound. The papain solution with its few counter ions was passed through this column as the last step before Hg analysis. In the X-ray analysis of papain [15] an excess of HgC12 was present, which meant that no dimer could have been formed, even if the crystal packing had allowed it. It is known from X-ray analysis of crystals [15] that the thiol group is located at the bottom of the cleft of the active centre, which is about 0.8 nm deep. A single mercuric ion cannot bridge a distance of 1.6 nm, even a molecule of reagent I is too short. Two explanations are possible : either the spatial structure of papain in crystals is not the same as in solution, or the structure of the enzyme is flexible enough locally to allow a conformational change to bring the thiol group to the surface. Papain inside crystals suspended in substrate solution was found to be as active towards two substrates as dissolved papain [16], indicating the same conformation of the active site in both phases. Hence a change of conformation is required to bring the thiol group of cysteine-25 to the surface. In this case, therefore, the occurrence of dimerization is a direct demonstration of a conformational change and of a certain flexibility of the active site. Examination of the model shows that little more than one turn of the central helix needs be unwound to bring the thiol group outside, with a number of changes in the preceding part of the main chain. It is not to be expected that such a small change in conformation will cause a significant change in circular dichroism. In fact, no such change was observed. Other methods of detecting conformational changes
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L. A. AE. Sluyterman, J. Wijdenes, and G. Voorn
are not likely to distinguish between the effects of conformational change and these of dimerization. Only X-ray analysis can reveal the nature of the changes involved in the dimerization.
REFERENCES 1. Sluyterman, L. A. AE. & Wijdenes, J. (1976) Eur. J . Biochem. 71,383 - 391, 2. Sluyterman, L. A. AE. & Wijdenes, J. (1970) Biochim. Biophys. Acta, 200, 593 - 595. 3. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77. 4. Yphantis, D. A. (1960) Ann. N . Y . Acad. Sci. 88, 586-601. 5. Kolthoff, J. M., Stricks, W. & Morren, L. (1954) Anal. Chem. 26,366 - 372. 6. Andrews, P. (1970) Methods Biochem. Anal. 18, 1-53.
7. Fossum, K. & Whitaker, J. R. (1968) Arch. Biochem. Biophys. 125, 367 - 375. 8. Maggio, E. T. & Shafer, J. A. (1973) Anal. Biochem. 54, 616618. 9. McDowall, M. A. (1973) Biochim. Biophys. Acta, 293, 226231. 10. McDowall, M. A. (1970) Eur. J . Biochem. 14,214-221. 11. Pandit, M. W. & Narasinga Rao, M. S. (1974) Biochim. Biophys. Acta, 371, 211 -218. 12. Kimmel, J. R. & Smith, E. L. (1954) J . Biol. Chem. 207, 515531. 13. Smith, E. L., Kimmel, J. R. & Brown, D. M. (1954) J . B i d . Chem. 207,533- 549. 14. Finkle, B. J. & Smith, E. L. (1958) J . Bid. Chern. 230, 669690. 15. Drenth, J., Jansonius, J. N., Koekoek, R. & Wolthers, B. G. (1971) Adv. Protein Chem. 25, 79-115. 16. Sluyterman, L. A. AE. & de Graaf, M. J. M. (1968) Biochim. Biophys. Acta, 171, 277-287.
L. A. AE. Sluyterman, J. Wijdenes, and G. Voorn, Natuurkundig Laboratorium, N.V. Philips Gloeilampenfabrieken, Eindhoven, The Netherlands
