Biochemical SocietyTransactions ( 1 992) 20
Stability and unfolding studies on papain
257s
16 c
GEOFFREY S. BRIGGS', ROBERT B. FREEDMAN', PETER W. GOODENOUGH" and IAN G. SUMNER". ' Biological Laboratory, University of Kent at Canterbury, Canterbury, Kent. CT2 7NJ .* Department of Protein Engineering, AFRC Institute of Food Research, Shinfield, Reading, Berks. RG2 9AT
Papain (EC.3.4.22.2) is a cysteine proteinase extracted from the plant, Curicupupayu, which is used extensively in many industrial processes, due to its high stability and broad substrate range. The enzyme consists of a single polypeptide chain of 212 residues and has a molecular weight of 23,400 daltons [l]. The three dimensional structure has been determined to high resolution, and shows that papain is folded to form two domains of roughly equal size with a deep cleft between them. The active site residues, Cys25 and His-159, are positioned on either side of this cleft. We have investigated the stability of papain to the denaturant, guanidine-HCI, using fluorescence emission spectroscopy. Papain was purchased as a crystalline suspension from Boehringer Mannheim, and resuspended in 0.1 M acetate buffer, pH 4, in order to reduce papain activity and minimise autolysis. Although the inhibition of activity by acidic conditions is achieved by a disruption in orientation of residues at the active site, this did not effect our results, as we were looking for gross conformational changes. Papain was exposed to a range of guanidine-HCl concentrations and emission spectra were collected between 300 and 450 nm after excitation at 282 nm in a Perkin-Elmer U - 5 B Luminescence Spectrophotometer. The wavelength of maximum emission was determined for each scan, and the variation in this parameter with denaturant concentration indicated the formation of a stable intermediate between two unfolding transitions (Figure 1). It is hypothesised that, as papain is a two domain protein, the stable intermediate is a species where the first domain has unfolded (in the first transition), but the second domain is still folded. Previous work on the thermal denaturation of papain [2,3] also suggested that the two domains unfold independently, and could be effectively treated as two separate polypeptides in terms of their denaturation characteristics. This hypothesis is supported by the conformational stability which can be calculated from these data using the method of Pace er ul [4] (figure 2).
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...
0
COncentratim of Guamdine-HCl
(Molar)
Figure 1. The Unfolding of Papain in Guanidine-HCI.
2
3
5
4
Cmcentraticn of Gwnidine-HCI
6
(Molar)
Figure 2. Thermodynamic Analysis of Papain Denaturation.
+ 0
0
Transition from fully folded to fully unfolded state. Calculated AG,, value = 3.7 kcallmol Transition from fully folded state to intermediate. Calculated AGmo value = 12.0 kcallmol Transition from intermediate to fully unfolded state. Calculated AGmo value = 13.9 kcallmol
If we treat the intermediate as a definite species in distinct equilibria with the fully unfolded and fully folded species, conformational stabilities can be calculated for each individual transition, giving a total conformational stability of 25.9 kcal/mol. Analysis of the whole transition as a single step from folded to unfolded gives a conformational stability of 3.7 kcallmol. The dometrically determined value for papain's conformational stability is 22.9 kcal/mol [5], which agrees with the value obtained when the two transitions are treated separately. Hence, we conclude that the two domains of papain do unfold independently. This work was supported by a Science and Engineering Research Council CASE studentship to G.S.B.
1.
Kamphuis, I.G., Kalk, K.H., Swarte, M.B.A., & Drenth, J. (1984) J. Mol. Biol. 179,253-256
2.
Tiktopulo, E.I., & Privalov, P.L. (1978) FEBS Letts. 91,57-58
3.
Hernandez-Arana, A . , & Soriano-Garcia, M. (1988) Biochim. Biophys. Acta 954,170-175
4.
Pace, C.N., Shirley, B.A., & Thomson, J.A. (1990) in Protein Structure (Creighton, T.E., ed.), pp. 311-330, IRL Press, Oxford.
5.
Privalov, P.L., & Gill, S.J. (1988) Adv. Prot. Chem. 39,191-234
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1
Stability and unfolding studies on papain
257s
16 c
GEOFFREY S. BRIGGS', ROBERT B. FREEDMAN', PETER W. GOODENOUGH" and IAN G. SUMNER". ' Biological Laboratory, University of Kent at Canterbury, Canterbury, Kent. CT2 7NJ .* Department of Protein Engineering, AFRC Institute of Food Research, Shinfield, Reading, Berks. RG2 9AT
Papain (EC.3.4.22.2) is a cysteine proteinase extracted from the plant, Curicupupayu, which is used extensively in many industrial processes, due to its high stability and broad substrate range. The enzyme consists of a single polypeptide chain of 212 residues and has a molecular weight of 23,400 daltons [l]. The three dimensional structure has been determined to high resolution, and shows that papain is folded to form two domains of roughly equal size with a deep cleft between them. The active site residues, Cys25 and His-159, are positioned on either side of this cleft. We have investigated the stability of papain to the denaturant, guanidine-HCI, using fluorescence emission spectroscopy. Papain was purchased as a crystalline suspension from Boehringer Mannheim, and resuspended in 0.1 M acetate buffer, pH 4, in order to reduce papain activity and minimise autolysis. Although the inhibition of activity by acidic conditions is achieved by a disruption in orientation of residues at the active site, this did not effect our results, as we were looking for gross conformational changes. Papain was exposed to a range of guanidine-HCl concentrations and emission spectra were collected between 300 and 450 nm after excitation at 282 nm in a Perkin-Elmer U - 5 B Luminescence Spectrophotometer. The wavelength of maximum emission was determined for each scan, and the variation in this parameter with denaturant concentration indicated the formation of a stable intermediate between two unfolding transitions (Figure 1). It is hypothesised that, as papain is a two domain protein, the stable intermediate is a species where the first domain has unfolded (in the first transition), but the second domain is still folded. Previous work on the thermal denaturation of papain [2,3] also suggested that the two domains unfold independently, and could be effectively treated as two separate polypeptides in terms of their denaturation characteristics. This hypothesis is supported by the conformational stability which can be calculated from these data using the method of Pace er ul [4] (figure 2).
/-+
_--__---
...
0
COncentratim of Guamdine-HCl
(Molar)
Figure 1. The Unfolding of Papain in Guanidine-HCI.
2
3
5
4
Cmcentraticn of Gwnidine-HCI
6
(Molar)
Figure 2. Thermodynamic Analysis of Papain Denaturation.
+ 0
0
Transition from fully folded to fully unfolded state. Calculated AG,, value = 3.7 kcallmol Transition from fully folded state to intermediate. Calculated AGmo value = 12.0 kcallmol Transition from intermediate to fully unfolded state. Calculated AGmo value = 13.9 kcallmol
If we treat the intermediate as a definite species in distinct equilibria with the fully unfolded and fully folded species, conformational stabilities can be calculated for each individual transition, giving a total conformational stability of 25.9 kcal/mol. Analysis of the whole transition as a single step from folded to unfolded gives a conformational stability of 3.7 kcallmol. The dometrically determined value for papain's conformational stability is 22.9 kcal/mol [5], which agrees with the value obtained when the two transitions are treated separately. Hence, we conclude that the two domains of papain do unfold independently. This work was supported by a Science and Engineering Research Council CASE studentship to G.S.B.
1.
Kamphuis, I.G., Kalk, K.H., Swarte, M.B.A., & Drenth, J. (1984) J. Mol. Biol. 179,253-256
2.
Tiktopulo, E.I., & Privalov, P.L. (1978) FEBS Letts. 91,57-58
3.
Hernandez-Arana, A . , & Soriano-Garcia, M. (1988) Biochim. Biophys. Acta 954,170-175
4.
Pace, C.N., Shirley, B.A., & Thomson, J.A. (1990) in Protein Structure (Creighton, T.E., ed.), pp. 311-330, IRL Press, Oxford.
5.
Privalov, P.L., & Gill, S.J. (1988) Adv. Prot. Chem. 39,191-234
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1
