3 14
BIOCHEMICAL SOCIETY TRANSACTIONS
velocities in the forward and reverse directions was 54.5: 1, somewhat lower than that previously reported (McQuate & Utter, 1959). Product-inhibition experiments with a non-saturating concentration of the constant substrate showed that in every case competitive inhibition occurs. This is consistent with a rapidequilibrium random Bi Bi mechanism for the reverse reaction, a similar mechanism to that proposed for the forward reaction (Reynard et al., 1961 ; Ainsworth & MacFarlane, 1973). Analysis of the slope replots showed that there was linear competitive inhibition between ATP and both products. When pyruvate is the variable substrate, however, parabolic competitive inhibition occurred with both products. The experimental data for each of these plots were fitted to the relevant rate equation by the leastsquares method of Cleland (1967) and apparent constants were obtained. Fig. 2 shows the experimental data with pyruvate as the variable substrate and phosphoenolpyruvate as the product inhibitor. Parabolic slope effects are normally the result of multiple combinations of 'the inhibitor with the enzyme. It is curious that the parabolic effects were only seen with one substrate and not both, as would be the case if complexes of the form enzyme-productproduct occurred in a rapid equilibrium mechanism. One explanation is that the binding of pyruvate is not at complete thermodynamic equilibrium, and that the reaction possesses some non-rapid-equilibrium random Bi Bi character. Isotopicexchange experiments at equilibrium should confirm whether or not the enzyme really does catalyse a rapid equilibrium mechanism. Ainsworth, S. & MacFarlane, N. (1973) Biochem. J. 131,223-236 Cleland, W. W. (1967) Advan. Enzymol. Relat. Areas Mol. Biol. 29, 1-32 Jonson, C.A. & Cleland, W. W. (1974) J. Biol. Chem. 249, 2567-2571 Krimskey, I. (1959) J. Biol. Chem. 234,232-236 MacFarlane, N. & Ainsworth, S. (1972) Biochem. J. 129,1035-1047 McQuate, J . T. & Utter, M. F. (1959)J. Biol. Chem. 234, 2151-2157 Reynard, A. M., Hass, L. F., Jacobson, D. D. &. Boyer, P. D. (1961) J. Biol. Chem. 236,22772283
Enzyme Inhibition by Sodium Alkyl Sulphates J. C. MARSDEN Polytechnic of Central London, 115 New Cavendish Street, London WlM8JS,
U.K.
The irreversible denaturation of proteins by anionic detergents, particularly sodium dodecyl sulphate, has been well documented (Tanford, 1968,1970). As the concentration of detergent in a protein solution is increased, the protein undergoes a relatively sudden and irreversible transition from the native to the denatured state. The nature of both protein and detergent affect the concentration at which denaturation takes place, and it has been suggested that the more hydrophobic the non-polar region of the detergent, the lower the concentration required to cause denaturation (Decker & Foster, 1966). In this study, theeffectsof a variety of sodium alkyl sulphates, esterified at C-1, on two enzyme activities present in the digestive juices of the snail Helix pomatia (supplied by Industrie Biologique FranGaise, Gennevilliers, France) are reported. Glucuronidase activity was measured by the method of Fishman et al. (1948), which uses phenolphthalein glucuronideas substrate; sulphataseactivity was assessed by the same method, by substituting only the dipotassium salt of phenolphthalein disulphate as substrate. Results are shown graphically in Figs. 1 and 2. The pattern of inactivation of the enzymes by the detergents was irreversible and, where it occurred, resembled certain physical changes (in colligative properties and electrical conductivity) which take place in anionic detergent solutions alone in the neighbourhood of the critical micelle concentration of the detergent (Preston, 1948). However, the detergent concentrations causing enzyme inhibition proved to be considerably below the critical micellar concentrations
1975
554th MEETING, LONDON
315
a
=+?----
3
\
6
2
9
Concn. of detergent (mi)
Fig. 1. Effectof sodium alkyl sulphates on the sulphatase of Helix pomatia Symbols for alkyl-group chain length: v , Clo; m, Cll; A , ClZ;0 , Cia; 0, C14;A , CIS; 0,CI6; v, C17,CIS;Q, solutions cloudy.
Concn. of detergent (mM)
Fig. 2. Effect of sodium alkyI sulphates on the glucuronidase of Helix pomatia For symbols see Fig. 1.
Vol. 3
316
BIOCHEMICAL SOCLETY TRANSACTIONS
of the detergents used (Evans, 1956), although the sharp cut-off in activity evident lends support to the idea that protein denaturation by anionic detergents involves the formation of protein-detergent micelles (Tanford, 1970). The most effective inhibitor of glucuronidase activity was sodium tridecyl sulphate, whereas sodium pentadecyl sulphate exerted the strongest effects on the sulphatase. In the latter case, inhibition by CI6,CI7and CISalkyl sulphates was limited by the insolubilities of these detergents at the temperature used (37°C). With glucuronidase activity, complete inhibition was not evident even at the highest detergent concentration tested ( l o r n ) , suggesting the possibility of traces of resistant enzyme in the mixture; however, resolution of the crude enzyme mixture by gel filtration on Sephadex G-75 (Pharmacia, Uppsala, Sweden) revealed only a single peak of glucuronidase activity separated from two sulphatase activities. A noteworthy feature of the results relates to the effects of sodium pentadecyl sulphate on the two enzyme activities. At a concentration of 1rn,inactivation of sulphatase by this detergent was complete, whereas glucuronidase activity remained unimpaired by concentrations as high as 10mM. Such a selective action suggests more general applications of sodium alkyl sulphates to problems associated with biochemical separations in that choice of a particular alkyl sulphate and its concentration may permit the irreversible inactivation of unwanted enzyme activities, leaving others unscathed. There may be possibilities here, too, in isoenzyme estimations. These studies also indicate that earlier observations suggesting a relationship between side-chain hydrophobicity and the effectiveness of anionic detergents in protein denaturation may need reconsideration and that the interactions between these detergents and proteins may be more specific than has been supposed. I thank Dr. M. J. How of Unilever Research, Shambrook, Beds., for providing some of the sodium alkyl sulphatesand Miss Gillian Atkinson and Mr. Brian Tetlow and Mr. Peter Sirey for valuable assistance.
Decker, R. V. & Foster, J. F. (1966) Biochemistry 5, 1242-1254 Evans, H. C. (1956) J. Chem. Sac. London 579-586 Fishman, W. H., Springer, B. & Brunetti, R. (1948) J. Biol. Chem. 173,449-456 Preston, W. C. (1948) J. Phys. Colloid Chem. 52, 84-97 Tanford, C. (1968) Aduun. Protein Chem. 23, 121-282 Tanford, C. (1970) Aduun. Protein Chem. 24,2-95
Inhibition of Cytochrome c Oxidase by Sulphide PETER NICHOLLS Institute of Biochemistry, University of Odense, DK-5000 Odense, Denmark
Cyanide, azide and sulphide have all been used as terminal inhibitors of respiration. Cyanide, the classical inhibitor, was later abandoned because of its slow and complex inhibition kinetics(Chance, 1952;NicholIsetal.,1972).Azide, whose kineticsaresimpler, was found to induce a shift in the a peak of reduced cytochrome a, from 605 to 603nm, after its binding to ferric a3 (Wilson & Chance, 1967; Nicholls & Kimelberg, 1968). Sulphide was therefore used in later studies (Chance & Schoener, 1696). Observations with isolated cytochrome au3preparations prepared as described by van Buuren (1972) now show that there are also complications in the inhibition pattern given by sulphide. Fig. 1 shows the a-peak region of cytochrome aa3 both in the fully reduced state ( a * + ~ ~and ~ + in ) the form of the reduced sulphide complex (d+aJ3+H2S). Although the shift in the peak is less marked than for azide, approx. 1nm instead of 2nm, there is no doubt that an effect is seen in the cytochrome a spectrum after the presumed binding to the a3haem. The Soret band of the sulphide-inhibited enzyme is 1975
BIOCHEMICAL SOCIETY TRANSACTIONS
velocities in the forward and reverse directions was 54.5: 1, somewhat lower than that previously reported (McQuate & Utter, 1959). Product-inhibition experiments with a non-saturating concentration of the constant substrate showed that in every case competitive inhibition occurs. This is consistent with a rapidequilibrium random Bi Bi mechanism for the reverse reaction, a similar mechanism to that proposed for the forward reaction (Reynard et al., 1961 ; Ainsworth & MacFarlane, 1973). Analysis of the slope replots showed that there was linear competitive inhibition between ATP and both products. When pyruvate is the variable substrate, however, parabolic competitive inhibition occurred with both products. The experimental data for each of these plots were fitted to the relevant rate equation by the leastsquares method of Cleland (1967) and apparent constants were obtained. Fig. 2 shows the experimental data with pyruvate as the variable substrate and phosphoenolpyruvate as the product inhibitor. Parabolic slope effects are normally the result of multiple combinations of 'the inhibitor with the enzyme. It is curious that the parabolic effects were only seen with one substrate and not both, as would be the case if complexes of the form enzyme-productproduct occurred in a rapid equilibrium mechanism. One explanation is that the binding of pyruvate is not at complete thermodynamic equilibrium, and that the reaction possesses some non-rapid-equilibrium random Bi Bi character. Isotopicexchange experiments at equilibrium should confirm whether or not the enzyme really does catalyse a rapid equilibrium mechanism. Ainsworth, S. & MacFarlane, N. (1973) Biochem. J. 131,223-236 Cleland, W. W. (1967) Advan. Enzymol. Relat. Areas Mol. Biol. 29, 1-32 Jonson, C.A. & Cleland, W. W. (1974) J. Biol. Chem. 249, 2567-2571 Krimskey, I. (1959) J. Biol. Chem. 234,232-236 MacFarlane, N. & Ainsworth, S. (1972) Biochem. J. 129,1035-1047 McQuate, J . T. & Utter, M. F. (1959)J. Biol. Chem. 234, 2151-2157 Reynard, A. M., Hass, L. F., Jacobson, D. D. &. Boyer, P. D. (1961) J. Biol. Chem. 236,22772283
Enzyme Inhibition by Sodium Alkyl Sulphates J. C. MARSDEN Polytechnic of Central London, 115 New Cavendish Street, London WlM8JS,
U.K.
The irreversible denaturation of proteins by anionic detergents, particularly sodium dodecyl sulphate, has been well documented (Tanford, 1968,1970). As the concentration of detergent in a protein solution is increased, the protein undergoes a relatively sudden and irreversible transition from the native to the denatured state. The nature of both protein and detergent affect the concentration at which denaturation takes place, and it has been suggested that the more hydrophobic the non-polar region of the detergent, the lower the concentration required to cause denaturation (Decker & Foster, 1966). In this study, theeffectsof a variety of sodium alkyl sulphates, esterified at C-1, on two enzyme activities present in the digestive juices of the snail Helix pomatia (supplied by Industrie Biologique FranGaise, Gennevilliers, France) are reported. Glucuronidase activity was measured by the method of Fishman et al. (1948), which uses phenolphthalein glucuronideas substrate; sulphataseactivity was assessed by the same method, by substituting only the dipotassium salt of phenolphthalein disulphate as substrate. Results are shown graphically in Figs. 1 and 2. The pattern of inactivation of the enzymes by the detergents was irreversible and, where it occurred, resembled certain physical changes (in colligative properties and electrical conductivity) which take place in anionic detergent solutions alone in the neighbourhood of the critical micelle concentration of the detergent (Preston, 1948). However, the detergent concentrations causing enzyme inhibition proved to be considerably below the critical micellar concentrations
1975
554th MEETING, LONDON
315
a
=+?----
3
\
6
2
9
Concn. of detergent (mi)
Fig. 1. Effectof sodium alkyl sulphates on the sulphatase of Helix pomatia Symbols for alkyl-group chain length: v , Clo; m, Cll; A , ClZ;0 , Cia; 0, C14;A , CIS; 0,CI6; v, C17,CIS;Q, solutions cloudy.
Concn. of detergent (mM)
Fig. 2. Effect of sodium alkyI sulphates on the glucuronidase of Helix pomatia For symbols see Fig. 1.
Vol. 3
316
BIOCHEMICAL SOCLETY TRANSACTIONS
of the detergents used (Evans, 1956), although the sharp cut-off in activity evident lends support to the idea that protein denaturation by anionic detergents involves the formation of protein-detergent micelles (Tanford, 1970). The most effective inhibitor of glucuronidase activity was sodium tridecyl sulphate, whereas sodium pentadecyl sulphate exerted the strongest effects on the sulphatase. In the latter case, inhibition by CI6,CI7and CISalkyl sulphates was limited by the insolubilities of these detergents at the temperature used (37°C). With glucuronidase activity, complete inhibition was not evident even at the highest detergent concentration tested ( l o r n ) , suggesting the possibility of traces of resistant enzyme in the mixture; however, resolution of the crude enzyme mixture by gel filtration on Sephadex G-75 (Pharmacia, Uppsala, Sweden) revealed only a single peak of glucuronidase activity separated from two sulphatase activities. A noteworthy feature of the results relates to the effects of sodium pentadecyl sulphate on the two enzyme activities. At a concentration of 1rn,inactivation of sulphatase by this detergent was complete, whereas glucuronidase activity remained unimpaired by concentrations as high as 10mM. Such a selective action suggests more general applications of sodium alkyl sulphates to problems associated with biochemical separations in that choice of a particular alkyl sulphate and its concentration may permit the irreversible inactivation of unwanted enzyme activities, leaving others unscathed. There may be possibilities here, too, in isoenzyme estimations. These studies also indicate that earlier observations suggesting a relationship between side-chain hydrophobicity and the effectiveness of anionic detergents in protein denaturation may need reconsideration and that the interactions between these detergents and proteins may be more specific than has been supposed. I thank Dr. M. J. How of Unilever Research, Shambrook, Beds., for providing some of the sodium alkyl sulphatesand Miss Gillian Atkinson and Mr. Brian Tetlow and Mr. Peter Sirey for valuable assistance.
Decker, R. V. & Foster, J. F. (1966) Biochemistry 5, 1242-1254 Evans, H. C. (1956) J. Chem. Sac. London 579-586 Fishman, W. H., Springer, B. & Brunetti, R. (1948) J. Biol. Chem. 173,449-456 Preston, W. C. (1948) J. Phys. Colloid Chem. 52, 84-97 Tanford, C. (1968) Aduun. Protein Chem. 23, 121-282 Tanford, C. (1970) Aduun. Protein Chem. 24,2-95
Inhibition of Cytochrome c Oxidase by Sulphide PETER NICHOLLS Institute of Biochemistry, University of Odense, DK-5000 Odense, Denmark
Cyanide, azide and sulphide have all been used as terminal inhibitors of respiration. Cyanide, the classical inhibitor, was later abandoned because of its slow and complex inhibition kinetics(Chance, 1952;NicholIsetal.,1972).Azide, whose kineticsaresimpler, was found to induce a shift in the a peak of reduced cytochrome a, from 605 to 603nm, after its binding to ferric a3 (Wilson & Chance, 1967; Nicholls & Kimelberg, 1968). Sulphide was therefore used in later studies (Chance & Schoener, 1696). Observations with isolated cytochrome au3preparations prepared as described by van Buuren (1972) now show that there are also complications in the inhibition pattern given by sulphide. Fig. 1 shows the a-peak region of cytochrome aa3 both in the fully reduced state ( a * + ~ ~and ~ + in ) the form of the reduced sulphide complex (d+aJ3+H2S). Although the shift in the peak is less marked than for azide, approx. 1nm instead of 2nm, there is no doubt that an effect is seen in the cytochrome a spectrum after the presumed binding to the a3haem. The Soret band of the sulphide-inhibited enzyme is 1975
