724
BIOCHEMICAL SOCIETY TRANSACTIONS
Nachmansohn, D. (1 959) Chemical and Molecular Basis of Nerve Acfiuity, Academic Press, New York and London Pennington, R. T. (1969) Disorders of VolunfuryMuscle, p. 385, Churchill, London Rodan, S. B., Hintz, R. L.,Sha’afi, R. I. &Rodan,G. A. (1974) Nuture(London)252,589-591 Roses, A. D., Herbstreith, M. H. & Appel, S. H. (1975) Nature (London)254,350-351 Walton, J. N. (1969) Br. Med. J . i, 1271-1274
Assay of Enzyme Activity by Polarography P. D. J. WEITZMAN Department of Biochemistry, School of Biological Sciences, University of Leicester, Leicester LE1 I R H , U.K.
Polarography is a powerful electroanalytical technique, but, except for measurements made with the oxygen electrode, it has not been fully exploited in biochemical studies. Polarographic equipment is rarely encountered in biochemical laboratories and the technique is frequently viewed as requiring considerable skill and experience for the interpretation of results. Although there are, as with other techniques, problems that may beset the newcomer, a working familiarity with the method and the ability to make reliable measurements may readily be acquired. It is the purpose of this communication to show that polarography may usefully and advantageously be applied to the determination of the activities of a variety of enzymes. The technique relies on examination of the current produced at a polarizable microelectrode as a function of the applied potential. The circuit is completed with a nonpolarizable reference electrode (both electrodes being in contact with the test solution) and a sensitive instrument for applying the potential and measuring the small currents. The micro-electrode generally used in polarographic studies is the dropping mercury electrode, which consists of a very fine capillary tube from which mercury emerges regularly as tiny drops; a saturated calomel electrode usually serves as the reference electrode. Reduction or oxidation of substances at the micro-electrode give rise respectively to cathodic or anodic currents. If the solution contains an electroactive substance, the dependence of current on applied potential is characteristically of the form shown in Fig. 1. As the potential is varied a limiting current is reached at which the substance is electrolysed as fast as it arrives at the electrode surface by diffusion, and this limiting current is therefore directly proportional to the concentration of the substance. Continuous and automatic recording of the change in limiting current at an appropriate fixed potential provides a measure of the rate of formation or consumption of an electroactive species. Many enzyme-catalysed reactions involve substrates or products which are electroactive and such enzymes should lend themselves to polarographic assay. I previously reported that citrate synthase and malate synthase may be assayed polarographically and described a suitable apparatus and procedure (Weitzman, 1966,1969).The technique of polarographic assay has now been extended to a number of enzymes of different reaction types. The assays for citrate synthase and malate synthase are based on the fact that CoA, but not its S-acyl derivatives, gives an anodic polarographic wave whose magnitude is proportional to CoA concentration. The cleavage of acetyl-CoA to CoA by the citrate synthase or malate synthase reactions may thus be followed by monitoring the limiting current at -0.2V to -0.3V. For malate synthase, the polarographic method is particularly advantageous as the chromogenic thiol-specific reagent 5,5’-dithiobis-(2-nitrobenzoate), which might in principle be used for the continuous spectrophotomctric measurement of CoA production, inactivates the enzyme. Some citrate synthases are also inactivated or desensitized to regulatory effectors by 5,5’-dithiobis-(2-nitrobenzoate) (Weitzman & Danson, 1976). In all these cases, polarographic measurement of activities does not affect the enzymes. 1976
563rd MEETING, LONDON
725
Potential (-)
Fig. 1. Characteristic polarographic waves The limiting current of the cathodic wave (a)is proportional to the concentration of the electroreduciblespecies,X,andthelimitingcurrent of theanodic wave (6) is proportional to the concentration of the electro-oxidizable species, Y.
The method may be extended to any other acyl-CoAICoA reaction system. Examples of other enzymes conveniently assayed in this way are acetate thiokinase and succinate thiokinase. Advantages of the polarographic assay for these enzymes include the ability to follow the reactions in either direction and the non-interference of the adenine or guanine nucleotide substrate with the measurements (in contrast with the spectrophotometric procedure). The cathodic and anodic waves produced respectively by lipoamide and dihydrolipoamide (measurable at -0.75V and -0.2V) form the basis of an assay for (dihydro)lipoamide dehydrogenase activity. Although this enzyme may be assayed spectrophotometrically at 340nm, examination of its inhibition by NADH may more conveniently be studied polarographically, since the presence of NADH does not interfere with the lipoamide waves (Parker & Weitzman, 1973). The anodic wave of thiocholine has also been utilized by Fiserova-Bergerova (1963) and Ridgway & Mark (1965) for the polarographic assay of cholinesterase, with acetylthiocholine as substrate. Another electroactive functional group frequently encountered in substrates or products of enzyme-catalysed reactions is the carbonyl group. For example, pyruvate gives a well-defined cathodic wave whose limiting current may be measured at -1.4V. This provides the basis for a direct assay of the enzyme pyruvate kinase, the production of pyruvate being followed continuously by monitoring the increase of cathodic current. Unlike other assays generally used for pyruvate kinase, no coupling enzyme is required. Another class of enzymes amenable to polarographic assay are the NAD(P)-linked
VOl. 4
726
BIOCHEMICAL SOCIETY TRANSACTIONS
dehydrogenases. NAD+ and NADP+ are reducible at the dropping mercury electrode, and the cathodic waves may be measured in the region -1.1 V to -1.3 V. The reduced nucleotides NADH and NADPH are not electroactive at these potentials and thus do not interferewith themeasurements. Under somecircumstances,this polarographic assay for a dehydrogenase may offer advantages over the conventional spectrophotometric method. Such situations include the presence of turbid suspensions,e.g. in the examination of enzyme activities in cells made permeable (Weitzman, 1973), and the presence of NADH oxidase activity. The latter interferes with the normal assay for dehydrogenases, but, under the anaerobic conditions used in polarographic measurements, is inoperative. The enzymes measurable by the polarographic procedure may also, of course, serve as coupling enzymes and thereby extend the method to other reactions and the general approach to polarographic assays outlined here should prove applicable to many other enzymes. The ability of polarographs to measure very small currents renders these assay methods comparable in sensitivity with other available (generally spectrophotometric) procedures. In conclusion, a number of enzyme activities may be determined polarographically. The method is continuous and sensitive, may be performed under a wide range of experimental conditions and is unaffected by solution turbidity. In particular cases, additional advantages may be presented. I thank the Science Research Council for support.
Fiserova-Bergerova, V. (1963) Coll. Czech. Chem. Commun. 28, 331 1-3325 Parker, M. G.& Weitman, P. D. J. (1973)Biochem. J. 135,215-223 Ridgway, T. H.& Mark, H. B. (1965) Anal. Biochem. 12,357-366 Weitzrnan, P. D.J. (1966)Biochem. J. 99, 1 8 ~ Weitman, P. D. J. (1969)Methods Enzymol. 13, 365-368 Weitman, P. D. J. (1973)FEBS Lert. 32,247-250 Weitzrnan, P.D.J. & Danson, M. J. (1976) Curr. Top. Cell. Regul. 10, 161-204
The Metabolism of Labetalol in Animals and Man ROBERT HOPKINS, LESLIE E. MARTIN and ROBERT BLAND
Allen and Hanburys Research Ltd., Ware, Herts. SG12 ODJ, U.K. Labetalol (Trandate; Allen and Hanburys Ltd.) is a combined a- and ,!?-receptor antagonist that is effective in the treatment of hypertension in man (Prichard et al., 1975).
The metabolism of ['4C]labetalol (2-hydroxy-5-{1-hydroxy-2-[(1-methyl-3-phenyl[3-14C]propyl)amino]thyl}benzamide hydrochloride) and [3H]labetalol (2-hydroxy-5{ 1-hydroxy-2-[(1-methyl-3-phenylpropyl)aminoI[ 1-3H]ethyl}benzamide hydrochloride) has been studied in rat, rabbit, dog and man. Radioautographic examination of the normal and the pregnant rat given [14C]labetalolorally (50-200mg/kg) or intravenously (25mg/kg) showed that the radioactivity was quickly taken up into the tissues and rapidly cleared from the body via both the kidney and bile. Radiochemical analysis of the plasma and tissues after oral doses to rat, rabbit (50mg of rH]- or [14C]-labetalol/kg) and dog (20mg of ["C]- or [3H]-labetalol/kg)showed that the drug was well absorbed. During absorption, labetalol is extensively metabolized. The difference between the plasma radioactivity and labetalol concentration (Table 1) has been shown by chromatography of plasma extracts to be due mainly to circulating glucuronide conjugates of labetalol. The highest concentrations of radioactivity were found in the liver, kidneys and bile. The rat excreted 37 %, the rabbit 61 % and the dog 66 % of the dose in the 0-96 h urine. The remainder of the dose of radioactivity was excreted in the faeces. T.1.c. of the urine from rat, rabbit and dog (Fig. l a ) showed that the rat excreted 11 % of the dose as labetalol, 5 % of the dose as metabolite (I30 ), % of the dose as metabolite 01)and 1.5 % 1976
BIOCHEMICAL SOCIETY TRANSACTIONS
Nachmansohn, D. (1 959) Chemical and Molecular Basis of Nerve Acfiuity, Academic Press, New York and London Pennington, R. T. (1969) Disorders of VolunfuryMuscle, p. 385, Churchill, London Rodan, S. B., Hintz, R. L.,Sha’afi, R. I. &Rodan,G. A. (1974) Nuture(London)252,589-591 Roses, A. D., Herbstreith, M. H. & Appel, S. H. (1975) Nature (London)254,350-351 Walton, J. N. (1969) Br. Med. J . i, 1271-1274
Assay of Enzyme Activity by Polarography P. D. J. WEITZMAN Department of Biochemistry, School of Biological Sciences, University of Leicester, Leicester LE1 I R H , U.K.
Polarography is a powerful electroanalytical technique, but, except for measurements made with the oxygen electrode, it has not been fully exploited in biochemical studies. Polarographic equipment is rarely encountered in biochemical laboratories and the technique is frequently viewed as requiring considerable skill and experience for the interpretation of results. Although there are, as with other techniques, problems that may beset the newcomer, a working familiarity with the method and the ability to make reliable measurements may readily be acquired. It is the purpose of this communication to show that polarography may usefully and advantageously be applied to the determination of the activities of a variety of enzymes. The technique relies on examination of the current produced at a polarizable microelectrode as a function of the applied potential. The circuit is completed with a nonpolarizable reference electrode (both electrodes being in contact with the test solution) and a sensitive instrument for applying the potential and measuring the small currents. The micro-electrode generally used in polarographic studies is the dropping mercury electrode, which consists of a very fine capillary tube from which mercury emerges regularly as tiny drops; a saturated calomel electrode usually serves as the reference electrode. Reduction or oxidation of substances at the micro-electrode give rise respectively to cathodic or anodic currents. If the solution contains an electroactive substance, the dependence of current on applied potential is characteristically of the form shown in Fig. 1. As the potential is varied a limiting current is reached at which the substance is electrolysed as fast as it arrives at the electrode surface by diffusion, and this limiting current is therefore directly proportional to the concentration of the substance. Continuous and automatic recording of the change in limiting current at an appropriate fixed potential provides a measure of the rate of formation or consumption of an electroactive species. Many enzyme-catalysed reactions involve substrates or products which are electroactive and such enzymes should lend themselves to polarographic assay. I previously reported that citrate synthase and malate synthase may be assayed polarographically and described a suitable apparatus and procedure (Weitzman, 1966,1969).The technique of polarographic assay has now been extended to a number of enzymes of different reaction types. The assays for citrate synthase and malate synthase are based on the fact that CoA, but not its S-acyl derivatives, gives an anodic polarographic wave whose magnitude is proportional to CoA concentration. The cleavage of acetyl-CoA to CoA by the citrate synthase or malate synthase reactions may thus be followed by monitoring the limiting current at -0.2V to -0.3V. For malate synthase, the polarographic method is particularly advantageous as the chromogenic thiol-specific reagent 5,5’-dithiobis-(2-nitrobenzoate), which might in principle be used for the continuous spectrophotomctric measurement of CoA production, inactivates the enzyme. Some citrate synthases are also inactivated or desensitized to regulatory effectors by 5,5’-dithiobis-(2-nitrobenzoate) (Weitzman & Danson, 1976). In all these cases, polarographic measurement of activities does not affect the enzymes. 1976
563rd MEETING, LONDON
725
Potential (-)
Fig. 1. Characteristic polarographic waves The limiting current of the cathodic wave (a)is proportional to the concentration of the electroreduciblespecies,X,andthelimitingcurrent of theanodic wave (6) is proportional to the concentration of the electro-oxidizable species, Y.
The method may be extended to any other acyl-CoAICoA reaction system. Examples of other enzymes conveniently assayed in this way are acetate thiokinase and succinate thiokinase. Advantages of the polarographic assay for these enzymes include the ability to follow the reactions in either direction and the non-interference of the adenine or guanine nucleotide substrate with the measurements (in contrast with the spectrophotometric procedure). The cathodic and anodic waves produced respectively by lipoamide and dihydrolipoamide (measurable at -0.75V and -0.2V) form the basis of an assay for (dihydro)lipoamide dehydrogenase activity. Although this enzyme may be assayed spectrophotometrically at 340nm, examination of its inhibition by NADH may more conveniently be studied polarographically, since the presence of NADH does not interfere with the lipoamide waves (Parker & Weitzman, 1973). The anodic wave of thiocholine has also been utilized by Fiserova-Bergerova (1963) and Ridgway & Mark (1965) for the polarographic assay of cholinesterase, with acetylthiocholine as substrate. Another electroactive functional group frequently encountered in substrates or products of enzyme-catalysed reactions is the carbonyl group. For example, pyruvate gives a well-defined cathodic wave whose limiting current may be measured at -1.4V. This provides the basis for a direct assay of the enzyme pyruvate kinase, the production of pyruvate being followed continuously by monitoring the increase of cathodic current. Unlike other assays generally used for pyruvate kinase, no coupling enzyme is required. Another class of enzymes amenable to polarographic assay are the NAD(P)-linked
VOl. 4
726
BIOCHEMICAL SOCIETY TRANSACTIONS
dehydrogenases. NAD+ and NADP+ are reducible at the dropping mercury electrode, and the cathodic waves may be measured in the region -1.1 V to -1.3 V. The reduced nucleotides NADH and NADPH are not electroactive at these potentials and thus do not interferewith themeasurements. Under somecircumstances,this polarographic assay for a dehydrogenase may offer advantages over the conventional spectrophotometric method. Such situations include the presence of turbid suspensions,e.g. in the examination of enzyme activities in cells made permeable (Weitzman, 1973), and the presence of NADH oxidase activity. The latter interferes with the normal assay for dehydrogenases, but, under the anaerobic conditions used in polarographic measurements, is inoperative. The enzymes measurable by the polarographic procedure may also, of course, serve as coupling enzymes and thereby extend the method to other reactions and the general approach to polarographic assays outlined here should prove applicable to many other enzymes. The ability of polarographs to measure very small currents renders these assay methods comparable in sensitivity with other available (generally spectrophotometric) procedures. In conclusion, a number of enzyme activities may be determined polarographically. The method is continuous and sensitive, may be performed under a wide range of experimental conditions and is unaffected by solution turbidity. In particular cases, additional advantages may be presented. I thank the Science Research Council for support.
Fiserova-Bergerova, V. (1963) Coll. Czech. Chem. Commun. 28, 331 1-3325 Parker, M. G.& Weitman, P. D. J. (1973)Biochem. J. 135,215-223 Ridgway, T. H.& Mark, H. B. (1965) Anal. Biochem. 12,357-366 Weitzrnan, P. D.J. (1966)Biochem. J. 99, 1 8 ~ Weitman, P. D. J. (1969)Methods Enzymol. 13, 365-368 Weitman, P. D. J. (1973)FEBS Lert. 32,247-250 Weitzrnan, P.D.J. & Danson, M. J. (1976) Curr. Top. Cell. Regul. 10, 161-204
The Metabolism of Labetalol in Animals and Man ROBERT HOPKINS, LESLIE E. MARTIN and ROBERT BLAND
Allen and Hanburys Research Ltd., Ware, Herts. SG12 ODJ, U.K. Labetalol (Trandate; Allen and Hanburys Ltd.) is a combined a- and ,!?-receptor antagonist that is effective in the treatment of hypertension in man (Prichard et al., 1975).
The metabolism of ['4C]labetalol (2-hydroxy-5-{1-hydroxy-2-[(1-methyl-3-phenyl[3-14C]propyl)amino]thyl}benzamide hydrochloride) and [3H]labetalol (2-hydroxy-5{ 1-hydroxy-2-[(1-methyl-3-phenylpropyl)aminoI[ 1-3H]ethyl}benzamide hydrochloride) has been studied in rat, rabbit, dog and man. Radioautographic examination of the normal and the pregnant rat given [14C]labetalolorally (50-200mg/kg) or intravenously (25mg/kg) showed that the radioactivity was quickly taken up into the tissues and rapidly cleared from the body via both the kidney and bile. Radiochemical analysis of the plasma and tissues after oral doses to rat, rabbit (50mg of rH]- or [14C]-labetalol/kg) and dog (20mg of ["C]- or [3H]-labetalol/kg)showed that the drug was well absorbed. During absorption, labetalol is extensively metabolized. The difference between the plasma radioactivity and labetalol concentration (Table 1) has been shown by chromatography of plasma extracts to be due mainly to circulating glucuronide conjugates of labetalol. The highest concentrations of radioactivity were found in the liver, kidneys and bile. The rat excreted 37 %, the rabbit 61 % and the dog 66 % of the dose in the 0-96 h urine. The remainder of the dose of radioactivity was excreted in the faeces. T.1.c. of the urine from rat, rabbit and dog (Fig. l a ) showed that the rat excreted 11 % of the dose as labetalol, 5 % of the dose as metabolite (I30 ), % of the dose as metabolite 01)and 1.5 % 1976
