- Email: [email protected]
Flow injection determination of chlorpromazine by inhibition of glutamate dehydrogenase
Analytica Chimica Acta 387 (1999) 47±51
Flow injection determination of chlorpromazine by inhibition of glutamate dehydrogenase Tahseen Ghous1, Alan Townshend* Department of Chemistry, University of Hull, HU6 7RX, Hull, UK Received 14 August 1998; received in revised form 17 December 1998; accepted 30 December 1998
Abstract A double injection ¯ow procedure is used for the determination of chlorpromazine (1010ÿ5 M, in 44 ml), by its inhibition of glutamate dehydrogenase (GlDH). The enzyme is injected into pH 7.5 phosphate buffer containing substrate (110ÿ3 M aketoglutaric acid) and 110ÿ3 M NH4Cl, and drug/NADH (810ÿ5 M) solution is injected from a second valve in series into the enzyme zone. The relative standard deviation for the determination of 610ÿ5 M drug (n6) was 5.0%; the 3 limit of detection was 210ÿ5 M. Although GlDH was successfully immobilised on controlled pore glass, and showed good activity between pH 7.0 and 8.5 in a single channel manifold, it was not inhibited by chlorpromazine. Possible reasons for this behaviour are given. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Enzyme inhibition; Glutamate dehydrogenase; Spectrophotometry; Flow injection; Chlorpromazine
1. Introduction Chlorpromazine, 2-chloro-10-(3-dimethylaminopropyl)phenothiazine, is an antipsychotic drug used as a major tranquilizer for patients suffering from schizophrenia and other psychoses, particularly during behavioural disturbances. The drug has been used in the short term to treat anxiety, to soothe patients who are dying and as premedication prior to surgery [1]. A number of studies have indicated that chlorpromazine might be useful in the treatment of disturbed and mentally retarded patients [2]. *Corresponding author. Tel.: +44-1482-465027; fax: +44-1482466416; e-mail: [email protected] 1 Present address: Girls College, Khoiratta, Kotli (AK), Pakistan.
Drugs which are used for psychotics, like chlorpromazine and other isoesters of phenothiazines, are known to inhibit glutamate dehydrogenase (GlDH). Of these chlorpromazine is the most potent inhibitor of this enzyme [3]. The biochemical action of the drug is related to its pharmacological behaviour. Although a number of drugs which do not have antipsychotic
0003-2670/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S0003-2670(99)00034-3
48
T. Ghous, A. Townshend / Analytica Chimica Acta 387 (1999) 47±51
actions have been shown not to inhibit GlDH, a range of other compounds including steroids, thyroxine and anionic dyes have also been shown to be inhibitors of GlDH [4]. Chlorpromazine is an allosteric inhibitor of GlDH. The enzyme is composed of six identical subunits whose amino acid sequence is known [5], each of which has two binding sites for NADH, an active site and an inactive site. At the latter site NADH binds at high concentrations as an inhibitor, bringing conformational change and resulting in the inactivation of the enzyme. The enzyme also has two binding sites for allosteric inhibitors. Chlorpromazine binds at such a binding site and also causes a change in enzyme conformation. Chlorpromazine is more inhibitory if the enzyme is already in an inactive state, that is, in the presence of a high concentration of NADH, and is less inhibitory if the enzyme is in its active conformation in the presence of NAD [6]. The vast number of phenothiazine derivatives and their continuous introduction as drugs has encouraged many workers to explore new methods for their determination. Many spectrophotometric methods have been developed, some lacking sensitivity and selectivity [7,8] or needing a long heating time [9]. Less often, spectro¯uorimetric methods are used for their determination. Most of these methods are based on chemical oxidation reactions [10,11]. Chromatographic methods are the most common for phenothiazine determination [12,13]. All require the use of sophisticated equipment and are time consuming. A few ¯ow injection (FI) methods have been reported. One is based on chemical oxidation [14] and others on UV photochemical oxidation of these compounds [15,16]. Recently a FI biamperometric method was developed [17], which achieved a detection limit of 0.4 mg mlÿ1 with 450 ml of sample. In this paper a FI method for the determination of chlorpromazine (1.010ÿ4 M) based on the inhibition of soluble GlDH is described. The inhibitor and NADH are injected into the ¯owing enzyme zone. When GlDH was immobilised on controlled pore glass, it showed good activity with its substrate aketoglutaric acid at pH 7.0±8.5. However, its use in a ¯ow system did not result in any measurable inhibition by chlorpromazine.
2. Experimental 2.1. Chemicals and reagents L-Glutamate dehydrogenase (EC. 1.4.1.3) type III from bovine liver (40 U per mg protein) was purchased from Sigma (Poole, Dorset, UK). A stock solution (0.2 mg mlÿ1) was prepared by dissolving 20 mg of protein in 50 ml of 0.05 M sodium phosphate buffer, pH 7.5. This was stored for up to one week at 48C, and diluted in appropriate buffer immediately before use. All other reagents were prepared fresh every day. aKetoglutaric acid (crystalline free acid) was obtained from Sigma; 0.0365 g was dissolved in 250 ml of the phosphate buffer to make a 110ÿ3 M solution. A stock solution (410ÿ3 M) of chlorpromazine hydrochloride (Sigma) was prepared by dissolving 0.071 g in 50 ml of ice-cold pH 6.0 0.05 M sodium phosphate buffer. This was kept in an ice bath and protected from light in an amber-coloured bottle. The stock solution was prepared in pH 6.0 sodium phosphate buffer because the solution became turbid at alkaline pH. It was adjusted to pH 7.5 with dilute NaOH solution as required. NADH, disodium salt (Sigma) stock solution was made by dissolving 0.073 g in the sodium phosphate buffer (pH 7.5). NH4Cl and all other reagents were of analytical reagent grade.
2.2. Enzyme immobilisation GlDH (2.5 mg mlÿ1) was immobilised on controlled pore glass (CPG) (0.2 g) by the method described by Leon-Gonzales and Townshend [18]. 2.3. Instrumentation and procedures Injections were made by Rheodyne RH 5020 rotary injection valves (Anachem), the peristaltic pump was a Gilson Minipuls 2, the ¯ow tubing was 0.5 mm i.d. PTFE and the detector was a Cecil CE 272 spectrophotometer, with a 30 ml, 10 mm light path ¯ow cell. 2.3.1. Soluble enzyme The FI manifold used comprised two injection valves connected in series. Enzyme solution (10 mg mlÿ1) was injected from the ®rst injection valve (70 ml) into the carrier stream of sodium phosphate buffer (0.05 M, pH 7.5) containing a-ketoglu-
T. Ghous, A. Townshend / Analytica Chimica Acta 387 (1999) 47±51
taric acid (110ÿ3 M) and NH4Cl (110ÿ3 M) ¯owing at 1.0 ml minÿ1. When the enzyme reached the second injection valve after exactly 10 s (timed with a stop watch), mixed NADH drug-solution (44 ml) was injected. The NADH peak aborbance was monitored at 340 nm. The % inhibition (%I) was calculated on the basis of the following equation: %I 100 As ÿ Ae ÿ As ÿ Ai = As ÿ Ae 100 Ai ÿ As = As ÿ Ae ; where Assubstrate peak absorbance alone, Aisubstrate absorbance in the presence of the enzyme and inhibitor and Aesubstrate absorbance in the presence of enzyme only. 2.3.2. Immobilised enzyme A column of immobilised enzyme (2.5 mm long2.5 mm i.d.) was used in a single channel manifold. Inhibition studies were carried out by injecting a mixture of NADH and chlorpromazine (50 ml) into the carrier stream (1.0 ml minÿ1) of sodium phosphate buffer (0.05 M, pH 7.5) containing 110ÿ3 M NH4Cl and 510ÿ4 M a-ketoglutaric acid. The NADH peak absorbance was measured at 340 nm in the presence and absence of the drug, in order to measure %I. 3. Results and discussion 3.1. Basic description of the procedure The purpose of the investigation was to develop a FI method for the determination of chlorpromazine on the basis of its inhibition of GlDH. Such procedures have been shown to be applicable to other drugs, such as metrifonate [19], neostigmine and galanthamine
49
[20] based on inhibition of acetylcholinesterase (AChE), as well as to pesticides which are also AChE inhibitors [18,21,22]. It was intended to study both the soluble and immobilised enzyme, but only the former provided useful results. GlDH catalyses the reaction: HOOCCH2 CH2 COCOOH NADH NH 4 HOOCCH2 CH2 CH NH2 COOH NAD H2 O For the study of the soluble enzyme, to minimise the consumption of enzyme and coenzyme NADH, a double injection procedure was selected, in which a small volume of the enzyme solution was injected into a carrier stream comprising a-ketoglutarate, ammonium chloride and sodium phosphate buffer, and NADH plus drug was injected from a second value downstream to coincide with the passage of the enzyme zone. Accurate timing between the two injections (in this case exactly 10 s, measured by a stop watch) was necessary to achieve reproducible results. 3.2. Optimisation The variables and ranges studied, and the consequent recommended values, are summarised in Table 1. As described previously [20], a drug has less effect in assays involving higher concentrations of the enzyme. This can also be seen in Fig. 1. The per cent inhibition decreased with increase in enzyme concentration. This might be due to the loss of substrate inhibition which occurs at high enzyme concentration, which could be due to the masking of the NADH modi®er site, or due to the inability of chlorpromazine to promote conformational changes when the enzyme is at high concentration. An increase in % inhibition was observed down to 0.4 mg mlÿ1, but 4.0 mg mlÿ1
Table 1 Optimisation study for chlorpromazine determination by inhibition of soluble GlDH Variable
Range studied
Recommended value
Results shown in
pH GlDH (mg mlÿ1) NADH concentration (M) a-Ketoglutarate concentration (M)
5.5±8.0 0.2±100 2±1010ÿ5 2±2010ÿ4
7.5 4.0 810ÿ5 1010ÿ4
Fig. 3 Fig. 1 Fig. 2
810ÿ5 M chlorpromazine, other parameters at the recommended values unless being varied.
50
T. Ghous, A. Townshend / Analytica Chimica Acta 387 (1999) 47±51
Fig. 3. Effect of pH on per cent inhibition. (810ÿ5 M NADH, 44 ml volume, 810ÿ5 M drug and 4 mg mlÿ1 enzyme).
Fig. 1. Effect of enzyme concentration on per cent inhibition. Drug and NADH concentrations were 810ÿ5 M.
was selected for further work as the enzyme gave too little response at concentrations as low as 0.4 mg mlÿ1. The effect of NADH concentration on inhibition was studied at two concentrations of enzyme (4.0 and 20 ml mlÿ1). As can be seen in Fig. 2, % inhibition increased with increase in NADH concentration at both enzyme concentrations, but the increase was greater at the lower enzyme concentration. Thus 8 10ÿ5 M NADH was selected for further study, which gave 33% inhibition with 4.0 mg mlÿ1 enzyme. Maximum enzyme activity was found at pH 7.0, but inhibition was greatest at pH 7.5 (Fig. 3). The % inhibition increased with increase in substrate concentration up to 110ÿ3 M, above which it had no effect. 3.3. Chlorpromazine determination From the results obtained under the recommended conditions (Table 1), a calibration graph was constructed for chlorpromazine which was linear in the
Fig. 2. Effect of NADH concentration on per cent inhibition. Enzyme concentration was (a) 4.0 and (b) 20 mg mlÿ1; drug concentration was 810ÿ5 M.
range 2±1010ÿ5 M (7±42 %I). The least squares linear equation was %Iÿ2.14 4.42105 (drug, M), r0.998 (n7). The limit of detection (calculated as the blank signal plus three standard deviations of the blank signal) was 210ÿ5 M. A relative standard deviation of 5% was obtained for 610ÿ5 M drug (n6). 3.4. Immobilised enzyme Immobilisation of GlDH on controlled pore glass was successful. When incorporated into the FI system shown in Fig. 2, the enzyme showed good activity between pH 7.0 and 8.5, with maximum activity at pH 7.5, a little higher pH than the soluble enzyme. Good responses to the substrate (510ÿ4 M a-ketoglutarate/110ÿ3 M NH4Cl), were achieved at 7± 20104 M NADH (30±90 ml) at a ¯ow rate of 0.6± 2.0 ml minÿ1. Under such conditions, when NADH and chlorpromazine were injected as a 50 ml mixed solution, as for the soluble enzyme, no inhibition was observed. Reasons for this lack of inhibition were considered. GlDH exists in solution in an association±dissociation equilibrium. At a protein concentration lower than 0.05 mg mlÿ1 dissociation is favoured and the enzyme exists in the monomeric form. Above 0.05 mg mlÿ1 association of monomeric units occurs. It has been suggested [6] that nucleotides and allosteric effectors preferably bind either to the monomeric or polymeric form. As chlorpromazine binds preferably to the monomeric form it means there will be more inhibition produced by chlorpromazine when the enzyme is monomeric [23]. In this work when the enzyme was immobilised its concentration was initially 2.5 ml g mlÿ1, which shows that the enzyme was in the polymeric form when immobilised and less sus-
T. Ghous, A. Townshend / Analytica Chimica Acta 387 (1999) 47±51
ceptible to inhibition. However, when enzyme at two other, much lower concentrations (0.02 and 0.0004 mg mlÿ1) was immobilised, no inhibition was again obtained. This means that the enzyme concentration and association was not the source of the problem. The lack of inhibition was probably a consequence of the immobilisation procedure. The effect of the cross-linking agent glutaraldehyde on the allosteric properties of the enzyme has been discussed in detail by Josephs et al. [6]. It may be that binding of the enzyme with glutaraldehyde makes it dif®cult for the enzyme to exist in the monomeric form, and is therefore less susceptible to inhibition. There is another possibility, that the inhibitor binding site was not affected during immobilisation but the inactive binding site for NADH was masked so that interaction of NADH with this site was ineffective. As inhibition by the drug is consequent on an induced change in the enzyme conformation by the interaction of NADH with this inactive site, the lack of such change will lead to less or no inhibition of the enzyme by its inhibitors. Acknowledgements Thanks are due to Dr. Wali Muhammed and the Charles Wallace Trust for their partial ®nancial support to TG. References [1] H. Graham, H. Shapiro, Medicines, a comprehensive guide, Bloomsbury, London, 1991.
51
[2] L.S. Goodman, A. Gilman, The pharmacological basis of therapeutics, 4th ed., Macmillan, London, 1970. [3] O.A. Shemisa, L.A. Fahien, Mol. Pharmacol. 7 (1971) 8. [4] I. Couee, K.F. Timpton, Bioochem. Pharmacol. 39 (1990) 827 and references therein. [5] J.H. Julliard, E.L. Smith, J. Biochem. 254 (1979) 3427. [6] R. Josephs, H. Eisenberg, E. Reisler, Biochemistry 12 (1973) 4060. [7] P.G. Ramappa, H.S. Gowda, A.N. Nayak, Analyst 105 (1980) 663. [8] P.G. Ramappa, K. Basaviah, Indian J. Pharm. Sci. 47 (1985) 125. [9] M.S. Mahrous, M.M. Abdel Khalik, Talanta 31 (1984) 289. [10] J.B. Rangland, V.J. Kinross-Wright, Anal. Chem. 36 (1964) 1357. [11] T.J. Millinger, C.E. Keeler, Anal. Chem. 36 (1964) 1841. [12] L.S. Chagonda, J.S. Millership, Analyst 113 (1988) 233. [13] H. Maurer, K. Pfleger, J. Chromatogr., Biomed. Appl. 306 (1984) 125. [14] M.A. Koupparis, A. Barcuchova, Analyst 306 (1986) 313. [15] D. Chen, A. Rios, M.D. Luque de Castro, M. Valcarcel, Analyst 116 (1991) 171. [16] J. Martinez Calatayud, C. Gomez Benito, Anal. Chim. Acta 256 (1992) 105. [17] J. Michalowski, A. Kojlo, B. Magnuszewska, M. Trojanowicz, Anal. Chim. Acta 289 (1994) 339. [18] M.E. Leon-Gonzales, A. Townshend, Anal. Chim. Acta 236 (1990) 267. [19] T. Ghous, A. Townshend, Anal. Chim. Acta 332 (1996) 179. [20] T. Ghous, A. Townshend, Anal. Chim. Acta 372 (1998) 379. [21] A.M. Almuaibed, I.A. Takruni, A. Townshend, Anal. Chim. Acta 282 (1993) 312. [22] C. Garcia de Maria, T.M. Munoz, A. Townshend, Anal. Chim. Acta 295 (1994) 287. [23] K.F. Tipton, I. Couee, Glutamate dehydrogenase, in: E. Kvamme (Ed.), Glutamine and Glutamate in Mammals, CRC Press, Boca Raton, FL, 1988.
Flow injection determination of chlorpromazine by inhibition of glutamate dehydrogenase Tahseen Ghous1, Alan Townshend* Department of Chemistry, University of Hull, HU6 7RX, Hull, UK Received 14 August 1998; received in revised form 17 December 1998; accepted 30 December 1998
Abstract A double injection ¯ow procedure is used for the determination of chlorpromazine (1010ÿ5 M, in 44 ml), by its inhibition of glutamate dehydrogenase (GlDH). The enzyme is injected into pH 7.5 phosphate buffer containing substrate (110ÿ3 M aketoglutaric acid) and 110ÿ3 M NH4Cl, and drug/NADH (810ÿ5 M) solution is injected from a second valve in series into the enzyme zone. The relative standard deviation for the determination of 610ÿ5 M drug (n6) was 5.0%; the 3 limit of detection was 210ÿ5 M. Although GlDH was successfully immobilised on controlled pore glass, and showed good activity between pH 7.0 and 8.5 in a single channel manifold, it was not inhibited by chlorpromazine. Possible reasons for this behaviour are given. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Enzyme inhibition; Glutamate dehydrogenase; Spectrophotometry; Flow injection; Chlorpromazine
1. Introduction Chlorpromazine, 2-chloro-10-(3-dimethylaminopropyl)phenothiazine, is an antipsychotic drug used as a major tranquilizer for patients suffering from schizophrenia and other psychoses, particularly during behavioural disturbances. The drug has been used in the short term to treat anxiety, to soothe patients who are dying and as premedication prior to surgery [1]. A number of studies have indicated that chlorpromazine might be useful in the treatment of disturbed and mentally retarded patients [2]. *Corresponding author. Tel.: +44-1482-465027; fax: +44-1482466416; e-mail: [email protected] 1 Present address: Girls College, Khoiratta, Kotli (AK), Pakistan.
Drugs which are used for psychotics, like chlorpromazine and other isoesters of phenothiazines, are known to inhibit glutamate dehydrogenase (GlDH). Of these chlorpromazine is the most potent inhibitor of this enzyme [3]. The biochemical action of the drug is related to its pharmacological behaviour. Although a number of drugs which do not have antipsychotic
0003-2670/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved. PII: S0003-2670(99)00034-3
48
T. Ghous, A. Townshend / Analytica Chimica Acta 387 (1999) 47±51
actions have been shown not to inhibit GlDH, a range of other compounds including steroids, thyroxine and anionic dyes have also been shown to be inhibitors of GlDH [4]. Chlorpromazine is an allosteric inhibitor of GlDH. The enzyme is composed of six identical subunits whose amino acid sequence is known [5], each of which has two binding sites for NADH, an active site and an inactive site. At the latter site NADH binds at high concentrations as an inhibitor, bringing conformational change and resulting in the inactivation of the enzyme. The enzyme also has two binding sites for allosteric inhibitors. Chlorpromazine binds at such a binding site and also causes a change in enzyme conformation. Chlorpromazine is more inhibitory if the enzyme is already in an inactive state, that is, in the presence of a high concentration of NADH, and is less inhibitory if the enzyme is in its active conformation in the presence of NAD [6]. The vast number of phenothiazine derivatives and their continuous introduction as drugs has encouraged many workers to explore new methods for their determination. Many spectrophotometric methods have been developed, some lacking sensitivity and selectivity [7,8] or needing a long heating time [9]. Less often, spectro¯uorimetric methods are used for their determination. Most of these methods are based on chemical oxidation reactions [10,11]. Chromatographic methods are the most common for phenothiazine determination [12,13]. All require the use of sophisticated equipment and are time consuming. A few ¯ow injection (FI) methods have been reported. One is based on chemical oxidation [14] and others on UV photochemical oxidation of these compounds [15,16]. Recently a FI biamperometric method was developed [17], which achieved a detection limit of 0.4 mg mlÿ1 with 450 ml of sample. In this paper a FI method for the determination of chlorpromazine (1.010ÿ4 M) based on the inhibition of soluble GlDH is described. The inhibitor and NADH are injected into the ¯owing enzyme zone. When GlDH was immobilised on controlled pore glass, it showed good activity with its substrate aketoglutaric acid at pH 7.0±8.5. However, its use in a ¯ow system did not result in any measurable inhibition by chlorpromazine.
2. Experimental 2.1. Chemicals and reagents L-Glutamate dehydrogenase (EC. 1.4.1.3) type III from bovine liver (40 U per mg protein) was purchased from Sigma (Poole, Dorset, UK). A stock solution (0.2 mg mlÿ1) was prepared by dissolving 20 mg of protein in 50 ml of 0.05 M sodium phosphate buffer, pH 7.5. This was stored for up to one week at 48C, and diluted in appropriate buffer immediately before use. All other reagents were prepared fresh every day. aKetoglutaric acid (crystalline free acid) was obtained from Sigma; 0.0365 g was dissolved in 250 ml of the phosphate buffer to make a 110ÿ3 M solution. A stock solution (410ÿ3 M) of chlorpromazine hydrochloride (Sigma) was prepared by dissolving 0.071 g in 50 ml of ice-cold pH 6.0 0.05 M sodium phosphate buffer. This was kept in an ice bath and protected from light in an amber-coloured bottle. The stock solution was prepared in pH 6.0 sodium phosphate buffer because the solution became turbid at alkaline pH. It was adjusted to pH 7.5 with dilute NaOH solution as required. NADH, disodium salt (Sigma) stock solution was made by dissolving 0.073 g in the sodium phosphate buffer (pH 7.5). NH4Cl and all other reagents were of analytical reagent grade.
2.2. Enzyme immobilisation GlDH (2.5 mg mlÿ1) was immobilised on controlled pore glass (CPG) (0.2 g) by the method described by Leon-Gonzales and Townshend [18]. 2.3. Instrumentation and procedures Injections were made by Rheodyne RH 5020 rotary injection valves (Anachem), the peristaltic pump was a Gilson Minipuls 2, the ¯ow tubing was 0.5 mm i.d. PTFE and the detector was a Cecil CE 272 spectrophotometer, with a 30 ml, 10 mm light path ¯ow cell. 2.3.1. Soluble enzyme The FI manifold used comprised two injection valves connected in series. Enzyme solution (10 mg mlÿ1) was injected from the ®rst injection valve (70 ml) into the carrier stream of sodium phosphate buffer (0.05 M, pH 7.5) containing a-ketoglu-
T. Ghous, A. Townshend / Analytica Chimica Acta 387 (1999) 47±51
taric acid (110ÿ3 M) and NH4Cl (110ÿ3 M) ¯owing at 1.0 ml minÿ1. When the enzyme reached the second injection valve after exactly 10 s (timed with a stop watch), mixed NADH drug-solution (44 ml) was injected. The NADH peak aborbance was monitored at 340 nm. The % inhibition (%I) was calculated on the basis of the following equation: %I 100 As ÿ Ae ÿ As ÿ Ai = As ÿ Ae 100 Ai ÿ As = As ÿ Ae ; where Assubstrate peak absorbance alone, Aisubstrate absorbance in the presence of the enzyme and inhibitor and Aesubstrate absorbance in the presence of enzyme only. 2.3.2. Immobilised enzyme A column of immobilised enzyme (2.5 mm long2.5 mm i.d.) was used in a single channel manifold. Inhibition studies were carried out by injecting a mixture of NADH and chlorpromazine (50 ml) into the carrier stream (1.0 ml minÿ1) of sodium phosphate buffer (0.05 M, pH 7.5) containing 110ÿ3 M NH4Cl and 510ÿ4 M a-ketoglutaric acid. The NADH peak absorbance was measured at 340 nm in the presence and absence of the drug, in order to measure %I. 3. Results and discussion 3.1. Basic description of the procedure The purpose of the investigation was to develop a FI method for the determination of chlorpromazine on the basis of its inhibition of GlDH. Such procedures have been shown to be applicable to other drugs, such as metrifonate [19], neostigmine and galanthamine
49
[20] based on inhibition of acetylcholinesterase (AChE), as well as to pesticides which are also AChE inhibitors [18,21,22]. It was intended to study both the soluble and immobilised enzyme, but only the former provided useful results. GlDH catalyses the reaction: HOOCCH2 CH2 COCOOH NADH NH 4 HOOCCH2 CH2 CH NH2 COOH NAD H2 O For the study of the soluble enzyme, to minimise the consumption of enzyme and coenzyme NADH, a double injection procedure was selected, in which a small volume of the enzyme solution was injected into a carrier stream comprising a-ketoglutarate, ammonium chloride and sodium phosphate buffer, and NADH plus drug was injected from a second value downstream to coincide with the passage of the enzyme zone. Accurate timing between the two injections (in this case exactly 10 s, measured by a stop watch) was necessary to achieve reproducible results. 3.2. Optimisation The variables and ranges studied, and the consequent recommended values, are summarised in Table 1. As described previously [20], a drug has less effect in assays involving higher concentrations of the enzyme. This can also be seen in Fig. 1. The per cent inhibition decreased with increase in enzyme concentration. This might be due to the loss of substrate inhibition which occurs at high enzyme concentration, which could be due to the masking of the NADH modi®er site, or due to the inability of chlorpromazine to promote conformational changes when the enzyme is at high concentration. An increase in % inhibition was observed down to 0.4 mg mlÿ1, but 4.0 mg mlÿ1
Table 1 Optimisation study for chlorpromazine determination by inhibition of soluble GlDH Variable
Range studied
Recommended value
Results shown in
pH GlDH (mg mlÿ1) NADH concentration (M) a-Ketoglutarate concentration (M)
5.5±8.0 0.2±100 2±1010ÿ5 2±2010ÿ4
7.5 4.0 810ÿ5 1010ÿ4
Fig. 3 Fig. 1 Fig. 2
810ÿ5 M chlorpromazine, other parameters at the recommended values unless being varied.
50
T. Ghous, A. Townshend / Analytica Chimica Acta 387 (1999) 47±51
Fig. 3. Effect of pH on per cent inhibition. (810ÿ5 M NADH, 44 ml volume, 810ÿ5 M drug and 4 mg mlÿ1 enzyme).
Fig. 1. Effect of enzyme concentration on per cent inhibition. Drug and NADH concentrations were 810ÿ5 M.
was selected for further work as the enzyme gave too little response at concentrations as low as 0.4 mg mlÿ1. The effect of NADH concentration on inhibition was studied at two concentrations of enzyme (4.0 and 20 ml mlÿ1). As can be seen in Fig. 2, % inhibition increased with increase in NADH concentration at both enzyme concentrations, but the increase was greater at the lower enzyme concentration. Thus 8 10ÿ5 M NADH was selected for further study, which gave 33% inhibition with 4.0 mg mlÿ1 enzyme. Maximum enzyme activity was found at pH 7.0, but inhibition was greatest at pH 7.5 (Fig. 3). The % inhibition increased with increase in substrate concentration up to 110ÿ3 M, above which it had no effect. 3.3. Chlorpromazine determination From the results obtained under the recommended conditions (Table 1), a calibration graph was constructed for chlorpromazine which was linear in the
Fig. 2. Effect of NADH concentration on per cent inhibition. Enzyme concentration was (a) 4.0 and (b) 20 mg mlÿ1; drug concentration was 810ÿ5 M.
range 2±1010ÿ5 M (7±42 %I). The least squares linear equation was %Iÿ2.14 4.42105 (drug, M), r0.998 (n7). The limit of detection (calculated as the blank signal plus three standard deviations of the blank signal) was 210ÿ5 M. A relative standard deviation of 5% was obtained for 610ÿ5 M drug (n6). 3.4. Immobilised enzyme Immobilisation of GlDH on controlled pore glass was successful. When incorporated into the FI system shown in Fig. 2, the enzyme showed good activity between pH 7.0 and 8.5, with maximum activity at pH 7.5, a little higher pH than the soluble enzyme. Good responses to the substrate (510ÿ4 M a-ketoglutarate/110ÿ3 M NH4Cl), were achieved at 7± 20104 M NADH (30±90 ml) at a ¯ow rate of 0.6± 2.0 ml minÿ1. Under such conditions, when NADH and chlorpromazine were injected as a 50 ml mixed solution, as for the soluble enzyme, no inhibition was observed. Reasons for this lack of inhibition were considered. GlDH exists in solution in an association±dissociation equilibrium. At a protein concentration lower than 0.05 mg mlÿ1 dissociation is favoured and the enzyme exists in the monomeric form. Above 0.05 mg mlÿ1 association of monomeric units occurs. It has been suggested [6] that nucleotides and allosteric effectors preferably bind either to the monomeric or polymeric form. As chlorpromazine binds preferably to the monomeric form it means there will be more inhibition produced by chlorpromazine when the enzyme is monomeric [23]. In this work when the enzyme was immobilised its concentration was initially 2.5 ml g mlÿ1, which shows that the enzyme was in the polymeric form when immobilised and less sus-
T. Ghous, A. Townshend / Analytica Chimica Acta 387 (1999) 47±51
ceptible to inhibition. However, when enzyme at two other, much lower concentrations (0.02 and 0.0004 mg mlÿ1) was immobilised, no inhibition was again obtained. This means that the enzyme concentration and association was not the source of the problem. The lack of inhibition was probably a consequence of the immobilisation procedure. The effect of the cross-linking agent glutaraldehyde on the allosteric properties of the enzyme has been discussed in detail by Josephs et al. [6]. It may be that binding of the enzyme with glutaraldehyde makes it dif®cult for the enzyme to exist in the monomeric form, and is therefore less susceptible to inhibition. There is another possibility, that the inhibitor binding site was not affected during immobilisation but the inactive binding site for NADH was masked so that interaction of NADH with this site was ineffective. As inhibition by the drug is consequent on an induced change in the enzyme conformation by the interaction of NADH with this inactive site, the lack of such change will lead to less or no inhibition of the enzyme by its inhibitors. Acknowledgements Thanks are due to Dr. Wali Muhammed and the Charles Wallace Trust for their partial ®nancial support to TG. References [1] H. Graham, H. Shapiro, Medicines, a comprehensive guide, Bloomsbury, London, 1991.
51
[2] L.S. Goodman, A. Gilman, The pharmacological basis of therapeutics, 4th ed., Macmillan, London, 1970. [3] O.A. Shemisa, L.A. Fahien, Mol. Pharmacol. 7 (1971) 8. [4] I. Couee, K.F. Timpton, Bioochem. Pharmacol. 39 (1990) 827 and references therein. [5] J.H. Julliard, E.L. Smith, J. Biochem. 254 (1979) 3427. [6] R. Josephs, H. Eisenberg, E. Reisler, Biochemistry 12 (1973) 4060. [7] P.G. Ramappa, H.S. Gowda, A.N. Nayak, Analyst 105 (1980) 663. [8] P.G. Ramappa, K. Basaviah, Indian J. Pharm. Sci. 47 (1985) 125. [9] M.S. Mahrous, M.M. Abdel Khalik, Talanta 31 (1984) 289. [10] J.B. Rangland, V.J. Kinross-Wright, Anal. Chem. 36 (1964) 1357. [11] T.J. Millinger, C.E. Keeler, Anal. Chem. 36 (1964) 1841. [12] L.S. Chagonda, J.S. Millership, Analyst 113 (1988) 233. [13] H. Maurer, K. Pfleger, J. Chromatogr., Biomed. Appl. 306 (1984) 125. [14] M.A. Koupparis, A. Barcuchova, Analyst 306 (1986) 313. [15] D. Chen, A. Rios, M.D. Luque de Castro, M. Valcarcel, Analyst 116 (1991) 171. [16] J. Martinez Calatayud, C. Gomez Benito, Anal. Chim. Acta 256 (1992) 105. [17] J. Michalowski, A. Kojlo, B. Magnuszewska, M. Trojanowicz, Anal. Chim. Acta 289 (1994) 339. [18] M.E. Leon-Gonzales, A. Townshend, Anal. Chim. Acta 236 (1990) 267. [19] T. Ghous, A. Townshend, Anal. Chim. Acta 332 (1996) 179. [20] T. Ghous, A. Townshend, Anal. Chim. Acta 372 (1998) 379. [21] A.M. Almuaibed, I.A. Takruni, A. Townshend, Anal. Chim. Acta 282 (1993) 312. [22] C. Garcia de Maria, T.M. Munoz, A. Townshend, Anal. Chim. Acta 295 (1994) 287. [23] K.F. Tipton, I. Couee, Glutamate dehydrogenase, in: E. Kvamme (Ed.), Glutamine and Glutamate in Mammals, CRC Press, Boca Raton, FL, 1988.