45
Journal of Biochemical and Biophysical Methods, 23 (1991) 45-52 0 1991 Elsevier Science Publishers B.V. 0165-022X/91/$03.50 ADONIS 0165022X9100078E
JBBM
00887
etermination of hemoglobin through its peroxidase activity on chlorpromazine A. Vhzquez I, J. Tudela 2, R. Varh
’ and F. Garcia-CBnovas
2
’ Gitedra de Quimica, Escuela Universitaria Polit&zica de Albacete, Unioersidad de Castilla-La Mancha, Spain and 2 Departamento
de Bioquimica,
Facultad de Biologia, Universidad de Murcia, Spain
(Received 1 August 1990) (Revision received 3 December 1990) (Accepted 22 February 1991)
Chlorpromazine is an excellent chromogen for determining micro-quantities of hemoglobin. The oxidation of chlorpromazine by peroxidase activity of hemoglobin is coupled to a non-enzymatic reaction of second-order. Kinetic analysis of the overall system leads to a discussion about the optimal assay conditions. Spectrophotometric progress curves for the accumulation of the chlorpromazine cation radical during the reaction have been obtained, and further analyzed by non-linear regression. The use of a linear calibration curve of the enzymatic reaction rate against hemoglobin concentration is proposed for its determination. Key words: Hemoglobin;
Peroxidase activity; Enzyme kinetics; Chlorpromazine;
Phenothiazine
Introduction
Almost all hemoproteins and hemin compounds show peroxidase activity. Thus, hemoglobin (Hb) and some of its degradation products possess properties comparable to those of peroxidase enzymes [l]. The peroxidase activity of Hb is about lo3 times lower than that of the activity of horseradish peroxidase [2]. Various methods have been proposed to determine Hb in serum and urine based on the peroxidase activity of Hb using either benzidine [3-71, or o-tolidine [X] as hydrogen donors. These reagents are carcinogenic, and some authors have sought other chromogens [9]. Correspondence address: Dr. Francisco Garcia C&novas, Departamento de Biologia; Edificio de Bioquimica, Planta 2, Ala C, Universidad Murcia, Spain.
de Bioquimica, Unidad Docente de Murcia. E-30100 Espinardo,
46 Thus, the peroxidase activity of Hb has been used with chlorpromazine (CPZ) as reductant [10]. This analytical method is based on the measurement of the rate of formation of the coloured cation radical (CPZ +') formed during the enzymatic oxidation of CPZ. The use of CPZ has been reported as being suitable for the detection of occult blood in feces [11 !. The estimation Of micro-quantities of H b in aqueous solution and urine, previously dialyzed, confirms that this method is more precise and sensitive than the o-tolidine method [91. Several different phenothiazines have also been used as reagents for developing tests for the demonstration of the peroxidase activity of hemoglobin [12]. A study of six different phenothiazines was carried out [13] and it was concluded that chlorpromazine and proch!orperazine were more sensitive than the other four phenothiazines. Other authors [14] have described an assay for the determination of Hb with C P Z in tissue homogenates, using an apparent linear calibration curve relating the m a x i m u m level of the cation radical with the H b concentration. The above reports did not bear in mind that the oxidation of CPZ by any peroxidase is coupled to a non-enzymatic reaction of second-order [15] causing the breakdown of the intermediate CPZ +" Both reactions are affected by the assay conditions [16,17] and this determines the speed of the appearance and disappearance of CPZ+; as well as the m a x i m u m level of its accumulation. In this paper a kinetic analysis of two coupled reactions the enzymatic oxidation of CPZ by peroxidase action of Hb, and the non-enzymatic breakdown of CPZ +" has been carried out. T!"As leads to discussion of the optimal conditions for the experimental assays, as well as to improvement of the kinetic data analysis of determination of Hb.
Materials and Methods Hemoglobin (Hb) and chlorpromazine hydrochloride ( C P Z - HC1) were purchased from Sigma (U.S.A.). Other reagents were of analytical reagent grade and were supplied by E. Merck (F.R.G.). The stock solution of H b was prepared i m g / m l and the exact H b content was determined by the Hartree method [18]. The H202 and CPZ solutions were freshly prepared every day. The reaction mixture contained E D T A 0.83 m M to inhibit Fe z+ catalytic activity, without affecting peroxidase activity [10]. The barfer used was 0.1 M sodium phosphate at different p H values to increase the stability of CPZ +° [191. Spectrophotometric measurements were carried out using a Beckman D U - 7 spectrophotometer. Temperature was controlled by a Hetofrig circulating b a t h with a precision of + 0.1 ° C. The CPZ +" accumulation was monitored spectrophotometrically at 530 nm, the corresponding molar absorptivity being 12 380 M - 1 c m - 1 [20]. The progress curves of As30 vs. t consisting of 100 data points were automatically acquired by the spectrophotometer and transferred by means of a serial RS232C interface to an IBM P C / X T microcomputer for storage, and for regression analysis later. The data points were fitted to Eqn. 2 by a two-parameter non-linear regression
47 algorithm [21] implemented in a powerful BASIC program [22]. Thus, the program uses the sum of the relative errors of the parameters as convergence criterion, numerical derivatives of the function regarding to the parameters, as well as robust weighting factors to remove bias due to outliers [22]. The program yields the values ! of the parameters (V0 a n d kapp) and their respective variances, whose reciprocals are used as weighting factors in further regression analysis, such as that applied to ~ 0 vs. [CPZ] or V0 vs. THbl data. Statistical parameters describing the precision and accuracy of the method have been calculated [21,23].
Results and Discussion
Kinetic analysis The oxidation of chlorpromazine (CPZ) by H202, catalyzed by hemoglobin, involves two c~upled reactions [15]: \ 2 CPZ + H202 'H-~b2 CPZ +'+ 2 H 2 0 2 CPZ +" kapp>CPZ-sulfoxide + CPZ (Scheme 1) where V0 is the enzymatic reaction rate and kap p is the apparent second order constant of radical decomposition. In the kinetic analysis of the overall system (Scheme 1), it has been considered that the consumption of CPZ and H202 is negligible during the assay time, and that the levels in the reaction medium of the intermediate CPZ ÷" and of the product (CPZ-sulfoxide) do not inhibit the peroxidase activity of Hb. These conditions have been controlled and verified in the experimental assays. The accumulation of [CPZ +'] is described by: d{CPZ+'l t +. 2 dt - NO- kapp [CPZ ]
(1)
/
where kap p = 2 kap p. Integrating this differential equation, with [CPZ +'] = 0 for t = 0, the following analytical expression for the accumulation of [CPZ +'] with time can be obtained [241:
1-e-2~t [CPZ+'] =
V°
X
ap~-rk 7
When 2
~
l+e-2~t
(2)
t >> 1, Eqn. 2 is transformed into:
/ [CPZ+']max = 1/ V
Vo
a-Gp
(3)
48
Thus, the maximum level of the intermediate is related to kinetic parameters corresponding to both the enzymatic and the non-enzymatic reactions of Scheme 1.
Data analysis of experimental recordings The experimental recordings of [CPZ +'] vs. time (Fig. 1) permit the calculation of V0 from the slope of the progress curve at low time values (t ~ 0). These data lead also to the evaluation of [CPZ +] .... from data with zero slope at the plateau (Fig. 1). Then, kap p c a n be calculated by using Eqn. 3. The values of V0 and kap p can be introduced in Eqn. 2, but poor agreement between experimental and calculated data is obtained (Fig. 1). These values, however, may be used as initial estimations for a non-linear regression fitting [21,22] of the experimental data to Eqn. 2. The final estimations of both kinetic parameters provide a successful overlay between experimental and calculated data (Fig. 1), Therefore, the transient phase approach used in the kinetic analysis, coupled to a non-linear regression fitting of the complete progress curve, improves the reliability of V0 and katpp values for Scheme 1, compared to the slope method for the initial and final portions of the experimental recordings. t
t
Assay conditions The increase of [CPZ], [ H 2 0 2 ] and pH enhances V0 but decreases the lifetime of the plateau, of which the maximum level [CPZ+'[m~.~ is higher at low pH values (Fig. 2). The enzymatic reaction rate shows a hyperbofic dependence of V0 on [CPZ] and [H202]. These data have been fitted, by non-linear regression [21,22], to the Michaelis equation, yielding K c p z = 1.1 ± 0.5 mM and K ~ 2°2 = 1.5 _+ 0.6 M. Initial
0.3
E 0.2
0.1
////
///
I
10
20
30 t (rain) Fig. 1. Data analysis of a progress curve. (O) experimental resutts; (. . . . . . ) data calculated using initial estimations of the kinetic parameters; V0 = 0.017/~M.s -1 and ka~pp= 60.0 M -1 s - I by the steady state approach; ( ) data calculated using final estimations of the kinetic parameters, obtained by non-linear regression: V0 = 0.0199 ~ M . s -1 a n d k~pp = 65.6 M - i s -~. The reaction medium at 25 ° C contained 0.1 M sodium phosphate buffer, pH 3.0, 0.83 mM EDTA, 16 mM CPZ, 90 mM H202 and 3.3 /~g/ml Hb.
49
Journal of Biochemical and Biophysical Methods, 23 (1991) 45-52 0 1991 Elsevier Science Publishers B.V. 0165-022X/91/$03.50 ADONIS 0165022X9100078E
JBBM
00887
etermination of hemoglobin through its peroxidase activity on chlorpromazine A. Vhzquez I, J. Tudela 2, R. Varh
’ and F. Garcia-CBnovas
2
’ Gitedra de Quimica, Escuela Universitaria Polit&zica de Albacete, Unioersidad de Castilla-La Mancha, Spain and 2 Departamento
de Bioquimica,
Facultad de Biologia, Universidad de Murcia, Spain
(Received 1 August 1990) (Revision received 3 December 1990) (Accepted 22 February 1991)
Chlorpromazine is an excellent chromogen for determining micro-quantities of hemoglobin. The oxidation of chlorpromazine by peroxidase activity of hemoglobin is coupled to a non-enzymatic reaction of second-order. Kinetic analysis of the overall system leads to a discussion about the optimal assay conditions. Spectrophotometric progress curves for the accumulation of the chlorpromazine cation radical during the reaction have been obtained, and further analyzed by non-linear regression. The use of a linear calibration curve of the enzymatic reaction rate against hemoglobin concentration is proposed for its determination. Key words: Hemoglobin;
Peroxidase activity; Enzyme kinetics; Chlorpromazine;
Phenothiazine
Introduction
Almost all hemoproteins and hemin compounds show peroxidase activity. Thus, hemoglobin (Hb) and some of its degradation products possess properties comparable to those of peroxidase enzymes [l]. The peroxidase activity of Hb is about lo3 times lower than that of the activity of horseradish peroxidase [2]. Various methods have been proposed to determine Hb in serum and urine based on the peroxidase activity of Hb using either benzidine [3-71, or o-tolidine [X] as hydrogen donors. These reagents are carcinogenic, and some authors have sought other chromogens [9]. Correspondence address: Dr. Francisco Garcia C&novas, Departamento de Biologia; Edificio de Bioquimica, Planta 2, Ala C, Universidad Murcia, Spain.
de Bioquimica, Unidad Docente de Murcia. E-30100 Espinardo,
46 Thus, the peroxidase activity of Hb has been used with chlorpromazine (CPZ) as reductant [10]. This analytical method is based on the measurement of the rate of formation of the coloured cation radical (CPZ +') formed during the enzymatic oxidation of CPZ. The use of CPZ has been reported as being suitable for the detection of occult blood in feces [11 !. The estimation Of micro-quantities of H b in aqueous solution and urine, previously dialyzed, confirms that this method is more precise and sensitive than the o-tolidine method [91. Several different phenothiazines have also been used as reagents for developing tests for the demonstration of the peroxidase activity of hemoglobin [12]. A study of six different phenothiazines was carried out [13] and it was concluded that chlorpromazine and proch!orperazine were more sensitive than the other four phenothiazines. Other authors [14] have described an assay for the determination of Hb with C P Z in tissue homogenates, using an apparent linear calibration curve relating the m a x i m u m level of the cation radical with the H b concentration. The above reports did not bear in mind that the oxidation of CPZ by any peroxidase is coupled to a non-enzymatic reaction of second-order [15] causing the breakdown of the intermediate CPZ +" Both reactions are affected by the assay conditions [16,17] and this determines the speed of the appearance and disappearance of CPZ+; as well as the m a x i m u m level of its accumulation. In this paper a kinetic analysis of two coupled reactions the enzymatic oxidation of CPZ by peroxidase action of Hb, and the non-enzymatic breakdown of CPZ +" has been carried out. T!"As leads to discussion of the optimal conditions for the experimental assays, as well as to improvement of the kinetic data analysis of determination of Hb.
Materials and Methods Hemoglobin (Hb) and chlorpromazine hydrochloride ( C P Z - HC1) were purchased from Sigma (U.S.A.). Other reagents were of analytical reagent grade and were supplied by E. Merck (F.R.G.). The stock solution of H b was prepared i m g / m l and the exact H b content was determined by the Hartree method [18]. The H202 and CPZ solutions were freshly prepared every day. The reaction mixture contained E D T A 0.83 m M to inhibit Fe z+ catalytic activity, without affecting peroxidase activity [10]. The barfer used was 0.1 M sodium phosphate at different p H values to increase the stability of CPZ +° [191. Spectrophotometric measurements were carried out using a Beckman D U - 7 spectrophotometer. Temperature was controlled by a Hetofrig circulating b a t h with a precision of + 0.1 ° C. The CPZ +" accumulation was monitored spectrophotometrically at 530 nm, the corresponding molar absorptivity being 12 380 M - 1 c m - 1 [20]. The progress curves of As30 vs. t consisting of 100 data points were automatically acquired by the spectrophotometer and transferred by means of a serial RS232C interface to an IBM P C / X T microcomputer for storage, and for regression analysis later. The data points were fitted to Eqn. 2 by a two-parameter non-linear regression
47 algorithm [21] implemented in a powerful BASIC program [22]. Thus, the program uses the sum of the relative errors of the parameters as convergence criterion, numerical derivatives of the function regarding to the parameters, as well as robust weighting factors to remove bias due to outliers [22]. The program yields the values ! of the parameters (V0 a n d kapp) and their respective variances, whose reciprocals are used as weighting factors in further regression analysis, such as that applied to ~ 0 vs. [CPZ] or V0 vs. THbl data. Statistical parameters describing the precision and accuracy of the method have been calculated [21,23].
Results and Discussion
Kinetic analysis The oxidation of chlorpromazine (CPZ) by H202, catalyzed by hemoglobin, involves two c~upled reactions [15]: \ 2 CPZ + H202 'H-~b2 CPZ +'+ 2 H 2 0 2 CPZ +" kapp>CPZ-sulfoxide + CPZ (Scheme 1) where V0 is the enzymatic reaction rate and kap p is the apparent second order constant of radical decomposition. In the kinetic analysis of the overall system (Scheme 1), it has been considered that the consumption of CPZ and H202 is negligible during the assay time, and that the levels in the reaction medium of the intermediate CPZ ÷" and of the product (CPZ-sulfoxide) do not inhibit the peroxidase activity of Hb. These conditions have been controlled and verified in the experimental assays. The accumulation of [CPZ +'] is described by: d{CPZ+'l t +. 2 dt - NO- kapp [CPZ ]
(1)
/
where kap p = 2 kap p. Integrating this differential equation, with [CPZ +'] = 0 for t = 0, the following analytical expression for the accumulation of [CPZ +'] with time can be obtained [241:
1-e-2~t [CPZ+'] =
V°
X
ap~-rk 7
When 2
~
l+e-2~t
(2)
t >> 1, Eqn. 2 is transformed into:
/ [CPZ+']max = 1/ V
Vo
a-Gp
(3)
48
Thus, the maximum level of the intermediate is related to kinetic parameters corresponding to both the enzymatic and the non-enzymatic reactions of Scheme 1.
Data analysis of experimental recordings The experimental recordings of [CPZ +'] vs. time (Fig. 1) permit the calculation of V0 from the slope of the progress curve at low time values (t ~ 0). These data lead also to the evaluation of [CPZ +] .... from data with zero slope at the plateau (Fig. 1). Then, kap p c a n be calculated by using Eqn. 3. The values of V0 and kap p can be introduced in Eqn. 2, but poor agreement between experimental and calculated data is obtained (Fig. 1). These values, however, may be used as initial estimations for a non-linear regression fitting [21,22] of the experimental data to Eqn. 2. The final estimations of both kinetic parameters provide a successful overlay between experimental and calculated data (Fig. 1), Therefore, the transient phase approach used in the kinetic analysis, coupled to a non-linear regression fitting of the complete progress curve, improves the reliability of V0 and katpp values for Scheme 1, compared to the slope method for the initial and final portions of the experimental recordings. t
t
Assay conditions The increase of [CPZ], [ H 2 0 2 ] and pH enhances V0 but decreases the lifetime of the plateau, of which the maximum level [CPZ+'[m~.~ is higher at low pH values (Fig. 2). The enzymatic reaction rate shows a hyperbofic dependence of V0 on [CPZ] and [H202]. These data have been fitted, by non-linear regression [21,22], to the Michaelis equation, yielding K c p z = 1.1 ± 0.5 mM and K ~ 2°2 = 1.5 _+ 0.6 M. Initial
0.3
E 0.2
0.1
////
///
I
10
20
30 t (rain) Fig. 1. Data analysis of a progress curve. (O) experimental resutts; (. . . . . . ) data calculated using initial estimations of the kinetic parameters; V0 = 0.017/~M.s -1 and ka~pp= 60.0 M -1 s - I by the steady state approach; ( ) data calculated using final estimations of the kinetic parameters, obtained by non-linear regression: V0 = 0.0199 ~ M . s -1 a n d k~pp = 65.6 M - i s -~. The reaction medium at 25 ° C contained 0.1 M sodium phosphate buffer, pH 3.0, 0.83 mM EDTA, 16 mM CPZ, 90 mM H202 and 3.3 /~g/ml Hb.
49
