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A new rapid fluorimetric method for the determination of carbonic anhydrase
A.lwwTIcA CHIMICA
AC’L4 ELSEVIER
Analytica
Chimica Acta 317 (1995) 353-357
A new rapid fluorimetric method for the determination of carbonic anhydrase Huai-Na Li ‘, Yun-Xiang Ci
*
Department of Chemistry, Peking Uniuersity, Beijing 100871, China Received 28 February
1995; revised 19 July 1995; accepted
19 July 1995
Abstract A new assay procedure for carbonic anhydrase activity has been developed, based on the formation of a fluorescent inclusion complex of carbonic anhydrase with dansylamide. The excitation and emission wavelengths of the fluorescent complex are at 280 nm and 460 nm, respectively. The method can be used at 20 f l”C, with buffered media over a pH range of 7.0-8.5. The detection limit for carbonic anhydrase is 29 ng ml-‘. The recommended method is very sensitive, simple and rapid, making it ideal for routine determinations of carbonic anhydrase activity. Keywords:
Fluorimetry;
Carbonic
anhydrase
1. Introduction Carbonic anhydrase common and important This enzyme catalyses CO, in solution: CO, + H,O
(CA) is one of the most enzymes in life processes. the reversible hydration of
= H++ HCO;
(1)
Because of its biological importance, carbonic anhydrase has been studied for many decades. Despite the interest in this enzyme, all of the means of assaying its activity have serious disadvantages [l]. Perhaps the Wilbur-Anderson method [2] is the most widely used assay for carbonic anhydrase. This method does not give a linear response over a broad enzyme range
* Corresponding author. ’ Permanent address: Department of Chemistry, University, Q&u, Shandong 273165, China.
Qufu Normal
0003-2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved S.SDIOOO3-2670(95)00389-4
and cannot accurately measure small differences in activity. The mass spectrometric method [3] is sensitive and accurate but is slow and requires the dedicated use of a mass spectrometer. Stopped-flow [4,5] and calorimetric assays [6,7] have been developed to measure this activity. However, these assays are typically about lOOO-fold less sensitive and inconvenient. There have been some reported [8,9] radiotracer assays for carbonic anhydrase. Although radiotracer methods show some advantages (e.g., high sensitivity) and are inexpensive, the disadvantages are the production of radioactivity and inconvenience. Chen and Kernohan [lo] have found that 5dimethylaminonaphthalene-l-sulfonamide (dansylamide, DNSA) bound as an inhibitor to the zinc in the active site of carbonic anhydrase resulted in an enhancement in the fluorescence emission at 460 nm when excited at 326 nm. Thompson and Jones [ll] used this fluorimetric method to assay zinc in sea
354
H.-N. Li, Y.-X. Ci /Analytica
water. We found that the fluorescence emission of CA-DNSA, which was maximal at 460 nm, showed a higher sensitivity with excitation at 280 nm and therefore excitation at 280 nm is used in this study. The procedure overcomes many of the disadvantages of the methods described above. The method is inexpensive and requires simple equipment, can be used at 20 f l”C, with buffered media over a pH range of 7.0-8.5. The technology makes the method ideal for routine determination of carbonic anhydrase.
Chimica Acta 317 (1995) 353-357
cence intensities of the CA-DNSA complex were measured at 460 nm with excitation at 280 run. Fluorescence readings are given as net fluorescence intensities in arbitrary units of the instrument. Background fluorescence has been subtracted for each value reported except for excitation and emission spectra.
3. Results and discussion 3.1. Spectral characteristic
2. Experimental 2.1. Materials and reagents Carbonic anhydrase from bovine erythrocytes was purchased from Dong Feng Biochemical, Shanghai, China (activity 2000 U mg-r). It was dissolved in 0.1 M phosphate buffer (pH 7.5) and its concentration was estimated spectrophotometrically, with l2s0 = 5.7 X lo4 1 mol-’ cm-’ [lo]; human serum aibumin (HSA) was a crystalline sample obtained from Shanghai Biochemical Reagent, China; 5-dimethylaminonaphthalene-l-sulfonamide (DNSA) was purchased from Sigma, a 1 X 10e3 M stock solution was made by dissolving a weighed amount of the sulfonamide in 1 X lo-’ M HCl. This solution was kept in a refrigerator at 4°C. All chemicals were of analytical grade and all aqueous solutions were made up in deionized water from a Millipore system.
The fluorescence excitation and emission spectra of DNSA, CA and CA-DNSA are shown in Fig. 1. The excitation spectrum of DNSA showed a peak at about 326 nm. The excitation spectrum of CA-DNSA showed a new maximum peak at 280 nm and a shoulder at 326 nm, which is the excitation peak of DNSA. The emission spectrum showed that the fluorescence emission maximum for DNSA is at 526 nm (A,, = 326 mn) and the CA-DNSA complex showed maximum emission at 460 nm with excitation at 280
n
60
Ex
Em
2
2.2. Apparatus The fluorescence intensities and spectra were measured with a Shimadzu spectrofluorimeter RF540. Absorbance was measured with a Shimadzu UV-265 UV-visible spectrometer.
4
2.3. Procedure 0
Typically, solutions of CA and DNSA were added to a 5.0 ml calibrated flask and the mixture was diluted to the 5.0 ml mark with 0.1 M phosphate buffer (pH 7.5) at 20 It 1°C. The entrance and exit slits were maintained at 5 nm for fluorescence measurements (unless otherwise indicated). The fluores-
Wavelength Fig. 1. Excitation and emission spectra of DNSA, CA and CADNSA complex. 1.1’ = CA, 2.2’ = DNSA, 3.3’ = CA-DNSA complex; A,, = 460 nm, h,, = 326 nm. 4’ = CA-DNSA complex, A,, = 280 nm, A,, = 460 nm. DNSA: 1 PM, CA: 0.27 PM.
H.-N. Li, Y.-X. Ci/Analytica
Chimica Acta 317 (1995) 353-357
355
intensity was obtained between pH 7.0 and 8.5 for CA-DNSA. The optimal pH is at 7.5. So all subsequent experiments were conducted in the 0.1 M phosphate buffer of pH 7.5. 3.4. Effect of DNSA concentration and composition of inclusion complex
Fig. 2. Binding of DNSA to CA. The N atom of the sulfonamide group of DNSA is bound as a 4th ligand to Zn.
nm or 326 nm. The emission intensity of CA-DNSA at 460 nm with excitation at 280 nm was much greater than that with excitation at 326 nm. This proves the formation of a fluorescent inclusion complex between DNSA and CA (Fig. 2). Therefore, the former was used for fluorescence intensity measurements. 3.2. Effect of temperature
The variation in the fluorescence intensity was investigated as a function of the concentration of DNSA in the presence of a constant concentration of CA (1.34 PM). When DNSA was added to a solution of CA, the CA fluorescence was quenched (h,, = 280 nm) and th e new emission peak (A,, = 460 nm) of the CA-DNSA complex appeared (Fig. 4). The fluorescence intensity of the CA-DNSA complex increased until a mole ratio of 1: 1 for CA and DNSA was reached [12]. Above this concentration the fluorescence intensity is constant, which suggests that only 1 mole of ligand (DNSA) is bound per mole of CA, as was reported by Chen and Kernohan [lo] when using an excitation wavelength of 326 nm. 3.5. Calibration graph for CA The calibration graph for CA was obtained under the optimal conditions. The relationship between the
A solution of 100 ~1 (1.01 X lop5 M) CA and 100 pl(5 X 10e5 M) DNSA were added to a 5.0 ml calibrated flask and diluted to the mark with 0.1 M phosphate buffer. The flask was then immersed in a thermostatically controlled water-bath at various temperatures. After 5 min, the change in fluorescence intensity with temperature was measured at an emission maximum of 460 nm with excitation of 280 nm. The results showed that the fluorescence intensity of the CA-DNSA complex decreases slightly as the solution is warmed up. Therefore, all subsequent experiments were conducted at 20 + 1°C. 3.3. Effect of pH and buffer
1 6
Assays can be conduced in buffered media over a wide pH range which are shown in Fig. 3. Here, 0.1 M sodium phosphate buffer and 0.1 M sodium carbonate buffer were used. The maximum fluorescence
7
6
9
10
11
m
Fig. 3. Effect of pH on the fluorescence intensity. 1 = DNSA, 2 = CA-DNSA complex. DNSA: 1 PM, CA: 0.27 FM. A,, = 280 nm, A,, = 460 nm.
H.-N. Li, Y.-X. Ci/Analytica
1
I
Chimica Acta 317 (1995) 353-357 Table 2 Recoveries
I
of CA from human serum
CA added
Recovery
(pg ml-‘)
(a)
Coefficient variation
2.6 5.2
101 104
4.8% (n = 6) 2.5% (n = 6)
listed in Table 1. The determination tained by the method of Hernandez
of
limit was oband Escriche
[131. The advantages of the recommended method are its high sensitivity and accuracy (Table 1). It has a detection limit of 29 ng ml-’ CA, which corresponds to 9.8 X lo- I3 M, close to the radiolabeled hydrogencarbonate method [9]. The assay gives a wide linear response from 1 to 100 nM CA. 3.6. Effect of interfering materials A potential interferent, human serum albumin, was studied. The fluorescence intensity of CA-DNSA was not affected by HSA up to ca. 0.67 PM. The fluorescence intensity of CA-DNSA was also not affected by inorganic salts, for example, a 1200-fold molar excess of NaCl, NaNO, or IU.
300
400
500
3.7. Application
600
Wavelength
Application of the recommended method for the determination of CA added to human serum showed satisfactory results (Table 2).
Fig. 4. Effect of DNSA concentration on CA-DNSA complex formation. A solution of 1.34 PM CA was used. pH 7.5, DNSA (PM): (1) 0, (2) 0.167, (3) 0.33, (4) 0.67, (5) 1.00, (6) 1.33, (7) 1.67, (8) 2.00, (9) 2.50, (10) 3.00, (11) 4.00, (12) 5.00. A,, = 280 nm, h,, = 460 nm.
4. Conclusions Compared with other methods for the determination of carbonic anhydrase, the recommended method is rapid, simple, sensitive and accurate.
concentration of CA and fluorescence intensity is linear in the range O-61 X 10e9 M CA. The linear regression equations and other calibration data are
Table 1 Analytical
parameters
Regression
equation
I,
for the determination
= 0.58 + 1.53 x lO’[CA]
a Obtained
for the determination
of CA as a fluorescence
Linear range O-61 X lo-’
Correlation M
of 8.73 nM CA.
complex with DNSA coefficient
0.9991 (n = 10)
(r)
Detection limit
Coefficient
of variation
29 ng ml-’
1.8% (n = 8)
’
H.-N. Li, Y.-X. Ci/Analytica
[41 Q.H. Gibson and F.J.W. Roughton,
Acknowledgements This work was supported by the National Science Foundation of China.
Chimica Acta 317 (1995) 353-357
Natural
References [l] R.E. Forster, in S.J. Dodson, R.E. Tashian, G. Gros and N.D. Carter (Eds.), Carbonic Anhydrase: Cellular Physiology and Molecular Genetics, Plenum, New York, 1991, pp. 59-68. [2] KM. Wilbur and N.G. Anderson, J. Biol. Chem., 176 (1948) 147. [3] G. Hock and B. Kok, Arch. Biochem. Biophys., 101 (1963) 160.
357
Proc. R. Sot. B, 143 (1955) 310. [Sl Q.H. Gibson and L. Milnes, Biochem. J., 91 (1964) 161. 161Y. Packer and J.T. Stone, Biochemistry, 6 (1967) 668. [71 J.W. Wells, S.I. Kandel, M. Kandel and A.G. Gornal, J. Biol. Chem., 250 (1975) 3522. 181G.D. Hodgen and R.J. Falk, Int. J. Appl. Radiat. Isot., 22 (1971) 492. [91 A. Stemler, Anal. Biochem., 210 (1993) 32X. DO1 R.F. Chen and J.C. Kemohan, J. Biol. Chcm.. 242 (1967) 5813. [Ill R.B. Thompson and E.R. Jones, Anal. Chem., 65 (1993) 730. WI V. Jukka, S. Anders and L. Anders,Int. J. Biol. Macromol., 15 (1993) 97. [131F.H. Hernandez and J.M. Escriche, Analyst. 109 (1984) 1585.
AC’L4 ELSEVIER
Analytica
Chimica Acta 317 (1995) 353-357
A new rapid fluorimetric method for the determination of carbonic anhydrase Huai-Na Li ‘, Yun-Xiang Ci
*
Department of Chemistry, Peking Uniuersity, Beijing 100871, China Received 28 February
1995; revised 19 July 1995; accepted
19 July 1995
Abstract A new assay procedure for carbonic anhydrase activity has been developed, based on the formation of a fluorescent inclusion complex of carbonic anhydrase with dansylamide. The excitation and emission wavelengths of the fluorescent complex are at 280 nm and 460 nm, respectively. The method can be used at 20 f l”C, with buffered media over a pH range of 7.0-8.5. The detection limit for carbonic anhydrase is 29 ng ml-‘. The recommended method is very sensitive, simple and rapid, making it ideal for routine determinations of carbonic anhydrase activity. Keywords:
Fluorimetry;
Carbonic
anhydrase
1. Introduction Carbonic anhydrase common and important This enzyme catalyses CO, in solution: CO, + H,O
(CA) is one of the most enzymes in life processes. the reversible hydration of
= H++ HCO;
(1)
Because of its biological importance, carbonic anhydrase has been studied for many decades. Despite the interest in this enzyme, all of the means of assaying its activity have serious disadvantages [l]. Perhaps the Wilbur-Anderson method [2] is the most widely used assay for carbonic anhydrase. This method does not give a linear response over a broad enzyme range
* Corresponding author. ’ Permanent address: Department of Chemistry, University, Q&u, Shandong 273165, China.
Qufu Normal
0003-2670/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved S.SDIOOO3-2670(95)00389-4
and cannot accurately measure small differences in activity. The mass spectrometric method [3] is sensitive and accurate but is slow and requires the dedicated use of a mass spectrometer. Stopped-flow [4,5] and calorimetric assays [6,7] have been developed to measure this activity. However, these assays are typically about lOOO-fold less sensitive and inconvenient. There have been some reported [8,9] radiotracer assays for carbonic anhydrase. Although radiotracer methods show some advantages (e.g., high sensitivity) and are inexpensive, the disadvantages are the production of radioactivity and inconvenience. Chen and Kernohan [lo] have found that 5dimethylaminonaphthalene-l-sulfonamide (dansylamide, DNSA) bound as an inhibitor to the zinc in the active site of carbonic anhydrase resulted in an enhancement in the fluorescence emission at 460 nm when excited at 326 nm. Thompson and Jones [ll] used this fluorimetric method to assay zinc in sea
354
H.-N. Li, Y.-X. Ci /Analytica
water. We found that the fluorescence emission of CA-DNSA, which was maximal at 460 nm, showed a higher sensitivity with excitation at 280 nm and therefore excitation at 280 nm is used in this study. The procedure overcomes many of the disadvantages of the methods described above. The method is inexpensive and requires simple equipment, can be used at 20 f l”C, with buffered media over a pH range of 7.0-8.5. The technology makes the method ideal for routine determination of carbonic anhydrase.
Chimica Acta 317 (1995) 353-357
cence intensities of the CA-DNSA complex were measured at 460 nm with excitation at 280 run. Fluorescence readings are given as net fluorescence intensities in arbitrary units of the instrument. Background fluorescence has been subtracted for each value reported except for excitation and emission spectra.
3. Results and discussion 3.1. Spectral characteristic
2. Experimental 2.1. Materials and reagents Carbonic anhydrase from bovine erythrocytes was purchased from Dong Feng Biochemical, Shanghai, China (activity 2000 U mg-r). It was dissolved in 0.1 M phosphate buffer (pH 7.5) and its concentration was estimated spectrophotometrically, with l2s0 = 5.7 X lo4 1 mol-’ cm-’ [lo]; human serum aibumin (HSA) was a crystalline sample obtained from Shanghai Biochemical Reagent, China; 5-dimethylaminonaphthalene-l-sulfonamide (DNSA) was purchased from Sigma, a 1 X 10e3 M stock solution was made by dissolving a weighed amount of the sulfonamide in 1 X lo-’ M HCl. This solution was kept in a refrigerator at 4°C. All chemicals were of analytical grade and all aqueous solutions were made up in deionized water from a Millipore system.
The fluorescence excitation and emission spectra of DNSA, CA and CA-DNSA are shown in Fig. 1. The excitation spectrum of DNSA showed a peak at about 326 nm. The excitation spectrum of CA-DNSA showed a new maximum peak at 280 nm and a shoulder at 326 nm, which is the excitation peak of DNSA. The emission spectrum showed that the fluorescence emission maximum for DNSA is at 526 nm (A,, = 326 mn) and the CA-DNSA complex showed maximum emission at 460 nm with excitation at 280
n
60
Ex
Em
2
2.2. Apparatus The fluorescence intensities and spectra were measured with a Shimadzu spectrofluorimeter RF540. Absorbance was measured with a Shimadzu UV-265 UV-visible spectrometer.
4
2.3. Procedure 0
Typically, solutions of CA and DNSA were added to a 5.0 ml calibrated flask and the mixture was diluted to the 5.0 ml mark with 0.1 M phosphate buffer (pH 7.5) at 20 It 1°C. The entrance and exit slits were maintained at 5 nm for fluorescence measurements (unless otherwise indicated). The fluores-
Wavelength Fig. 1. Excitation and emission spectra of DNSA, CA and CADNSA complex. 1.1’ = CA, 2.2’ = DNSA, 3.3’ = CA-DNSA complex; A,, = 460 nm, h,, = 326 nm. 4’ = CA-DNSA complex, A,, = 280 nm, A,, = 460 nm. DNSA: 1 PM, CA: 0.27 PM.
H.-N. Li, Y.-X. Ci/Analytica
Chimica Acta 317 (1995) 353-357
355
intensity was obtained between pH 7.0 and 8.5 for CA-DNSA. The optimal pH is at 7.5. So all subsequent experiments were conducted in the 0.1 M phosphate buffer of pH 7.5. 3.4. Effect of DNSA concentration and composition of inclusion complex
Fig. 2. Binding of DNSA to CA. The N atom of the sulfonamide group of DNSA is bound as a 4th ligand to Zn.
nm or 326 nm. The emission intensity of CA-DNSA at 460 nm with excitation at 280 nm was much greater than that with excitation at 326 nm. This proves the formation of a fluorescent inclusion complex between DNSA and CA (Fig. 2). Therefore, the former was used for fluorescence intensity measurements. 3.2. Effect of temperature
The variation in the fluorescence intensity was investigated as a function of the concentration of DNSA in the presence of a constant concentration of CA (1.34 PM). When DNSA was added to a solution of CA, the CA fluorescence was quenched (h,, = 280 nm) and th e new emission peak (A,, = 460 nm) of the CA-DNSA complex appeared (Fig. 4). The fluorescence intensity of the CA-DNSA complex increased until a mole ratio of 1: 1 for CA and DNSA was reached [12]. Above this concentration the fluorescence intensity is constant, which suggests that only 1 mole of ligand (DNSA) is bound per mole of CA, as was reported by Chen and Kernohan [lo] when using an excitation wavelength of 326 nm. 3.5. Calibration graph for CA The calibration graph for CA was obtained under the optimal conditions. The relationship between the
A solution of 100 ~1 (1.01 X lop5 M) CA and 100 pl(5 X 10e5 M) DNSA were added to a 5.0 ml calibrated flask and diluted to the mark with 0.1 M phosphate buffer. The flask was then immersed in a thermostatically controlled water-bath at various temperatures. After 5 min, the change in fluorescence intensity with temperature was measured at an emission maximum of 460 nm with excitation of 280 nm. The results showed that the fluorescence intensity of the CA-DNSA complex decreases slightly as the solution is warmed up. Therefore, all subsequent experiments were conducted at 20 + 1°C. 3.3. Effect of pH and buffer
1 6
Assays can be conduced in buffered media over a wide pH range which are shown in Fig. 3. Here, 0.1 M sodium phosphate buffer and 0.1 M sodium carbonate buffer were used. The maximum fluorescence
7
6
9
10
11
m
Fig. 3. Effect of pH on the fluorescence intensity. 1 = DNSA, 2 = CA-DNSA complex. DNSA: 1 PM, CA: 0.27 FM. A,, = 280 nm, A,, = 460 nm.
H.-N. Li, Y.-X. Ci/Analytica
1
I
Chimica Acta 317 (1995) 353-357 Table 2 Recoveries
I
of CA from human serum
CA added
Recovery
(pg ml-‘)
(a)
Coefficient variation
2.6 5.2
101 104
4.8% (n = 6) 2.5% (n = 6)
listed in Table 1. The determination tained by the method of Hernandez
of
limit was oband Escriche
[131. The advantages of the recommended method are its high sensitivity and accuracy (Table 1). It has a detection limit of 29 ng ml-’ CA, which corresponds to 9.8 X lo- I3 M, close to the radiolabeled hydrogencarbonate method [9]. The assay gives a wide linear response from 1 to 100 nM CA. 3.6. Effect of interfering materials A potential interferent, human serum albumin, was studied. The fluorescence intensity of CA-DNSA was not affected by HSA up to ca. 0.67 PM. The fluorescence intensity of CA-DNSA was also not affected by inorganic salts, for example, a 1200-fold molar excess of NaCl, NaNO, or IU.
300
400
500
3.7. Application
600
Wavelength
Application of the recommended method for the determination of CA added to human serum showed satisfactory results (Table 2).
Fig. 4. Effect of DNSA concentration on CA-DNSA complex formation. A solution of 1.34 PM CA was used. pH 7.5, DNSA (PM): (1) 0, (2) 0.167, (3) 0.33, (4) 0.67, (5) 1.00, (6) 1.33, (7) 1.67, (8) 2.00, (9) 2.50, (10) 3.00, (11) 4.00, (12) 5.00. A,, = 280 nm, h,, = 460 nm.
4. Conclusions Compared with other methods for the determination of carbonic anhydrase, the recommended method is rapid, simple, sensitive and accurate.
concentration of CA and fluorescence intensity is linear in the range O-61 X 10e9 M CA. The linear regression equations and other calibration data are
Table 1 Analytical
parameters
Regression
equation
I,
for the determination
= 0.58 + 1.53 x lO’[CA]
a Obtained
for the determination
of CA as a fluorescence
Linear range O-61 X lo-’
Correlation M
of 8.73 nM CA.
complex with DNSA coefficient
0.9991 (n = 10)
(r)
Detection limit
Coefficient
of variation
29 ng ml-’
1.8% (n = 8)
’
H.-N. Li, Y.-X. Ci/Analytica
[41 Q.H. Gibson and F.J.W. Roughton,
Acknowledgements This work was supported by the National Science Foundation of China.
Chimica Acta 317 (1995) 353-357
Natural
References [l] R.E. Forster, in S.J. Dodson, R.E. Tashian, G. Gros and N.D. Carter (Eds.), Carbonic Anhydrase: Cellular Physiology and Molecular Genetics, Plenum, New York, 1991, pp. 59-68. [2] KM. Wilbur and N.G. Anderson, J. Biol. Chem., 176 (1948) 147. [3] G. Hock and B. Kok, Arch. Biochem. Biophys., 101 (1963) 160.
357
Proc. R. Sot. B, 143 (1955) 310. [Sl Q.H. Gibson and L. Milnes, Biochem. J., 91 (1964) 161. 161Y. Packer and J.T. Stone, Biochemistry, 6 (1967) 668. [71 J.W. Wells, S.I. Kandel, M. Kandel and A.G. Gornal, J. Biol. Chem., 250 (1975) 3522. 181G.D. Hodgen and R.J. Falk, Int. J. Appl. Radiat. Isot., 22 (1971) 492. [91 A. Stemler, Anal. Biochem., 210 (1993) 32X. DO1 R.F. Chen and J.C. Kemohan, J. Biol. Chcm.. 242 (1967) 5813. [Ill R.B. Thompson and E.R. Jones, Anal. Chem., 65 (1993) 730. WI V. Jukka, S. Anders and L. Anders,Int. J. Biol. Macromol., 15 (1993) 97. [131F.H. Hernandez and J.M. Escriche, Analyst. 109 (1984) 1585.