ANALYTICAL
BIOCHEMISTRY
Rapid
97, 277-281 (1979)
Purification and Direct Spectrophotometric 5Aminolevulinic Acid Dehydratase D. H~CKEL University
of Bremen,
Assay for
AND D. BEYERSMANN
NW II, D 2800 Bremen,
Federal
Republic
of Germany
Received December 28, 1978 A two-step purification procedure for 5-aminolevulinic acid dehydratase (EC 4.2.1.24) from human red blood cells has been developed. It involves one ion exchange and one gel filtration step. The purification is about lOOO-fold, and the yield is more than 85%. With the purified enzyme a direct spectrophotometric assay of product formation without subsequent reaction with Ehrlich’s reagent is described.
5Aminolevulinic acid dehydratase (EC 4.2.1.24) catalyzes the conversion of 2 mol of 5aminolevulinic acid to 1 mol of porphobilinogen in the biosynthetic pathway leading to porphyrins: yztH y-y CH2
fH2 CH I
2 ,,.....Cd,2 12 I
OS. , ..NAcHZ FH2
2
b,i ,‘“2
H
The activity of the enzyme serves as an indicator for early recognition of a possible uptake of lead doses which do not yet produce clinical symptoms of intoxication. We have been interested in the mechanism of lead action on human heme synthesis and have studied the enzyme from human red blood cells. The enzyme has been highly purified from various sources, mostly from bovine liver (l-3), but also from human blood (4,5). Since human red blood cells contain relatively few kinds of protein, we were able to develop a simplified two-step purification procedure for 5aminolevulinic acid dehydratase. For kinetic studies on the enzyme, the assay method used previously (6) was rather inconvenient and inexact. It in-
volved incubation of the enzyme with the substrate and interruption of the reaction by adding a mixture of acid and heavy metal salts, followed by a further reaction with p-dimethylaminobenzaldehyde (Ehrlich’s reagent) to give a colored complex with the product (porphobilinogen) of the enzymic reaction. This assay was time consuming, it was subject to errors due to the involvement of two subsequent reactions and the formation of an unstable colored product, and it did not allow one to register progress curves continuously. The reproducibility of the assay has been improved by applying automated procedures in flow systems (7,8). However, these systems are still based on a subsequent reaction with Ehrlich’s reagent. To follow the progress curves directly, we have developed a method to measure the formation of porphobilinogen by its own absorbance. MATERIALS
AND METHODS
Outdated human bank blood was a gift of Dr. Zockler, St. Jtirgen’s Hospital, Bremen. Chromatography media were purchased from Pharmacia, Uppsala, 5-aminolevulinic acid was from Serva, Heidelberg, and all other chemicals which were of reagent grade (p.a.) were from Merck, Darmstadt. All 277
0003-2697/79/120277-05$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
278
HiiCKEL
AND
solutions were prepared with bidistilled water. The standard buffer was 0.05 M Tris-HCl (pH 7.0), if not stated otherwise. Puri$cation dehydratase.
of
5-aminolevulinic
acid
Five hundred milliliters of human blood was centrifuged at 1OOOgand the sediment was washed twice with 300 ml of 0.03 M Tris-HCl (pH 7.0). The cells were lysed by freezing and thawing. The lysate (220 ml) was mixed with 200 ml of a suspension of 100 g DEAE-Sephacel in standard buffer and agitated gently for 30 min. The suspension was filtered by suction over a sintered glass filter, and the residue was washed twice with 100 ml of standard buffer. The residue was resuspended in 300 ml of the same buffer and poured into a chromatographic column (2.5 x 80 cm). The column was washed with standard buffer until most of the hemoglobin had been eluted. A linear gradient was applied (total volume 500 ml, 0.05-0.5 M Tris-HCl at pH 7.0). Active enzyme was eluted between 360 and 440 ml (at about 0.4 M Tris-HCl). The active fractions (80 ml) were collected and concentrated on an ultrafilter (Millipore PSED) to give 6 ml offraction I. This fraction was applied to a combination of two columns arranged in sequence: The first one (2.5 x 80 cm), filled with Sephadex G-100, was coupled to the second one (2.5 x 80 cm), filled with Sepharose CL-6B, both equilibrated with standard buffer. The columns were operated at 0.8 mhmin. The enzyme was eluted between 230 and 293 ml. The active fractions were collected to givefrac-
BEYERSMANN
lion ZZ. An examination
by discontinuous polyacrylamide gel electrophoresis showed that fraction II still contained 20% contamination by other proteins. This fraction had sufficient purity for kinetic studies. A further purification to 95% homogeneity was performed by chromatography on Sephacryl 200. Since this step results in a loss of 70% of the total activity, it is omitted for kinetic studies. The purification procedure is summarized in Table 1. Assays of enzyme activity. During the purification procedure the enzyme activity was determined by the conventional method as described by Granick and Mauzerall(6). For kinetic studies, a new assay was established. All measurements were performed in a Perkin-Elmer spectrophotometer, model 554, at 236 nm. One-tenth milliliter of enzyme (fraction II) was mixed with 0.2 ml of 10 mM glutathione in standard buffer. The mixture was preincubated for 10 min at 37°C in a quartz cuvette, and the reaction was started by addition of 0.2 ml of 3 mM aminolevulinic acid in standard buffer. The reference cuvette contained the same substances as the sample with the exception of enzyme protein which was substituted by an amount of bovine serum albumin giving the same absorbance at 236 nm as the enzyme protein in the sample. The substrate was added to the reference at the same time as it was given to the sample. One unit (U) of enzyme activity is the amount of enzyme catalyzing the condensation of 1 pmol of substrate per minute.
TABLE PURIFICATION
Fraction Hemolysate I. (DEAE-Sephacel)
II.
(G-lOO/CL-6B)
OF 5-AMINOLEVULINIC
ACID
1 DEHYDRATASE
Volume
Activity
(ml)
(U/ml)
220 6 63
0.008 0.245 0.024
FROM
HUMAN
Protein bd-4
210 22 0.6
BLOOD
CELLS Specific activity (Uimg x 10m3)
0.04 11.4 40.0
5-AMINOLEVULINIC
L
ACID DEHYDRATASE
8
16
12
20
PURIFICATION
2L
28
279
32 TIME
,m,n
FIG. 1. Enzymic formation of porphobilinogen from Saminolevulinic acid at various concentrations as measured by the absorbance of porphobilinogen at 236 nm. For details see under Materials and Methods.
Determination ofprotein. Protein concentrations were determined by the method of Lowry (9).
Assay of Enzyme Activity
RESULTS AND DISCUSSION
Purijication of 5-Aminolevuiinic Dehydratase
Acid
Table 1 summarizes the isolation procedure. This method results in a high degree of purification (more than lOOO-fold) in a high yield (about 85% of fraction II). Furthermore, it does not involve heat treatment. This is important for studies on the
-8
-7
-6
-5
-4
-3
-2
-7
mechanism of lead action since the lead sensitivity of the enzyme is modified by heat treatment (10).
1
With the purified enzyme, it is possible to measure porphobilinogen formation directly in a spectrophotometer at 236 nm. Since nearly all other compounds of the incubation mixture (enzyme, substrate, glutathione) have a considerable absorbance at this wavelength, it is essential to adjust the reference solution to nearly the same absorbance as that of the sample.
2
3
L
5
6
7
I SUBSTRATE
FIG. 2. Lineweaver-Burk
8
9 I-’
10 mM -’
plot of initial rate data obtained from the experiment described in Fig. 1.
280
HiiCKEL
L
8
12
AND BEYERSMANN
16
20
24
28
32
36 TIME,
LO min
FIG. 3. Effect of lead ions on the activity of 5-aminolevulinic acid dehydratase. (A) Lead acetate is added prior to substrate at a final concentration of 10m5M. (B) Lead acetate at the same concentration is added at the time indicated.
The linearity and sensitivity of the assay were investigated by addition of porphobilinogen to the complete, but inactive assay mixture. Inactivation was achieved by addition of lead acetate in a final concentration of 10P5 M prior to addition of substrate. The absorbance at 236 nm up to A = 0.7 is a linear function of porphobilinogen concentration up to 2.6 x 1O-3 M. The assay is sensitive down to a porphobilinogen concentration of 5 X lo-’ M, allowing the measurement of progress curves within a few seconds. Figure 1 demonstrates progress curves as a function of substrate concentration. Figure 2 shows a LineweaverBurk plot of the initial rate data. A value of K, = 1.58 x 10e4 is obtained. The direct assay allows the convenient investigation of the inhibition of the enzyme activity by lead ions. Figure 3 demonstrates that lead ions at 10e5 M inactivate the enzyme completely if administered prior to the substrate. If the same concentration of lead ions is added after the reaction has been started with substrate, the inhibition is much less pronounced. Thus, the substrate 5aminolevulinic acid seems to protect the enzyme against attack by lead ions partially. The methods described for the rapid
purification and the direct assay of 5aminolevulinic acid dehydratase are convenient tools for studies on the enzyme. The direct spectrophotometric assay offers several advantages as compared to the former assay employing a subsequent reaction with Ehrlich’s reagent (6). The new assay is simpler and therefore less subject to errors. It allows one to measure progress curves continuously, and its sensitivity is of the same order of magnitude as that of the former assay. The main restriction of the new assay is that it is confined to studies on the partially purified enzyme and is not applicable to measurements of enzyme activity in cell lysates directly. ACKNOWLEDGMENTS We thank Mrs. M. Cox for excellent technical help. We are indebted to Dr. Zockler of the St. Jiirgen’s Hospital for supplying us with outdated bank blood.
REFERENCES 1. Gibson, K. D., Neuberger, A., and Scott, J. J. (1965) Biochem. J. 61, 618. 2. Battle, A, M., Ferramola, A. M., and Grinstein, M. (1967) &o&em. J. 104, 244. 3. Wilson, E. L., Burger, P. E., and Dowdle, E. B. (1972) Eur. J. Biochem. 29, 563.
SAMINOLEVULINIC
ACID DEHYDRATASE
4. Weissberg, J. B., and Voytek, P. E. (1974) Biochim. Biophys. Acta 364, 304. 5. Calissano, P., Cartasegna, C., and Matteini, M. (1966) Ital. J. Biochem. 15, 18. 6. Granick, S., and Mauzerall, D. (1958) 1. Biol. Chem. 232, 1119. 7. Gore, M. G., Jordan, P. M., and Chaudhry, A. G. (1978) Anal. Biochem. 87,41.
PURIFICATION
281
8. Bodlaender, P., Ulmer, D. D., and Vallee, B. L. (1974) Anal. Biochem. 58, 500. 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265. 10. Vergnano, C., Cartasegna, C., and Matteini. (1968) Boll. Sot. Ztal. Bid. Sper. 44, 695.
M.
BIOCHEMISTRY
Rapid
97, 277-281 (1979)
Purification and Direct Spectrophotometric 5Aminolevulinic Acid Dehydratase D. H~CKEL University
of Bremen,
Assay for
AND D. BEYERSMANN
NW II, D 2800 Bremen,
Federal
Republic
of Germany
Received December 28, 1978 A two-step purification procedure for 5-aminolevulinic acid dehydratase (EC 4.2.1.24) from human red blood cells has been developed. It involves one ion exchange and one gel filtration step. The purification is about lOOO-fold, and the yield is more than 85%. With the purified enzyme a direct spectrophotometric assay of product formation without subsequent reaction with Ehrlich’s reagent is described.
5Aminolevulinic acid dehydratase (EC 4.2.1.24) catalyzes the conversion of 2 mol of 5aminolevulinic acid to 1 mol of porphobilinogen in the biosynthetic pathway leading to porphyrins: yztH y-y CH2
fH2 CH I
2 ,,.....Cd,2 12 I
OS. , ..NAcHZ FH2
2
b,i ,‘“2
H
The activity of the enzyme serves as an indicator for early recognition of a possible uptake of lead doses which do not yet produce clinical symptoms of intoxication. We have been interested in the mechanism of lead action on human heme synthesis and have studied the enzyme from human red blood cells. The enzyme has been highly purified from various sources, mostly from bovine liver (l-3), but also from human blood (4,5). Since human red blood cells contain relatively few kinds of protein, we were able to develop a simplified two-step purification procedure for 5aminolevulinic acid dehydratase. For kinetic studies on the enzyme, the assay method used previously (6) was rather inconvenient and inexact. It in-
volved incubation of the enzyme with the substrate and interruption of the reaction by adding a mixture of acid and heavy metal salts, followed by a further reaction with p-dimethylaminobenzaldehyde (Ehrlich’s reagent) to give a colored complex with the product (porphobilinogen) of the enzymic reaction. This assay was time consuming, it was subject to errors due to the involvement of two subsequent reactions and the formation of an unstable colored product, and it did not allow one to register progress curves continuously. The reproducibility of the assay has been improved by applying automated procedures in flow systems (7,8). However, these systems are still based on a subsequent reaction with Ehrlich’s reagent. To follow the progress curves directly, we have developed a method to measure the formation of porphobilinogen by its own absorbance. MATERIALS
AND METHODS
Outdated human bank blood was a gift of Dr. Zockler, St. Jtirgen’s Hospital, Bremen. Chromatography media were purchased from Pharmacia, Uppsala, 5-aminolevulinic acid was from Serva, Heidelberg, and all other chemicals which were of reagent grade (p.a.) were from Merck, Darmstadt. All 277
0003-2697/79/120277-05$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
278
HiiCKEL
AND
solutions were prepared with bidistilled water. The standard buffer was 0.05 M Tris-HCl (pH 7.0), if not stated otherwise. Puri$cation dehydratase.
of
5-aminolevulinic
acid
Five hundred milliliters of human blood was centrifuged at 1OOOgand the sediment was washed twice with 300 ml of 0.03 M Tris-HCl (pH 7.0). The cells were lysed by freezing and thawing. The lysate (220 ml) was mixed with 200 ml of a suspension of 100 g DEAE-Sephacel in standard buffer and agitated gently for 30 min. The suspension was filtered by suction over a sintered glass filter, and the residue was washed twice with 100 ml of standard buffer. The residue was resuspended in 300 ml of the same buffer and poured into a chromatographic column (2.5 x 80 cm). The column was washed with standard buffer until most of the hemoglobin had been eluted. A linear gradient was applied (total volume 500 ml, 0.05-0.5 M Tris-HCl at pH 7.0). Active enzyme was eluted between 360 and 440 ml (at about 0.4 M Tris-HCl). The active fractions (80 ml) were collected and concentrated on an ultrafilter (Millipore PSED) to give 6 ml offraction I. This fraction was applied to a combination of two columns arranged in sequence: The first one (2.5 x 80 cm), filled with Sephadex G-100, was coupled to the second one (2.5 x 80 cm), filled with Sepharose CL-6B, both equilibrated with standard buffer. The columns were operated at 0.8 mhmin. The enzyme was eluted between 230 and 293 ml. The active fractions were collected to givefrac-
BEYERSMANN
lion ZZ. An examination
by discontinuous polyacrylamide gel electrophoresis showed that fraction II still contained 20% contamination by other proteins. This fraction had sufficient purity for kinetic studies. A further purification to 95% homogeneity was performed by chromatography on Sephacryl 200. Since this step results in a loss of 70% of the total activity, it is omitted for kinetic studies. The purification procedure is summarized in Table 1. Assays of enzyme activity. During the purification procedure the enzyme activity was determined by the conventional method as described by Granick and Mauzerall(6). For kinetic studies, a new assay was established. All measurements were performed in a Perkin-Elmer spectrophotometer, model 554, at 236 nm. One-tenth milliliter of enzyme (fraction II) was mixed with 0.2 ml of 10 mM glutathione in standard buffer. The mixture was preincubated for 10 min at 37°C in a quartz cuvette, and the reaction was started by addition of 0.2 ml of 3 mM aminolevulinic acid in standard buffer. The reference cuvette contained the same substances as the sample with the exception of enzyme protein which was substituted by an amount of bovine serum albumin giving the same absorbance at 236 nm as the enzyme protein in the sample. The substrate was added to the reference at the same time as it was given to the sample. One unit (U) of enzyme activity is the amount of enzyme catalyzing the condensation of 1 pmol of substrate per minute.
TABLE PURIFICATION
Fraction Hemolysate I. (DEAE-Sephacel)
II.
(G-lOO/CL-6B)
OF 5-AMINOLEVULINIC
ACID
1 DEHYDRATASE
Volume
Activity
(ml)
(U/ml)
220 6 63
0.008 0.245 0.024
FROM
HUMAN
Protein bd-4
210 22 0.6
BLOOD
CELLS Specific activity (Uimg x 10m3)
0.04 11.4 40.0
5-AMINOLEVULINIC
L
ACID DEHYDRATASE
8
16
12
20
PURIFICATION
2L
28
279
32 TIME
,m,n
FIG. 1. Enzymic formation of porphobilinogen from Saminolevulinic acid at various concentrations as measured by the absorbance of porphobilinogen at 236 nm. For details see under Materials and Methods.
Determination ofprotein. Protein concentrations were determined by the method of Lowry (9).
Assay of Enzyme Activity
RESULTS AND DISCUSSION
Purijication of 5-Aminolevuiinic Dehydratase
Acid
Table 1 summarizes the isolation procedure. This method results in a high degree of purification (more than lOOO-fold) in a high yield (about 85% of fraction II). Furthermore, it does not involve heat treatment. This is important for studies on the
-8
-7
-6
-5
-4
-3
-2
-7
mechanism of lead action since the lead sensitivity of the enzyme is modified by heat treatment (10).
1
With the purified enzyme, it is possible to measure porphobilinogen formation directly in a spectrophotometer at 236 nm. Since nearly all other compounds of the incubation mixture (enzyme, substrate, glutathione) have a considerable absorbance at this wavelength, it is essential to adjust the reference solution to nearly the same absorbance as that of the sample.
2
3
L
5
6
7
I SUBSTRATE
FIG. 2. Lineweaver-Burk
8
9 I-’
10 mM -’
plot of initial rate data obtained from the experiment described in Fig. 1.
280
HiiCKEL
L
8
12
AND BEYERSMANN
16
20
24
28
32
36 TIME,
LO min
FIG. 3. Effect of lead ions on the activity of 5-aminolevulinic acid dehydratase. (A) Lead acetate is added prior to substrate at a final concentration of 10m5M. (B) Lead acetate at the same concentration is added at the time indicated.
The linearity and sensitivity of the assay were investigated by addition of porphobilinogen to the complete, but inactive assay mixture. Inactivation was achieved by addition of lead acetate in a final concentration of 10P5 M prior to addition of substrate. The absorbance at 236 nm up to A = 0.7 is a linear function of porphobilinogen concentration up to 2.6 x 1O-3 M. The assay is sensitive down to a porphobilinogen concentration of 5 X lo-’ M, allowing the measurement of progress curves within a few seconds. Figure 1 demonstrates progress curves as a function of substrate concentration. Figure 2 shows a LineweaverBurk plot of the initial rate data. A value of K, = 1.58 x 10e4 is obtained. The direct assay allows the convenient investigation of the inhibition of the enzyme activity by lead ions. Figure 3 demonstrates that lead ions at 10e5 M inactivate the enzyme completely if administered prior to the substrate. If the same concentration of lead ions is added after the reaction has been started with substrate, the inhibition is much less pronounced. Thus, the substrate 5aminolevulinic acid seems to protect the enzyme against attack by lead ions partially. The methods described for the rapid
purification and the direct assay of 5aminolevulinic acid dehydratase are convenient tools for studies on the enzyme. The direct spectrophotometric assay offers several advantages as compared to the former assay employing a subsequent reaction with Ehrlich’s reagent (6). The new assay is simpler and therefore less subject to errors. It allows one to measure progress curves continuously, and its sensitivity is of the same order of magnitude as that of the former assay. The main restriction of the new assay is that it is confined to studies on the partially purified enzyme and is not applicable to measurements of enzyme activity in cell lysates directly. ACKNOWLEDGMENTS We thank Mrs. M. Cox for excellent technical help. We are indebted to Dr. Zockler of the St. Jiirgen’s Hospital for supplying us with outdated bank blood.
REFERENCES 1. Gibson, K. D., Neuberger, A., and Scott, J. J. (1965) Biochem. J. 61, 618. 2. Battle, A, M., Ferramola, A. M., and Grinstein, M. (1967) &o&em. J. 104, 244. 3. Wilson, E. L., Burger, P. E., and Dowdle, E. B. (1972) Eur. J. Biochem. 29, 563.
SAMINOLEVULINIC
ACID DEHYDRATASE
4. Weissberg, J. B., and Voytek, P. E. (1974) Biochim. Biophys. Acta 364, 304. 5. Calissano, P., Cartasegna, C., and Matteini, M. (1966) Ital. J. Biochem. 15, 18. 6. Granick, S., and Mauzerall, D. (1958) 1. Biol. Chem. 232, 1119. 7. Gore, M. G., Jordan, P. M., and Chaudhry, A. G. (1978) Anal. Biochem. 87,41.
PURIFICATION
281
8. Bodlaender, P., Ulmer, D. D., and Vallee, B. L. (1974) Anal. Biochem. 58, 500. 9. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265. 10. Vergnano, C., Cartasegna, C., and Matteini. (1968) Boll. Sot. Ztal. Bid. Sper. 44, 695.
M.
