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Partial purification of sulfite oxidase from Thiobacillus neapolitanus
FEM~ Microbiologyletters 67 (1990) 59-62 Published by Elsevier
59
FEMSLE 03855
Partial purification of sulfite oxidase from Thiobacillus neapolitanus A l e k s a n d r a Sldodowska
Instituteof Microbiology.Universityof Warsaw. Warsaw.Ptdand Received 18 September 1989 Accepted 22 September 1989 Key words: Cytochrome c: Flavoprotein; Difference spectrum
I. S U M M A R Y
3. MATERIALS A N D M E T H O D S
Sulfite oxidase was purified 172-fold from cell extract of Thiobacillus neapolitanus (protein A). The enzymatic activity was stimulated by protein B which shows properties of cytochrome c and flavoprolein. Sulfite oxidase was inhibited 20~ by the addition of AMP.
3.1. Bacterial strain and growth conditions T. oeapolitanus obtained from the Department of Microbiology, University of Groningen, Netherlands, was grown as described previously [2].
2. I N T R O D U C T I O N
Thiobacillus neapol#anus is a sulfur bacterium which utilizes thiosulphate as an energy source. Sulfite oxidase was purified 100-fold from an extract of T. neapolitanus by Hempfling et al. [1] who showed that the enzyme activity is stimulated by AMP. This fact indicated that a single enzyme is responsible for the oxidation of sulfite in the presence and absence of AMP. Reduced glutathlone inhibited activity and prevented A M P stimulation. This communication contains information about the isolation and partial purification of A M P independent sulfite oxidase. Correspondenceao: A. Sklodowska. (Present add~ss) Departfacial of Photography and Image Information, Warsaw University, uL Nowy ~wiat 67, PL 00-046 Warsaw, Poland.
3.2. Purification of sulfite oxidase Cell free extract from T. neapolitanus in 0.05 M phosphate buffer, pH 7.5 containing 3.7 protein per 1 ml was obtained according to the procedure of Witholt et aL [3]. Cell free extract was fractionated by the addition of increasing concentration of ( N H 4)2SO4. Precipitates obtained after centrifugation at 20000 X g for 20 rain. at + 4 ° C , were dissolved in 0.05 M phosphate buffer, pH 7.5 at temperature + 4 o C. Then the solution was placed on a column containing DEAE-celhilose (2 x 16 cm) which had be*n equilibrated with 0.05 M phosphate buffer, pH 7.5 and eluted with the same buffer containing increasing concentrations of NaCI: 0.0 M, 0.1 M0 0.2 M, 0.3 M, 0.4 M with an elution rate of 60 ml per hour. The volume of collected fractions was 5 ml. The active fractions were combined and then loaded on CM-celhilose column (2 x 10 cm) equilibrated with 0.05 M phosphate buffer, pH 6.5 and eluted with the same buffer (pH 6.5) containing
0378.1097/90/$03.50 © 1990 Federation oI European MicrobiologicalSocieties
60 increasing concentrations of NaCI as in the case of DEAE-cellulose, at the same rate. The protein content and enzyme activity was assayed in each fraction. The enzyme activity of 'combined fraction' fro:' example one fraction from DEAE and one from CM-cellulose) was also examined.
3. 3. Protein determination Protein content was assayed according to the procedure of Lowry. Light absorption at 280 nm was also examined.
03
3.4. Assay of enzyme activity
o
?Ca i a 4 M N a g l
i~INa
Sulfite oxidase activity was measured by manometric Warburg method.
3.5. Spectrophotometry
o2
a. C M - c e l l u l o s e
Difference spectra were recorded by Beckman Acta IlI speetrophotometer. For experimental data see descriptions under the figures.
a4MNaCI
02MIVaCI
1MNoCI 4. RESULTS AND DISCUSSION Purification of sulfite oxidase is presented in Table l and Fig. IA, B. The active fraction was obtained in the DEAE-cellulose eluate at NaCI concentration 03 M and it was called protein A. Table 1 Purificationof sulffieoxidase Fraction
Total activity pM 02 / min
Dialysed cell-free extract 267.0 0-15 ** 0 15-25 0 25-35 0 35-45 9.6 45-55 0.36 55-65 0.60 DE-cellulose 0.1 M NaCi 29.0
Proteins Specific Specific contents activity activity Ing/min * in the pl~sence of AMP *
5g.0
4.8 4.7 2.8
0.096 0.19 0 0 0 0.at 0.012 0.004 0,005 0.0ll 0.015
0.1"/5
16.6
* gM O2/min×rag protein; • * % (NH4)2SO4concentration.
3~m
'ml
Fig. 1. Fracfionafionof cell free extract on the column with DEAF,cellulose(A) and CM-ce0ulose(B).
The enzyme was purified 172-fold. Protein obtained in the eluate from CM-cellnlose column at NaCI concentration 0.1 M was called protein B. The activity of protein A was stimulated 2-fold by protein B. Protein B was not able to oxidize sulfite alone. Cyanide and azide completely inhibited the activity of protein. This enzyme lost 50% of activity after 10 min. at 6 0 ° C and 100~ after 2 rain at 100°C. It did not lose activity at + 4 ° C during two weeks. Protein A carried electrons immediately to oxygen as did the sulfite oxidase isolated by Hempfling et al. [1]. Protein B shows some properties of peroxidase (positive benzidine and o-dianizidine test) but the lack of flavoprotein autooxidation was opposite to these. Protein B had no properties of cytochrome oxidase type a (negative Patho Tec CO test). The difference spectrum of protein B shows a maximum at 550 nm, 520 nm, 418-420 nm (as
61
ASJO +prOleJnA 002~
+so/$
J
!IdAO.OI
and a t t e m p t s at further purification of the enzyme were unsuccessful. Similar problems were encountered by Lu and Kelly [4] hecause the single enzyme lost its activity and renewed c o m b i n a t i o n of enzymes in an enzyme complex was not always successful [8]. As for protein A, it was probably salfite oxidase or oxygenas¢. It seems that the isolated enzyme complex was one of the terminal parts o f the thiosnlphate oxidizing system in T. neapolitanus, which was described previously [2]. It is unlikely that T. neapolitanus cells which did not t a k e up sulfite [9l had a separate sulfite oxidation mechanism.
ACKNOWLEDGEMENT
4O0 5OO Fig. 2. Diffcxen4~ sl~trum of protein B (al and reduction of protein B by protein A and sulfit¢ (b). { . . . . . . ). reduced by SO32- ; (-- -- --), protein A + SO~-, I mira I ). protein A+SO 2" . 10 rain. cytochrome c), a small p e a k at 590 nm and minim u m about 460 nm. (Fig. 2a). T h e difference s p e c t r u m o f protein B in the presence o f sulfite a n d protein A was similar to the dithionite: H 2 0 2 difference spectrum except for the peak at 590 nm. Such results show that protein B contained cytochrume c a n d flavoprotein. Lu and Kelly isolated sulfite oxidas¢ from T. versutus {4l, which was d e p e n d e n t on cytochrome c a n d the enzyme system isolated in the present w o r k seems to be similar to this oxidase. In a d d i t i o n other a t t e m p t s to isolate snlfite oxidnse according to the methods of Charles a d Suzuki [5] and Lu and Kelly [4,6-8]
T h i s w o r k was s u p p o r t e d by Central Research Program C P B P 0308 a n d 0402.
REFERENCES It] Hempflin 8, W.P.. Trudin~r. P.A. and Vishniac. W. (1967) Arch. Mierobiol. 59. 149-157. [2] Sldodowska, A. (|988) Can. J. Microbiol. 34,1283-1287. 13] WitholL B.. Boekhout, H., Brock, M., Kingma, J.. Herrkhulzen, H.V. and Leij, L. (1976) Anal. Biochem. 74, 160-170. [4] Lu, W.P. and Kelly, D.P. (1984) J. Gun. Microbiol. 130, 1683-1692. [51 Charles, A.M. and Suzuki, L (1966) Bicchim. Biophys. A¢la. 128, 522-534. [6] Lu, W.P. and Kelly, D.P. {1983) J. Gen. Microbiol. 129. 1661-1671. [71 Lu, W.P. and Kelly. D.P. 0983) J. Gun. Microbiol. 129, 1673-1681. [8} Lu, W.P. and Kelly, D.P. (1983) J. Gun. MicrobioL 129, 3549-3564. 19] Sklodowska, A. (1985) Acla MicrobioL Polon. 34, 271-276.
59
FEMSLE 03855
Partial purification of sulfite oxidase from Thiobacillus neapolitanus A l e k s a n d r a Sldodowska
Instituteof Microbiology.Universityof Warsaw. Warsaw.Ptdand Received 18 September 1989 Accepted 22 September 1989 Key words: Cytochrome c: Flavoprotein; Difference spectrum
I. S U M M A R Y
3. MATERIALS A N D M E T H O D S
Sulfite oxidase was purified 172-fold from cell extract of Thiobacillus neapolitanus (protein A). The enzymatic activity was stimulated by protein B which shows properties of cytochrome c and flavoprolein. Sulfite oxidase was inhibited 20~ by the addition of AMP.
3.1. Bacterial strain and growth conditions T. oeapolitanus obtained from the Department of Microbiology, University of Groningen, Netherlands, was grown as described previously [2].
2. I N T R O D U C T I O N
Thiobacillus neapol#anus is a sulfur bacterium which utilizes thiosulphate as an energy source. Sulfite oxidase was purified 100-fold from an extract of T. neapolitanus by Hempfling et al. [1] who showed that the enzyme activity is stimulated by AMP. This fact indicated that a single enzyme is responsible for the oxidation of sulfite in the presence and absence of AMP. Reduced glutathlone inhibited activity and prevented A M P stimulation. This communication contains information about the isolation and partial purification of A M P independent sulfite oxidase. Correspondenceao: A. Sklodowska. (Present add~ss) Departfacial of Photography and Image Information, Warsaw University, uL Nowy ~wiat 67, PL 00-046 Warsaw, Poland.
3.2. Purification of sulfite oxidase Cell free extract from T. neapolitanus in 0.05 M phosphate buffer, pH 7.5 containing 3.7 protein per 1 ml was obtained according to the procedure of Witholt et aL [3]. Cell free extract was fractionated by the addition of increasing concentration of ( N H 4)2SO4. Precipitates obtained after centrifugation at 20000 X g for 20 rain. at + 4 ° C , were dissolved in 0.05 M phosphate buffer, pH 7.5 at temperature + 4 o C. Then the solution was placed on a column containing DEAE-celhilose (2 x 16 cm) which had be*n equilibrated with 0.05 M phosphate buffer, pH 7.5 and eluted with the same buffer containing increasing concentrations of NaCI: 0.0 M, 0.1 M0 0.2 M, 0.3 M, 0.4 M with an elution rate of 60 ml per hour. The volume of collected fractions was 5 ml. The active fractions were combined and then loaded on CM-celhilose column (2 x 10 cm) equilibrated with 0.05 M phosphate buffer, pH 6.5 and eluted with the same buffer (pH 6.5) containing
0378.1097/90/$03.50 © 1990 Federation oI European MicrobiologicalSocieties
60 increasing concentrations of NaCI as in the case of DEAE-cellulose, at the same rate. The protein content and enzyme activity was assayed in each fraction. The enzyme activity of 'combined fraction' fro:' example one fraction from DEAE and one from CM-cellulose) was also examined.
3. 3. Protein determination Protein content was assayed according to the procedure of Lowry. Light absorption at 280 nm was also examined.
03
3.4. Assay of enzyme activity
o
?Ca i a 4 M N a g l
i~INa
Sulfite oxidase activity was measured by manometric Warburg method.
3.5. Spectrophotometry
o2
a. C M - c e l l u l o s e
Difference spectra were recorded by Beckman Acta IlI speetrophotometer. For experimental data see descriptions under the figures.
a4MNaCI
02MIVaCI
1MNoCI 4. RESULTS AND DISCUSSION Purification of sulfite oxidase is presented in Table l and Fig. IA, B. The active fraction was obtained in the DEAE-cellulose eluate at NaCI concentration 03 M and it was called protein A. Table 1 Purificationof sulffieoxidase Fraction
Total activity pM 02 / min
Dialysed cell-free extract 267.0 0-15 ** 0 15-25 0 25-35 0 35-45 9.6 45-55 0.36 55-65 0.60 DE-cellulose 0.1 M NaCi 29.0
Proteins Specific Specific contents activity activity Ing/min * in the pl~sence of AMP *
5g.0
4.8 4.7 2.8
0.096 0.19 0 0 0 0.at 0.012 0.004 0,005 0.0ll 0.015
0.1"/5
16.6
* gM O2/min×rag protein; • * % (NH4)2SO4concentration.
3~m
'ml
Fig. 1. Fracfionafionof cell free extract on the column with DEAF,cellulose(A) and CM-ce0ulose(B).
The enzyme was purified 172-fold. Protein obtained in the eluate from CM-cellnlose column at NaCI concentration 0.1 M was called protein B. The activity of protein A was stimulated 2-fold by protein B. Protein B was not able to oxidize sulfite alone. Cyanide and azide completely inhibited the activity of protein. This enzyme lost 50% of activity after 10 min. at 6 0 ° C and 100~ after 2 rain at 100°C. It did not lose activity at + 4 ° C during two weeks. Protein A carried electrons immediately to oxygen as did the sulfite oxidase isolated by Hempfling et al. [1]. Protein B shows some properties of peroxidase (positive benzidine and o-dianizidine test) but the lack of flavoprotein autooxidation was opposite to these. Protein B had no properties of cytochrome oxidase type a (negative Patho Tec CO test). The difference spectrum of protein B shows a maximum at 550 nm, 520 nm, 418-420 nm (as
61
ASJO +prOleJnA 002~
+so/$
J
!IdAO.OI
and a t t e m p t s at further purification of the enzyme were unsuccessful. Similar problems were encountered by Lu and Kelly [4] hecause the single enzyme lost its activity and renewed c o m b i n a t i o n of enzymes in an enzyme complex was not always successful [8]. As for protein A, it was probably salfite oxidase or oxygenas¢. It seems that the isolated enzyme complex was one of the terminal parts o f the thiosnlphate oxidizing system in T. neapolitanus, which was described previously [2]. It is unlikely that T. neapolitanus cells which did not t a k e up sulfite [9l had a separate sulfite oxidation mechanism.
ACKNOWLEDGEMENT
4O0 5OO Fig. 2. Diffcxen4~ sl~trum of protein B (al and reduction of protein B by protein A and sulfit¢ (b). { . . . . . . ). reduced by SO32- ; (-- -- --), protein A + SO~-, I mira I ). protein A+SO 2" . 10 rain. cytochrome c), a small p e a k at 590 nm and minim u m about 460 nm. (Fig. 2a). T h e difference s p e c t r u m o f protein B in the presence o f sulfite a n d protein A was similar to the dithionite: H 2 0 2 difference spectrum except for the peak at 590 nm. Such results show that protein B contained cytochrume c a n d flavoprotein. Lu and Kelly isolated sulfite oxidas¢ from T. versutus {4l, which was d e p e n d e n t on cytochrome c a n d the enzyme system isolated in the present w o r k seems to be similar to this oxidase. In a d d i t i o n other a t t e m p t s to isolate snlfite oxidnse according to the methods of Charles a d Suzuki [5] and Lu and Kelly [4,6-8]
T h i s w o r k was s u p p o r t e d by Central Research Program C P B P 0308 a n d 0402.
REFERENCES It] Hempflin 8, W.P.. Trudin~r. P.A. and Vishniac. W. (1967) Arch. Mierobiol. 59. 149-157. [2] Sldodowska, A. (|988) Can. J. Microbiol. 34,1283-1287. 13] WitholL B.. Boekhout, H., Brock, M., Kingma, J.. Herrkhulzen, H.V. and Leij, L. (1976) Anal. Biochem. 74, 160-170. [4] Lu, W.P. and Kelly, D.P. (1984) J. Gun. Microbiol. 130, 1683-1692. [51 Charles, A.M. and Suzuki, L (1966) Bicchim. Biophys. A¢la. 128, 522-534. [6] Lu, W.P. and Kelly, D.P. {1983) J. Gen. Microbiol. 129. 1661-1671. [71 Lu, W.P. and Kelly. D.P. 0983) J. Gun. Microbiol. 129, 1673-1681. [8} Lu, W.P. and Kelly, D.P. (1983) J. Gun. MicrobioL 129, 3549-3564. 19] Sklodowska, A. (1985) Acla MicrobioL Polon. 34, 271-276.