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Carbonic anhydrase: A new method of detection on polyacrylamide gels using low-temperature fluorescence
ANALYTICAL
BIOCHEMISTRY
44, 388-391 (1971)
Carbonic A New
Method
of Detection
Using
low-Temperature
B. D. PATTERSON,’ AND
Plant
An hydrase: on Polyacrylamide
Gels
Fluorescence
C. A. ATKINS2 R. B. H. WILLS
D. GRAHAM,
Physiology Unit, CUR0 Division of Food Research, School of Biological Sciences, Macquarie University, North Ryde, 2113, Sydney, Australia
Received February
and
3, 1971
Carbonic anhydrase catalyzes the reversible hydration dioxide :
of carbon
COz + Hz0 ti HCOa- + H+
The increased rate of production of hydrogen ions in the presence of the enzyme and carbon dioxide can be used to locate the enzyme on polyacrylamide gels if a suitable indicator such as bromothymol blue is used (1). Bands of carbonic anhydrase appear yellow against a blue background. However, even when the reaction is carried out at 0”, the bands showing the enzyme activity are only transient. The rapid loss of contrast is caused by two factors: rapid diffusion of buffer through the gel which blurs the edges of bands, and the appreciable nonenzymic rate of hydration of carbon dioxide. This loss of contrast, which occurs within a few minutes, makes examination and photography of the gels difficult. As a consequence,animal carbonic anhydrases have usually been detected on gels by their associated e&erase activity (2). However, Tobin found no esterase activity in purified carbonic anhydrase from parsley leaves (3), and so the esterase method is unsatisfactory for detecting the plant enzyme. We have devised a new method that depends on the enzyme-catalyzed increase in the rate of CO, hydration, and the resultant change in pH. With this method, using low-temperature fluorescence, the stained bands have a life of many hours and can be readily photographed. ‘Present address: East Malling Research ’ Rothmans Post-doctoral Fellow.
388
Station,
Maidstone,
Kent,
England.
CARBONIC
ANHYDRASE
MSTERIALS
ON
AND
POLYACRYLAMIDE
389
METHODS
Gradipore gradient gels (426% concave gradient of acrylamide (4) were used with a Gradipore eleetrophoresis apparatus (Townson and Mercer Pty. Ltd., Lane Cove, N.S.W., Australia). The electrophoresis buffer, pH 8.28 at 20”, contained tris (hydroxymethyl) methylamine (10.75 gm) , disodium ethylenediaminetetraacetic acid (0.93 gm) , and boric acid (5.94 gm) per liter (5). Purified carbonic anhydrase from bovine erythrocytes was obtained from Sigma Chemical Co., St. Louis, MO., and 2 ~1 of a solution containing 2 pg protein was applied to the gel. Plant material was extracted at 0” by grinding it in a mortar with sand and 2 vol electrophoresis buffer containing 10 m&I dithiothreitol. The extracts were filtered through nylon mesh (60 pm hole size) and centrifuged at 14,900g for 20 min; sucrose was added to the supernatants to give a concentration of 5% (w/v). Between 5 ~1 and 25 ~1 was applied to each origin on the gel under a layer of buffer. After electrophoresis for 20 hr at 5” and 75V, the gel was split into two halves with a wire gel slicer (Townson and Mercer). With the cut surface uppermost, the gel was cooled to 0’ on an aluminum plate and flooded with a cold (0”) solution of bromocresol purple (0.1%) in electrophoresis buffer containing 10 mM dithiothreitol. After 1 min, the cut surface was blotted free of all excess liquid, and covered for 0.5 to 2 min with an inverted filter funnel attached to a source of pure carbon dioxide. The precise time was determined in preliminary experiments by a blank run with pieces of gel and indicator, because at -70” the indicator-buffer mixture changes color at a higher apparent pH than at 0”. Immediately after CO, treatment, the gels were frozen in dry ice and examined under ultraviolet light (x,,, = 366 nm) . The yellow form of the bromocresol purple in zones of carbonic anhydrase activity fluoresced bright yellow against a pink background. The gels were photographed using Polaroid Land film type 107, through a green filter (see Fig. 1 for details). Gels were stained for protein with amido black. RESULTS
AND
DISCUSSION
Figure 1 shows the patterns of carbonic anhydrase isozymes from bovine erythrocytes, and the leaves of Petroselinum horteme Hoflm. (parsley) and Trudesmntiu aEb$ora K&h. (spiderwort) . The gels could be stored with little loss of contrast in solid CO, for several hours, or for much longer in liquid nitrogen. After longer periods of storage, the gels could be thawed and redeveloped with indicator solution and carbon dioxide. The method has been used routinely to follow the purification of car-
390
PATTERSON
a
ET
b
AL.
c
origin
-0
-2 _
0
3
-4
-6 -I FIG. 1. Acrylamide gel electrophoresis (2) of carbonic anhydrases of plant and animal origin: (a) Parsley leaves, (b) spiderwort leaves, (c) purified carbonic anhydrase from bovine erythrocytes. Photographic conditions: gel illuminated with longwavelength UV light (ChromateVue, U-V Products Inc.), Wratten filter 74, 10 set
exposure,f = 8, American StandardsAssociation3000. bonic anhydrase isozymes from Tradescantia albiflora. Purified preparations of high specific activity gave stained protein bands which were coincident with the carbonic anhydrase activity on the gels. we have used bromothymol blue for the quantitative assay of carbonic anhydrase (6) in plant extracts, but on the gels at -70” bromocresol purple gave better results. SUMMARY
Carbonic anhydrase was located on polyacrylamide gels with carbon dioxide as the substrate and bromocresol purple to indicate hydrogen ion formation. The developed gels were fixed by freezing to -7O”, and the enzyme bands were detected by their low-temperature fluorescence in ultraviolet light. ACKNOWLEDGMENTS The authors wish to thank Mr. D. Hockley Maguire for photography.
for technical assistance and Mr. P. R.
CARBONIC
ANHYDRASE
ON
POLYACRYLAMIDE
391
REFERENCES 1. 2. 3.
M. L., Ph.D. Thesis, University of Sydney, 1970. TASHIAN, R. E., Amer. J. Hum. Genet. 17, 257 (1965). TOBIN, A. J., J. Bid. Chem. 245, 2656 (1970). 4. MARGOLIS, J., AND KENRICK, K. G., Anal. Biochem. 25, 347 (1968). 5. KITCHIN, F. D., Proc. Sot. Ezp. Biol. Med. 119, 1153 (1965). 6. RICXLI, E. E., GHAZANFAR, S. A. S., GIBBONS, B. H., AND EDSALL, J. T., d. Rioi. Chem. 239, 1065 (1964). REED,
BIOCHEMISTRY
44, 388-391 (1971)
Carbonic A New
Method
of Detection
Using
low-Temperature
B. D. PATTERSON,’ AND
Plant
An hydrase: on Polyacrylamide
Gels
Fluorescence
C. A. ATKINS2 R. B. H. WILLS
D. GRAHAM,
Physiology Unit, CUR0 Division of Food Research, School of Biological Sciences, Macquarie University, North Ryde, 2113, Sydney, Australia
Received February
and
3, 1971
Carbonic anhydrase catalyzes the reversible hydration dioxide :
of carbon
COz + Hz0 ti HCOa- + H+
The increased rate of production of hydrogen ions in the presence of the enzyme and carbon dioxide can be used to locate the enzyme on polyacrylamide gels if a suitable indicator such as bromothymol blue is used (1). Bands of carbonic anhydrase appear yellow against a blue background. However, even when the reaction is carried out at 0”, the bands showing the enzyme activity are only transient. The rapid loss of contrast is caused by two factors: rapid diffusion of buffer through the gel which blurs the edges of bands, and the appreciable nonenzymic rate of hydration of carbon dioxide. This loss of contrast, which occurs within a few minutes, makes examination and photography of the gels difficult. As a consequence,animal carbonic anhydrases have usually been detected on gels by their associated e&erase activity (2). However, Tobin found no esterase activity in purified carbonic anhydrase from parsley leaves (3), and so the esterase method is unsatisfactory for detecting the plant enzyme. We have devised a new method that depends on the enzyme-catalyzed increase in the rate of CO, hydration, and the resultant change in pH. With this method, using low-temperature fluorescence, the stained bands have a life of many hours and can be readily photographed. ‘Present address: East Malling Research ’ Rothmans Post-doctoral Fellow.
388
Station,
Maidstone,
Kent,
England.
CARBONIC
ANHYDRASE
MSTERIALS
ON
AND
POLYACRYLAMIDE
389
METHODS
Gradipore gradient gels (426% concave gradient of acrylamide (4) were used with a Gradipore eleetrophoresis apparatus (Townson and Mercer Pty. Ltd., Lane Cove, N.S.W., Australia). The electrophoresis buffer, pH 8.28 at 20”, contained tris (hydroxymethyl) methylamine (10.75 gm) , disodium ethylenediaminetetraacetic acid (0.93 gm) , and boric acid (5.94 gm) per liter (5). Purified carbonic anhydrase from bovine erythrocytes was obtained from Sigma Chemical Co., St. Louis, MO., and 2 ~1 of a solution containing 2 pg protein was applied to the gel. Plant material was extracted at 0” by grinding it in a mortar with sand and 2 vol electrophoresis buffer containing 10 m&I dithiothreitol. The extracts were filtered through nylon mesh (60 pm hole size) and centrifuged at 14,900g for 20 min; sucrose was added to the supernatants to give a concentration of 5% (w/v). Between 5 ~1 and 25 ~1 was applied to each origin on the gel under a layer of buffer. After electrophoresis for 20 hr at 5” and 75V, the gel was split into two halves with a wire gel slicer (Townson and Mercer). With the cut surface uppermost, the gel was cooled to 0’ on an aluminum plate and flooded with a cold (0”) solution of bromocresol purple (0.1%) in electrophoresis buffer containing 10 mM dithiothreitol. After 1 min, the cut surface was blotted free of all excess liquid, and covered for 0.5 to 2 min with an inverted filter funnel attached to a source of pure carbon dioxide. The precise time was determined in preliminary experiments by a blank run with pieces of gel and indicator, because at -70” the indicator-buffer mixture changes color at a higher apparent pH than at 0”. Immediately after CO, treatment, the gels were frozen in dry ice and examined under ultraviolet light (x,,, = 366 nm) . The yellow form of the bromocresol purple in zones of carbonic anhydrase activity fluoresced bright yellow against a pink background. The gels were photographed using Polaroid Land film type 107, through a green filter (see Fig. 1 for details). Gels were stained for protein with amido black. RESULTS
AND
DISCUSSION
Figure 1 shows the patterns of carbonic anhydrase isozymes from bovine erythrocytes, and the leaves of Petroselinum horteme Hoflm. (parsley) and Trudesmntiu aEb$ora K&h. (spiderwort) . The gels could be stored with little loss of contrast in solid CO, for several hours, or for much longer in liquid nitrogen. After longer periods of storage, the gels could be thawed and redeveloped with indicator solution and carbon dioxide. The method has been used routinely to follow the purification of car-
390
PATTERSON
a
ET
b
AL.
c
origin
-0
-2 _
0
3
-4
-6 -I FIG. 1. Acrylamide gel electrophoresis (2) of carbonic anhydrases of plant and animal origin: (a) Parsley leaves, (b) spiderwort leaves, (c) purified carbonic anhydrase from bovine erythrocytes. Photographic conditions: gel illuminated with longwavelength UV light (ChromateVue, U-V Products Inc.), Wratten filter 74, 10 set
exposure,f = 8, American StandardsAssociation3000. bonic anhydrase isozymes from Tradescantia albiflora. Purified preparations of high specific activity gave stained protein bands which were coincident with the carbonic anhydrase activity on the gels. we have used bromothymol blue for the quantitative assay of carbonic anhydrase (6) in plant extracts, but on the gels at -70” bromocresol purple gave better results. SUMMARY
Carbonic anhydrase was located on polyacrylamide gels with carbon dioxide as the substrate and bromocresol purple to indicate hydrogen ion formation. The developed gels were fixed by freezing to -7O”, and the enzyme bands were detected by their low-temperature fluorescence in ultraviolet light. ACKNOWLEDGMENTS The authors wish to thank Mr. D. Hockley Maguire for photography.
for technical assistance and Mr. P. R.
CARBONIC
ANHYDRASE
ON
POLYACRYLAMIDE
391
REFERENCES 1. 2. 3.
M. L., Ph.D. Thesis, University of Sydney, 1970. TASHIAN, R. E., Amer. J. Hum. Genet. 17, 257 (1965). TOBIN, A. J., J. Bid. Chem. 245, 2656 (1970). 4. MARGOLIS, J., AND KENRICK, K. G., Anal. Biochem. 25, 347 (1968). 5. KITCHIN, F. D., Proc. Sot. Ezp. Biol. Med. 119, 1153 (1965). 6. RICXLI, E. E., GHAZANFAR, S. A. S., GIBBONS, B. H., AND EDSALL, J. T., d. Rioi. Chem. 239, 1065 (1964). REED,