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A manifold and oxygen-removing apparatus for preparing anaerobic enzymes
ANALYTICAL
BIOCHEMISTRY
121, 335-338 (1982)
A Manifold
and Oxygen-Removing Apparatus Preparing Anaerobic Enzymes
JAY B. PETERSON, R. A. GLENISTER, Boyce
Thompson
Institute
for
Plant
Research,
Tower
for
AND D. L. ESKEW Road,
Ithaca,
New
York
14853
Received November 30, 1981 An apparatus is described which is useful in preparations for anaerobic enzymology and physiology experiments. It has two columns which effectively remove O2 from standard Nz and Ar. Ar with 9.4 ppm O2 was passed through the columns at 2 liters per minute and the O2 concentration was lowered to less than 0.2 ppm. Purified gas can be used in an attached manifold system for replacing the gas phase in rubber-stoppered serum vials. The apparatus has several improvements over previously described systems. It is made primarily of metal, making it more durable and able to withstand higher gas pressures than glass systems. It also contains an indicator in one of the 02-removing columns to warn of contaminating OX.
Anaerobic techniques are widely used in enzymology, because some enzymes, enzyme substrates, and reaction products react directly with O2 in undesirable reactions. Such work requires the preparation of 02-free gases and solutions as well as anaerobic reaction vessels. Standard laboratory gases, such as Ar and NZ, frequently contain unacceptably high levels of OZ. Techniques used for removing the O2 include passing the gas over hot copper turnings or over BASF catalyst or through solutions of reduced methyl viologen or Na,S,04. Techniques used for making solutions or vessels anaerobic consist of either extensive flushing or alternate evaculation and refilling, using a manifold system. Various laboratories have apparatuses with 02-removing and manifold gas-replacement capabilities. Manifolds are usually made of glass, such as the systems described by Burgess et al. ( 1) and Eady (2). Here we describe the construction of an apparatus (Fig. 1) which has improved capabilities.’ ’ Reprints and details on construction are available from J. Peterson. 335
MATERIALS AND METHODS
Valves, tube fittings, conversion fittings, pipe fittings, weld fittings, and flexible tubing were purchased from Rochester Valve and Fitting Company, Rochester, New York. Support rings, clamps, insulated heating tape, and Variac were from Arthur H. Thomas Company, Philadelphia, Pennsylvania. The high-capacity indicating OxyTrap column was from Alltech Associates, Deerfield, Illinois. Butyl rubber tubing and Flexaframe support were from Fisher Scientific Company, Rochester, New York. Needle-tubing connectors and Teflon Tape were from Aldrich Chemical Company, Milwaukee, Wisconsin. The side dial thermometer was from Cole-Parmer Instrument Company, Chicago, Illinois. Gas-Pure catalyst (BASF catalyst R3- 11) was from Ace Scientific Company, Linden, New Jersey. The brass pressure gauge was a product of Union Carbide, New York, New York. The reaction vessel for housing the Gas-Pure catalyst was a steel gas lecture bottle, 2 X 12 in. (volume 0.42 liter). Copper and stainless steel tubing (% in.), angle iron, aluminum sheeting (l/s in. thick), and materials for the 0003-2697/82/060335-04$02.00/O Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
336
PETERSON,
GLENISTER,
AND ESKEW
FIG. 1. Frontal view of the complete oxygen-removing and manifold device.
base were standard products purchased locally. Torr Seal was a product of Varian Associates, Palo Alto, California. For testing the oxygen-removing capacity of the columns, a mixture of 9.4 ppm O2 in argon (Union Carbide, Linde Division) was purchased from Ames, Ithaca, New York. Assays. Oxygen content of gases was measured by bubbling the gas through alkaline pyrogallol containing ferrous iron (6) for 20 min and then measuring absorption at 520 nm. The reaction flask and a flow-through cuvette were connected by narrow-gauge plastic tubing. The tubing connection served first to purge the cuvette with oxygen-free gas and then to transfer the pyrogallol solution without contact with the atmosphere, using pressure inside the reaction flask to force the transfer. The color development was approximately linear over the O2 range examined and was sensitive enough to detect 0.2 ppm Oz. Nitrogenase activity of bacteria and bacterial extracts was measured by standard techniques (2,3). Assays were performed in rubber-stoppered serum vials under an atmosphere (by volume) of 90% argon and 10% acetylene.
Construction. The lecture bottle used for the reaction vessel has a Gin. NPT hole at one end. A second hole, %-in. NPT, was drilled in the opposite end to serve as gas outlet. Fittings attached to the reaction vessel were stainless steel since they would be subjected to large temperature variations. The manifold was constructed by welding stainless steel fittings and tubing together to form a one-piece, vacuum-tight unit between valves (Fig. 2). The manifold was set up for attaching serum vials by mounting short pieces of copper tubing to the front of the valves. Butyl tubing was then used for attaching needle tubing connectors and syringe needles were mounted on these. A piece of flexible steel tubing was required at one end of the Oxy-Trap column (Fig. 2). When copper tubing was used it was difficult to mount the column without breaking it, since input and output connections were difficult to line up properly. Leaks were sealed with Teflon tape (for pipe fittings) or Torr Seal (for Swagelok fittings). Safety. H2 is used for reducing the BASF catalyst and the usual precautions must be used with this gas. The whole device should
APPARATUS
FOR ANAEROBIC
iZ!l Toggle
q
Regulating
On-Off
PREPARATIONS
AND ENZYME
ASSAYS
337
valve
valve
Thermometer
column
! II
vapor
Outlet
t Manifold
FIG. 2. Diagrammatic
representation of the oxygen-removing and manifold device.
be grounded, since the heating tape can break or its insulating cover can wear through. The frontal valves should not be opened when the manifold is pressurized over several pounds per square inch, since the attachments (e.g., syringe needles) may be blown off. As a further precaution, it is suggested that a shield be built and attached to prevent the laboratory worker from being directly in front of the frontal valves. Safety glasses should be worn. Although the OxyTrap column is rated at 100 psi maximum, we have found that 30 psi is adequate to test for leaks on the entire apparatus and cannot recommend increasing the pressure above that level. PERFORMANCE
The finished apparatus (Figs. 1, 2) has been used extensively to assay crude Rhizobium japonicum and Azotobacter vinelandii nitrogenases in vitro in the presence of Na2S204, which protects against O2 inactivation. Nitrogenase is very O2 labile in the absence of protecting agents (2,4,5). We have successfully worked with crude R. japonicum nitrogenase, chromatographed anaerobically to remove Na2S204. The system was also used to assay nitrogenase activity of intact R. japonicum bacteroids and A.
vinelandii cells. Here, a low level of O2 is
permissible. For such uses, the apparatus has a bypass to the manifold which permits preparation of assay mixtures without use of the 02-removing columns. In all such assays, a large number of anaerobic assay mixtures was prepared rapidly. The manifold has eight ports to which rubber-stoppered serum vials are connected. Extensive evacuations with regassing can be accomplished in several minutes. If desired, one port can be left free and used to bring the manifold to atmospheric pressure. The oxygen-removing ability of the apparatus was measured. A standard mixture of 9.4 ppm O2 in argon was passed through the columns at a flow rate of 2.0 liters/min. The reaction vessel temperature was 120°C. The oxygen concentration was reduced to less than 0.2 ppm. An analogous measurement was made with standard laboratory N2 (rated by the manufacturer at
338
PETERSON,
GLENISTER,
Materials and Methods) to test for leaks. The additional 02-removing and -detecting column will signal a loss of O,-removing capacity of the BASF catalyst and provide additional 02-removing capacity. In addition, the apparatus can be easily modified (e.g., addition of flow meters, manometer) should the need arise. ACKNOWLEDGMENT
AND ESKEW
REFERENCES I. Burgess, B. K., Jacobs, D. B., and Stiefel, E. I. (1980)
Biochem.
We thank Dr. T. A. LaRue for his support of this project.
Biochim.
Biophys.
Acia
614,
196-209.
2. Eady, R. R. (1980) in Methods for Evaluating Biological Nitrogen Fixation (Bergerson, F. J., ed.), pp. 213-264, Wiley, New York. 3. Emerich, D. W., Ruiz-Argueso, T., Ching, T.-M., and Evans, H. J. (1979) J. Bacterial. 137, 153160. 4. Klucas, R. V., Koch, B., Russell, S. A., and Evans, H. J. (1968) Plant Physiol. 43, 1906-1912. 5. Winter, H. C., and Burris, R. H. (1976) Annu. Rev. 45, 409-426.
6. Wolfe, If. H., Zander, R., and Lang, W. ( 1976) Anal. Biochem. 74, 5855591.
BIOCHEMISTRY
121, 335-338 (1982)
A Manifold
and Oxygen-Removing Apparatus Preparing Anaerobic Enzymes
JAY B. PETERSON, R. A. GLENISTER, Boyce
Thompson
Institute
for
Plant
Research,
Tower
for
AND D. L. ESKEW Road,
Ithaca,
New
York
14853
Received November 30, 1981 An apparatus is described which is useful in preparations for anaerobic enzymology and physiology experiments. It has two columns which effectively remove O2 from standard Nz and Ar. Ar with 9.4 ppm O2 was passed through the columns at 2 liters per minute and the O2 concentration was lowered to less than 0.2 ppm. Purified gas can be used in an attached manifold system for replacing the gas phase in rubber-stoppered serum vials. The apparatus has several improvements over previously described systems. It is made primarily of metal, making it more durable and able to withstand higher gas pressures than glass systems. It also contains an indicator in one of the 02-removing columns to warn of contaminating OX.
Anaerobic techniques are widely used in enzymology, because some enzymes, enzyme substrates, and reaction products react directly with O2 in undesirable reactions. Such work requires the preparation of 02-free gases and solutions as well as anaerobic reaction vessels. Standard laboratory gases, such as Ar and NZ, frequently contain unacceptably high levels of OZ. Techniques used for removing the O2 include passing the gas over hot copper turnings or over BASF catalyst or through solutions of reduced methyl viologen or Na,S,04. Techniques used for making solutions or vessels anaerobic consist of either extensive flushing or alternate evaculation and refilling, using a manifold system. Various laboratories have apparatuses with 02-removing and manifold gas-replacement capabilities. Manifolds are usually made of glass, such as the systems described by Burgess et al. ( 1) and Eady (2). Here we describe the construction of an apparatus (Fig. 1) which has improved capabilities.’ ’ Reprints and details on construction are available from J. Peterson. 335
MATERIALS AND METHODS
Valves, tube fittings, conversion fittings, pipe fittings, weld fittings, and flexible tubing were purchased from Rochester Valve and Fitting Company, Rochester, New York. Support rings, clamps, insulated heating tape, and Variac were from Arthur H. Thomas Company, Philadelphia, Pennsylvania. The high-capacity indicating OxyTrap column was from Alltech Associates, Deerfield, Illinois. Butyl rubber tubing and Flexaframe support were from Fisher Scientific Company, Rochester, New York. Needle-tubing connectors and Teflon Tape were from Aldrich Chemical Company, Milwaukee, Wisconsin. The side dial thermometer was from Cole-Parmer Instrument Company, Chicago, Illinois. Gas-Pure catalyst (BASF catalyst R3- 11) was from Ace Scientific Company, Linden, New Jersey. The brass pressure gauge was a product of Union Carbide, New York, New York. The reaction vessel for housing the Gas-Pure catalyst was a steel gas lecture bottle, 2 X 12 in. (volume 0.42 liter). Copper and stainless steel tubing (% in.), angle iron, aluminum sheeting (l/s in. thick), and materials for the 0003-2697/82/060335-04$02.00/O Copyright 0 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.
336
PETERSON,
GLENISTER,
AND ESKEW
FIG. 1. Frontal view of the complete oxygen-removing and manifold device.
base were standard products purchased locally. Torr Seal was a product of Varian Associates, Palo Alto, California. For testing the oxygen-removing capacity of the columns, a mixture of 9.4 ppm O2 in argon (Union Carbide, Linde Division) was purchased from Ames, Ithaca, New York. Assays. Oxygen content of gases was measured by bubbling the gas through alkaline pyrogallol containing ferrous iron (6) for 20 min and then measuring absorption at 520 nm. The reaction flask and a flow-through cuvette were connected by narrow-gauge plastic tubing. The tubing connection served first to purge the cuvette with oxygen-free gas and then to transfer the pyrogallol solution without contact with the atmosphere, using pressure inside the reaction flask to force the transfer. The color development was approximately linear over the O2 range examined and was sensitive enough to detect 0.2 ppm Oz. Nitrogenase activity of bacteria and bacterial extracts was measured by standard techniques (2,3). Assays were performed in rubber-stoppered serum vials under an atmosphere (by volume) of 90% argon and 10% acetylene.
Construction. The lecture bottle used for the reaction vessel has a Gin. NPT hole at one end. A second hole, %-in. NPT, was drilled in the opposite end to serve as gas outlet. Fittings attached to the reaction vessel were stainless steel since they would be subjected to large temperature variations. The manifold was constructed by welding stainless steel fittings and tubing together to form a one-piece, vacuum-tight unit between valves (Fig. 2). The manifold was set up for attaching serum vials by mounting short pieces of copper tubing to the front of the valves. Butyl tubing was then used for attaching needle tubing connectors and syringe needles were mounted on these. A piece of flexible steel tubing was required at one end of the Oxy-Trap column (Fig. 2). When copper tubing was used it was difficult to mount the column without breaking it, since input and output connections were difficult to line up properly. Leaks were sealed with Teflon tape (for pipe fittings) or Torr Seal (for Swagelok fittings). Safety. H2 is used for reducing the BASF catalyst and the usual precautions must be used with this gas. The whole device should
APPARATUS
FOR ANAEROBIC
iZ!l Toggle
q
Regulating
On-Off
PREPARATIONS
AND ENZYME
ASSAYS
337
valve
valve
Thermometer
column
! II
vapor
Outlet
t Manifold
FIG. 2. Diagrammatic
representation of the oxygen-removing and manifold device.
be grounded, since the heating tape can break or its insulating cover can wear through. The frontal valves should not be opened when the manifold is pressurized over several pounds per square inch, since the attachments (e.g., syringe needles) may be blown off. As a further precaution, it is suggested that a shield be built and attached to prevent the laboratory worker from being directly in front of the frontal valves. Safety glasses should be worn. Although the OxyTrap column is rated at 100 psi maximum, we have found that 30 psi is adequate to test for leaks on the entire apparatus and cannot recommend increasing the pressure above that level. PERFORMANCE
The finished apparatus (Figs. 1, 2) has been used extensively to assay crude Rhizobium japonicum and Azotobacter vinelandii nitrogenases in vitro in the presence of Na2S204, which protects against O2 inactivation. Nitrogenase is very O2 labile in the absence of protecting agents (2,4,5). We have successfully worked with crude R. japonicum nitrogenase, chromatographed anaerobically to remove Na2S204. The system was also used to assay nitrogenase activity of intact R. japonicum bacteroids and A.
vinelandii cells. Here, a low level of O2 is
permissible. For such uses, the apparatus has a bypass to the manifold which permits preparation of assay mixtures without use of the 02-removing columns. In all such assays, a large number of anaerobic assay mixtures was prepared rapidly. The manifold has eight ports to which rubber-stoppered serum vials are connected. Extensive evacuations with regassing can be accomplished in several minutes. If desired, one port can be left free and used to bring the manifold to atmospheric pressure. The oxygen-removing ability of the apparatus was measured. A standard mixture of 9.4 ppm O2 in argon was passed through the columns at a flow rate of 2.0 liters/min. The reaction vessel temperature was 120°C. The oxygen concentration was reduced to less than 0.2 ppm. An analogous measurement was made with standard laboratory N2 (rated by the manufacturer at
338
PETERSON,
GLENISTER,
Materials and Methods) to test for leaks. The additional 02-removing and -detecting column will signal a loss of O,-removing capacity of the BASF catalyst and provide additional 02-removing capacity. In addition, the apparatus can be easily modified (e.g., addition of flow meters, manometer) should the need arise. ACKNOWLEDGMENT
AND ESKEW
REFERENCES I. Burgess, B. K., Jacobs, D. B., and Stiefel, E. I. (1980)
Biochem.
We thank Dr. T. A. LaRue for his support of this project.
Biochim.
Biophys.
Acia
614,
196-209.
2. Eady, R. R. (1980) in Methods for Evaluating Biological Nitrogen Fixation (Bergerson, F. J., ed.), pp. 213-264, Wiley, New York. 3. Emerich, D. W., Ruiz-Argueso, T., Ching, T.-M., and Evans, H. J. (1979) J. Bacterial. 137, 153160. 4. Klucas, R. V., Koch, B., Russell, S. A., and Evans, H. J. (1968) Plant Physiol. 43, 1906-1912. 5. Winter, H. C., and Burris, R. H. (1976) Annu. Rev. 45, 409-426.
6. Wolfe, If. H., Zander, R., and Lang, W. ( 1976) Anal. Biochem. 74, 5855591.