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Anaerobic spectrophotometry: An improved cuvette
ANALYTICAL
5, 470-473
BIOCHEMISTRY
Anaerobic
(1973)
Spectrophotometry:
E. K. HODGSON, of
Department
AND
Biochemist,.y, Duke Cniversity Durham, North Carolina 27710
Received A cuvette is any gas mixture. which can be Thunberg head the cuvette and can be achieved for at least 12
J. M. McCORD,
An Improved
June
26,
1972;
accepted
August
Cuvettel
I. FRIDOVICH Medical
30,
Center.
1972
described which allows purging of the reaction solution with The cuvette design incorporates a gas inlet and outlet simultaneously sealed by a small angular rotation of the and a gassing tube which is an integral part of the body of which is, therefore, not easily broken. Anaerobic conditions in 10 min and can be maintained, in the sealed cuvette, hr.
Spectrophotometry under controlled atmospheres has generally been achieved by two methods. The first of these depends upon alternate evacuation and refilling of the cuvette with the desired gas (l-3). The slowness with which liquid and gas phases equilibrates places a limitation on the utility of this method. The second procedure, which allows bubbling the gas through the liquid (4,5), was devised to facilitate equilibration between the liquid and the gas phases. The cuvettes, which have been designed to permit sweeping the liquid phase with the gas, have been both cumbersome and fragile. Stopcocks to close the gas inlet and outlet tubes and a thin glass gassing tube to conduct the gas down into the liquid phase, have seemed t#o be indispensible components of such cuvettes. Cuvettes with retractable metal gassing tubes have been constructed (Hellma Cells, Inc.) but these have a packing gland which is prone to gas leakage. Dixon (5) has circumvented these problems by working under a continuous stream of N2 and thoroughly described the problems in achieving and maintaining anaerobic conditions in spectrophotometer cuvettes. If the thin tube which conducts the gas into the liquid phase could be made an integral part, of the body of the cuvette, problems of fragility would be solved and if the gas inlet and gas outlet closures could be incorporated into the stopper of the cuvette, extraneous stopcocks could be eliminated. This report describes a cuvette which achieves these ends. ‘This stitutes Copyright All rights
work was supported of Health, Bethesda,
in full Maryland.
@ 1973 by Academic Press. of reproduction in any form
by
Grant
470 Inc. reserved.
GM-10287
from
the
National
In-
ANAEROBIC
MATERIALS
CUVETTE
AND
471
METHODS
The quartz anaerobic cuvette, described below, was fabricated by Precision Cells, Inc., of Hicksville, New York. Santhine oxidase was prepared from raw cream by a procedure which avoided exposure to proteolytic agents (6). Prepurified N, was obtained from the Matheson Co. and was freed of traces of 0, by passage over a column of hot colloidal copper dispersed on an inert support (7). The oxidation of xanthine to mate was folowed at 295 nm in a Gilford Model 2000 recording spectrophometer. RESULTS
Figure 1 presents a diagram of the cuvette. The stream of gas enters the cuvette through the side arm and is conducted, through a groove in the male member of the ground joint, to the gassing tube; which carries the gas to within 3 mm of the bottom of the cuvette. The gassing tube
Fm. 1. Anaerobic cuvette. The bottom part of this cuvette has the dimensions of an ordinary rectangular cuvette with a l.O-cm light path. The height of the complete assembly was 10.0 cm, the grooves cut in the male and female members of the ground joint are 1.5 mm n-ide and 1.0 mm deep. A top view of the rectangular bottom part of the cuvette is provided to show the position of the gassing tube.
472
HODGSON,
MCCORD,
AND
FRIDOVICH
did not interfere with the light beam of the spectrophotometer and because it was sealed onto the inside of the cuvette, as shown, it was not subject to breakage during assembly and cleaning of the cuvette. After rising through the liquid phase, the gas escapes through a hole in the male member of the joint which connects with a groove in the female member of the ground joint. A slight angular displacement of the hollow stopper simultaneously seals both the gas inlet and the gas outlet. In practice, the ground joint was lightly greased with Sisco 300 to provide a gas-tight seal and to facilitate rotation of the hollow stopper. The practicality of this design was tested with the xanthine oxidase reaction. Reaction mixtures, containing 5 X 1O-5M xanthine, 1 X 1O-4M EDTA, and 0.05M potassium phosphate in a total volume of 3.0 ml at pH 7.8 and at 24°C were placed in the cuvette and 10 ~1 of 9.1 X 1CP M xanthine oxidase was placed in the hollow stopper. The cuvette was then assembled and gassed with N, before being sealed. The reaction was started by tipping and thus rinsing the enzyme from the hollow stopper into the body of the cuvette. A gentle stream of N, was used during the gassing period to avoid frothing and loss of solution. A taller cuvette of otherwise identical design would have allowed more rapid gassing and thus a more rapid elimination of 0,. Figure 2 demonstrates that 10 min of gassing accomplished the complete elimination of O,, as judged by a prevention of the xanthine oxidase reaction. Removal of OZ from the
0
2
4
6 Minutes
8 IO of Purging
12
14
I6
FIG. 2. Elimination of 0, as a function of time of purging. Reaction mixtures containing 5 X lo-‘M xanthine, 1 X lo-‘M EDTA, and 0.05 M potassium phosphate in a total volume of 3.0 ml at pH 7.8 and at 24°C were swept with a gentle stream of N, for variable periods of time. The cuvette was then sealed, 10 ~1 of 9.1 x 10e6M xanthine oxidase was tipped in, and the rate of urate accumulation was followed at 295 nm. The ordinate is in terms of increase in absorbancy at 295 nm/min X 10.
ANAEROBIC
CUVETTE
473
enzyme solution, which was in the hollow stopper, was dependent upon diffusion. It is, therefore, desirable to keep to a minimum the volume of enzyme solution placed in the hollow stopper. The cuvette, once having been gassed with N, and sealed, was able to exclude atmospheric 0, for long periods of time. Thus, the interval of time between sealing and tipping-in could be extended to 12 hr without noticeable entry of oxygen. DISCUSSION
The cuvette described above allows rapid sweeping of oxygen from the reaction solution, effective and facile sealing of the cuvette contents from atmospheric O,, and initiation of the reaction at any time subsequent to sealing. The complete cuvette assembly is small and no more fragile than a standard rectangular quartz cuvette. The cuvette described above does not provide for sequential addition of reagents without breaching the gas-tight seal but this feature could be added in the form of a rubber-sealed injection port. Such an injection port would also allow the enzyme to be added after bubbling with NZ had commenced. This might be of importance in work with oxygen-labile enzymes. The xanthine oxidase reaction, which was used to test the workings of this cuvette, has a K, for 0, of approximately 2.7 X lo-” N (81, under the conditions used. Clearly, an enzyme with a higher affinity for 0, would have provided a more stringent test of oxygen exclusion. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
DIXON, M., AND KENWORTHY, P. (1967) Biochim. Biopkys Acta 146, 54. MASSEY, V., GIBSON, Q. H., AND VEIXER, C. (1960) Biochem. J. 77, 341. VELICK. S. F., AND STRITTMATTER, P. (1956) J. Biol. Chem. 221, 265. LAZAROW, A., AND COOPERSTEIN, S. J. (1954) Science 120, 674. DIXON, M. (1971) Biochim. Biophys. Acta 226, 241. BRADY, F. 0. (1969) Ph.D. thesis, Duke University. MEYER, F. B., AND RONGE, G. (1939) 2. Angetu. Chem. 52, 637. FRIDOVICH, I., AND HANDLER, P. (1962) J. Bid. Chem. 237, 916.
5, 470-473
BIOCHEMISTRY
Anaerobic
(1973)
Spectrophotometry:
E. K. HODGSON, of
Department
AND
Biochemist,.y, Duke Cniversity Durham, North Carolina 27710
Received A cuvette is any gas mixture. which can be Thunberg head the cuvette and can be achieved for at least 12
J. M. McCORD,
An Improved
June
26,
1972;
accepted
August
Cuvettel
I. FRIDOVICH Medical
30,
Center.
1972
described which allows purging of the reaction solution with The cuvette design incorporates a gas inlet and outlet simultaneously sealed by a small angular rotation of the and a gassing tube which is an integral part of the body of which is, therefore, not easily broken. Anaerobic conditions in 10 min and can be maintained, in the sealed cuvette, hr.
Spectrophotometry under controlled atmospheres has generally been achieved by two methods. The first of these depends upon alternate evacuation and refilling of the cuvette with the desired gas (l-3). The slowness with which liquid and gas phases equilibrates places a limitation on the utility of this method. The second procedure, which allows bubbling the gas through the liquid (4,5), was devised to facilitate equilibration between the liquid and the gas phases. The cuvettes, which have been designed to permit sweeping the liquid phase with the gas, have been both cumbersome and fragile. Stopcocks to close the gas inlet and outlet tubes and a thin glass gassing tube to conduct the gas down into the liquid phase, have seemed t#o be indispensible components of such cuvettes. Cuvettes with retractable metal gassing tubes have been constructed (Hellma Cells, Inc.) but these have a packing gland which is prone to gas leakage. Dixon (5) has circumvented these problems by working under a continuous stream of N2 and thoroughly described the problems in achieving and maintaining anaerobic conditions in spectrophotometer cuvettes. If the thin tube which conducts the gas into the liquid phase could be made an integral part, of the body of the cuvette, problems of fragility would be solved and if the gas inlet and gas outlet closures could be incorporated into the stopper of the cuvette, extraneous stopcocks could be eliminated. This report describes a cuvette which achieves these ends. ‘This stitutes Copyright All rights
work was supported of Health, Bethesda,
in full Maryland.
@ 1973 by Academic Press. of reproduction in any form
by
Grant
470 Inc. reserved.
GM-10287
from
the
National
In-
ANAEROBIC
MATERIALS
CUVETTE
AND
471
METHODS
The quartz anaerobic cuvette, described below, was fabricated by Precision Cells, Inc., of Hicksville, New York. Santhine oxidase was prepared from raw cream by a procedure which avoided exposure to proteolytic agents (6). Prepurified N, was obtained from the Matheson Co. and was freed of traces of 0, by passage over a column of hot colloidal copper dispersed on an inert support (7). The oxidation of xanthine to mate was folowed at 295 nm in a Gilford Model 2000 recording spectrophometer. RESULTS
Figure 1 presents a diagram of the cuvette. The stream of gas enters the cuvette through the side arm and is conducted, through a groove in the male member of the ground joint, to the gassing tube; which carries the gas to within 3 mm of the bottom of the cuvette. The gassing tube
Fm. 1. Anaerobic cuvette. The bottom part of this cuvette has the dimensions of an ordinary rectangular cuvette with a l.O-cm light path. The height of the complete assembly was 10.0 cm, the grooves cut in the male and female members of the ground joint are 1.5 mm n-ide and 1.0 mm deep. A top view of the rectangular bottom part of the cuvette is provided to show the position of the gassing tube.
472
HODGSON,
MCCORD,
AND
FRIDOVICH
did not interfere with the light beam of the spectrophotometer and because it was sealed onto the inside of the cuvette, as shown, it was not subject to breakage during assembly and cleaning of the cuvette. After rising through the liquid phase, the gas escapes through a hole in the male member of the joint which connects with a groove in the female member of the ground joint. A slight angular displacement of the hollow stopper simultaneously seals both the gas inlet and the gas outlet. In practice, the ground joint was lightly greased with Sisco 300 to provide a gas-tight seal and to facilitate rotation of the hollow stopper. The practicality of this design was tested with the xanthine oxidase reaction. Reaction mixtures, containing 5 X 1O-5M xanthine, 1 X 1O-4M EDTA, and 0.05M potassium phosphate in a total volume of 3.0 ml at pH 7.8 and at 24°C were placed in the cuvette and 10 ~1 of 9.1 X 1CP M xanthine oxidase was placed in the hollow stopper. The cuvette was then assembled and gassed with N, before being sealed. The reaction was started by tipping and thus rinsing the enzyme from the hollow stopper into the body of the cuvette. A gentle stream of N, was used during the gassing period to avoid frothing and loss of solution. A taller cuvette of otherwise identical design would have allowed more rapid gassing and thus a more rapid elimination of 0,. Figure 2 demonstrates that 10 min of gassing accomplished the complete elimination of O,, as judged by a prevention of the xanthine oxidase reaction. Removal of OZ from the
0
2
4
6 Minutes
8 IO of Purging
12
14
I6
FIG. 2. Elimination of 0, as a function of time of purging. Reaction mixtures containing 5 X lo-‘M xanthine, 1 X lo-‘M EDTA, and 0.05 M potassium phosphate in a total volume of 3.0 ml at pH 7.8 and at 24°C were swept with a gentle stream of N, for variable periods of time. The cuvette was then sealed, 10 ~1 of 9.1 x 10e6M xanthine oxidase was tipped in, and the rate of urate accumulation was followed at 295 nm. The ordinate is in terms of increase in absorbancy at 295 nm/min X 10.
ANAEROBIC
CUVETTE
473
enzyme solution, which was in the hollow stopper, was dependent upon diffusion. It is, therefore, desirable to keep to a minimum the volume of enzyme solution placed in the hollow stopper. The cuvette, once having been gassed with N, and sealed, was able to exclude atmospheric 0, for long periods of time. Thus, the interval of time between sealing and tipping-in could be extended to 12 hr without noticeable entry of oxygen. DISCUSSION
The cuvette described above allows rapid sweeping of oxygen from the reaction solution, effective and facile sealing of the cuvette contents from atmospheric O,, and initiation of the reaction at any time subsequent to sealing. The complete cuvette assembly is small and no more fragile than a standard rectangular quartz cuvette. The cuvette described above does not provide for sequential addition of reagents without breaching the gas-tight seal but this feature could be added in the form of a rubber-sealed injection port. Such an injection port would also allow the enzyme to be added after bubbling with NZ had commenced. This might be of importance in work with oxygen-labile enzymes. The xanthine oxidase reaction, which was used to test the workings of this cuvette, has a K, for 0, of approximately 2.7 X lo-” N (81, under the conditions used. Clearly, an enzyme with a higher affinity for 0, would have provided a more stringent test of oxygen exclusion. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
DIXON, M., AND KENWORTHY, P. (1967) Biochim. Biopkys Acta 146, 54. MASSEY, V., GIBSON, Q. H., AND VEIXER, C. (1960) Biochem. J. 77, 341. VELICK. S. F., AND STRITTMATTER, P. (1956) J. Biol. Chem. 221, 265. LAZAROW, A., AND COOPERSTEIN, S. J. (1954) Science 120, 674. DIXON, M. (1971) Biochim. Biophys. Acta 226, 241. BRADY, F. 0. (1969) Ph.D. thesis, Duke University. MEYER, F. B., AND RONGE, G. (1939) 2. Angetu. Chem. 52, 637. FRIDOVICH, I., AND HANDLER, P. (1962) J. Bid. Chem. 237, 916.