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[151] Bacterial luciferase
[151]
BACTERIAL LUCIFERASE
857
[151] B a c t e r i a l L u c i f e r a s e O D P N H ~- F M N + RC
-{- Os -~ Light
\ H
or
O FMNH2 ~- RC
-[- O, --~ Light
\ H
By ARDA A. GREEN and WILLIA~ D. MCELROY Assay Method
Bacterial luciferase is a flavoprotein which catalyzes the oxidation of reduced D P N by numerous oxidants such as FMN, ferricyanide, quinones, and various dyes. In the presence of F M N and a long-chain fatty aldehyde the oxidation of D P N H is accompanied by light emission. ~Pyridine nucleotides are not essential for light emission, since chemically reduced F M N and aldehyde will support luminescence. The intensity of the light emitted depends on the concentration of these various components. Quantitative measurements can be made by a suitable photocell arrangement such as the Farrand photofluorometer. Qualitative estimations can be made with the naked eye. Procedure. The reaction mixture consists of 0.5 ml. of 0.1 M phosphate buffer, pH 6.8, 1.0 ml. of saturated dodecyl aldehyde, 0.2 ml. of riboflavin phosphate (2 X 10-4 M), 0.05 ml. of 1% bovine albumin, 0.2 ml. of D P N H (7.0 × 10-4 M for maximum luminescence), 0.05 or 0.1 ml. of diluted enzyme, and water to a total of 2.5 ml. The reaction is initiated with D P N H , and the light intensity recorded for various time periods. Definition of Unit. One unit of enzyme is defined as that amount which gives 1 unit of light in arbitrary values under the above stated conditions. Specific activity is expressed as light units per milligram of protein. Protein is determined by the method of Lowry et al3 Growth of the Organism. The salt-water bacterium Achromobacter fischeri was grown under forced aeration or by shaking in the following 1 W. D. McElroy and B. L. Strehler, Bacteriol Revs. 18, 177 (1954). 20. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193~ 265 (1951).
858
RESPIRATORY ENZYMES
[151]
medium: NaC1, 30 g.; Na2HP04, 5.3 g.; KH~P04, 2.1 g.; (NH4)~HPO4, 0.5 g.; MgSO4, 0.1 g.; glycerol, 3 ml.; peptone, 1 g.; and water, 1 1. The pH was adjusted to 7.1 to 7.3 with NaOH. At the peak of luminescence (15 to 20 hours) the cells were harvested by high-speed centrifugation. With good aeration approximately 4 g. (wet weight) of cells per liter is obtained. Purification Procedure
The following procedures are based in part on the original report of McElroy et al2 A crude bacterial extract is obtained by lysing the cells in distilled water (1 g. wet weight per 15 ml. of water). After thoroughly mixing in the cold, the debris and unlysed cells are removed by centrifugation at high speed in a Servall centrifuge. An active isoelectric precipitate is formed by adjusting the pH to 4.0 to 4.3 with HC1. The precipitate is separated by centrifugation in the cold, then suspended in water (one-tenth the volume used for lysing) and dissolved by the addition of N/IO NaOH to pH 6.8 to 7.0. This materially increases the volume and brings the final protein concentration to between 15 and 20 mg./ml. The centrifuged supernatant of the lysed cells contains about one-half of the total protein in the cells, and 85 to 90% of this protein is found in the isoelectric precipitate. Thus, the isoelectric precipitation is a concentration rather than a purification procedure, except for the removal of certain inhibitors, especially riboflavin. These inhibitors make it impossible to properly assay the initial extract. Ammonium sulfate precipitation has proved to be the only practical method of purification. To the solution of the isoelectric precipitate, fraction A, is added 0.1 vol. of Na4P~07 (0.2 M adjusted to pH 7 with HC1). Solid ammonium sulfate is then added with stirring, and the final pH is adjusted to 6.8. All pH values are read on the Beckman glass electrode pH meter without correction for salt concentration. Successive precipitates are separated by centrifugation in the high-speed Servall centrifuge, dissolved in 0.02 M pyrophosphate, and the pH readjusted to 6.8. The initial fractionation procedure yields four fractions. Fraction B. The precipitate obtained at 1.35 M ammonium sulfate (0.33 saturation) is voluminous and contains about 70% of the total protein. It is washed once with 1/~ vol. of 1.35 M ammonium sulfate and the precipitate discarded, since it contains a negligible amount of activity. Fraction C. The combined supernatants from the previous step are brought to 2.05 M ammonium sulfate (0.50 saturation). The precipitate contains about 5 % of the protein and 5 % of the activity. 3 W. D. McElroy, J. W. Hastings, V. Sonnenfeld, and J. Coulombre, J. Bacteriol. 67, 402 (1954).
[151]
BACTERIAL LUCIFERASE
859
Fraction D. The supernatant from fraction C is brought to 2.67 M ammonium sulfate (0.65 saturation). This fraction contains 15 to 20% of the total protein and 90 % of the activity. In fact, it may assay substantially higher total activity than fraction A, owing to assay in a more concentrated solution and to the removal of inhibitors. Fraction E. A fraction at 2.87 M ammonium sulfate contains so little protein and so little activity that it is not worth taking. Fraction D thus contains essentially all the luciferase with a specific activity which may vary from 1200 to 3000 light units per milligram of protein but is about five times the specific activity of fraction A. The variation is due largely to the particular strain of bacterium used. Besides protein impurity this preparation contains large amounts of nucleic acid. Attempts to remove this by protamine sulfate or manganese ion were unsuccessful. The fractionation was followed by optical density measurements at 280 and 260 mu. It was found that the nucleic acid tended to precipitate in the higher ammonium sulfate fractions. Thus, after repeated fractionation the worst fractions are almost pure nucleic acid and the best have a "280 to 260 ratio" of about 1.2. Fraction D is repeatedly refractionated in ammonium sulfate. The point of firstprecipitation depends on the concentration of the proteins and the character of the protein impurities but usually occurs around 2 M ammonium sulfate. Small increments of the solid salt are added so that the protein is divided into convenient amounts. All fractions are analyzed for protein, enzymatic activity, and ultraviolet absorption. Similar fractions are combined and refractionated. The table presents the results from a single run including the isoelectric precipitate, A, the first active ammonium sulFRACTIONATION OF LUCIFERASE BY AMMONIUM SULFATE
Fraction A D Dl D~ D3 D4 D5 86
Ml.
Total L.U. ~
186 26 2.5 5 9 8 3
986,000 1,639,000 54,000 210,000 864,000 384,000 3,000
Total protein, E280 b E ..... mg. L . U . / m g . protein ~ E . . . . /1000 L. U. mtL 2880 670 58 101 230 182 28
341 2420 925 2080 3760 2100 107 5200
0.705 0.712 0.89 0.78 0.79 0.74 0.57 1.24
9.8 1.41 1.7 1.04 0.62 1.75 11.8 0.29
260 260 265 265 263 260 260 275
L. U. = light units as defined in text. b E = optical density determined in a Beckman D U spectrophotometer in a 1-cm. cell.
860
RESPIRATORY ENZYMES
[151]
fate precipitate, D, and a series, D1 to Ds, derived from D. If the per cent protein and activity of the subfractions of D are calculated with the values for D taken as 100 %, 90 % of the protein and 93 % of the activity are recovered. Further fractionation may be accompanied by loss of activity. We have tried stabilizing the enzymes by the addition of cysteine, FMN, or the aldehyde without marked success. In fact, cysteine and the aldehyde, in the presence of the enzyme, form an inhibitor. In the last row of the table are given the characteristics of one of the best fractions we have obtained. About 70 % of this protein is in a symmetrical peak on electrophoresis, and activity seems to be correlated with this peak. The sedimentation constant is consistent with a molecular weight of about 100,000. Properties
Stability. Bacterial luciferase slowly loses activity even in the frozen state. It is rapidly inactivated at temperatures above 40 °. The enzyme can be dialyzed in the cold without great loss of activity with 0.02 M pyrophosphate buffer and 2 X 10-5 M FMN. Various attempts to demonstrate a metal requirement by dialysis against metal-free buffers have been unsuccessful. Effect of Inhibitors. The enzyme is particularly sensitive to various SH reagents, p-Chloromercuribenzoic acid at 4 X 10-e M inhibits light emission approximately 50%. This inhibition can be reversed by glutathione. Riboflavin is a potent inhibitor of luminescence (2 X 10 -e M gives approximately 50 % inhibition). It appears to compete with FMN. Cyanide and Versene likewise inhibit, as does copper, iron, and other heavy metals. Various quinones and ferricyanide inhibit light emission by removing reduced DPN. As indicated below, this inhibition is due to the rapid reduction of these compounds by D P N H in the presence of bacterial luciferase. Reduction of Dyes and FMN. In the absence of aldehyde bacterial luciferase catalyzes the reduction of methylene blue, various quinones, ferricyanide, and F M N by D P N H without light emission. In the presence of aldehyde there is a competition between light emission and the reduction of the various compounds." F M N does not appear to be required for the reduction of quinones and ferricyanide. 4 Requirements for Light Emission. Bacterial luciferase catalyzes a lightemitting reaction in the presence of oxygen, reduced FMN, and a longchain aldehyde. There is an absolute requirement for all these compo4 W. D. McElroy and A. A. Green, Arch. Biochem. and Biophys. §6t 240 (1955).
[152]
ASSAY AND PROPERTIES OF HYDROGENASES
861
nents, and all are utilized during the reaction. Cormier and Strehler 5 have shown that a number of long-chain aldehydes will function in the reaction. Dodecyl or tetradecyl aldehyde are excellent substrates. With F M N various reducing agents will support light emission. Reduced safranin T, indigotrisulfonate, and rosindulin GG are all effective. Strehler et al. e reported that reduced riboflavin would support luminescence in crude extracts. Our studies with the purified enzyme indicate, however, that F M N is an absolute requirement. Reduced D P N and T P N are both effective reducing agents and appear to be the natural substrates for light emission. Chemically reduced F M N is the most effective substrate for light emission and the evidence shows quite clearly that pyridine nucleotides are not required for luminescence. Kinetics. The kinetics of light emission starting with reduced F M N indicate that two F1VINH~ molecules combine with luciferase. It seems likely that a complex interaction between oxygen, two reduced F M N molecules, and aldehyde is necessary for luminescence. The concentration of reduced F M N which gives approximately one-half maximum light intensity is 2.5 X 10-8 M. The relationship between light intensity and enzyme concentration is proportional when reduced F M N is used but is nonlinear when D P N H and F M N is used. This latter effect appears to be due to the autoxidation of reduced F M N which is formed from D P N H and FMN. pH and Temperature Optimum. Light emission has been observed over a pH range of 5.5 to 8.5, with a peak at 6.8. At the latter pH the temperature optimum is approximately 27 °, which is remarkably similar to that observed in the intact bacterium. 5 M. J. Cormier and B. L. Strehler, J. Am. Chem. Soc. 75, 4864 (1953). 6 B. L. Strehler, E. N. Harvey, J. J. Cheng, and M. C. Cormier, Proc. Natl. Acad. Sci.
U.S. 40, 10 (1954).
[152] Assay and Properties of Hydrogenases By ANTHONY SAN PIETRO
Hydrogenase activity may be defined as the enzymatic activation of molecular hydrogen. The main methods which have been employed to assay hydrogenase activity, in cell-free preparations, are as follows: (a) The reduction of some substrate by molecular hydrogen. Various acceptors which have been used in this assay system include methylene
BACTERIAL LUCIFERASE
857
[151] B a c t e r i a l L u c i f e r a s e O D P N H ~- F M N + RC
-{- Os -~ Light
\ H
or
O FMNH2 ~- RC
-[- O, --~ Light
\ H
By ARDA A. GREEN and WILLIA~ D. MCELROY Assay Method
Bacterial luciferase is a flavoprotein which catalyzes the oxidation of reduced D P N by numerous oxidants such as FMN, ferricyanide, quinones, and various dyes. In the presence of F M N and a long-chain fatty aldehyde the oxidation of D P N H is accompanied by light emission. ~Pyridine nucleotides are not essential for light emission, since chemically reduced F M N and aldehyde will support luminescence. The intensity of the light emitted depends on the concentration of these various components. Quantitative measurements can be made by a suitable photocell arrangement such as the Farrand photofluorometer. Qualitative estimations can be made with the naked eye. Procedure. The reaction mixture consists of 0.5 ml. of 0.1 M phosphate buffer, pH 6.8, 1.0 ml. of saturated dodecyl aldehyde, 0.2 ml. of riboflavin phosphate (2 X 10-4 M), 0.05 ml. of 1% bovine albumin, 0.2 ml. of D P N H (7.0 × 10-4 M for maximum luminescence), 0.05 or 0.1 ml. of diluted enzyme, and water to a total of 2.5 ml. The reaction is initiated with D P N H , and the light intensity recorded for various time periods. Definition of Unit. One unit of enzyme is defined as that amount which gives 1 unit of light in arbitrary values under the above stated conditions. Specific activity is expressed as light units per milligram of protein. Protein is determined by the method of Lowry et al3 Growth of the Organism. The salt-water bacterium Achromobacter fischeri was grown under forced aeration or by shaking in the following 1 W. D. McElroy and B. L. Strehler, Bacteriol Revs. 18, 177 (1954). 20. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem. 193~ 265 (1951).
858
RESPIRATORY ENZYMES
[151]
medium: NaC1, 30 g.; Na2HP04, 5.3 g.; KH~P04, 2.1 g.; (NH4)~HPO4, 0.5 g.; MgSO4, 0.1 g.; glycerol, 3 ml.; peptone, 1 g.; and water, 1 1. The pH was adjusted to 7.1 to 7.3 with NaOH. At the peak of luminescence (15 to 20 hours) the cells were harvested by high-speed centrifugation. With good aeration approximately 4 g. (wet weight) of cells per liter is obtained. Purification Procedure
The following procedures are based in part on the original report of McElroy et al2 A crude bacterial extract is obtained by lysing the cells in distilled water (1 g. wet weight per 15 ml. of water). After thoroughly mixing in the cold, the debris and unlysed cells are removed by centrifugation at high speed in a Servall centrifuge. An active isoelectric precipitate is formed by adjusting the pH to 4.0 to 4.3 with HC1. The precipitate is separated by centrifugation in the cold, then suspended in water (one-tenth the volume used for lysing) and dissolved by the addition of N/IO NaOH to pH 6.8 to 7.0. This materially increases the volume and brings the final protein concentration to between 15 and 20 mg./ml. The centrifuged supernatant of the lysed cells contains about one-half of the total protein in the cells, and 85 to 90% of this protein is found in the isoelectric precipitate. Thus, the isoelectric precipitation is a concentration rather than a purification procedure, except for the removal of certain inhibitors, especially riboflavin. These inhibitors make it impossible to properly assay the initial extract. Ammonium sulfate precipitation has proved to be the only practical method of purification. To the solution of the isoelectric precipitate, fraction A, is added 0.1 vol. of Na4P~07 (0.2 M adjusted to pH 7 with HC1). Solid ammonium sulfate is then added with stirring, and the final pH is adjusted to 6.8. All pH values are read on the Beckman glass electrode pH meter without correction for salt concentration. Successive precipitates are separated by centrifugation in the high-speed Servall centrifuge, dissolved in 0.02 M pyrophosphate, and the pH readjusted to 6.8. The initial fractionation procedure yields four fractions. Fraction B. The precipitate obtained at 1.35 M ammonium sulfate (0.33 saturation) is voluminous and contains about 70% of the total protein. It is washed once with 1/~ vol. of 1.35 M ammonium sulfate and the precipitate discarded, since it contains a negligible amount of activity. Fraction C. The combined supernatants from the previous step are brought to 2.05 M ammonium sulfate (0.50 saturation). The precipitate contains about 5 % of the protein and 5 % of the activity. 3 W. D. McElroy, J. W. Hastings, V. Sonnenfeld, and J. Coulombre, J. Bacteriol. 67, 402 (1954).
[151]
BACTERIAL LUCIFERASE
859
Fraction D. The supernatant from fraction C is brought to 2.67 M ammonium sulfate (0.65 saturation). This fraction contains 15 to 20% of the total protein and 90 % of the activity. In fact, it may assay substantially higher total activity than fraction A, owing to assay in a more concentrated solution and to the removal of inhibitors. Fraction E. A fraction at 2.87 M ammonium sulfate contains so little protein and so little activity that it is not worth taking. Fraction D thus contains essentially all the luciferase with a specific activity which may vary from 1200 to 3000 light units per milligram of protein but is about five times the specific activity of fraction A. The variation is due largely to the particular strain of bacterium used. Besides protein impurity this preparation contains large amounts of nucleic acid. Attempts to remove this by protamine sulfate or manganese ion were unsuccessful. The fractionation was followed by optical density measurements at 280 and 260 mu. It was found that the nucleic acid tended to precipitate in the higher ammonium sulfate fractions. Thus, after repeated fractionation the worst fractions are almost pure nucleic acid and the best have a "280 to 260 ratio" of about 1.2. Fraction D is repeatedly refractionated in ammonium sulfate. The point of firstprecipitation depends on the concentration of the proteins and the character of the protein impurities but usually occurs around 2 M ammonium sulfate. Small increments of the solid salt are added so that the protein is divided into convenient amounts. All fractions are analyzed for protein, enzymatic activity, and ultraviolet absorption. Similar fractions are combined and refractionated. The table presents the results from a single run including the isoelectric precipitate, A, the first active ammonium sulFRACTIONATION OF LUCIFERASE BY AMMONIUM SULFATE
Fraction A D Dl D~ D3 D4 D5 86
Ml.
Total L.U. ~
186 26 2.5 5 9 8 3
986,000 1,639,000 54,000 210,000 864,000 384,000 3,000
Total protein, E280 b E ..... mg. L . U . / m g . protein ~ E . . . . /1000 L. U. mtL 2880 670 58 101 230 182 28
341 2420 925 2080 3760 2100 107 5200
0.705 0.712 0.89 0.78 0.79 0.74 0.57 1.24
9.8 1.41 1.7 1.04 0.62 1.75 11.8 0.29
260 260 265 265 263 260 260 275
L. U. = light units as defined in text. b E = optical density determined in a Beckman D U spectrophotometer in a 1-cm. cell.
860
RESPIRATORY ENZYMES
[151]
fate precipitate, D, and a series, D1 to Ds, derived from D. If the per cent protein and activity of the subfractions of D are calculated with the values for D taken as 100 %, 90 % of the protein and 93 % of the activity are recovered. Further fractionation may be accompanied by loss of activity. We have tried stabilizing the enzymes by the addition of cysteine, FMN, or the aldehyde without marked success. In fact, cysteine and the aldehyde, in the presence of the enzyme, form an inhibitor. In the last row of the table are given the characteristics of one of the best fractions we have obtained. About 70 % of this protein is in a symmetrical peak on electrophoresis, and activity seems to be correlated with this peak. The sedimentation constant is consistent with a molecular weight of about 100,000. Properties
Stability. Bacterial luciferase slowly loses activity even in the frozen state. It is rapidly inactivated at temperatures above 40 °. The enzyme can be dialyzed in the cold without great loss of activity with 0.02 M pyrophosphate buffer and 2 X 10-5 M FMN. Various attempts to demonstrate a metal requirement by dialysis against metal-free buffers have been unsuccessful. Effect of Inhibitors. The enzyme is particularly sensitive to various SH reagents, p-Chloromercuribenzoic acid at 4 X 10-e M inhibits light emission approximately 50%. This inhibition can be reversed by glutathione. Riboflavin is a potent inhibitor of luminescence (2 X 10 -e M gives approximately 50 % inhibition). It appears to compete with FMN. Cyanide and Versene likewise inhibit, as does copper, iron, and other heavy metals. Various quinones and ferricyanide inhibit light emission by removing reduced DPN. As indicated below, this inhibition is due to the rapid reduction of these compounds by D P N H in the presence of bacterial luciferase. Reduction of Dyes and FMN. In the absence of aldehyde bacterial luciferase catalyzes the reduction of methylene blue, various quinones, ferricyanide, and F M N by D P N H without light emission. In the presence of aldehyde there is a competition between light emission and the reduction of the various compounds." F M N does not appear to be required for the reduction of quinones and ferricyanide. 4 Requirements for Light Emission. Bacterial luciferase catalyzes a lightemitting reaction in the presence of oxygen, reduced FMN, and a longchain aldehyde. There is an absolute requirement for all these compo4 W. D. McElroy and A. A. Green, Arch. Biochem. and Biophys. §6t 240 (1955).
[152]
ASSAY AND PROPERTIES OF HYDROGENASES
861
nents, and all are utilized during the reaction. Cormier and Strehler 5 have shown that a number of long-chain aldehydes will function in the reaction. Dodecyl or tetradecyl aldehyde are excellent substrates. With F M N various reducing agents will support light emission. Reduced safranin T, indigotrisulfonate, and rosindulin GG are all effective. Strehler et al. e reported that reduced riboflavin would support luminescence in crude extracts. Our studies with the purified enzyme indicate, however, that F M N is an absolute requirement. Reduced D P N and T P N are both effective reducing agents and appear to be the natural substrates for light emission. Chemically reduced F M N is the most effective substrate for light emission and the evidence shows quite clearly that pyridine nucleotides are not required for luminescence. Kinetics. The kinetics of light emission starting with reduced F M N indicate that two F1VINH~ molecules combine with luciferase. It seems likely that a complex interaction between oxygen, two reduced F M N molecules, and aldehyde is necessary for luminescence. The concentration of reduced F M N which gives approximately one-half maximum light intensity is 2.5 X 10-8 M. The relationship between light intensity and enzyme concentration is proportional when reduced F M N is used but is nonlinear when D P N H and F M N is used. This latter effect appears to be due to the autoxidation of reduced F M N which is formed from D P N H and FMN. pH and Temperature Optimum. Light emission has been observed over a pH range of 5.5 to 8.5, with a peak at 6.8. At the latter pH the temperature optimum is approximately 27 °, which is remarkably similar to that observed in the intact bacterium. 5 M. J. Cormier and B. L. Strehler, J. Am. Chem. Soc. 75, 4864 (1953). 6 B. L. Strehler, E. N. Harvey, J. J. Cheng, and M. C. Cormier, Proc. Natl. Acad. Sci.
U.S. 40, 10 (1954).
[152] Assay and Properties of Hydrogenases By ANTHONY SAN PIETRO
Hydrogenase activity may be defined as the enzymatic activation of molecular hydrogen. The main methods which have been employed to assay hydrogenase activity, in cell-free preparations, are as follows: (a) The reduction of some substrate by molecular hydrogen. Various acceptors which have been used in this assay system include methylene