BIOTECHNOLOGY AND BIOENGINEERING
VOL. XVIII (1976)
Immobilization of Nitrate Reductase within Polyacrylamide Gels INTRODUCTION Although there are a host of organochemical methods suited to the analysis of nitrate ion, each method is limited to some extent in regard to its specificity or detection limits. The present work in our laboratories is directed toward the use of immobilized enzymes as agents that will very specifically detect their substrates and also allow quantitation of the substrate and/or its reaction product. To this end, nitrate reductase from Escherichia coli (E.C. 1.9.6.1) has been immobilized on controlled pore glass by Senn and Carr' for the purpose of quantitating trace amounts of nitrate ion in aqueous samples. The results of our studies show that the enzyme may also be entrapped in active form within the matrix of polyacrylamide gels. MATERIALS AND METHODS Nitrate reductase from E . coli (E.C. 1.9.6.1) was purchased as a lyophilized powder from the Worthington Biochemical Corporation, Freehold, New Jersey. The enzyme was assayed either in the free or immobilized form as described by Lowe and Evans2using methyl viologen in place of benzyl viologen as the electron donor. The enzyme was entrapped in acrylamide gels3 that were cast around 1.5 X 1.5 cm pieces of basket woven platinum screen. Each enzyme grid contained 0.035 enzyme units entrapped in 230-260 mg of gel. The enzyme grids were assayed by suspending them in 10 ml of incubation medium a t 23°C from which aliquots were withdrawn for assays at given intervals. The enzyme grids were rotated every 5 min to ensure adequate mixing in the reaction vessel. The enzyme grids were stored in O.l;iM potassium phosphate buffer, pH 7.0 between incubations. RESULTS AND DISCUSSION Nitrate reductase was immobilized in polyacrylamide gels cast around thin pieces of platinum gauze. The enzyme grids were stored in phosphate buffer between assays. In this study, the effect of storage on the immobilized enzyme grids a t 6 and 23°C was evaluated. Also, the reusability of the enzyme grid was determined. This was done by first assaying the enzyme grid after the second day of storage followed by a second assay on the same grid after nine days of storage. Figures 1 and 2 present our experimental results. These results indicate that the immobilized enzyme is more stable a t 6°C than a t 23°C. Also, the assay procedure itself probably leads to some enzyme inactivation. Assays performed on gel-coated platinum grids in the absence of enzyme showed little or no background when assayed as described. It was observed that the immobilized enzyme lost twice as much activity over a nine day period as did the 1643
@ 1976 by John Wiley & Sons, Inc.
1644 BIOTECHNOLOGY AND BIOENGINEERING VOL. XVIII (1976)
Fig. 1. The effect of storage a t 6°C on the activity of immobilized nitrate assay performed reductme. ( A ) Assay performed after 2 days of storage, (0) result of second assay performed after 9 days of after 9 days of storage, (0) storage. First assay performed after second day of storage ( A ) .
0
0
60 Incubation Time (min.)
Fig. 2. The effect of storage a t 23°C on the activity of immobilized nitrate assay performed reductase. ( A ) Assay performed after 2 days of storage, (0) result of second assay performed after 9 days of after 9 days of storage, (0) storage. First assay performed after second day of storage ( A ) .
COMMUNICATIONS TO T H E EDITOR
1645
free enzyme in solution under otherwise comparable conditions. It is unlikely that there was any significant leakage of the nitrate reductase, molecular weight 1 x 108 daltons,' from the gel. This rationale is based upon the observation that a much smaller enzyme, glucose oxidase, with molecular weight 1.5 X lo6 daltons,5 when immobilized in the same manner showed no loss of activity over a 20 day period of time.6 Based upon the results obtained, we conclude that immobilized nitrate reductase may be used to detect nitrate ions in a system similar to the one described by Senn and Carr.1 However, the prohibitively high cost of nitrate reductase suggests that direct preparation of the enzyme from cells of E . coli is mandatory in order to make this application economically acceptable. The authors are indebted to Ms. S. Wozniak and Mr. L. Vassilaros for their technical assistance during the course of this work. This study was supported by Lewis Research Center, NASA, under the research grant NSG-3002. The discussion and comments of Dr. J. S. Fordyce of Lewis Research Center are gratefully appreciated.
References 1. I). It. Senn and P. W. Carr, Anal. Chem., 48, 954 (1976) 2. 13. H.Lowe and H. J. Evans, Biochem. Biophys. Acta, 85, 377 (1964). 3. J . G. Schiller and C. C. Liu, Biotechnol Bioeng., 18, 1405 (1976). 4. S. Taniguchi and E. Stagaki, Biochem. Biophys. Acta, 44. 263 (1960). 3. J . H. Pazur, K. Kleppe, and A. Cepure, rlrch. Biochem. Bilophys., 111, 351 (1965). 6 . J. G.Schiller and C. C. Liu, unpublished observation.
JULIANG. SCHILLER C. C. LIU* Department of Chemical and Petroleum Engineering University of Pittsburgh Pittsburgh, Pennsylvania 1.5261 Accepted for Publication July 2, 1976
* To whom correspondence should be addressed.
VOL. XVIII (1976)
Immobilization of Nitrate Reductase within Polyacrylamide Gels INTRODUCTION Although there are a host of organochemical methods suited to the analysis of nitrate ion, each method is limited to some extent in regard to its specificity or detection limits. The present work in our laboratories is directed toward the use of immobilized enzymes as agents that will very specifically detect their substrates and also allow quantitation of the substrate and/or its reaction product. To this end, nitrate reductase from Escherichia coli (E.C. 1.9.6.1) has been immobilized on controlled pore glass by Senn and Carr' for the purpose of quantitating trace amounts of nitrate ion in aqueous samples. The results of our studies show that the enzyme may also be entrapped in active form within the matrix of polyacrylamide gels. MATERIALS AND METHODS Nitrate reductase from E . coli (E.C. 1.9.6.1) was purchased as a lyophilized powder from the Worthington Biochemical Corporation, Freehold, New Jersey. The enzyme was assayed either in the free or immobilized form as described by Lowe and Evans2using methyl viologen in place of benzyl viologen as the electron donor. The enzyme was entrapped in acrylamide gels3 that were cast around 1.5 X 1.5 cm pieces of basket woven platinum screen. Each enzyme grid contained 0.035 enzyme units entrapped in 230-260 mg of gel. The enzyme grids were assayed by suspending them in 10 ml of incubation medium a t 23°C from which aliquots were withdrawn for assays at given intervals. The enzyme grids were rotated every 5 min to ensure adequate mixing in the reaction vessel. The enzyme grids were stored in O.l;iM potassium phosphate buffer, pH 7.0 between incubations. RESULTS AND DISCUSSION Nitrate reductase was immobilized in polyacrylamide gels cast around thin pieces of platinum gauze. The enzyme grids were stored in phosphate buffer between assays. In this study, the effect of storage on the immobilized enzyme grids a t 6 and 23°C was evaluated. Also, the reusability of the enzyme grid was determined. This was done by first assaying the enzyme grid after the second day of storage followed by a second assay on the same grid after nine days of storage. Figures 1 and 2 present our experimental results. These results indicate that the immobilized enzyme is more stable a t 6°C than a t 23°C. Also, the assay procedure itself probably leads to some enzyme inactivation. Assays performed on gel-coated platinum grids in the absence of enzyme showed little or no background when assayed as described. It was observed that the immobilized enzyme lost twice as much activity over a nine day period as did the 1643
@ 1976 by John Wiley & Sons, Inc.
1644 BIOTECHNOLOGY AND BIOENGINEERING VOL. XVIII (1976)
Fig. 1. The effect of storage a t 6°C on the activity of immobilized nitrate assay performed reductme. ( A ) Assay performed after 2 days of storage, (0) result of second assay performed after 9 days of after 9 days of storage, (0) storage. First assay performed after second day of storage ( A ) .
0
0
60 Incubation Time (min.)
Fig. 2. The effect of storage a t 23°C on the activity of immobilized nitrate assay performed reductase. ( A ) Assay performed after 2 days of storage, (0) result of second assay performed after 9 days of after 9 days of storage, (0) storage. First assay performed after second day of storage ( A ) .
COMMUNICATIONS TO T H E EDITOR
1645
free enzyme in solution under otherwise comparable conditions. It is unlikely that there was any significant leakage of the nitrate reductase, molecular weight 1 x 108 daltons,' from the gel. This rationale is based upon the observation that a much smaller enzyme, glucose oxidase, with molecular weight 1.5 X lo6 daltons,5 when immobilized in the same manner showed no loss of activity over a 20 day period of time.6 Based upon the results obtained, we conclude that immobilized nitrate reductase may be used to detect nitrate ions in a system similar to the one described by Senn and Carr.1 However, the prohibitively high cost of nitrate reductase suggests that direct preparation of the enzyme from cells of E . coli is mandatory in order to make this application economically acceptable. The authors are indebted to Ms. S. Wozniak and Mr. L. Vassilaros for their technical assistance during the course of this work. This study was supported by Lewis Research Center, NASA, under the research grant NSG-3002. The discussion and comments of Dr. J. S. Fordyce of Lewis Research Center are gratefully appreciated.
References 1. I). It. Senn and P. W. Carr, Anal. Chem., 48, 954 (1976) 2. 13. H.Lowe and H. J. Evans, Biochem. Biophys. Acta, 85, 377 (1964). 3. J . G. Schiller and C. C. Liu, Biotechnol Bioeng., 18, 1405 (1976). 4. S. Taniguchi and E. Stagaki, Biochem. Biophys. Acta, 44. 263 (1960). 3. J . H. Pazur, K. Kleppe, and A. Cepure, rlrch. Biochem. Bilophys., 111, 351 (1965). 6 . J. G.Schiller and C. C. Liu, unpublished observation.
JULIANG. SCHILLER C. C. LIU* Department of Chemical and Petroleum Engineering University of Pittsburgh Pittsburgh, Pennsylvania 1.5261 Accepted for Publication July 2, 1976
* To whom correspondence should be addressed.
