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Radial diffusion assay of lipoamide dehydrogenase and its use to assess riboflavin deficiency
ANALYTICAL
Radial
BIOCHEMISTRY
89, 103- 109 (1978)
Diffusion Assay of Lipoamide Its Use to Assess Riboflavin
Dehydrogenase Deficiency1,2
and
JASON C. H. SHIH Department
of Poultry Raleigh.
Science, North
North Carolina State Carolina 27650
University.
Received December 14, 1977 A new, simple, and quantitative method was developed to determine lipoamide dehydrogenase (E.C. 1.6.4.3, NADH:lipoamide oxidoreductase). The principle of this technique is to allow the enzyme or tissue extracts to diffuse in an agarose gel containing lipoate. The enzyme, after 24 hr of diffusion and 2 hr of reaction with NADH, can be determined by the size of a dark or fluorescence-quenching zone in the gel when illuminated with uv light. The diameter of the quenching zone which indicates the enzymatic oxidation of NADH is linearly proportional to the logarithm of enzyme concentration. Since lipoamide dehydrogenase is a FAD-enzyme, the activity was decreased in liver homogenates of riboflavin-deficient chicks, as measured by the new method. This demonstrated the potential importance of this new technique in nutritional and clinical applications.
The activities of several kinds of hydrolytic enzymes can be followed and quantitated visually by the radial diffusion of an enzyme in an agar gel where appropriate substrate has been incorporated. Protease was determined by including heat-denatured fibrinogen or casein in the gel (1). Trypsin and anti-trypsin activities were measured by the solubilization of calcium caseinate in the agar plates (2). Amylase was determined in agarcontaining starch or dextran blue-conjugated starch (3,4), and lipase was assayed in agar including emulsified triglycerides (5).3 A plate assay for determination of cellulase production by a fungus was devised by including acid-swollen cellulose and Phosfon D in the agar (6). The advantages of the enzyme diffusion technique are the simplicity of the procedure, the nonrequirement for sophisticated instruments, and, especially, the application of it to the screening tests for a large number of samples. In this report, the radial diffusion technique is extended to assay an ’ Paper No. 5463 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina. Z The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Experiment Station of the product named nor criticism of similar ones not mentioned. 3 Sandholm, M., and Scott, M. L. (1977) Submitted for publication. 103
0003-2697/78/0891-0103$02.00/0 Copyright Q 1978 by Academic Ress. Inc. All rights of reproduction in any form reserved.
104
JASON C. H. SHIH
oxidoreductase. Based on the oxidation of NADH with the simultaneous loss of its fluorescence in the presence of the substrate and the enzyme, a diffusion zone can be measured and an enzyme level can be estimated. EXPERIMENTAL
Lipoate agar plates. One gram of low EEO agarose (Sigma Chemical) was dissolved in 95 ml of 0.05 M potassium phosphate buffer, pH 6.6, with 1 mM EDTA, in a boiling water bath. Simultaneously, 100 mg (0.49 mmol) of dl-lipoic acid (Sigma) was dissolved in 1 ml of 1 M NaHCO, and 4 ml of HzO. The lipoate solution was then mixed thoroughly into the agarose solution at 50 to 60°C. The mixture was poured rapidly onto a clean glass plate (20 x 20 cm) and spread evenly. After the agar solidified, small wells were punched with a glass tubing (7.0 mm od). The small well could then be filled with 50 ~1 of an enzyme source to be assayed. The preparation of the lipoate agar plate was conducted in a semidark room to minimize photolysis and polymerization of lipoate (7). For a smaller number of enzyme samples, the agar plate can be made smaller in a plastic petri dish with the reagents reduced proportionately. Enzyme assay. Fifty microliters of an enzyme preparation, either a solution of standard purified enzyme or a tissue homogenate, were pipetted into the well in the agar gel. The plate was kept in a dark moist chamber at room temperature (20°C) for 24 hr to allow the enzyme to diffuse radially. At the end of the diffusion period, the wells in the plate were filled with a molten 1% agarose solution (50 ~1 in each well). When the filling agar solidified, a solution of 35 mg (0.05 mmol) of NADH (Sigma) in 2.5 ml of 0.2 M potassium phosphate buffer, pH 8.0, was evenly spread over the plate with a glass rod. The agar plate, spread with NADH and incubated for additional 2 hr in the moist chamber, was placed under a long-wavelength uv light (Ultra-Violet Products, Black-ray lamp, Model Xx-15) in a dark room. The gel exhibited a yellowish green fluorescence owing to the presence of reduced nicotinamide adenine dinucleotide (NADH), but around the wells where enzyme was added there were dark or quenched circles. The diameters of the circular zone were measured with a caliper. The results could be photographed for a permanent record. Enzyme preparations. Purified yeast lipoamide dehydrogenase (Sigma) was used as the standard enzyme. A series of dilutions of enzyme in 0.05 M potassium phosphate buffer, pH 6.6, with 1 mM EDTA, was made and pipetted into the wells on a lipoate agar plate for the activity assay. The standard curve was established by plotting the diameters of the quenched zones against the enzyme concentration on a logarithmic scale. Tissue homogenates were prepared from the freshly excised liver from 2-week-old chicks. One gram of liver tissue was ground with 4 ml of icecold 0.05 M potassium phosphate buffer, pH 6.6, with 1 mM EDTA, for
DIFFUSION
ASSAY OF LIPOAMIDE
DEHYDROGENASE
105
FIG. 1. Time course of the radial diffusion assay of lipoamide dehydrogenase. Left: various diffusion times at a fixed reaction time; right: various reaction times at a fixed diffusion time.
1 min with a Teflon tissue homogenizer at 0 to 5°C. Drops of Triton X-100 were added to the homogenate to give a concentration of 0.5% of this detergent. Detergent is necessary to rupture the mitochondrial membrane and to release lipoamide dehydrogenase. Before the assay, the tissue homogenate went through one cycle of freezing (- 15C) and thawing. After thawing, a series of four twofold dilutions of the homogenate was made, and aliquots of the dilutions were added to the agar plate for the assay. Riboflavin dejiciency . Triplicate lots of 10 day-old male chicks were fed with either control or riboflavin-deficient diet. The diets were based on isolated soy protein and glucose, as used by Gries and Scott (8) to produce riboflavin deficiency. All chicks were caged in electrically heated, thermostatically controlled, wire-floor brooders. Water and feed were available ad libitum. At 2 weeks of age, when 40% of the experimental group died from riboflavin deficiency, the remaining chicks were killed to prepare the liver homogenates for the enzyme assay. RESULTS
Levels of substrate and coenzyme. To determine the appropriate levels of lipoate and NADH to be used in the assay, agar plates (5-cm diameter petri dishes) containing various levels oflipoate from 0, 1.25,2.5, to 5.0 mM in the gels were prepared. After 50 ~1 of purified yeast lipoamide dehydrogenase (0.2 mg/ml) was added into each well in each plate and diffusion was carried out, NADH at various levels from 0,0.5, 1.0, to 2.0 mM was applied to each dish. The enzymatic reaction, as indicated by a zone of quenched fluorescence, was observed under the uv lamp. The most distinct and easily read zones were observed in the plates containing 5.0 mM lipoate and 0.5 or 1.0 mM NADH. Hence, 5.0 mM lipoate and 0.5 mM NADH were selected for the standard procedure used in later experiments. Diffusion time and reaction time. Various diffusion and reaction times were tested with four levels of yeast lipoamide dehydrogenase. The results are shown in Fig. 1. Although larger zones were produced with
106
JASON C. H. SHIH
FIG. 2. The radial diffusion zones of a series of twofold dilutions of yeast lipoamide dehydrogenase (original concentration: 0.2 mg/ml).
longer diffusion and longer reaction times, they were less distinct against the fluorescent background. Twenty-four hours of diffusion and 2 hr of reaction were selected for the standard procedure. Standard curves. Purified yeast lipoamide dehydrogenase was used to establish the relationship between the enzyme concentrations and the sizes of the fluorescence-quenching zones (Fig. 2). A semilogarithmic-linear relationship is demonstrated in Fig. 3. A similar linear relationship was obtained with various levels of chick liver homogenate (Fig. 4). In this case, Triton X-100 permitted the detection of a higher level of enzyme. The enzyme activity of the preparations was also measured by the conventional spectrophotometric method (9,lO) to ensure that the activities were proportional to the concentrations. Substrate and coenzyme specificities. To test the substrate specificity, lipoate, lipoamide, oxidized glutathione, cystine, and pantothine (all from Sigma) were used to prepare separate agar plates. Either NADH or NADPH were spread on each plate to test the specificity for the coenzyme. From the experiments with the purified enzyme, it is evident that that the enzyme reaction is specific for lipoate or lipoamide and NADH. From the experiments with the liver homogenate, different enzymatic reactions, namely,
FIG. 3. A semilogarithmic relationship between the diameters of the diffusion zones and the concentrations of yeast lipoamide dehydrogenase. Vertical bars on data points are SEM.
DIFFUSION
ASSAY
FIG. 4. Effect of Triton X-100 level of lipoamide dehydrogenase
OF
LIPOAMIDE
107
DEHYDROGENASE
(0.5% in tissue homogenate) in the chick liver.
in the determination
of the
lipoamide dehydrogenase and NADPH-dependent glutathione reductase, were detected according to the different kinds of substrates and cofactors incorporated in the agar gel. Riboj?avin deficiency. At 2 weeks of age, the deficient group had a mortality of 40% and an average body weight of 114.5 g, compared to 221.0 g for the control group. Liver homogenates from individual chicks in each nutritional treatment were prepared, diluted, and analyzed on the lipoate agar plates. The results are summarized in Fig. 5. Since the deficient tissue at the 0.2 g/ml level produced a diffusion zone the same size as the control tissue at approximately 0.08 g/ml, one can estimate that lipoamide dehydrogenase in the deficient group is about 40% of that in the control. DISCUSSION
A new method for analyzing lipoamide dehydrogenase has been developed based on the diffusion of the enzyme in the agar gel and disappearance of fluorescence when reduced nicotinamide adenine dinucleotide is oxidized. The method is simple, inexpensive, and time-saving. No sophisticated equipment is needed and a large number of samples can be screened in a short period of time.
FIG.
deficient
5. Graphic chicks.
analyses
of lipoamide
dehydrogenase
in the control
and the riboflavin-
108
JASON
C. H. SHIH
The determination of dehydrogenase activity by radial diffusion may have potential importance in clinical and nutritional applications. In this report, riboflavin deficiency was detected by measuring decreased lipoamide dehydrogenase activity in the liver of experimental chicks. Recently, an incident of riboflavin deficiency broke out at a poultry farm in North Carolina. The deficiency was quickly detected by this method and the error was corrected by adding riboflavin into the drinking water for the chicks.4 These results have demonstrated that the technique can serve as an enzymatic diagnosis for riboflavin deficiency. The decreased activity of lipoamide dehydrogenase, however, could be due to the deficiency of FAD alone, or to the lower levels of FAD and enzyme protein both. Distinguishing between these two possibilities was not attempted. Lipoamide dehydrogenase, like other NAD- or NADP-dependent oxidoreductases, could be analyzed by either a forward or reverse reaction. When reduced lipoate prepared from the sodium borohydride reduction (11) was included in the agar gel and when NAD was spread on the gel after diffusion, a fluorescent zone indicating enzymatic reduction of NAD against a dark background could be detected under the uv lamp. To avoid the additional step of the reduction of lipoate to prepare the gel, however, the lipoate-NADH-quenching (substrate-coenzyme-detection) procedure is recommended and described here as the standard method for the assay of lipoamide dehydrogenase. The radial diffusion assay was approximately 50 times less sensitive than the conventional spectrophotometric method (9,lO). Hence, the new method is a supplement rather than a replacement for the spectrophotometric method, which is highly sensitive and more accurate. On the other hand, this method is extremely useful for large-scale screening. Also, lower sensitivity can be an advantage because no exhaustive dilution is necessary in preparing the tissue samples. The tissue homogenate (20%) can be directly assayed on the gel. For additional accuracy, the homogenate may be diluted to two-, four-, and eight-fold prior to analyses on the gel, as the results shown in Fig. 5 illustrate. It should be remembered that a level of enzyme in the chick liver should not be compared for its absolute concentration with the standard curve of a yeast enzyme. The enzymes of two species are different in physical properties, especially their molecular weights (12,13), and therefore possess different diffusion rate or mobilities in the gel. When the two zones of enzymes from different origins are of the same size, they may not be at the same concentration. Since lipoamide dehydrogenase is located in the mitochondria, a surfactant such as Triton X-100 which solubilized the membrane released more of the enzyme from the tissue homogenate (Fig. 4). Part of lipoamide 4 The author’s
unpublished
results
(1977).
DIFFUSION
ASSAY OF LIPOAMIDE
DEHYDROGENASE
109
dehydrogenase, however, could still be complexed with a-keto acid dehydrogenases and diffused more slowly. With respect to the specificities, the purified yeast lipoamide dehydrogenase was specific for the lipoate or lipoamide and unreactive toward the other disulfide compounds tested. Since lipoamide was suspended and not totally soluble in the gel, it was not the substrate of choice to be used in the standard procedure. NADH, but not NADPH, was reactive with the purified enzyme. The chick liver homogenate is not only reactive toward lipoate or lipoamide with NADH but also reactive toward oxidized glutathione with NADPH. This reactivity is due to the presence ofglutathione reductase (E.C. 1.6.4.2, NAD(P)H: oxidized glutathione oxidoreductase) in the tissue. When a different substrate is incorporated into the agar gel, a different enzyme in the tissue homogenate can be detected accordingly. ACKNOWLEDGMENT The author wishes to thank Drs. M. Sandholm and M. L. Scott with whom this work was first initiated and to Ms. Elizabeth Teulings for her technical assistance.
REFERENCES 1. Fossum, K. (1970) Acta Pathol. Microbial. Stand. Sect. B. 78, 350-362. 2. Sandholm, M., Smith, R. R., Shih, J. C. H., and Scott, M. L. (1976) J. Nutr. 106, 761-766. 3. Fossum, K., and Whitaker, J. R. (1974) J. Nutr. 104, 930-936. 4. Ceska, M. (1971) C/in. Chim. Acta 33, 135-145. 5. Goldberg, J. M., and Pagast, P. (1976) C/in. Chem. 22, 633-637. 6. Montenecourt. B. S., and Eveleigh. D. E. (1977) App. Environ. Microbial. 33, 17% 183. 7. Wagner, A. F., Walton, E., Boxer, G. E., Pruss, M. P., Holly, F. W., and Folkers, K. (1956) J. Amer. Chem. Sot. 78, 5079-5081. 8. Gries, C. L., and Scott, M. L. (1972) J. Nutr. 102, 1269-1286. 9. Koike, M., and Hayakawa, T. (1970) in Methods in Enzymology (McCormick, D. B., and Wright, L. D., eds.) Vol. 18, Part A, pp. 298-307, Academic Press, New York. 10. Shih, J. C. H., Sandholm, M., and Scott, M. L. (1977) J. Nutr. 107, 1583-1589. 11. Gunsalus. I. C., Barton, L. S.. and Gruber. W. (1956) J. Amer. Chem. Sot. 78, 17631766. 12. Massey, V. (1963) in The Enzymes (Boyer, P. D., ed.), 2nd ed., Vol. 7. Part A, pp. 275-306, Academic Press, New York. 13. Williams, C. H., Jr. (1976) in The Enzymes (Boyer, P. D. ed.) 3rd ed.. Vol. 13. Part C, pp. 89-173, Academic Press, New York.
Radial
BIOCHEMISTRY
89, 103- 109 (1978)
Diffusion Assay of Lipoamide Its Use to Assess Riboflavin
Dehydrogenase Deficiency1,2
and
JASON C. H. SHIH Department
of Poultry Raleigh.
Science, North
North Carolina State Carolina 27650
University.
Received December 14, 1977 A new, simple, and quantitative method was developed to determine lipoamide dehydrogenase (E.C. 1.6.4.3, NADH:lipoamide oxidoreductase). The principle of this technique is to allow the enzyme or tissue extracts to diffuse in an agarose gel containing lipoate. The enzyme, after 24 hr of diffusion and 2 hr of reaction with NADH, can be determined by the size of a dark or fluorescence-quenching zone in the gel when illuminated with uv light. The diameter of the quenching zone which indicates the enzymatic oxidation of NADH is linearly proportional to the logarithm of enzyme concentration. Since lipoamide dehydrogenase is a FAD-enzyme, the activity was decreased in liver homogenates of riboflavin-deficient chicks, as measured by the new method. This demonstrated the potential importance of this new technique in nutritional and clinical applications.
The activities of several kinds of hydrolytic enzymes can be followed and quantitated visually by the radial diffusion of an enzyme in an agar gel where appropriate substrate has been incorporated. Protease was determined by including heat-denatured fibrinogen or casein in the gel (1). Trypsin and anti-trypsin activities were measured by the solubilization of calcium caseinate in the agar plates (2). Amylase was determined in agarcontaining starch or dextran blue-conjugated starch (3,4), and lipase was assayed in agar including emulsified triglycerides (5).3 A plate assay for determination of cellulase production by a fungus was devised by including acid-swollen cellulose and Phosfon D in the agar (6). The advantages of the enzyme diffusion technique are the simplicity of the procedure, the nonrequirement for sophisticated instruments, and, especially, the application of it to the screening tests for a large number of samples. In this report, the radial diffusion technique is extended to assay an ’ Paper No. 5463 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina. Z The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Experiment Station of the product named nor criticism of similar ones not mentioned. 3 Sandholm, M., and Scott, M. L. (1977) Submitted for publication. 103
0003-2697/78/0891-0103$02.00/0 Copyright Q 1978 by Academic Ress. Inc. All rights of reproduction in any form reserved.
104
JASON C. H. SHIH
oxidoreductase. Based on the oxidation of NADH with the simultaneous loss of its fluorescence in the presence of the substrate and the enzyme, a diffusion zone can be measured and an enzyme level can be estimated. EXPERIMENTAL
Lipoate agar plates. One gram of low EEO agarose (Sigma Chemical) was dissolved in 95 ml of 0.05 M potassium phosphate buffer, pH 6.6, with 1 mM EDTA, in a boiling water bath. Simultaneously, 100 mg (0.49 mmol) of dl-lipoic acid (Sigma) was dissolved in 1 ml of 1 M NaHCO, and 4 ml of HzO. The lipoate solution was then mixed thoroughly into the agarose solution at 50 to 60°C. The mixture was poured rapidly onto a clean glass plate (20 x 20 cm) and spread evenly. After the agar solidified, small wells were punched with a glass tubing (7.0 mm od). The small well could then be filled with 50 ~1 of an enzyme source to be assayed. The preparation of the lipoate agar plate was conducted in a semidark room to minimize photolysis and polymerization of lipoate (7). For a smaller number of enzyme samples, the agar plate can be made smaller in a plastic petri dish with the reagents reduced proportionately. Enzyme assay. Fifty microliters of an enzyme preparation, either a solution of standard purified enzyme or a tissue homogenate, were pipetted into the well in the agar gel. The plate was kept in a dark moist chamber at room temperature (20°C) for 24 hr to allow the enzyme to diffuse radially. At the end of the diffusion period, the wells in the plate were filled with a molten 1% agarose solution (50 ~1 in each well). When the filling agar solidified, a solution of 35 mg (0.05 mmol) of NADH (Sigma) in 2.5 ml of 0.2 M potassium phosphate buffer, pH 8.0, was evenly spread over the plate with a glass rod. The agar plate, spread with NADH and incubated for additional 2 hr in the moist chamber, was placed under a long-wavelength uv light (Ultra-Violet Products, Black-ray lamp, Model Xx-15) in a dark room. The gel exhibited a yellowish green fluorescence owing to the presence of reduced nicotinamide adenine dinucleotide (NADH), but around the wells where enzyme was added there were dark or quenched circles. The diameters of the circular zone were measured with a caliper. The results could be photographed for a permanent record. Enzyme preparations. Purified yeast lipoamide dehydrogenase (Sigma) was used as the standard enzyme. A series of dilutions of enzyme in 0.05 M potassium phosphate buffer, pH 6.6, with 1 mM EDTA, was made and pipetted into the wells on a lipoate agar plate for the activity assay. The standard curve was established by plotting the diameters of the quenched zones against the enzyme concentration on a logarithmic scale. Tissue homogenates were prepared from the freshly excised liver from 2-week-old chicks. One gram of liver tissue was ground with 4 ml of icecold 0.05 M potassium phosphate buffer, pH 6.6, with 1 mM EDTA, for
DIFFUSION
ASSAY OF LIPOAMIDE
DEHYDROGENASE
105
FIG. 1. Time course of the radial diffusion assay of lipoamide dehydrogenase. Left: various diffusion times at a fixed reaction time; right: various reaction times at a fixed diffusion time.
1 min with a Teflon tissue homogenizer at 0 to 5°C. Drops of Triton X-100 were added to the homogenate to give a concentration of 0.5% of this detergent. Detergent is necessary to rupture the mitochondrial membrane and to release lipoamide dehydrogenase. Before the assay, the tissue homogenate went through one cycle of freezing (- 15C) and thawing. After thawing, a series of four twofold dilutions of the homogenate was made, and aliquots of the dilutions were added to the agar plate for the assay. Riboflavin dejiciency . Triplicate lots of 10 day-old male chicks were fed with either control or riboflavin-deficient diet. The diets were based on isolated soy protein and glucose, as used by Gries and Scott (8) to produce riboflavin deficiency. All chicks were caged in electrically heated, thermostatically controlled, wire-floor brooders. Water and feed were available ad libitum. At 2 weeks of age, when 40% of the experimental group died from riboflavin deficiency, the remaining chicks were killed to prepare the liver homogenates for the enzyme assay. RESULTS
Levels of substrate and coenzyme. To determine the appropriate levels of lipoate and NADH to be used in the assay, agar plates (5-cm diameter petri dishes) containing various levels oflipoate from 0, 1.25,2.5, to 5.0 mM in the gels were prepared. After 50 ~1 of purified yeast lipoamide dehydrogenase (0.2 mg/ml) was added into each well in each plate and diffusion was carried out, NADH at various levels from 0,0.5, 1.0, to 2.0 mM was applied to each dish. The enzymatic reaction, as indicated by a zone of quenched fluorescence, was observed under the uv lamp. The most distinct and easily read zones were observed in the plates containing 5.0 mM lipoate and 0.5 or 1.0 mM NADH. Hence, 5.0 mM lipoate and 0.5 mM NADH were selected for the standard procedure used in later experiments. Diffusion time and reaction time. Various diffusion and reaction times were tested with four levels of yeast lipoamide dehydrogenase. The results are shown in Fig. 1. Although larger zones were produced with
106
JASON C. H. SHIH
FIG. 2. The radial diffusion zones of a series of twofold dilutions of yeast lipoamide dehydrogenase (original concentration: 0.2 mg/ml).
longer diffusion and longer reaction times, they were less distinct against the fluorescent background. Twenty-four hours of diffusion and 2 hr of reaction were selected for the standard procedure. Standard curves. Purified yeast lipoamide dehydrogenase was used to establish the relationship between the enzyme concentrations and the sizes of the fluorescence-quenching zones (Fig. 2). A semilogarithmic-linear relationship is demonstrated in Fig. 3. A similar linear relationship was obtained with various levels of chick liver homogenate (Fig. 4). In this case, Triton X-100 permitted the detection of a higher level of enzyme. The enzyme activity of the preparations was also measured by the conventional spectrophotometric method (9,lO) to ensure that the activities were proportional to the concentrations. Substrate and coenzyme specificities. To test the substrate specificity, lipoate, lipoamide, oxidized glutathione, cystine, and pantothine (all from Sigma) were used to prepare separate agar plates. Either NADH or NADPH were spread on each plate to test the specificity for the coenzyme. From the experiments with the purified enzyme, it is evident that that the enzyme reaction is specific for lipoate or lipoamide and NADH. From the experiments with the liver homogenate, different enzymatic reactions, namely,
FIG. 3. A semilogarithmic relationship between the diameters of the diffusion zones and the concentrations of yeast lipoamide dehydrogenase. Vertical bars on data points are SEM.
DIFFUSION
ASSAY
FIG. 4. Effect of Triton X-100 level of lipoamide dehydrogenase
OF
LIPOAMIDE
107
DEHYDROGENASE
(0.5% in tissue homogenate) in the chick liver.
in the determination
of the
lipoamide dehydrogenase and NADPH-dependent glutathione reductase, were detected according to the different kinds of substrates and cofactors incorporated in the agar gel. Riboj?avin deficiency. At 2 weeks of age, the deficient group had a mortality of 40% and an average body weight of 114.5 g, compared to 221.0 g for the control group. Liver homogenates from individual chicks in each nutritional treatment were prepared, diluted, and analyzed on the lipoate agar plates. The results are summarized in Fig. 5. Since the deficient tissue at the 0.2 g/ml level produced a diffusion zone the same size as the control tissue at approximately 0.08 g/ml, one can estimate that lipoamide dehydrogenase in the deficient group is about 40% of that in the control. DISCUSSION
A new method for analyzing lipoamide dehydrogenase has been developed based on the diffusion of the enzyme in the agar gel and disappearance of fluorescence when reduced nicotinamide adenine dinucleotide is oxidized. The method is simple, inexpensive, and time-saving. No sophisticated equipment is needed and a large number of samples can be screened in a short period of time.
FIG.
deficient
5. Graphic chicks.
analyses
of lipoamide
dehydrogenase
in the control
and the riboflavin-
108
JASON
C. H. SHIH
The determination of dehydrogenase activity by radial diffusion may have potential importance in clinical and nutritional applications. In this report, riboflavin deficiency was detected by measuring decreased lipoamide dehydrogenase activity in the liver of experimental chicks. Recently, an incident of riboflavin deficiency broke out at a poultry farm in North Carolina. The deficiency was quickly detected by this method and the error was corrected by adding riboflavin into the drinking water for the chicks.4 These results have demonstrated that the technique can serve as an enzymatic diagnosis for riboflavin deficiency. The decreased activity of lipoamide dehydrogenase, however, could be due to the deficiency of FAD alone, or to the lower levels of FAD and enzyme protein both. Distinguishing between these two possibilities was not attempted. Lipoamide dehydrogenase, like other NAD- or NADP-dependent oxidoreductases, could be analyzed by either a forward or reverse reaction. When reduced lipoate prepared from the sodium borohydride reduction (11) was included in the agar gel and when NAD was spread on the gel after diffusion, a fluorescent zone indicating enzymatic reduction of NAD against a dark background could be detected under the uv lamp. To avoid the additional step of the reduction of lipoate to prepare the gel, however, the lipoate-NADH-quenching (substrate-coenzyme-detection) procedure is recommended and described here as the standard method for the assay of lipoamide dehydrogenase. The radial diffusion assay was approximately 50 times less sensitive than the conventional spectrophotometric method (9,lO). Hence, the new method is a supplement rather than a replacement for the spectrophotometric method, which is highly sensitive and more accurate. On the other hand, this method is extremely useful for large-scale screening. Also, lower sensitivity can be an advantage because no exhaustive dilution is necessary in preparing the tissue samples. The tissue homogenate (20%) can be directly assayed on the gel. For additional accuracy, the homogenate may be diluted to two-, four-, and eight-fold prior to analyses on the gel, as the results shown in Fig. 5 illustrate. It should be remembered that a level of enzyme in the chick liver should not be compared for its absolute concentration with the standard curve of a yeast enzyme. The enzymes of two species are different in physical properties, especially their molecular weights (12,13), and therefore possess different diffusion rate or mobilities in the gel. When the two zones of enzymes from different origins are of the same size, they may not be at the same concentration. Since lipoamide dehydrogenase is located in the mitochondria, a surfactant such as Triton X-100 which solubilized the membrane released more of the enzyme from the tissue homogenate (Fig. 4). Part of lipoamide 4 The author’s
unpublished
results
(1977).
DIFFUSION
ASSAY OF LIPOAMIDE
DEHYDROGENASE
109
dehydrogenase, however, could still be complexed with a-keto acid dehydrogenases and diffused more slowly. With respect to the specificities, the purified yeast lipoamide dehydrogenase was specific for the lipoate or lipoamide and unreactive toward the other disulfide compounds tested. Since lipoamide was suspended and not totally soluble in the gel, it was not the substrate of choice to be used in the standard procedure. NADH, but not NADPH, was reactive with the purified enzyme. The chick liver homogenate is not only reactive toward lipoate or lipoamide with NADH but also reactive toward oxidized glutathione with NADPH. This reactivity is due to the presence ofglutathione reductase (E.C. 1.6.4.2, NAD(P)H: oxidized glutathione oxidoreductase) in the tissue. When a different substrate is incorporated into the agar gel, a different enzyme in the tissue homogenate can be detected accordingly. ACKNOWLEDGMENT The author wishes to thank Drs. M. Sandholm and M. L. Scott with whom this work was first initiated and to Ms. Elizabeth Teulings for her technical assistance.
REFERENCES 1. Fossum, K. (1970) Acta Pathol. Microbial. Stand. Sect. B. 78, 350-362. 2. Sandholm, M., Smith, R. R., Shih, J. C. H., and Scott, M. L. (1976) J. Nutr. 106, 761-766. 3. Fossum, K., and Whitaker, J. R. (1974) J. Nutr. 104, 930-936. 4. Ceska, M. (1971) C/in. Chim. Acta 33, 135-145. 5. Goldberg, J. M., and Pagast, P. (1976) C/in. Chem. 22, 633-637. 6. Montenecourt. B. S., and Eveleigh. D. E. (1977) App. Environ. Microbial. 33, 17% 183. 7. Wagner, A. F., Walton, E., Boxer, G. E., Pruss, M. P., Holly, F. W., and Folkers, K. (1956) J. Amer. Chem. Sot. 78, 5079-5081. 8. Gries, C. L., and Scott, M. L. (1972) J. Nutr. 102, 1269-1286. 9. Koike, M., and Hayakawa, T. (1970) in Methods in Enzymology (McCormick, D. B., and Wright, L. D., eds.) Vol. 18, Part A, pp. 298-307, Academic Press, New York. 10. Shih, J. C. H., Sandholm, M., and Scott, M. L. (1977) J. Nutr. 107, 1583-1589. 11. Gunsalus. I. C., Barton, L. S.. and Gruber. W. (1956) J. Amer. Chem. Sot. 78, 17631766. 12. Massey, V. (1963) in The Enzymes (Boyer, P. D., ed.), 2nd ed., Vol. 7. Part A, pp. 275-306, Academic Press, New York. 13. Williams, C. H., Jr. (1976) in The Enzymes (Boyer, P. D. ed.) 3rd ed.. Vol. 13. Part C, pp. 89-173, Academic Press, New York.