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[15] Fructose-diphosphate aldolase, pyruvate kinase, and pyridine nucleotide-linked activities after electrophoresis
66
ENZYME ASSAY PROCEDURES
[15]
ance at 290 nm. Thus, 10 t~l of 1 mM E D T A can mimic the reaction of 0.15 unit of epimerase. Consequently, the absence of artifacts should be checked by testing, in the assay, the buffers used to dissolve the enzymes. 6. The method for isomerase gave values close to those obtained by the 340 nm assay with undialyzed extracts of rat muscle, heart, kidney, intestine, brain, lung, spleen, and uterus, but spuriously high values were obtained with extracts of rat liver and rat blood hemolysates2
Applications The isomerase assay has been used to follow the purification of the enzyme from spinach 7 and other sources, 8 in the determination of Michaelis constants, 7,s and for the assay of isomerase activity in animal tissues, s,9 The epimerase assay has been used to follow the purification of the enzyme and determine its Michaelis constant, s 7 F. C. Knowles and N. G. Pon, Anal. Biochem. 24, 305 (1968). 8 M. E. Kiely, A. L. Stuart, and T. Wood, Biochim. Biophys. Acta 293, 534 (1973). F. C. Kauffman, J. Neurochem. 19, 1 (1972).
[15] F r u c t o s e - d i p h o s p h a t e A l d o l a s e , P y r u v a t e K i n a s e , a n d Pyridine Nucleotide-Linked Activities after Electrophoresis
By
WALTER A. SUSOR, EDWARD PENHOET, a n d WILLIAM J. RUTTER
A number of methods have been used to localize specific enzyme activities after electrophoresis. The widely used method of choice of most enzymes that can be coupled to the reduction of NAD or NADP is the coupled reduction of a tetrazolium dye to form an insoluble pigment. This method as applied to the detection of fructose-diphosphate aldolase 1 is described in detail below. There are in addition several enzymes which are more readily coupled to the oxidation of NADH or NADPH. These can be assayed by the disappearance of fluorescence or UV absorbance of the reduced nucleotides. The second method presented here was designed for the detection of pyruvate kinase, ~ an enzyme that can be coupled to the oxidation of 1 E. Penhoet, T. l~ajkumar, and W. J. Rutter, Proc. Nat. Acad. Sci. U.S. 56, 1275 (1966). 2 W. A. Susor and W. J. Rutter, Anal. Biochem. 43, 147 (1971).
[15]
ALDOLASE AND PYRUVATE KINASE DETECTION
67
NADtt. The method can also be applied to the assay of aldolase and other enzyme systems which result in a net oxidation of NADH or NADPH to NAD or NADP.
D e t e c t i o n of Aldolase b y Tetrazolium D y e Reduction
Proteins in the sample with differing electrophoretic mobilities are resolved by electrophoresis on cellulose polyacetate strips. Bands of aldolase activities may then be detected by placing the strip in contact with an agar film containing the assay reagents. The aldolase on the strip comes in contact with its substrate fructose 1,6-diphosphate and catalyzes its cleavage to 3-phosphoglyceraldehyde and dihydroxyacetone phosphate. In the presence of arsenate, 3-phosphoglyceraldehyde is then oxidized to 1-arseno-3-phosphoglycerate by glyceraldehyde-3-phosphate dehydrogenase with the concomitant reduction of NAD to NADH. The NADH produced then reduces oxidized phenazine methosulfate; this in turn reduces oxidized nitroblue tetrazolium to the reduced, highly colored blue state. Thus, wherever aldolase is present on the strip, a blue band appears in the adjacent agar. Electrophoresis of Aldolase. The aldolase sample (tissue extract or purified enzyme) is prepared as described by Penhoet and Rutter2 Electrophoresis is carried out on 1 X 6 inch cellulose polyacetate strips (Gelman Sepraphore III or equivalent) in a chamber having about 15 cm between the anode and cathode buffers (Gelman No. 51101). The electrophoresis buffer consists of 60 mM barbital-sodium, 10 mM 2-mercaptoethanol, pH 8.6; it is prepared by dissolving 10.8 g of sodium barbital and 1.5 g of barbituric acid to 1000 ml with distilled water, cooling to 4 °, and adding 0.7 ml of neat 2-mercaptoethanol immediately prior to use. The cellulose polyacetate strips are "activated" prior to use by floating the dry strips on the surface of the buffer until they are thoroughly wetted and sink into the buffer. After about 20 min, the strip is placed on a piece of white photo blotter, and excess buffer is allowed to drain into the blotter. About 3 ~1 of sample is loaded in a band midway between the ends of the paper with a sample applicator similar to Gelman No. 51220. The sample application site can be conveniently marked by gently touching a freshly sharpened No. 1 lead pencil to the edge of the strip. The strip is placed in the chamber with the sample band midway between the electrodes and electrophoresis carried out for 90 min at 250 V, constant voltage, at 4% s This series, Vol. 42 [38].
68
ENZYME ASSAY PROCEDURES
[15]
Enzyme Detection. Bands of aldolase activity are detected by placing the strips containing the electrophoresed sample on the surface of an agar plate. The assay plates are prepared by dissolving 125 mg of Noble agar in 20 ml of 50 mM tris(hydroxymethyl)aminomethane, 10 mM sodium arsenate, pH 8.0, in a boiling water bath. The solution is immediately cooled to 42-45 ° in a 42 ° water bath, and the following ingredients are rapidly mixed in: 2 ml of 100 mM sodium fructose 1,6-diphosphate, 1 ml of 10-mg/ml nitroblue tetrazolium chloride, 2 ml of 15-mg/ml NAD, 0.6 ml of 1-mg/ml phenazine methosulfate, and 0.1 ml of 10-mg/ml glyceraldehyde-3-phosphate dehydrogenase. The phenazine methosulfate is light sensitive; it should be made up fresh and kept in the dark. After adding the last ingredient, pour the mixture into five or six 100-mm plastic petri plates and place in a refrigerator as they solidify. The plates may be stored in the dark for up to 24 hr before use. After electrophoresis, the strips are removed from the chamber with a forceps and placed on the agar surface. The plates are then incubated at 37 ° for 10-15 min for color development. Record the results by photographing the inverted plates. The principal artifacts observed with this method come from particlebound activities, which do not move from the origin, and alcohol dehydrogenase, which uses the alcohol of crystallization of the nitroblue tetrazolium as substrate. Particulate activities can be removed by adequate centrifugation. Alcohol dehydrogenase bands can be detected on a control plate prepared without fructose diphosphate. Detection by UV Absorbance Pyruvate kinase activity is detected by NADH oxidation produced by coupling the enzyme with lactate dehydrogenase. Aldolase may be detected either by NADH oxidation or NAD reduction by coupling with triosephosphate isomerase and a-glycerophosphate dehydrogenase or glyceraldehyde-3-phosphate dehydrogenase, respectively. The enzymes are first resolved by electrophoresis on cellulose polyacetate strips, then the strips are applied to a thin agar film containing the assay reagents. The change in the oxidation state of the pyridine nueleotide substrate is followed visually by observing changes in fuorescence excited by 340 nm light, and the result is recorded by contact printing on photographic paper. Reduced pyridine nueleotides, which absorb at 340 nm and fluoresce yellow, appear as light areas on the photographic paper, while oxidized pyridine nueleotides transmit the light, exposing the photographic paper to produce darkened areas. Electrophoresis of Pyruvate Kinase. The pyruvate kinase sample is prepared from rat tissue extract in 0.25 M sucrose, 25 mM tris(hydroxy-
[15]
ALDOLASE AND PYRUVATE KINASE DETECTION
69
methyl)aminomethane (Tris.C1), pH 7.5, 2.5 mM EDTA, and 0.1 M dithiothreitol and assayed spectrophotometrically for enzyme activity ~ in the presence of 1 mM 2-phosphoenolpyruvic acid, tricyclohexylammonium salt (PEP), 1 mM NADP, and 0.2 mM fructose 1,6-disphosphate (FDP). The sample should be diluted to 0.5-5 enzyme units/ml with 0.5 M sucrose, 20 mM Tris.C1 (pH 7.5), 0.1 M 2-mercaptoethanol, and 1 mg of fraction V bovine serum albumin per milliliter. An enzyme unit is defined as that amount which will oxidize 1 ~mole of NADH per minute at 25 ° in the spectrophotometric assay. Cellulose polyacetate strips (Sephraphore III, Gelman Instrument Co., work well) are "activated" prior to use by soaking for about 30 rain in electrophoresis buffer containing 1 mg of fraction V bovine serum albumin per milliliter. A strip is then placed on a piece of blotter paper (white photo blotter serves well), and 3 ~l of the sample is applied in a band midway between the ends of the strip with a sample applicator similar to Gelman No. 51220. Electrophoresis is carried out at 4 ° for 3 hr at 250 V (17 V/cm) in a buffer containing 0.5 M sucrose, 20 mM Tris.HC1 (pH 7.5), 0.1 mM F D P and 0.1 M 2-mercaptoethanol. F D P is added to effect separation of pyruvate kinase A and C, which have nearly identical mobilities in its absence) In the presence of FDP, pyruvate kinase C migrates farther toward the cathode while the mobility of pyruvate kinase A is essentially unchanged; 0.1 mM F D P is sufficient to give good separation with rat tissue, although higher concentrations may occasionally be indicated (1 mM F D P gives better resolution with rabbit extracts). Sucrose and 2-mercaptoethanol are required to stabilize the enzyme during electrophoresis. Serum albumin is added to the activation buffer to reduce tailing and to permit electrophoresis of purified pyruvate kinase since pyruvate kinase adheres tightly to the cellulose polyacetate strip in the absence of other proteins. Preparation o] Assay Plates. The position of the enzyme activity after electrophoresis is determined by placing the strip in contact with an agar film containing the components of the spectrophotometric assay medium. Glass lantern slide covers (3.25 X 4 inches) are used as (1) .support and (2) as cover glass to press the agar into a thin film. The support glasses for the agar film are prepared by immersing the slide covers for 2 min in a fresh silicone (Siliclad, Clay-Adams, Inc.) solution diluted 1 part to 50 parts distilled water. They are then rinsed with distilled water, and dried at 100 °. These Siliclad-treated glasses are wrapped with 2 lb test nylon monofilament fishing line, dividing the plates into three sections. The nylon line serves as a spacer for the agar film and allows the assay of three 1-inch cellulose acetate strips per plate. The cover-glass 4 T. Biicher and G. Pfleiderer, this series, Vol. 1, p. 435. 5 W. A. Susor, Fed. Proc., Fed. Amer. Soc. Exp. Biol. 29, 729 (1970).
70
ENZYME ASSAY PROCEDURES
[15]
plates are left untreated. The treated and wrapped support glasses and untreated cover glasses are warmed to 45 ° a short time prior to use. The assay medium for pyruvate kinase consists of a solution of 9 mg of Noble agar (Difeo) per milliliter, 50 mM Tris.C1 (pH 7.5), 10 mM MgCI~, 60 mM KC1, 5 mM EDTA, 2 mM PEP, 2 mM ADP, 1 mM NADH, 0.2 tLM FDP and 30 units of pig heart lactate dehydrogenase per milliliter (Sigma, type VI). The agar is dissolved in distilled water to one-half of the final volume, in a boiling water bath, then cooled to 45 °. The remaining regents, at 40 °, are then added to the agar solution, mixed, and immediately applied to the support glass. About 2 ml of this assay mixture is pipetted onto the surface of a wrapped silicone-treated glass, and an untreated glass cover is carefully placed over the still-fluid agar solution, to form a silicone-treated glass-agar-glass sandwich. The solidified agar solution will adhere more firmly to the silicone-treated glass than to the untreated cover, allowing the latter to be removed, exposing a thin, uniform agar surface. Completed plates can be kept for several days in a humid chamber at 4 °. The upper cover glass is removed immediately prior to use. Enzyme Detection. After electrophoresis, the cellulose acetate strips are placed on the surface of the agar-rcagent assay plate and covered with a sheet of plastic film, such as Handi-Wrap (Dow Chemical Co., Inc.). (When Handi-Wrap is used it should be freshly removed from the spool since the film slowly oxidizes on exposure to air to form an UVabsorbing material which interferes with the assay.) The course of the reaction is followed visually by observing the decrease (or increase) in fluorescence of the pyridine nueleotide on illumination with 340-nm light. After distinct bands have appeared (1-5 min), they are recorded by contact-printing on photographic enlarging paper (Kodabromide F-5, Eastman Kodak Company), with the Handi-Wrap side of the assay plate in contact with the enlarging paper. Exposure is made with a long-wave UV lamp of the hand-held type (Blak-Ray UVL-22) fitted with a 340-nm broad band-pass filter (Corning 7-60). The lamp should be secured about 30 cm above the sensitized paper on a mount having a 5 mm diameter hole between the bulb and the sensitized paper. Under these conditions, about 15-see exposure is required. Figure 1 is a sketch showing the physical relationship of the components of the assay system. Aldolase and Other Enzymes Aldolase may be detected by the above method, both by coupling to NAD reduction through glyeeraldehyde-3-phosphate dehydrogenase
[15]
ALDOLASE AND PYRUVATE KINASE DETECTION
71
/11\\ Ill/\ t/ I \ \ // I \\
Fia. 1. Cross sections showing arrangement of assay components: (a) 340 nm light source, (b) dark slide with 5 mm diameter opening, (c) silicone-treated lantern slide glass, (d) reagents in agar film, (e) electrophoresis strip, (f) plastic film, (g) enlarging paper. The relative dimensions of the drawing are exaggerated.
(NAD method) and by coupling to NADH oxidation through triosephosphate isomerase and a-glycerophosphate dehydrogenase (NADH method). The detection method for NAD and NADH consists of an agar film prepared as described above for pyruvate kinase. The detection medium for the NAD method contains 9 mg of Noble agar per milliliter 50 mM Tris.C1 (pH 7.8), 10 InM NaAsO4 (pH 7.8), 5 mM NaFDP, l mM NAD, and 0.1 mg of glyceraldehyde-3-phosphate dehydrogenase per milliliter. The detection medium for the NADH method contains 9 mg of Noble agar per milliliter, 50 mM Tris.C1 (pH 7.5), 2.5 mM NaFI)P, 1 mM NADH, and 0.1 mg of a-glycerophosphate dehydrogenase-triosephosphate dehydrogenase per milliliter (Calbiochem). The resolution and detection of aldolase A-C hybrid set of enzymes by the three methods is shown in Fig. 2. The resolution and sensitivity of these methods are similar. The NBT method retains the advantage, however, that the colored formazan product is a precipitate and does not diffuse; thus the plates can be read several days later. The plates produced by the N A D H and NAD methods, on the other hand, must be read and recorded within a few minutes since the products remain in solution and diffuse rather rapidly. Resolution of closely spaced bands can be further enhanced, or the identification of members of a hybrid set in the absence of a complete set simplified, by the simultaneous application of the test sample to half of the electrophoresis strip and a control sample to the other half. The
72
[15]
ENZYME ASSAY PROCEDURES
(+)
(4-)
---~-0
O ~
iii!!:if!i!!i!!!i!::ii!¸!if!!i!i (-)
(-) NADH
NAD
NBT
FIG. 2. Electropherograms of rat brain aldolase. NADH: Aldolase detected by coupling with triosephosphate isomerase and a-glyeerophosphate dehydrogenase to NADH oxidation. NAD: Aldolase detected by coupling with glyceraldehyde-3-phosphate dehydrogenase to NAD reduction. NBT: Aldolase detected by coupling with glyceraldehyde-3-phosphate dehydrogenase to NAD reduction, which is in turn coupled with phenazine methosulfate to nitroblue tetrazolium chloride (NBT) reduction. Adult rat brain tissue was homogenized in 2 vol of 0.25 M sucrose, 25 mM Tris. C1, and 2.5 mM EDTA, pH 7.5, and centrifuged at 100,000 g for 30 rain. NADH, NAD, and NBT samples consisted of 1.2 × 10-3, 2.5 × 10-~ and 2.5 × 10-5 unit of aldolase activity, respectively. Note that one equivalent of reduced nucleotide is produced by the NAD and NBT assays, and two equivalents of oxidized nucleotide are produced by the NADH method.
two samples are placed on opposite halves of the sample applicator and are allowed to mix in the center of the applicator. The partial mixing provides a control for interfering components, for example, high salt or protein interactions in the homogenate which m a y affect the mobilities. A sample of this technique is shown in Fig. 3. The method for detection of pyruvate kinase described here is adapted from the spectrophotometric assay of Bficher and Pfleiderer. 4 A method which is similar in principle was also developed by yon Fellenberg, Richterich, and Aebi 6 for the detection of pyruvate kinase after agar-gel electrophoresis. The latter involves flooding the surface of the electrophoresis gel with the assay reagents dissolved in agar. Bockelmann et R. yon Fellenberg, R. Richterich, and H. Aebi, Enzymol. Biol. Clin. 3, 240 (1963).
[15]
73
ALDOLASE AND PYRLIVATE KINASE DETECTION
(+)
_
-q~-O
: = !! i! I¸¸! ! : :
i ¸ i! =
l-)
I A
B
C
FI~. 3. Electropherograms of 13- and 21-day fetal rat liver aldolase: (A) 13day fetal rat liver, (B) 13- and 21-day samples simultaneously applied to opposite halves of the strip, (C) 21-day fetal rat liver. Samples A and C contained 4.7 × 10-3 and 6.4 × 10 3 unit of aldolase activity, respectively. Although three bands are seen, in both strips A and C the nonidentity of the more cathode bands is clearly demonstrated by strip B.
were able to detect pyruvate kinase activity by coupling through hexokinase and glucose-6-phosphate dehydrogenase to NAD reduction, which was in turn coupled to the formation of an insoluble blue formazan from tetrazolium bromide. Adenylate kinase, however, interferes, requiring additional controls. Alcohol dehydrogenase also interferes with the tetrazolium method, since the reagent contains alcohol of crystallization,s The many enzymes whose reactions involve a pyridine nucleotide cofactor or whose products can be coupled to a pyridine nucleotide oxidation or reduction can also be detected by the incorporation of the specific assay mixture into the agar film, adjusting the pyridine nucleotide to 1 mM and adding sufficient substrate to allow complete oxidation or reduction of the pyridine nucleotide. The customary controls should be performed, such as testing the reactions on an assay plate prepared without substrate. al. 7
7 W. Bockelmann, U. Wolf, and H. Ritter, Humangenetik 6, 78 (1968). 8 C. R. Shaw and A. C. Koen, Science 156, 1516 (1967).
ENZYME ASSAY PROCEDURES
[15]
ance at 290 nm. Thus, 10 t~l of 1 mM E D T A can mimic the reaction of 0.15 unit of epimerase. Consequently, the absence of artifacts should be checked by testing, in the assay, the buffers used to dissolve the enzymes. 6. The method for isomerase gave values close to those obtained by the 340 nm assay with undialyzed extracts of rat muscle, heart, kidney, intestine, brain, lung, spleen, and uterus, but spuriously high values were obtained with extracts of rat liver and rat blood hemolysates2
Applications The isomerase assay has been used to follow the purification of the enzyme from spinach 7 and other sources, 8 in the determination of Michaelis constants, 7,s and for the assay of isomerase activity in animal tissues, s,9 The epimerase assay has been used to follow the purification of the enzyme and determine its Michaelis constant, s 7 F. C. Knowles and N. G. Pon, Anal. Biochem. 24, 305 (1968). 8 M. E. Kiely, A. L. Stuart, and T. Wood, Biochim. Biophys. Acta 293, 534 (1973). F. C. Kauffman, J. Neurochem. 19, 1 (1972).
[15] F r u c t o s e - d i p h o s p h a t e A l d o l a s e , P y r u v a t e K i n a s e , a n d Pyridine Nucleotide-Linked Activities after Electrophoresis
By
WALTER A. SUSOR, EDWARD PENHOET, a n d WILLIAM J. RUTTER
A number of methods have been used to localize specific enzyme activities after electrophoresis. The widely used method of choice of most enzymes that can be coupled to the reduction of NAD or NADP is the coupled reduction of a tetrazolium dye to form an insoluble pigment. This method as applied to the detection of fructose-diphosphate aldolase 1 is described in detail below. There are in addition several enzymes which are more readily coupled to the oxidation of NADH or NADPH. These can be assayed by the disappearance of fluorescence or UV absorbance of the reduced nucleotides. The second method presented here was designed for the detection of pyruvate kinase, ~ an enzyme that can be coupled to the oxidation of 1 E. Penhoet, T. l~ajkumar, and W. J. Rutter, Proc. Nat. Acad. Sci. U.S. 56, 1275 (1966). 2 W. A. Susor and W. J. Rutter, Anal. Biochem. 43, 147 (1971).
[15]
ALDOLASE AND PYRUVATE KINASE DETECTION
67
NADtt. The method can also be applied to the assay of aldolase and other enzyme systems which result in a net oxidation of NADH or NADPH to NAD or NADP.
D e t e c t i o n of Aldolase b y Tetrazolium D y e Reduction
Proteins in the sample with differing electrophoretic mobilities are resolved by electrophoresis on cellulose polyacetate strips. Bands of aldolase activities may then be detected by placing the strip in contact with an agar film containing the assay reagents. The aldolase on the strip comes in contact with its substrate fructose 1,6-diphosphate and catalyzes its cleavage to 3-phosphoglyceraldehyde and dihydroxyacetone phosphate. In the presence of arsenate, 3-phosphoglyceraldehyde is then oxidized to 1-arseno-3-phosphoglycerate by glyceraldehyde-3-phosphate dehydrogenase with the concomitant reduction of NAD to NADH. The NADH produced then reduces oxidized phenazine methosulfate; this in turn reduces oxidized nitroblue tetrazolium to the reduced, highly colored blue state. Thus, wherever aldolase is present on the strip, a blue band appears in the adjacent agar. Electrophoresis of Aldolase. The aldolase sample (tissue extract or purified enzyme) is prepared as described by Penhoet and Rutter2 Electrophoresis is carried out on 1 X 6 inch cellulose polyacetate strips (Gelman Sepraphore III or equivalent) in a chamber having about 15 cm between the anode and cathode buffers (Gelman No. 51101). The electrophoresis buffer consists of 60 mM barbital-sodium, 10 mM 2-mercaptoethanol, pH 8.6; it is prepared by dissolving 10.8 g of sodium barbital and 1.5 g of barbituric acid to 1000 ml with distilled water, cooling to 4 °, and adding 0.7 ml of neat 2-mercaptoethanol immediately prior to use. The cellulose polyacetate strips are "activated" prior to use by floating the dry strips on the surface of the buffer until they are thoroughly wetted and sink into the buffer. After about 20 min, the strip is placed on a piece of white photo blotter, and excess buffer is allowed to drain into the blotter. About 3 ~1 of sample is loaded in a band midway between the ends of the paper with a sample applicator similar to Gelman No. 51220. The sample application site can be conveniently marked by gently touching a freshly sharpened No. 1 lead pencil to the edge of the strip. The strip is placed in the chamber with the sample band midway between the electrodes and electrophoresis carried out for 90 min at 250 V, constant voltage, at 4% s This series, Vol. 42 [38].
68
ENZYME ASSAY PROCEDURES
[15]
Enzyme Detection. Bands of aldolase activity are detected by placing the strips containing the electrophoresed sample on the surface of an agar plate. The assay plates are prepared by dissolving 125 mg of Noble agar in 20 ml of 50 mM tris(hydroxymethyl)aminomethane, 10 mM sodium arsenate, pH 8.0, in a boiling water bath. The solution is immediately cooled to 42-45 ° in a 42 ° water bath, and the following ingredients are rapidly mixed in: 2 ml of 100 mM sodium fructose 1,6-diphosphate, 1 ml of 10-mg/ml nitroblue tetrazolium chloride, 2 ml of 15-mg/ml NAD, 0.6 ml of 1-mg/ml phenazine methosulfate, and 0.1 ml of 10-mg/ml glyceraldehyde-3-phosphate dehydrogenase. The phenazine methosulfate is light sensitive; it should be made up fresh and kept in the dark. After adding the last ingredient, pour the mixture into five or six 100-mm plastic petri plates and place in a refrigerator as they solidify. The plates may be stored in the dark for up to 24 hr before use. After electrophoresis, the strips are removed from the chamber with a forceps and placed on the agar surface. The plates are then incubated at 37 ° for 10-15 min for color development. Record the results by photographing the inverted plates. The principal artifacts observed with this method come from particlebound activities, which do not move from the origin, and alcohol dehydrogenase, which uses the alcohol of crystallization of the nitroblue tetrazolium as substrate. Particulate activities can be removed by adequate centrifugation. Alcohol dehydrogenase bands can be detected on a control plate prepared without fructose diphosphate. Detection by UV Absorbance Pyruvate kinase activity is detected by NADH oxidation produced by coupling the enzyme with lactate dehydrogenase. Aldolase may be detected either by NADH oxidation or NAD reduction by coupling with triosephosphate isomerase and a-glycerophosphate dehydrogenase or glyceraldehyde-3-phosphate dehydrogenase, respectively. The enzymes are first resolved by electrophoresis on cellulose polyacetate strips, then the strips are applied to a thin agar film containing the assay reagents. The change in the oxidation state of the pyridine nueleotide substrate is followed visually by observing changes in fuorescence excited by 340 nm light, and the result is recorded by contact printing on photographic paper. Reduced pyridine nueleotides, which absorb at 340 nm and fluoresce yellow, appear as light areas on the photographic paper, while oxidized pyridine nueleotides transmit the light, exposing the photographic paper to produce darkened areas. Electrophoresis of Pyruvate Kinase. The pyruvate kinase sample is prepared from rat tissue extract in 0.25 M sucrose, 25 mM tris(hydroxy-
[15]
ALDOLASE AND PYRUVATE KINASE DETECTION
69
methyl)aminomethane (Tris.C1), pH 7.5, 2.5 mM EDTA, and 0.1 M dithiothreitol and assayed spectrophotometrically for enzyme activity ~ in the presence of 1 mM 2-phosphoenolpyruvic acid, tricyclohexylammonium salt (PEP), 1 mM NADP, and 0.2 mM fructose 1,6-disphosphate (FDP). The sample should be diluted to 0.5-5 enzyme units/ml with 0.5 M sucrose, 20 mM Tris.C1 (pH 7.5), 0.1 M 2-mercaptoethanol, and 1 mg of fraction V bovine serum albumin per milliliter. An enzyme unit is defined as that amount which will oxidize 1 ~mole of NADH per minute at 25 ° in the spectrophotometric assay. Cellulose polyacetate strips (Sephraphore III, Gelman Instrument Co., work well) are "activated" prior to use by soaking for about 30 rain in electrophoresis buffer containing 1 mg of fraction V bovine serum albumin per milliliter. A strip is then placed on a piece of blotter paper (white photo blotter serves well), and 3 ~l of the sample is applied in a band midway between the ends of the strip with a sample applicator similar to Gelman No. 51220. Electrophoresis is carried out at 4 ° for 3 hr at 250 V (17 V/cm) in a buffer containing 0.5 M sucrose, 20 mM Tris.HC1 (pH 7.5), 0.1 mM F D P and 0.1 M 2-mercaptoethanol. F D P is added to effect separation of pyruvate kinase A and C, which have nearly identical mobilities in its absence) In the presence of FDP, pyruvate kinase C migrates farther toward the cathode while the mobility of pyruvate kinase A is essentially unchanged; 0.1 mM F D P is sufficient to give good separation with rat tissue, although higher concentrations may occasionally be indicated (1 mM F D P gives better resolution with rabbit extracts). Sucrose and 2-mercaptoethanol are required to stabilize the enzyme during electrophoresis. Serum albumin is added to the activation buffer to reduce tailing and to permit electrophoresis of purified pyruvate kinase since pyruvate kinase adheres tightly to the cellulose polyacetate strip in the absence of other proteins. Preparation o] Assay Plates. The position of the enzyme activity after electrophoresis is determined by placing the strip in contact with an agar film containing the components of the spectrophotometric assay medium. Glass lantern slide covers (3.25 X 4 inches) are used as (1) .support and (2) as cover glass to press the agar into a thin film. The support glasses for the agar film are prepared by immersing the slide covers for 2 min in a fresh silicone (Siliclad, Clay-Adams, Inc.) solution diluted 1 part to 50 parts distilled water. They are then rinsed with distilled water, and dried at 100 °. These Siliclad-treated glasses are wrapped with 2 lb test nylon monofilament fishing line, dividing the plates into three sections. The nylon line serves as a spacer for the agar film and allows the assay of three 1-inch cellulose acetate strips per plate. The cover-glass 4 T. Biicher and G. Pfleiderer, this series, Vol. 1, p. 435. 5 W. A. Susor, Fed. Proc., Fed. Amer. Soc. Exp. Biol. 29, 729 (1970).
70
ENZYME ASSAY PROCEDURES
[15]
plates are left untreated. The treated and wrapped support glasses and untreated cover glasses are warmed to 45 ° a short time prior to use. The assay medium for pyruvate kinase consists of a solution of 9 mg of Noble agar (Difeo) per milliliter, 50 mM Tris.C1 (pH 7.5), 10 mM MgCI~, 60 mM KC1, 5 mM EDTA, 2 mM PEP, 2 mM ADP, 1 mM NADH, 0.2 tLM FDP and 30 units of pig heart lactate dehydrogenase per milliliter (Sigma, type VI). The agar is dissolved in distilled water to one-half of the final volume, in a boiling water bath, then cooled to 45 °. The remaining regents, at 40 °, are then added to the agar solution, mixed, and immediately applied to the support glass. About 2 ml of this assay mixture is pipetted onto the surface of a wrapped silicone-treated glass, and an untreated glass cover is carefully placed over the still-fluid agar solution, to form a silicone-treated glass-agar-glass sandwich. The solidified agar solution will adhere more firmly to the silicone-treated glass than to the untreated cover, allowing the latter to be removed, exposing a thin, uniform agar surface. Completed plates can be kept for several days in a humid chamber at 4 °. The upper cover glass is removed immediately prior to use. Enzyme Detection. After electrophoresis, the cellulose acetate strips are placed on the surface of the agar-rcagent assay plate and covered with a sheet of plastic film, such as Handi-Wrap (Dow Chemical Co., Inc.). (When Handi-Wrap is used it should be freshly removed from the spool since the film slowly oxidizes on exposure to air to form an UVabsorbing material which interferes with the assay.) The course of the reaction is followed visually by observing the decrease (or increase) in fluorescence of the pyridine nueleotide on illumination with 340-nm light. After distinct bands have appeared (1-5 min), they are recorded by contact-printing on photographic enlarging paper (Kodabromide F-5, Eastman Kodak Company), with the Handi-Wrap side of the assay plate in contact with the enlarging paper. Exposure is made with a long-wave UV lamp of the hand-held type (Blak-Ray UVL-22) fitted with a 340-nm broad band-pass filter (Corning 7-60). The lamp should be secured about 30 cm above the sensitized paper on a mount having a 5 mm diameter hole between the bulb and the sensitized paper. Under these conditions, about 15-see exposure is required. Figure 1 is a sketch showing the physical relationship of the components of the assay system. Aldolase and Other Enzymes Aldolase may be detected by the above method, both by coupling to NAD reduction through glyeeraldehyde-3-phosphate dehydrogenase
[15]
ALDOLASE AND PYRUVATE KINASE DETECTION
71
/11\\ Ill/\ t/ I \ \ // I \\
Fia. 1. Cross sections showing arrangement of assay components: (a) 340 nm light source, (b) dark slide with 5 mm diameter opening, (c) silicone-treated lantern slide glass, (d) reagents in agar film, (e) electrophoresis strip, (f) plastic film, (g) enlarging paper. The relative dimensions of the drawing are exaggerated.
(NAD method) and by coupling to NADH oxidation through triosephosphate isomerase and a-glycerophosphate dehydrogenase (NADH method). The detection method for NAD and NADH consists of an agar film prepared as described above for pyruvate kinase. The detection medium for the NAD method contains 9 mg of Noble agar per milliliter 50 mM Tris.C1 (pH 7.8), 10 InM NaAsO4 (pH 7.8), 5 mM NaFDP, l mM NAD, and 0.1 mg of glyceraldehyde-3-phosphate dehydrogenase per milliliter. The detection medium for the NADH method contains 9 mg of Noble agar per milliliter, 50 mM Tris.C1 (pH 7.5), 2.5 mM NaFI)P, 1 mM NADH, and 0.1 mg of a-glycerophosphate dehydrogenase-triosephosphate dehydrogenase per milliliter (Calbiochem). The resolution and detection of aldolase A-C hybrid set of enzymes by the three methods is shown in Fig. 2. The resolution and sensitivity of these methods are similar. The NBT method retains the advantage, however, that the colored formazan product is a precipitate and does not diffuse; thus the plates can be read several days later. The plates produced by the N A D H and NAD methods, on the other hand, must be read and recorded within a few minutes since the products remain in solution and diffuse rather rapidly. Resolution of closely spaced bands can be further enhanced, or the identification of members of a hybrid set in the absence of a complete set simplified, by the simultaneous application of the test sample to half of the electrophoresis strip and a control sample to the other half. The
72
[15]
ENZYME ASSAY PROCEDURES
(+)
(4-)
---~-0
O ~
iii!!:if!i!!i!!!i!::ii!¸!if!!i!i (-)
(-) NADH
NAD
NBT
FIG. 2. Electropherograms of rat brain aldolase. NADH: Aldolase detected by coupling with triosephosphate isomerase and a-glyeerophosphate dehydrogenase to NADH oxidation. NAD: Aldolase detected by coupling with glyceraldehyde-3-phosphate dehydrogenase to NAD reduction. NBT: Aldolase detected by coupling with glyceraldehyde-3-phosphate dehydrogenase to NAD reduction, which is in turn coupled with phenazine methosulfate to nitroblue tetrazolium chloride (NBT) reduction. Adult rat brain tissue was homogenized in 2 vol of 0.25 M sucrose, 25 mM Tris. C1, and 2.5 mM EDTA, pH 7.5, and centrifuged at 100,000 g for 30 rain. NADH, NAD, and NBT samples consisted of 1.2 × 10-3, 2.5 × 10-~ and 2.5 × 10-5 unit of aldolase activity, respectively. Note that one equivalent of reduced nucleotide is produced by the NAD and NBT assays, and two equivalents of oxidized nucleotide are produced by the NADH method.
two samples are placed on opposite halves of the sample applicator and are allowed to mix in the center of the applicator. The partial mixing provides a control for interfering components, for example, high salt or protein interactions in the homogenate which m a y affect the mobilities. A sample of this technique is shown in Fig. 3. The method for detection of pyruvate kinase described here is adapted from the spectrophotometric assay of Bficher and Pfleiderer. 4 A method which is similar in principle was also developed by yon Fellenberg, Richterich, and Aebi 6 for the detection of pyruvate kinase after agar-gel electrophoresis. The latter involves flooding the surface of the electrophoresis gel with the assay reagents dissolved in agar. Bockelmann et R. yon Fellenberg, R. Richterich, and H. Aebi, Enzymol. Biol. Clin. 3, 240 (1963).
[15]
73
ALDOLASE AND PYRLIVATE KINASE DETECTION
(+)
_
-q~-O
: = !! i! I¸¸! ! : :
i ¸ i! =
l-)
I A
B
C
FI~. 3. Electropherograms of 13- and 21-day fetal rat liver aldolase: (A) 13day fetal rat liver, (B) 13- and 21-day samples simultaneously applied to opposite halves of the strip, (C) 21-day fetal rat liver. Samples A and C contained 4.7 × 10-3 and 6.4 × 10 3 unit of aldolase activity, respectively. Although three bands are seen, in both strips A and C the nonidentity of the more cathode bands is clearly demonstrated by strip B.
were able to detect pyruvate kinase activity by coupling through hexokinase and glucose-6-phosphate dehydrogenase to NAD reduction, which was in turn coupled to the formation of an insoluble blue formazan from tetrazolium bromide. Adenylate kinase, however, interferes, requiring additional controls. Alcohol dehydrogenase also interferes with the tetrazolium method, since the reagent contains alcohol of crystallization,s The many enzymes whose reactions involve a pyridine nucleotide cofactor or whose products can be coupled to a pyridine nucleotide oxidation or reduction can also be detected by the incorporation of the specific assay mixture into the agar film, adjusting the pyridine nucleotide to 1 mM and adding sufficient substrate to allow complete oxidation or reduction of the pyridine nucleotide. The customary controls should be performed, such as testing the reactions on an assay plate prepared without substrate. al. 7
7 W. Bockelmann, U. Wolf, and H. Ritter, Humangenetik 6, 78 (1968). 8 C. R. Shaw and A. C. Koen, Science 156, 1516 (1967).