[33]
MEASUREMENT OF LONG-CHAIN C o A
273
[33] Enzymatic Determination of Long-Chain Fatty Acyl-CoA B y D. VELOSO and R. L. VEECH
The method consists of two main steps: enzymatic hydrolysis of longchain acyl-CoA (fatty acyl-CoA + H 2 0 - ~ C o A ~- f a t t y acid) and subsequent determination of the CoA produced. Several enzymes have been reported to release CoA from long-chain acyl-CoA; viz., pigeon l and r a t 2 liver f a t t y acid synthase, pig brain pahnityl-CoA deacylase, ~ Escherichia coli palmityl thioesterases, 4,5 rat liver microsome acyl-CoA hydrolase, G and pancreatic lipase7 R a t liver f a t t y acid synthase can be prepared easily in a highly pure form 2 and offers a convenient basis for the assay of long-chain acyl-CoA. F a t t y acid synthase hydrolyzes palmityl- and stearyl -s and oleyl-CoA2 Its action on other long-chain acylCoA is not known. The concentration of long-chain acyl-CoA in the samples to be hydrolyzed should be below 5 t~M to avoid errors resulting from micelle f o r m a t i o n Y This low concentration of long-chain CoA necessitates the use of a cycling technique in order to measure the concentration conveniently in a spectrophotometer. The CoA released from longchain acyl-CoA is measured with an enzymatic cycling procedure using the reactions catalyzed by carnitine acetyltransferase [acetyl-CoA:carnitine 0-acetyltransferase, EC 2.3.1.7] (CAT) and citrate synthase [EC 4.1.3.7] (CS). The oxalacetate used in the citrate synthase reaction is generated by the malate dehydrogenase reaction [ L - m a l a t e : N A D oxidoreductase, EC
CS
CAT
I S. Kumar, J. A. Dorsey, R. A. Muesing, and J. W. Porter, J. Biol. Chem. 245, 4732 (1970). 2S. Kumar, G. T. Phillips, and J. W. Porter, Int. J. Biochem. 3, 15 (1972). 3p. A. Srere, W. Seubert, and F. Lynen, Biochim. Biophys. Acta 33, 313 (1959). 4E. M. Barnes, Jr., and S. J. Wakil, J. Biol. Chem. 243, 2955 (1968). 5E. M. Barnes, Jr., A. C. Swindell, and S. J. Wakil, J. Biol. Chem. 245, 3122 (1970). 6 R. E. Barden and W. W. Cleland, J. Biol. Chem. 244, 3677 (1969). 7E. D. Barber and W. E. M. Lands, Bioehim. Biophys. Acta 250, 361 (1971). 8 G. T. Phillips, J. E. Nixon, J. A. Dorsey, P. H. W. Butterworth, C. J. Chesterton, and J. W. Porter, Arch. Biochem. Biophys. 138, 380 (1970). D. Veloso and R. L. Veech, in preparation.
274
GENERAL ANALYTICAL METHODS
[33]
1.1.1.37] establishing a connection between the enzymatic cycling reactions and a N A D H formation system: CoA + acetylcarnitine ~ acetyl-CoA + carnitine Acetyl-CoA + H20 + oxalacetate 2- ~ CoA + citrate 3- + H + Malate 2- + N A D + ~-- oxalacetate ~- + N A D H + H + For the CoA recycling to proceed, the formation of more oxalacetate is necessary and consequently more N A D H is formed. The rate of formation of N A D H concentration can be followed in a spectrophotometer (340 nm) or fluorometer. Similar to the enzymatic cycling procedure for measurement of pyridine nucleotides) ° the concentrations of CoA and acetylCoA must be below their K.,~ in order that the reaction rates be dependent on their concentrations. Coenzyme A is the standard for the cycling procedure because no reliable long-chain acyl-CoA preparations are commercially available.
Reagents F a t t y Acid Synthase. The enzyme is partially purified from rat l i v e r " and is suitable for use after dialysis of the second a m m o n i u m sulfate precipitate. F a t t y acid synthase activity is determined according to Nepokroeff et al. 11 and expressed as 1 unit = 1 t~mole malonyl-CoA used per minute per milligram of protein. Before use, the enzyme is activated by mixing it with an equal volume of a mixture of 0.8 M potassium phosphate buffer, p H 7.0, 0.02 M dithiothreitol, and 1.2 m M E D T A and incubating the mixture at 3.0° for 30 minutes. The diluted activated enzyme can be stored at - - 7 0 ° and reactivated once by immersion in a water bath (30 ° ) for 30 minutes. Coenzyme A Standard. Prepare a 3 m M solution of CoA in 10 m M dithiothreitol, standardize, and store in small portions at --20% Each tube should not be thawed more than 3 times. The concentration of CoA in the standard is determined by the method of Tubbs and Garland, 1-~ with 3-acetyl pyridine adenine nucleotide as coenzyme. Prior to use, dilute the enzyme 300-fold (spectrophotometry) or 750-fold (fluorometry) with a 1 m M dithiothreitol solution. Hydrolysis Mixture. This mixture, prepared according to K u m a r et al. 1 contains 10 ml of 1 M potassium phosphate buffer, p H 6.8, 1 ml 0.05 M E D T A , 1 ml 0.1 M dithiothreitol, and 38 ml of distilled water. The
lo0. H. Lowry, J. V. Pa~onneau, D. W. Schulz, and M. K. Rock, d. Biol. Chem. 236, 2746 (1961). 1~C. M. Nepokroeff, M. R. Lakshmanan, and J. W. Porter, this volume [6]. 12p. K. Tubbs and P. B. Garland, Vol. 13 [72].
[33]
MEASUREMENT OF LONG-CHAIN C o A
275
concentration of the reagents in this mixture must be altered according to the volume of sample to be hydrolyzed. The final reaction mixture after addition of sample contains the following concentration: 0.2 M phosphate, 1 mM EDTA, and 2 mM dithiothreitol. CoA Cycling Mixture I (SpectrophotometricAssay). The cycling mixture consists of 16 ml 0.1 M potassium phosphate buffer, pH 7.2, 1 ml 0.1 M L-malate, 0.2 ml 0.1 M NAD ~, 0.1 ml 0.05 M EDTA, 0.5 ml 0.02 M acetyl-DL-carnitine, 12.3 ml of distilled water, and 25/~l of malate dehydrogenase (1100 units/rag, 5 mg/ml). This mixture is prepared 10 minutes before the cycling assay and left at room temperature in order to equilibrate the malate dehydrogenase reaction. The reagents for the cycling assay must be neutralized with KOH and stored at --20 °. CoA Cycling Mixture II (Fluorometric Assay). For the fluorometric cycling assay, the spectrophotometric cycling mixture is modified as follows: 10 ml potassium phosphate buffer, 0.1 ml malate, 0.1 ml NAD +, 0.05 ml EDTA, 0.25 ml acetyl-DL-carnitine, 19.25 ml distilled water, and 20 t~l of malate dehydrogenase. Enzyme Mixture. Prior to use, mix gently 75 ~l of citrate synthase (70 units/mg, 2 mg/ml), 75 ~l of carnitine acetyltransferase (80 units/mg, 5 mg/ml), and 170 t~l of distilled water.
Procedure (Spectrophotometric Assay) Hydrolyses o] the Long-Chain Acyl-CoA. If the samples to be assayed are solutions that do not contain CoA or short-chain CoA derivatives or enzymes that interfere with the long-chain acyl-CoA determination (e.g., solutions of commercial long-chain acyl-CoA), incubate sample aliquots (2-5 nmoles of long-chain acyl-CoA) in 1 ml of the hydrolysis mixture and 0.04 unit of activated fatty acid synthase at 30 ° for 40 minutes in a shaking bath. If the concentration of long-chain acyl-CoA in the sample is very low and large volumes of sample are used for hydrolyses, a smaller volume of a more concentrated hydrolysis mixture should be used. After incubation, place the tubes in ice, add 0.1 ml of 2 N HC1 into each tube, and neutralize with N KOH. Blanks are handled in the same way except that the addition of fatty acid synthase is omitted. The use of a blank has two aims: (1) to detect any CoA or CoA derivative (e.g., acetyl-CoA) not hydrolyzed by fatty acid synthase that could react during the CoA cycling procedure, and (2) to allow a mixture similar to that used in the assay mixture to be added to the CoA standards in order to correct for any activator or inhibitor of the CoA cycling reactions present in the samples. If the material to be analyzed contains CoA or short acyl-CoA deriva-
276
GENERAL ANALYTICAL METHODS
[33]
tives (less than twelve carbons), a separation of these compounds from long-chain aeyl-CoA is possible with perehloric acid. 1~ If rat tissues are going to be assayed, homogenize pulverized frozen tissues in preweighed centrifuge tubes (1 g of brain of fed or starved rat, 1 g of liver of fed rat, and 0.4 g of 48 hour starved rat) with 8 ml of 0.85 M perehlorie acid. The frozen tissues can be stored at --70 ° without any loss of longchain acyl-CoA. Centrifuge the homogenates at 35,000 g and 0 °, for 15 minutes. Discard the supernatant and treat the pellet twice more with perehloric acid in order to remove adherent CoA or short-chain CoA. Weigh the tube plus the pellet and calculate the amount of liquid retained in the pellet: weight of tube plus pellet minus tube weight minus dry weight of pellet equals liquid in pellet. Add to the pellet 10 ml of the hydrolysis mixture, homogenize, and neutralize with KOH. For optimum results the amount of long-chain CoA should be from 2 to 5 ~ in this solution. For calculations V1 is defined as milliliters of volume retained in the pellet plus milliliters of hydrolysis mixture plus milliliters of KOH added. Mix very thoroughly and pipette 1 ml of the suspension (V~) into another centrifuge tube. Add fatty acid synthase (0.04 unit), mix, and incubate at 30 ° for 40 minutes. After the incubation, place the samples in ice, treat with 0.1 ml of 2 N HC1, and centrifuge at 35,000 g at 0 ° for 15 minutes (Va = V., 4-HC1 added). Pipette an aliquot sample of the supernatant (V~) and neutralize with N KOH (V~ = V4-4-KOH added). The sample is ready for cycling but can be stored at --70 °. Since the sample volumes are too small to be neutralized using a common pH meter, the amount of KOH necessary for neutralization is calculated from the preparation of tissue blank. For the preparation of the tissue blank, the remaining suspension (ca. 9.5 ml) left after pipetting 1 ml for hydrolysis of long-chain aeyl-CoA is treated with one-tenth of its volume of 2 N HC1 and centrifuged at 35,000 g at 0 ° for 15 minutes. Neutralize a measured volume of the supernatant with N KOH. The volume of KOH used is noted and from it the volume of KOH necessary to neutralize small volumes of samples can be calculated. The tissue blank is then ready for cycling. Measurement of the CoA Formed. Pipette 2.5 ml .of the CoA cycling mixture I into a series of 1 cm light path speetrophotometer cuvettes: 1 (zero reference); 5 (CoA standard curve) and the number of euvettes necessary for the samples and the tissue blank of each sample. For the CoA standard curve, pipette 5, 10, 15, 20, and 25 ~1 (Std,, Std~ . . . Std.~) of the diluted CoA standard and 50 ul of a blank of tissue and mix. If tissues with very different composition (such as protein content) are assayed, a CoA standard curve with the tissue blank of each type of tissue should be used. Add 50 ~l of each tissue blank (B~, B ~ , . . . B~) or sample
[33]
MEASUREMENT OF LONG-CHAIN C o A
277
($1, S ~ , . . . S,) (for calculations, defined as V6) to the group of cuvettes and mix. R e a d the cuvettes at 340 nm. Cycling reactions are initiated at timed intervals by the addition of 25 ~l of the enzyme mixture (CS and C A T ) . Leave the cuvettes in such a w a y t h a t all of them have the same temperature. In our experiments we have used a 10-cell cuvette holder, adapted for readings with the spectrophotometer Zeiss P M Q II. The cuvettes are read 30, 45, 60 minutes, etc., after initiation of the cycling reaction. For calculations AE1 = ±E of sample minus ±E of respective tissue blank; AE2 = AE of standard whose reading is the closest to the sample reading minus AE of the blank used. Figure 1 presents
0"2001 73 rnin
E
E
o re) c3
40rain
--- 0.100 c
0
27 min
o
Iv
,~
I
I
I
I
I0
15
20
25
p.l of CoA standard (9.48p, M)per cuvette FIG. 1. Plot of the volumes of diluted CoA standard against the E~. (spectrophotometry) obtained for each volume of standard. For conditions of experience see text. The time of readings are indicated.
278
[33]
GENERAL ANALYTICAL METHODS
a CoA standard curve used for determination of long-chain acyl-CoA in liver of fed rats. The concentration of long-chain acyl-CoA, expressed as mieromoles per gram wet weight tissue, is equal to: [diluted standard CoAl X volume standard (Stdl, Std2, Std3, Std4, or Stds) ×
hE~ total volume in euvette (sample) × hE2 × V6 total volume in euvette (standard) V5
V3
X
Vl
tissue w t g
For calculations all the volumes are expressed as milliliters.
Procedure (Fluorometric Assay) For measurement of smaller concentrations of long-chain acyl-CoA the fluorometric method is preferred. The standard curve was obtained in the following way: 1 ml of cycling mixture II was pipetted into fluorometric tubes and I, 5, 10, 15, 20, and 25/~i of the diluted CoA standard (4.25/~M) were added. After equilibration of the temperature, the tubes were read using a ratio fluorometer supplied by the Farrand Optical Company. Cycling reactions were initiated at timed intervals by the addition of 5/~i of the enzyme mixture (CS and CAT).
Remarks The method allows the estimation of a wide range of amounts of longchain acyl-CoA. There are three steps in the method that allow this flexibility: I. In the first step by allowing various amounts of long-chain acylCoA solution or varying amounts of tissue to be used. In our experiments similar results were obtained when amounts of a pooled rat liver powder from 0.4 to 2 g were treated with 8 ml of perchloric acid. 2. In the second step by using various volumes of hydrolysis mixture for diluting the solution of long-chain acyl-C~)A or making the suspension bf a tissue. For hydrolyses, concentrations of long-chain acyl-CoA between 2 and 5 ~ were used. Although concentrations higher than 5/~M cannot be used because of micelle formation, in our studies concentrations smaller than 2/~M were not assayed. 3. In the third step by using spectrophotometric or fluorometrie methods.
MEASUREMENT OF LONG-CHAIN C o A
273
[33] Enzymatic Determination of Long-Chain Fatty Acyl-CoA B y D. VELOSO and R. L. VEECH
The method consists of two main steps: enzymatic hydrolysis of longchain acyl-CoA (fatty acyl-CoA + H 2 0 - ~ C o A ~- f a t t y acid) and subsequent determination of the CoA produced. Several enzymes have been reported to release CoA from long-chain acyl-CoA; viz., pigeon l and r a t 2 liver f a t t y acid synthase, pig brain pahnityl-CoA deacylase, ~ Escherichia coli palmityl thioesterases, 4,5 rat liver microsome acyl-CoA hydrolase, G and pancreatic lipase7 R a t liver f a t t y acid synthase can be prepared easily in a highly pure form 2 and offers a convenient basis for the assay of long-chain acyl-CoA. F a t t y acid synthase hydrolyzes palmityl- and stearyl -s and oleyl-CoA2 Its action on other long-chain acylCoA is not known. The concentration of long-chain acyl-CoA in the samples to be hydrolyzed should be below 5 t~M to avoid errors resulting from micelle f o r m a t i o n Y This low concentration of long-chain CoA necessitates the use of a cycling technique in order to measure the concentration conveniently in a spectrophotometer. The CoA released from longchain acyl-CoA is measured with an enzymatic cycling procedure using the reactions catalyzed by carnitine acetyltransferase [acetyl-CoA:carnitine 0-acetyltransferase, EC 2.3.1.7] (CAT) and citrate synthase [EC 4.1.3.7] (CS). The oxalacetate used in the citrate synthase reaction is generated by the malate dehydrogenase reaction [ L - m a l a t e : N A D oxidoreductase, EC
CS
CAT
I S. Kumar, J. A. Dorsey, R. A. Muesing, and J. W. Porter, J. Biol. Chem. 245, 4732 (1970). 2S. Kumar, G. T. Phillips, and J. W. Porter, Int. J. Biochem. 3, 15 (1972). 3p. A. Srere, W. Seubert, and F. Lynen, Biochim. Biophys. Acta 33, 313 (1959). 4E. M. Barnes, Jr., and S. J. Wakil, J. Biol. Chem. 243, 2955 (1968). 5E. M. Barnes, Jr., A. C. Swindell, and S. J. Wakil, J. Biol. Chem. 245, 3122 (1970). 6 R. E. Barden and W. W. Cleland, J. Biol. Chem. 244, 3677 (1969). 7E. D. Barber and W. E. M. Lands, Bioehim. Biophys. Acta 250, 361 (1971). 8 G. T. Phillips, J. E. Nixon, J. A. Dorsey, P. H. W. Butterworth, C. J. Chesterton, and J. W. Porter, Arch. Biochem. Biophys. 138, 380 (1970). D. Veloso and R. L. Veech, in preparation.
274
GENERAL ANALYTICAL METHODS
[33]
1.1.1.37] establishing a connection between the enzymatic cycling reactions and a N A D H formation system: CoA + acetylcarnitine ~ acetyl-CoA + carnitine Acetyl-CoA + H20 + oxalacetate 2- ~ CoA + citrate 3- + H + Malate 2- + N A D + ~-- oxalacetate ~- + N A D H + H + For the CoA recycling to proceed, the formation of more oxalacetate is necessary and consequently more N A D H is formed. The rate of formation of N A D H concentration can be followed in a spectrophotometer (340 nm) or fluorometer. Similar to the enzymatic cycling procedure for measurement of pyridine nucleotides) ° the concentrations of CoA and acetylCoA must be below their K.,~ in order that the reaction rates be dependent on their concentrations. Coenzyme A is the standard for the cycling procedure because no reliable long-chain acyl-CoA preparations are commercially available.
Reagents F a t t y Acid Synthase. The enzyme is partially purified from rat l i v e r " and is suitable for use after dialysis of the second a m m o n i u m sulfate precipitate. F a t t y acid synthase activity is determined according to Nepokroeff et al. 11 and expressed as 1 unit = 1 t~mole malonyl-CoA used per minute per milligram of protein. Before use, the enzyme is activated by mixing it with an equal volume of a mixture of 0.8 M potassium phosphate buffer, p H 7.0, 0.02 M dithiothreitol, and 1.2 m M E D T A and incubating the mixture at 3.0° for 30 minutes. The diluted activated enzyme can be stored at - - 7 0 ° and reactivated once by immersion in a water bath (30 ° ) for 30 minutes. Coenzyme A Standard. Prepare a 3 m M solution of CoA in 10 m M dithiothreitol, standardize, and store in small portions at --20% Each tube should not be thawed more than 3 times. The concentration of CoA in the standard is determined by the method of Tubbs and Garland, 1-~ with 3-acetyl pyridine adenine nucleotide as coenzyme. Prior to use, dilute the enzyme 300-fold (spectrophotometry) or 750-fold (fluorometry) with a 1 m M dithiothreitol solution. Hydrolysis Mixture. This mixture, prepared according to K u m a r et al. 1 contains 10 ml of 1 M potassium phosphate buffer, p H 6.8, 1 ml 0.05 M E D T A , 1 ml 0.1 M dithiothreitol, and 38 ml of distilled water. The
lo0. H. Lowry, J. V. Pa~onneau, D. W. Schulz, and M. K. Rock, d. Biol. Chem. 236, 2746 (1961). 1~C. M. Nepokroeff, M. R. Lakshmanan, and J. W. Porter, this volume [6]. 12p. K. Tubbs and P. B. Garland, Vol. 13 [72].
[33]
MEASUREMENT OF LONG-CHAIN C o A
275
concentration of the reagents in this mixture must be altered according to the volume of sample to be hydrolyzed. The final reaction mixture after addition of sample contains the following concentration: 0.2 M phosphate, 1 mM EDTA, and 2 mM dithiothreitol. CoA Cycling Mixture I (SpectrophotometricAssay). The cycling mixture consists of 16 ml 0.1 M potassium phosphate buffer, pH 7.2, 1 ml 0.1 M L-malate, 0.2 ml 0.1 M NAD ~, 0.1 ml 0.05 M EDTA, 0.5 ml 0.02 M acetyl-DL-carnitine, 12.3 ml of distilled water, and 25/~l of malate dehydrogenase (1100 units/rag, 5 mg/ml). This mixture is prepared 10 minutes before the cycling assay and left at room temperature in order to equilibrate the malate dehydrogenase reaction. The reagents for the cycling assay must be neutralized with KOH and stored at --20 °. CoA Cycling Mixture II (Fluorometric Assay). For the fluorometric cycling assay, the spectrophotometric cycling mixture is modified as follows: 10 ml potassium phosphate buffer, 0.1 ml malate, 0.1 ml NAD +, 0.05 ml EDTA, 0.25 ml acetyl-DL-carnitine, 19.25 ml distilled water, and 20 t~l of malate dehydrogenase. Enzyme Mixture. Prior to use, mix gently 75 ~l of citrate synthase (70 units/mg, 2 mg/ml), 75 ~l of carnitine acetyltransferase (80 units/mg, 5 mg/ml), and 170 t~l of distilled water.
Procedure (Spectrophotometric Assay) Hydrolyses o] the Long-Chain Acyl-CoA. If the samples to be assayed are solutions that do not contain CoA or short-chain CoA derivatives or enzymes that interfere with the long-chain acyl-CoA determination (e.g., solutions of commercial long-chain acyl-CoA), incubate sample aliquots (2-5 nmoles of long-chain acyl-CoA) in 1 ml of the hydrolysis mixture and 0.04 unit of activated fatty acid synthase at 30 ° for 40 minutes in a shaking bath. If the concentration of long-chain acyl-CoA in the sample is very low and large volumes of sample are used for hydrolyses, a smaller volume of a more concentrated hydrolysis mixture should be used. After incubation, place the tubes in ice, add 0.1 ml of 2 N HC1 into each tube, and neutralize with N KOH. Blanks are handled in the same way except that the addition of fatty acid synthase is omitted. The use of a blank has two aims: (1) to detect any CoA or CoA derivative (e.g., acetyl-CoA) not hydrolyzed by fatty acid synthase that could react during the CoA cycling procedure, and (2) to allow a mixture similar to that used in the assay mixture to be added to the CoA standards in order to correct for any activator or inhibitor of the CoA cycling reactions present in the samples. If the material to be analyzed contains CoA or short acyl-CoA deriva-
276
GENERAL ANALYTICAL METHODS
[33]
tives (less than twelve carbons), a separation of these compounds from long-chain aeyl-CoA is possible with perehloric acid. 1~ If rat tissues are going to be assayed, homogenize pulverized frozen tissues in preweighed centrifuge tubes (1 g of brain of fed or starved rat, 1 g of liver of fed rat, and 0.4 g of 48 hour starved rat) with 8 ml of 0.85 M perehlorie acid. The frozen tissues can be stored at --70 ° without any loss of longchain acyl-CoA. Centrifuge the homogenates at 35,000 g and 0 °, for 15 minutes. Discard the supernatant and treat the pellet twice more with perehloric acid in order to remove adherent CoA or short-chain CoA. Weigh the tube plus the pellet and calculate the amount of liquid retained in the pellet: weight of tube plus pellet minus tube weight minus dry weight of pellet equals liquid in pellet. Add to the pellet 10 ml of the hydrolysis mixture, homogenize, and neutralize with KOH. For optimum results the amount of long-chain CoA should be from 2 to 5 ~ in this solution. For calculations V1 is defined as milliliters of volume retained in the pellet plus milliliters of hydrolysis mixture plus milliliters of KOH added. Mix very thoroughly and pipette 1 ml of the suspension (V~) into another centrifuge tube. Add fatty acid synthase (0.04 unit), mix, and incubate at 30 ° for 40 minutes. After the incubation, place the samples in ice, treat with 0.1 ml of 2 N HC1, and centrifuge at 35,000 g at 0 ° for 15 minutes (Va = V., 4-HC1 added). Pipette an aliquot sample of the supernatant (V~) and neutralize with N KOH (V~ = V4-4-KOH added). The sample is ready for cycling but can be stored at --70 °. Since the sample volumes are too small to be neutralized using a common pH meter, the amount of KOH necessary for neutralization is calculated from the preparation of tissue blank. For the preparation of the tissue blank, the remaining suspension (ca. 9.5 ml) left after pipetting 1 ml for hydrolysis of long-chain aeyl-CoA is treated with one-tenth of its volume of 2 N HC1 and centrifuged at 35,000 g at 0 ° for 15 minutes. Neutralize a measured volume of the supernatant with N KOH. The volume of KOH used is noted and from it the volume of KOH necessary to neutralize small volumes of samples can be calculated. The tissue blank is then ready for cycling. Measurement of the CoA Formed. Pipette 2.5 ml .of the CoA cycling mixture I into a series of 1 cm light path speetrophotometer cuvettes: 1 (zero reference); 5 (CoA standard curve) and the number of euvettes necessary for the samples and the tissue blank of each sample. For the CoA standard curve, pipette 5, 10, 15, 20, and 25 ~1 (Std,, Std~ . . . Std.~) of the diluted CoA standard and 50 ul of a blank of tissue and mix. If tissues with very different composition (such as protein content) are assayed, a CoA standard curve with the tissue blank of each type of tissue should be used. Add 50 ~l of each tissue blank (B~, B ~ , . . . B~) or sample
[33]
MEASUREMENT OF LONG-CHAIN C o A
277
($1, S ~ , . . . S,) (for calculations, defined as V6) to the group of cuvettes and mix. R e a d the cuvettes at 340 nm. Cycling reactions are initiated at timed intervals by the addition of 25 ~l of the enzyme mixture (CS and C A T ) . Leave the cuvettes in such a w a y t h a t all of them have the same temperature. In our experiments we have used a 10-cell cuvette holder, adapted for readings with the spectrophotometer Zeiss P M Q II. The cuvettes are read 30, 45, 60 minutes, etc., after initiation of the cycling reaction. For calculations AE1 = ±E of sample minus ±E of respective tissue blank; AE2 = AE of standard whose reading is the closest to the sample reading minus AE of the blank used. Figure 1 presents
0"2001 73 rnin
E
E
o re) c3
40rain
--- 0.100 c
0
27 min
o
Iv
,~
I
I
I
I
I0
15
20
25
p.l of CoA standard (9.48p, M)per cuvette FIG. 1. Plot of the volumes of diluted CoA standard against the E~. (spectrophotometry) obtained for each volume of standard. For conditions of experience see text. The time of readings are indicated.
278
[33]
GENERAL ANALYTICAL METHODS
a CoA standard curve used for determination of long-chain acyl-CoA in liver of fed rats. The concentration of long-chain acyl-CoA, expressed as mieromoles per gram wet weight tissue, is equal to: [diluted standard CoAl X volume standard (Stdl, Std2, Std3, Std4, or Stds) ×
hE~ total volume in euvette (sample) × hE2 × V6 total volume in euvette (standard) V5
V3
X
Vl
tissue w t g
For calculations all the volumes are expressed as milliliters.
Procedure (Fluorometric Assay) For measurement of smaller concentrations of long-chain acyl-CoA the fluorometric method is preferred. The standard curve was obtained in the following way: 1 ml of cycling mixture II was pipetted into fluorometric tubes and I, 5, 10, 15, 20, and 25/~i of the diluted CoA standard (4.25/~M) were added. After equilibration of the temperature, the tubes were read using a ratio fluorometer supplied by the Farrand Optical Company. Cycling reactions were initiated at timed intervals by the addition of 5/~i of the enzyme mixture (CS and CAT).
Remarks The method allows the estimation of a wide range of amounts of longchain acyl-CoA. There are three steps in the method that allow this flexibility: I. In the first step by allowing various amounts of long-chain acylCoA solution or varying amounts of tissue to be used. In our experiments similar results were obtained when amounts of a pooled rat liver powder from 0.4 to 2 g were treated with 8 ml of perchloric acid. 2. In the second step by using various volumes of hydrolysis mixture for diluting the solution of long-chain acyl-C~)A or making the suspension bf a tissue. For hydrolyses, concentrations of long-chain acyl-CoA between 2 and 5 ~ were used. Although concentrations higher than 5/~M cannot be used because of micelle formation, in our studies concentrations smaller than 2/~M were not assayed. 3. In the third step by using spectrophotometric or fluorometrie methods.
