Fluorometric Procedure for Measuring the Activity of Dehydrogenases GEORGE G. GUILBAULT and DAVID N. KRAMER Defensive Research Division, Chemical Research and Development laboratories, Edgewood Arsenal, Md.
b A simple, rapid fluorometric method is described for measuring the activity of dehydrogenases. The method is based upon the conversion of the nonfluorescent material, resazurin, to the highly fluorescent compound, resorufin, in conjunction with the nicotinamide adenine dinucleotide-reduced nicotinamide adenine dinucleotide (NAD+-NADH) system. By the procedures described, approximately 0.00010 to 0.1000 unit per ml. of lactic acid dehydrogenase (LDH), alcohol dehydrogenase (ADH), malic acid dehydrogenase (MADH), glutamic acid dehydrogenase (GADH), glucose-6phosphate dehydrogenase (G6PDH), L-a-glycerophosphate dehydrogenase (GPDH), and glycerol dehydrogenase (GDH) may b e determined with standard deviations of approximately =t1%. Likewise, diaphorase, 0.000400 to 0.0800 unit per ml., resazurin, 10-8 to lO-jM, and NADH 2 X lo-' to 2 X 1 O-5M, may b e determined with standard deviations of & O S , 0.8, and 0.570, respectively.
A
CLASS of enzymes are the dehydrogenases, which in the presence of a hydrogen acceptor such as nicotinamide adenine dinucleotide (SXD), effect the dehydrogenation of hydrosy compounds. h typical dehydrogenase, lactic acid dehydrogenase (LDH) , is responsible for the formation of lactic acid in glycolysis and for the oxidation of lactic acid in respiration. It is found in all glycolysing cells. Recently, n-e described a method for the determination of L D H (@, based upon the conversion of resazurin to t,he highly fluorescent resorufin. I n the present paper, a full description of this technique is given. I n addition, the application of this procedure to the determination of other dehydrogenases, diaphorase, S X D , N-LDH, ?;.1DPH, and resazurin is given. N niPoRT.mT
EXPERIMENTAL
Reagents. EXZYMES.Malic acid dehydrogenase ( N A D H ) , activity 670 enzyme units per mg. of protein (Calbiochem. Co., Los Angeles). One unit of activity is t h a t which causes a n initial rate of reduction of 1 pmole of KAD+ per minute a t 29" C. (Xote: for standard conditions of assay for
each enzyme, see any reference on methods of enzyme assay, such as the Enzyme Reagents Catalog, Korthington Biochemical Co., sections 1.1 to 1.7). Glutamic acid dehydrogenase (GADH), activity 3.3 enzyme units per mg. of protein (Calbiochem. Co., activity defined as above). L-a-glycerophosphate dehydrogenase (GPDH), activity 55 units per mg. of protein (Calbiochem. Co.) activity defined as above. Glycerol dehydrogenase (GDH) , activity 5.0 units per mg. of protein (Worthington Biochem. Co., Freehold, N. J.), activity defined as above. Glucose-6-phosphate dehydrogenase (GGPDH), activity 170 enzyme units per mg. of protein (Calbiochem. Co.), obtained from yeast. One unit of activity is that which causes the reduction of 1 pmole of NADP per minute. Diaphorase, Type I1 (Sigma Chemical Co., St. Louis), activity 16 units per mg. One unit equals a decrease in absorbancy of 1.00 per minute of 2,6dichloroindophenol a t 25" C. SVBITRATES. Resazurin (Eastman Organics Co., Rochester). -1stock 2 x 10-4M solution was prepared in methyl cellosolve. This solution should be nonfluorescent, with a bluish-red hue. A sample of resazurin that is contaminated with resorufin may be purified by acetylation of the compound with acetic anhydride and pyridine by conventional procedures ( I , 3 ) , followed by isolation of the ester in water and recrystallization. Addition of base to the ester will then give pure resazurin. Sodium Maleate, 0.1J1, and sodium lactate, 0.01J1, were prepared by dissolving the neutralized acid in 0.1J1 tris buffer, p H 9.0. Ethanol solution, 0.05.11, was prepared in 0.1JI tris buffer, p H 9.0. Sodium glutamate, 0.01.11. h stock solution was prepared by dissolving the purified compound in 0 . l M tris buffer, pH 8.0. Glucose-6-phosphate. h 10-3.11 solution was prepared in 0.1JI tris buffer, p H 8.5, with the purified material (Calbiochem. Go., 80.6YGpure). Glycerol and L ( -)-a-glycerol phosphate (Calbiochem. Co.). 10-331 solutions were prepared in 0 . 1 X tris buffer, p H 9.0. Sicotinamide Adenine Dinucleotide (SAD+) and its reduced form (KADH), obtained from Calbiochem. Co., were found to be 86.3 and 80.3YG pure. Stock 10-*.1.1 solutions were prepared in triply distilled water.
Sicotinamide adenine dinucleotide phosphate and its reduced form, N A D P + and NADPH. Stock 10-3111 solutions were prepared by dissolving the compound (Calbiochem. Co., 70.1 and 68.7% pure) in triply distilled water. Apparatus. All fluorescence measurements were made with a n XniincoBowman spectrophotofluorometer (SPF) , equipped with a thermoelectric cooler to maintain a constant temperature a t 29' C. T h e resorufin produced was measured a t excitation and emission wavelengths of 560 and 580 mp, respectively. Although the excitation and emission wavelengths are close together, scattered radiation was not found to be a problem, as was pointed out in previous papers (3, 6). KOblank correction was needed. Procedures. DETERMINATION OF DEHTDROGEKASES. Two milliliters of t h e appropriate substrate (sodium maleate for IZXADH, sodium glutamate for G X D H , etc.) of t h e concentration and pH specified in t h e reagents section, 0.1 ml. of 2 X 10-4M N A D + (?J;\DP+ in the determination of G-6-PDH), 0.1 ml. of 2 X 10-4AU resazurin, and 1 ml. of diaphorase (0.08 unit) are placed in the fluorescence cell in the SPF, and the instrument is adjusted to read zero. At zero time, 0.1 ml. of the solution of the dehydrogenase to be analyzed (containing approximately 0.0001 to 0.50 unit per ml.) is then added, and the change in fluorescence with time, AF/AL, is automatically recorded a t the excitation and emission wavelengths given. From calibration plots of AF'lniinute us. concentration, the quantity of the dehydrogenase present in solution is calculated. There should be no blank in this procedure-Le., no increase in fluorescence with time wit,hout the dehydrogenase added. If such an increase is observed, fresh solutions of the reagents, especially the NAD + and diaphorase, should be prepared. DETERMINATION O F DIAPHORASE. TO 3 ml. of a 2 X 10-4LUsolution of N A D H in tris buffer, O . l . l l , p H 8.0, is added 0.1 ml. of 2 X 10-4.V resazurin, and the instrument is adjusted to read zero fluorescence a t 560 and 580 mp. At zero time; 0.1 ml. of the unknown diaphorase solution is added (containing 0.001 to 0.10 unit). The amount of diaphorase present is then calculated from calibration curves of AF/At us. diaphorase concentration. DETERMINATION OF NADH. Three milliliters of N A D H to l O - 5 X ) VOL. 37, NO. 10, SEPTEMBER 1965
1219
Table I.
Determination of Various Dehydrogenases
LDH, units/ml. Error, Present Found % 0.000303 $1.0 0.000300 -2.0 0,000500 0.000490 0.0 0.00100 0.00100 $0.6 0.00500 0.00503 0.00990 -1.0 0.0100 f0.4 0.0500 0.0502 +l.O 0.100 0.101 Std. dev. f 1 1 I
hIADH, units/ml.
Present 0,00105 0.00450 0,0105 0.0450
0.108 0.223 0.510
Found 0.00104 0,00457 0.0105
%
-1.0 3-1.5 0.0
-0.9 0.0446 -1.0 0.107 +1.8 0.227 -0.2 0,509 Std. dev. 1 1 . 4
ADH, units/ml.
Present 0.000303 0.000606 0.00151 0.00606 0.0303 0.0606 0.151
Error,
Found 0,000301 0,000610 0.00149 0.00606 0.0302 0.0608
0.153 Std. dev.
Error,
% -0.7
$0.7
-1.3
0.0
-0.3 +0.4 $1.3
h0.8
GADH, units/ml. Error, Present Found 70 $1.0 0,000103 0.000104 -0.7 0,000330 0.000328 +0.5 0.000670 0.000673 0.0 0.00165 0.00165 $1.0 0.00330 0,00330 -0.6 0.0165 0.0164 Sl.5 0.0330 0.0335 Std. Dev. f 0 . 9 G-6-PDHJ unitslml. Error, Present Found 70 0.00202 0.00205 +1.5 0,00850 0.00850 0.0 0.0170 0.0169 -0.6 0.0430 0.0434 +1.0 +0.4 0.0850 0.0853 +1.7 0.170 0.173 -1.0 0.340 0.337 Std. dev. f l . 1
GPDH, unitslml. Present Found 0.01 0.5 0.0104 O.OZi0 0.0208 0.0550 0,0553 0.110 0.112 0.220 0.220 0.550 0.551 1.10 1.11 Std. dev.
Error,
% -1.0
-1.0 +0.5 +1.9
0.0
1-0.2
+0.9 h1.l
GDH, units/ml. Error, Present Found % + 0 .2 0.00501 0.00500 0.0111 +o. 9 0.0110 -1.5 0.0335 0.0340 0.0504 -0.2 0,0505 0,101 +1.0 0.100 0.504 -0.2 0.505 Std. dev. f 0 . 9
1220
ANALYTICAL CHEMISTRY
in 0.lM tris buffer, p H 8.0, is placed into a fluorescence cell in the SPF. One tenth of a ml. of 2 x 10-411f resazurin, and 0.1 ml. of diaphorase (0.08 unit) are added, and the change in fluorescence with time is recorded. The concentration of NADH is then calculated from calibration curves as above. DETECTIONAND DETERMINATION OF RESAZURIN. A 0.1-ml. solution of the unknown dye is added to 3 ml. of a 2 X lO-4M NADH solution containing 0.1 ml. of diaphorase (0.08 unit). If a red fluorescence is produced, with excitation and emission wavelengths at 560 and 580 mp, resazurin is present. From calibration plots of AF/At us. fluorescence, the concentration of resazurin (lo-* to 10-51V) may be calculated. RESULTS
The results of the determination of LDH, ADH, LIADH, GADH, G-6PDH, G D P H , and G D H are given in Table I. All results are based upon five or more determinations. By the procedures described, 0.000300 to 0.100 unit per ml. of LDH, 0.000303 to 0.151 unit per ml. of ilDH, 0.00105 to 0.510 unit per ml. of MADH, 0.0$103 to 0.0330 unit per ml. of GADH, 0.00202 to 0.340 unit per ml. of G-6-PDHJ 0.0105 to 1.10 unit per ml. of G P D H , and 0.00500 to 0.105 unit per ml. of G D H may be determined with standard deviations of =kl,l, 0.8, 1.4, 0.9, 1.1, 1.1, and 0.9%, respectively. Likewise, diaphorase, 0.000400 to 0.0800 unit per ml., resazurin, IO-* to 10-5M, and NADH, 2 X lo-’ to 2 X lO-5JI, may be determined with standard deviations of k0.5,0.8, and 0.5%, respectively (Table 11). DISCUSSION
The recent research in enzyme assay has been directed toward two immediate ends, to replace the long and tedious procedures required in most previous assays with simple, rapid measurements, and to use more sensitive procedures in all analyses. These have been our goals in the development of the present procedure for dehydrogenases. The previous indirect, lengthy methods (7, 8 ) have been replaced with the rapid measurement of the initial rate of production of resorufin, AF/At, which is a measure of the concentration of the enzymes present. Since resorufin is a highly fluorescent compound(fluorescence coefficient = 1.56 X lo1, compared to 1.40 X lo6 for quinine sulfate in 0.1N HPS04),the method is extremely sensitive, and small concentrations of enzymes may be determined unit). The fluorescence coefficient is defined as the fluorescence in units observed on the S P F divided by the concentration in moles per liter. See reference (4) for a discussion of this constant.
To ble II. Determination of Diaphorase, Resazurin, and NADH
Diaphorase, units/ml. Present Found 0.000400 0.000404 0.000800 0.000802 0,00400 0.00397 0.00800
0.00798
0.0800
0.0800
0.0200 0.0400
0,0202 0.0402
Std. dev. Resazurin, M X lo7 Present Found 0.100 0.101 0.300 0.302 1.50 1.48 3.00 3.01 6.00 5.97 12.0 12.0 60.0 60.6 Std. dev. NADH, M X lo6 Present Found 0.200 0.202 0,500 0.501 1.00 1.00 3.00 3.02 5.00 5.03 10.0 9.90 20.0 20.1 Std. dev.
Error,
70 +1.0 $0.2 -0.7 -0.2 +1.0 $0.5
0.0 f0.5
Error,
70
+1.0 +0.7 -1.3 +0.3 -0.5 0.0 +1.0 lt0.8 Error,
70
+1.0 +0.2 0.0
$0.7 +O. 6 -1.0
+0.5 f0.5
Since the rate of production of resorufin is proportional to the Concentration of diaphorase, NAD+, and resazurin, as well as to the dehydrogenases, these materials may be determined by this procedure. A method is not described for N d D + , since S X D H is a serious interference in attempts to quantitatively determine this coenzyme. If NADH is known to be absent (see below), N A D + in concentrations of to 10-41M may be determined by this procedure. Likewise, the concentration of NADH in N A D + can be accurately determined by this procedure. Also, this method provides a specific qualitative, as well as quantitative, test for resazurin. None of the reagents used will give a red fluorescence without resazurin. il search of the literature revealed that no other compound yields a red fluorescence in its reduced state, and is nonfluorescent in its oxidized state. The criterion of excitation and emission a t 560 and 580 mp, further imposes specificity but is not essential. Since glucose-6-phosphate dehydrogenase is active with the coenzyme XADPf and not N h D + , S . I D P + was used in the determination of this dehydrogenase. A concentration of 6.7 x 10-6Jf(O.l ml. of 2 X 10-4M added in the procedure) was optimum, and in the absence of NADPH, 5 x 10-6 t o 5 x lO-*Jf concentrations of NADP + produced a linear change in the rate of reaction, AF per minute, and hence can be determined.
For quantitative determination of diaphorase and resazurin, only the latter part of the coupled reaction was usedresazurin diaphorase. i.e., S A D H At a NADH concentration of 6.7 X 10-+3X,the rate of production of resorufin, AF per minute, is proportional to the concentration of diaphorase and resazurin with good accuracy and reproducibility (Table I I). At diaphorase and resazurin concentrations of 0.08 unit and 6.7 x 10-6M, the rate of reaction is proportional only to the concentration of NADH. More data on the kinetics of this reaction can be found in another publication(5). The application of this procedure to the determination of other dehydrogenases should be possible. Any enzymic reaction capable of reducing KAD + or K A D P + to NADH or K A D P H should be measurable in this system. Stability of Reagents. T h e stock resazurin solution, 2 X 10-4X in methyl cellosolve, is stable for a t least a
+
+
year, and the NA4D+and NADH solutions are stable for a week when stored at 5' C. The substrate solutions are stable for months a t room temperature. The only unstable reagent is diaphorase, and solutions must be prepared fresh each day. T h e calibration plots, however, need not be repeated daily, provided they are initially determined with a n enzyme solution that was freshly prepared. Effect of pH and Substrate Concentration. T h e rate of reaction is proportional t o t h e concentration of substrate used-Le., sodium lactate, etc.a t low concentrations. Generally, t h e rate becomes independent of t h e concentration of substrate a t concentrations > 1 O - * M , and this maximum concentration was used in the analysis (see Reagents for the concentration of each substrate used). Since the rate of all these reactions is higher a t pH's of 8-9 (the reverse reaction becoming predominant a t lower pH's), and since
the fluorescence of resorufin is also optimum a t p H 8-9 (S), this approximate range was used in all analyses. The p H optimum for each reaction is given in the Reagents section under the individual substrate. LITERATURE CITED
(1) Guilbault, G. G., Kramer, D. N.,
ANAL.CHEM.36,409 (1964). ( 2 ) Zhid., p. 2497. ( 3 ) Guilbault, G. G., Kramer, D. N., Zhid., 37,120 (1965). ( 4 ) Guilbault, G. G., Kramer, D. N.,
Edgewood Arsenal, Md., unpublished data, 1965. ( 5 ) Guilbault, G. G., Kramer, D. N., Goldberg, P., J . Phys. Chem., in press. ( 6 ) Kramer, D. N., Guilbault, G. G., ANAL.CHEM. 36. 1662 (1964). - - , (7) Lowry, 0. H.,'Roberts, N. R., Chang, M . , J . Biol. Chem. 222,97 (1956). (8) Lowry, 0. H., Roberts, N. R., Kapphahn, J. I., Zhid., 224, 1047 (1957). RECEIVEDfor review April 21, 1965. Accepted July 7, 1965. Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Pittsburgh, Pa., March 1965. \
A Constriction Response Analyzer and Its Application to Continuous Uranium Hexafluoride Determination C. W. WEBER
and W. S. PAPPAS
Technical Division, Oak Ridge Gaseous Diffusion Plant, Union Carbide Corp., Nuclear Division, Oak Ridge, Tenn. A continuous nondestructive analyzer which responds pneumatically to changes in density and viscosity of the fluid passing sequentially through an orifice and a capillary has been developed. It provides a pneumatic signal directly applicable to stream analysis and to feedback control systems. Good agreement with chemical analyses was obtained where applied to the determination of uranium hexafluoride in complex, corrosive, gas mixtures. A sensitivity of 0.03 mole % uranium hexafluoride in nitrogen was achieved, with a standard deviation of =t0.03 mole %. Little maintenance was required during a field application period exceeding three years.
C
and automatic control of the composition of gaseous and liquid systems are often required for efficient operation of modern chemical plants. This report describes a simple, low cost analyzer, responding to density and viscosity, that may fulfill some of those needs. The analyzer was designed especially for the continuous measurement of uranium hexafluoride in gas mixtures; its principles are applicable to any fluid system where the system value, density ONTINUOUS INDICATION
divided by the square of viscosity, varies with the concentration of the component sought. The authors recently reported another instrument for determining uranium hexafluoride, by the condensation pressure method ( 7 ) . While that analyzer was more specific for uranium hexafluoride, as applied to selected gas streams, the present constriction response approach has advantages: it responds more rapidly to marked, rapid shifts in uranium hexafluoride concentration; it handles a broader concentration range in a single instrument; since i t is based on different principles and does not require condensation of the constituent sought, its versatility of application is potentially greater; the instrument can be calibrated for any regions of sample pressure or flow, within the capabilities of the system components. THEORETICAL CONSlDERATlONS
If a constant flow of gas a t constant temperature and constant inlet pressure is passed through a n orifice which is operated noncritically, the pressure drop, AP, across the orifice varies with molecular weight ( 2 ) . I n the case where the orifice is operated critically, the forepressure will vary with the square root of the Gas Analysis.
rAP'l IAP1 p2 CAPILLARY
Pf
I ORIFICE
Figure 1. Series flow through a capillary and an orifice
molecular weight. If the flow passes through a capillary, selected to permit laminar flow, its pressure drop, AP', will be proportional to viscosity ( 2 ) . The relationships above indicate the responses that may be expected for a capillary or an orifice, independently applied when the flow is regulated by some device not affected by viscosity or molecular weight; volume displacement systems could be used for this purpose. For many analytical problems, however, a simplification is achieved by placing a capillary and an orifice in series (see Figure 1). The flow may be controlled by maintaining constant pressure conditions at one of the constrictions; the variable pressure at the other constriction serves as the analytical response. Actual flow may vary with concentration; but since identical flows pass through both restrictions, algebraic analysis reveals a useful relationship. For a flow restriction, at constant temperature, volumetric flow ( 2 ) is theoretically proportional to VOL. 37, NO. 10, SEPTEMBER 1965
1221
