AC Research
Articles Anal. Chem. 1997, 69, 1471-1476
Determination of Glucose in Blood Based on the Intrinsic Fluorescence of Glucose Oxidase Jose´ F. Sierra, Javier Galba´n,* and Juan R. Castillo
GEAS, Analytical Chemistry Department, Faculty of Sciences, University of Zaragoza, Zaragoza-50009, Spain
A method for the determination of glucose based on the alteration of the intrinsic fluorescence of glucose oxidase during the enzymatic reaction is proposed. Addition of glucose to a solution containing the enzyme does not immediately alter the fluorescence, which remains virtually constant over a given interval, after which it increases sharply and eventually levels off. The origin of the signal can be ascribed to the different fluorescence properties of the oxidized and reduced forms of the enzyme. Based on such differences, a mathematical model relating the concentrations of glucose, glucose oxidase, and oxygen in solution with the time at which the fluorescence change occurs was developed. The experimental results were consistent with the model’s predictions. The proposed method allows the determination of glucose over the range 5 × 10-4-2 × 10-2 M, with a precision of 2.1% as relative standard deviation. The method was used to determine the analyte in blood with good accuracy and precision. The determination of glucose is a key clinical analysis for diagnosing some metabolic disorders and controlling various food and biotechnological processing techniques. There are a wide variety of methods available for this purpose, prominent among which are those based on the enzymatic oxidation of the analyte with the enzyme glucose oxidase. From the previous studies, the mechanism of this reaction1 could be schematized as in Figure 1: glucose (G) reduces the FAD of glucose oxidase (GOx) to FADH2(GOxH2) with formation of gluconolactone (L), which is rapidly hydrolyzed to gluconic acid (AG); dissolved oxygen reoxidizes GOxH2 and produces H2O2 as a result. This last product is reconverted to O2 by the enzyme catalase. The species GOxH2L and GOxH2O2 in Figure 1 are reaction intermediates. This reaction is the basis for a host of determination methods for glucose,2 where the analyte is determined from the oxygen (1) Castner, J. F.; Wingard, L. B., Jr. Biochemistry 1984, 23 , 2203-2210. (2) Raba, J.; Mottola´, H. A. CRC Crit. Rev. Anal. Chem. 1995, 25, 1-42. S0003-2700(96)01132-8 CCC: $14.00
© 1997 American Chemical Society
Figure 1. Scheme of the enzymatic reaction. O2(s) and O2(i) denote the oxygen concentrations in the bulk solution and the interface, respectively. The following rate constants were determinated by Gibson et al.,11 at 13 °C, k1 ) 5500 M-1 s-1, k2 ) 3300 s-1, k3 ) 1,7 × 106 M-1 s-1, k4 ) 720 s-1.
uptake rate or the amount of H2O2 or AG produced by using amperometric measurements (potentiometric ones for AG) or an indirect procedure. Other methods modify the intrinsic properties of GOx. Thus, Degani and Heller3 used an enzyme electrode in which flavin residues were reoxidized by direct electron transfer between the enzyme and electrode via charge-transfer mediators bound to the enzyme. Trettnak and Wolfbeis4 developed a sensor based on the alteration of the fluorescence properties of flavin residues during its enzymatic reaction; the minimum determinable concentration is 450 mg/L, and the linear range very short (4501800 mg/L), which hinders application to many real samples. Most enzymes exhibit intrinsic fluorescence in the UV spectral region (at about 300-350 nm) that can be ascribed to amino acids (basically tyrosine and tryptophan). Some enzymes possess other types of fluorescence; thus, oxidases exhibit weak fluorescence in the visible zone by virtue of their flavin residues. The UV fluorescence is usually employed to derive information about the (3) Degani, Y.; Heller, A. J. Am. Chem. Soc. 1988, 110, 2615-2620. (4) Trettnak, W.; Wolfbeis, O. S. Anal. Chim. Acta 1989, 221, 196-203.
Analytical Chemistry, Vol. 69, No. 8, April 15, 1997 1471
enzyme configuration5,6 and bonding positions. However, we recently used changes in the fluorescence properties of cytochrome b2 to determine lactate by reaction with ferricyanide,7 thereby avoiding the shortcoming of the low molar absorptivity of these compounds or the need to couple the enzymatic reaction with another one. The ensuing method allows the determination of the analyte in fairly simple matrices such as milk samples. The fluorescence of other enzymes can also be used for determining lactate.8,9 In this work, we investigated the potential of using the intrinsic fluorescence of GOx for determining glucose. The method developed as a result is based on the differential fluorescence of the redox forms of the FAD bound to the enzyme and is thus applicable to any catalyzed reaction involving an enzyme of this type. The method, which involves very simple pretreatment, was applied to the determination of glucose in blood with good results. The theoretical study reported in this paper allows one to derive information on the origin of the analytical signal, as well as to determine some kinetic constants involved in the enzymatic reaction. In reality, we are introducing a new concept in this type of determination, making it applicable to any other enzymatic reaction using these types of enzymes (those containing FAD and possibly FMN), and making it unnecessary to know the analytical properties of coenzymes or analytes. EXPERIMENTAL SECTION Apparatus. All fluorescence measurements were made on a Perkin-Elmer LS-50 luminometer furnished with a sample compartment that allowed continuous stirring of the solution and temperature control (22 ( 1 °C) and with quartz cuvettes of 1 cm path length. A slit width of 5 nm was used for both excitation and emission. A Mikroprocessor OX-96 oxygen sensor from Macherey-Nagel Du¨ren (MN) was used. Molecular absorption measurements were performed on a Perkin-Elmer Lambda-5 spectrophotometer furnished with quartz cells of 1 cm path length. Ultrafree-MC, 1000 NMWL PTGC polysulfone filtration units of 400 µL free volume from Millipore were also employed. Reagents. A phosphate buffer of pH 6.5 was made fresh daily from 0.1 M H2KPO4 and 0.1 M HNa2PO4. Sigma G-7141 X-S Aspergillus niger (EC 1.1.3.4) GOx with an activity of 181 600 IU/g of solid was used to prepare solutions containing a preset amount of enzyme in the phosphate buffer; the solutions were kept in an ice bath during the tests. This commercially available reagent contains catalase. Sigma G-5250 β-D-(+)-glucose was also used to prepare solutions in distilled water that remained stable for several weeks. Procedure. The luminometer cuvette was filled with 1.6 mL of phosphate buffer and 200 µL of a GOx solution of 100 IU/mL. The mechanical stirrer was then started, and the fluorescence intensity was measured at 335 nm (with excitation at 278 nm). Next, 250 µL of a standard solution of glucose or the sample was added to the cuvette, and the fluorescence intensity was moni(5) Permyakov, A. Luminiscent Spectroscopy of Proteins; CRC Press Inc.: Boca Raton, FL, 1993; Chapter 4. (6) Lakowick, J. R. Topics in Fluorescence Spectroscopy, Vol. 2: Principles; Plenum: New York, 1991; Chapters 2 and 3. (7) Galban J.; de Marcos, S.; Castillo, J. R. Anal. Chem. 1993, 65, 3076-3080. (8) Galban, J.; de Marcos, S.; Segura, P.; Castillo, J. R. Anal. Chim. Acta 1994, 299, 277-284. (9) de Marcos, S.; Galban, J.; Castillo, J. R. Anal. Sci. 1995, 11, 233-238.
1472 Analytical Chemistry, Vol. 69, No. 8, April 15, 1997
Figure 2. Variation of the fluorescence intensity during the enzymatic reaction. Analytical conditions are pH 6.5 (phosphate buffer), [GOx]0 ) 5 IU/mL, [G]0 ) 95 mg/L, λex ) 278 nm, λem ) 334 nm. Top: full detected register. Bottom: time-scale expanded view of the highlighted time parameters.
tored. The time defined as tm - t0 (see below) was used as the analytical quantitation parameter. For the determination of glucose in blood, the sample was centrifuged at 1200g for 10 min, after which the supernatant was ultrafiltered at 4500g for 75 min. An aliquot of the resulting filtrate was subjected to the above-described procedure. RESULTS AND DISCUSSION Origin of the Analytical Signal. GOx is an oxidase and hence exhibits a very intense UV fluorescence (with an emission maximum at 334 nm and two excitation maxima at 224 and 278 nm), ascribed to tryptophan, as well as visible fluorescence (with an emission maximum at 520 nm and four excitation maxima at 206, 275, 375, and 460 nm), ascribed to the FAD and FADH2. The visible fluorescence is strongly quenched by adjacent amino acids, so a very high concentration is typically required for its detection (over 50 times higher than that needed to reveal its UV fluorescence), as previously found by other authors;10 as a result, the UV fluorescence is more useful for analytical purposes. Figure 2 shows the UV fluorescence signal obtained upon addition of glucose to a solution containing GOx. As can be seen, the fluorescence intensity initially remains constantsexcept for a small decrease resulting from dilutionsat a value I0; after some time tap, referred to as the “appearance time”, the fluorescence intensity increases gradually to a final value I1 at t1 that remains constant for some time and then decreases gradually again (t > t2). Changes in the fluorescence and tap are identical at both excitation wavelengths, of which we used 278 nm in this work. (10) Bastiaens, P. I. H.; Visser, A. J. W. G. In Fluorescence Spectroscopy: New Methods and Applications; Wolfbeiss, O. S., Ed.; Springer-Verlag: Heidelberg, 1993; Chapter 5.
Figure 3. Variation of the O2 concentration during the enzymatic reaction. Conditions: pH 6.5 (phosphate buffer), [GOx]0 ) 10 IU/ mL, [G]0 ) 50 mg/L. Note that the glucose concentration used is different from that employed in Figure 2, so the time parameters are not comparable. This is because of sensor sensitivity. Oxygen concentration increased continuously until the initial value, but only a part of this increase is shown.
The origin of the signal can only be established from the kinetics of the process. The authors11 who have studied these kinetics agree that the largest rate constant is that corresponding to the reoxidation of GOxH2 and the smallest one is the rate at which the solution takes up O2 from the surrounding atmosphere (named as mass transfer); however, reported values for such constants vary considerably, and some have not yet been reliably determined. A quantitative evaluation of the way the concentrations of the different species change during the reaction is quite important here. The continuous disappearance of glucose and formation of gluconolactone is accompanied by a somewhat more complex variation of the O2 concentration. Figure 3 shows such a variation as determined with the oxygen sensor. The changes can readily be accounted for on the basis of the kinetics of the process. Thus, taking into account the low enzyme concentrations used, the reaction initially involves a net uptake of O2, since the mass transfer rate is very low and catalase produces only onehalf of the oxygen consumed. As the reaction proceeds, the glucose concentration decreases, and so does the O2 uptake rate; therefore, the mass transfer rate becomes significant and eventually equals the oxygen uptake rate, so a steady state is reached, after which the mass transfer rate exceeds the uptake rate, and the dissolved O2 concentration gradually increases as a result. Regarding the concentration of the different enzyme forms, while the oxidation kinetics for species GOxH2 exceeds its production rate, the predominant species will be GOx; this statement relies both on the kinetic constants reported previously and on the fact that I0 is independent of the glucose concentration added at a given enzyme concentration. The variation of the GOxH2 concentration is somehow antiparallel to the change in the O2 concentration in solution; as the dissolved O2 concentration decreases, so does the rate of the step where GOx is regenerated. Also, by the time the O2 concentration decreases below a given level, such a step will be slow enough for GOxH2 to become the predominant species. The opposite process takes place when the O2 concentration starts to rise again by mass transfer. As shown below, the concentrations of species GOxH2O2 and GOxH2L remain at very low levels relative to those of the other two species throughout the process. (11) Gibson, Q. H.; Swoboda, B. E. P.; Massey, V. J. Biol. Chem. 1964, 239, 3927.
Based on the changes in the concentrations of the different species, we can put forward three different mechanisms to account for the fluorescence change observed in the GOx molecule: (1) Inner Filter. A differential internal filtering effect is noticed from some reagent or product, as previously observed in the determination of lactate.7 However, the molar absorptivities for the species involved are very low and quite similar, as confirmed by molecular absorption measurements. (2) O2 Quenching. If oxygen is assumed to have a quenching effect on the enzyme, then the variation of the fluorescence intensity must be a result of the above-described changes in the O2 concentration. The constant intensity observed prior to tap can be ascribed to the presence of fluorescent impurities masking the actual effect of O2 on the enzyme. However, the Stern-Volmer constant for the oxygen quenching on various enzymes6 never exceeds 20 M-1. Taking into account that the average concentration of dissolved O2 in distilled water is 2.2 × 10-4 M, as measured with an oxygen sensorsa value consistent with the saturation concentrationsthe maximum quenching one should expect is about 1%, which can never account for the intensity differences observed. In addition, if oxygen (over O2 saturation condition) or nitrogen (low [O2] condition) is bubbled over an enzyme solution (in the absence of reaction), the fluorescence intensity hardly changes, which confirms that the quenching effect of O2 on the enzyme fluorescence is negligible. (3) Energy Transfer. If energy transfer from tryptophan to GOx is stronger than that to GOxH2, then the intensity changes are consistent with the above-mentioned concentration changes in the two species. Experimental evidence for this assumption is difficult to obtain, but some facts are in support of this hypothesis: (a) I1 and I0 change with the GOx concentration (in UI/mL) as follows (the same glucose concentration was employed): I0 ) 24.8 + 13.43[GOx], r ) 0.998; I1 ) 23.2 + 19.06[GOx], r ) 0.998. Both intensities increase linearly with enzyme concentration, but the slopes of the two lines are different, so the fluorescent species must be different. (b) The molecular absorption spectra obtained at the reaction start and at the time I1 was measured suggest that the molar absorptivity of the enzyme increases above 278 nm, while the weak absorbance at 380 and 450 nm disappears. This result is consistent with reported findings that FAD exhibits several absorption maxima in that zones, whereas FADH2 does not. It should be noted that the absorbance increase observed is not correlated with the increase from I0 to I1, which suggests that GOx and GOxH2 also possess a different fluorescence quantum yield by virtue of conformational differences in the tryptophan moiety of the enzyme molecule, so energy transfer (from tryptophan to cofactor) is different in both species. (c) If the reaction is carried out after bubbling N2 or O2 over the cell, then tap increases with an increase in the O2 concentration, so such a time must be related to the oxygen uptake. Also, a comparison of Figures 2 and 3 reveals that intensity changes do not take place simultaneously with changes in the O2 concentration. For the above reasons, the variation of the signal observed during the process can be ascribed to this effect. Analytical Parameter and Mathematical Model. Based on the temporal variation of the fluorescence intensity in Figure 2, the determination of glucose can be accomplished by using two different parameters, viz., tap and the slope of the intensity rise Analytical Chemistry, Vol. 69, No. 8, April 15, 1997
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(dI/dt), since I1 was experimentally found to be independent of the glucose concentration. Appearance Time (Measuring Time). Based on the order of magnitude of the reported kinetic constants,1 if the steady state approximation is applied to species GOxH2O2, GOxH2L, and GOxH2 at a time t < tap, the concentrations of these intermediates are 500-1000 times lower than that of GOx and hence negligible. Therefore, before tap is reached, the enzyme is present as GOx; once the intensity rises and then levels off again (t1), the enzyme occurs in its reduced form. Therefore,
I0 ) KGOx[GOx] ) KGOx[GOx]0
(1)
I1 ) KGOxH2[GOxH2] ) KGOxH2[GOx]0
(2)
At any other time, the fluorescence intensity will be given by
It ) KGOx[GOx]t + KGOxH2[GOxH2]t
[G]tm ) [G]0 exp(-k1[GOx]0(tm - t0))
(4)
Therefore,
d[O2] 1 d[G] ) /2 dt dt
(9)
where substitution of the glucose concentration from eq 8 and integration yield
[O2]tm ) [O2]0 + 1/2[G]0[exp(-k1[GOx]0(tm - t0)) - 1] (10) The equation sought can now be obtained simply by substituting eqs 8 and 10 into eq 7. This expression was also derived on the assumption that k3 . k1:
tm - t0 )
(
)
[G]0 1 ln k1[GOx]0 [G]0 - 2[O2]0
Slope. Over the interval where the fluorescence intensity increases from I0 to I1, GOx and GOxH2 coexist at significant concentrations, so
[GOx]0 ) [GOx]t + [GOxH2]t
dI/dt ) (KGOxH2 - KGOx)(d[GOxH2]/dt) In such a close vicinity of tap, all kinetic approximations can be assumed to hold. The tm - t0 is referred to as the measuring time. Let us thus examine the relationship between the measuring time and the initial glucose concentration. Kinetically, the following assumptions can be made until tap is reached: (1) For the above-mentioned reasons, the steady state conditions can be applied to all the enzyme forms. Application of such conditions to the species GOxH2 and GOxH2L leads to
(5)
d[GOxH2] ) k2[GOxH2L] - k3[GOxH2][O2] ) 0 (6) dt Combination of these gives
[GOxH2]tm [GOx]tm
k1[G]tm )
k3[O2]tm
) 0.11
(7)
(2) The dependence of the glucose and oxygen concentrations on the time can be determined fairly simply. Thus, the glucose 1474
Analytical Chemistry, Vol. 69, No. 8, April 15, 1997
(11)
(12)
Solving this equation for [GOx]t, substituting into eq 3, and taking the derivative with respect to time yields
Itm ) I0 + 0.1(I1 - I0)
d[GOxH2L] ) k1[GOx][G] - k2[GOxH2L] ) 0 dt
(8)
whereas that for O2, based on the considerations made in the previous item, will be given by
(3)
where KGOx and KGOxH2 are the fluorescence proportionality constants for their respective species and [GOx]0 is the initial enzyme concentration. The experimental determination of tap can meet with some practical hindrances arising from the difficulty of establishing the moment where the fluorescence intensity starts to rise. This problem can be circumvented by replacing tap with tm - t0, viz., the time needed for the fluorescence intensity to reach a value equivalent to 10% of the overall increase, i.e., where 10% of the enzyme is in its GOxH2 form, so
[GOxH2]tm/[GOx]tm ) 0.11
concentration before and after the appearance time will be given by the corresponding second-order kinetics,
(13)
The accumulation rate for GOxH2 depends on the initial concentrations of glucose and GOx; however, because the steady state conditions are not obeyed in this situation, the expression relating such a slope with the concentrations of both species is difficult to obtain, and all efforts in this direction were unsuccessful. In practical terms, the maximum slope is the easiest to calculate in this zone since derivation provides a maximum in the curve. Experimentally, we found the maximum slope to vary linearly with the glucose concentration over a much more narrow concentration range than with measuring time, even though the detection limits achieved were similar in both cases. A critical analysis of which parameter was analytically more useful for the determination led to the following conclusions: (a) the slope is more empirical than the measuring time and hence more difficult to alter; (b) both parameters provide similar detection limits, even though the linear range is much wider for tm - t0, which also results in good sensitivity; and (c) unlike the fluorescence intensity, tm - t0 is not affected by the molecular environment. In practice, using tm - t0 could avoid potential interferences from weak internal filtering effects or fluorescent substances. Therefore, use of measuring time is definitely recommended.
Figure 5. Variation of tm - t0 versus ln([G]0/([G]0 - 2[O2])) at different GOx concentrations. Conditions: pH 6.0, H2PO4-/HPO42buffer, λex ) 278 nm, λem ) 334 nm. (2) 4 IU/mL GOx, ([) 7 IU/mL GOx, (b)10 IU/mL GOx, (]) 15 IU/mL GOx, (O)19 IU/mL GOx. Table 1. Glucose Concentration Results Obtained from Blood Samples (in mg/L)
Figure 4. Variation of tm - t0 (4) and dI/dt (O) as a function of pH and the type of buffer used. Conditions: [GOx]0 ) 10 IU/mL, [G]0 ) 175 mg/L, λex ) 278 nm, λem ) 334 nm. Top: citric acid/HPO42buffer. Bottom: H2PO4- / HPO42- buffer.
Optimization of Experimental Variables. Model Confirmation. The enzyme activity depends on pH and the type of buffer solution used, which affects the kinetics of the process.We tested various buffering solutions, including HPO42- /H2PO4- (pH ) 5-8), citric acid/HPO42- (pH ) 2.5-8), and OH-/HBO2 (pH ) 7-8.5). The results obtained by using both tm - t0 and (dI/ dt)max as analytical parameters are shown in Figure 4 for the first two buffer solutions (similar results were observed with the third buffer). As can be seen, there is an optimum pH zone between 4 and 7.5, where tm - t0 is the minimum. Outside this range, tm - t0 is longer because the reaction is slower; also, it disappears above pH 8.5 and below pH 3, where the reaction does not take place within a reasonably measurable time. tm - t0 is virtually independent of the type of buffer. The optimum pH zone for (dI/ dt)max is much more narrow and changes slightly with the type of buffer used; in addition, this parameter changes markedly with the nature of the buffer, which suggests that the molecular environment affects the intensity more strongly than it affects tm - t0 and that the latter parameter is thus of greater analytical use in terms of reliability and precision. The proposed model predicts that tm - t0 is influenced by the O2 and GOx concentrations. To text the model reliability with respect to enzyme concentration, we performed glucose calibrations curves (obtained using different glucose concentrations in each case) at different concentrations of GOx. For each GOx concentration, we represented (see Figure 5)
(
(tm - t0) versus ln
[G]0
)
[G]0 - 2[O2]0
Slopes of the calibration curves obtained change linearly with
sample
this method
NAD/G-6PDH/HK method
1 2 3
131 ( 4 107 ( 3 122 ( 5
132 ( 7 100 ( 8 118 ( 6
sample
this method
NAD/G-6DH/HK method
4 5 6
148 ( 6 179 ( 6 118 ( 4
143 ( 7 171 ( 6 115 ( 8
1/[GOx]0 as the model predicts (slope ) -56.5 + 1823(1/ [GOx]0), r ) 0.993). From these data and the O2 concentration measured in each test, k1 was calculated to be 29 300 ( 2400 M-1 s-1 at 22 °C. This value is comparable to that obtained from measurements with the O2 sensor, viz., 29 500 ( 2600 M-1 s-1, which was arrived at by applying the initial rate method to various test series where the concentration of glucose was varied while that of enzyme was kept constant. The result testifies to the potential of fluorescence measurements for determining kinetic constants for this type of reaction. To test the model reliability with varying O2 concentration, the tm - t0 value was obtained for solutions containing the same GOx (10 UI/mL) and glucose (1.4 × 10-3 M) concentrations and different O2 concentrations (obtained by bubbling N2 during different times; the O2 concentration was measured with the oxygen sensor). The tm - t0 values obtained for 8 × 10-5, 1.4 × 10-4, and 2.2 × 10-4 M O2 were 12.2, 23.0, and 44.2 s, which match very well with those predicted for the model (13.7, 21.2, and 42.6 s, respectively). The O2 concentration may seem difficult to control; however, the results in Table 1 suggest that the concentration obtained with a different method was similar, with an average of 2.18 × 10-4 M and an RSD of 2.0%. Analytical Figures of Merit. Below the lower limit of the linear response range for the method, the reaction kinetics is not fast enough to consume the amount of oxygen needed for GOxH2 to build up; above the upper limit, tm - t0 is independent of the glucose concentration. As predicted by the model, the linear response range and hence the detection limit depend on the GOx concentration used (Figure 5) and, especially, on the initial oxygen concentration present in solution. A concentration of 10 IU/mL at pH 6.5 (phosphate buffer) provided a linear response range from 95 to 1500 mg of glucose/L and a detection limit of 85 mg/ L. If the enzyme concentration was lowered to 4 IU/mL, then the linear response range expanded to 3500 mg/L, but the lower Analytical Chemistry, Vol. 69, No. 8, April 15, 1997
1475
limit was about 500 mg/L. The detection limit also depends on the O2 concentration used; tests with solutions deoxygenated by bubbling N2 for 2.5 min led to detection limits roughly one-half of those obtained at the O2 concentration in distilled water. As noted earlier, one of the advantages of using tm - t0 as the analytical parameter is its good reproducibility. In fact, the relative standard deviation obtained from eight determinations of 200 mg/L glucose was only 2.1%, which is good considering that fluorescence is characterized by its relatively high RSD values. Applications. The proposed method was applied to the determination of glucose in blood. Samples were centrifuged in the presence of a gel acting as the phase separator at 1200g for 10 min, which separated cells from all other materials. The supernatant was subjected to ultrafiltration at 4500g for 75 min, which provided better results than deproteination by acid precipitation and was operationally more simple. The sole problem faced in the determination can arise from the amount of O2 present in the sample. However, because the amount added is fairly small, the effect is minimal; in fact, measurements of the sample solution in the measuring cuvette gave oxygen values similar to those obtained in the absence of sample, which allows one to avoid the use of standard additions. The glucose recovery from blood samples thus obtained was 104 ( 5% (n ) 3). Six blood samples were subjected to the above-described procedure and the results compared with those provided by a Beckman CX-7 clinical analyzer based on the enzymatic reaction with hexokinase/glucose-6-PDH/NAD and molecular absorption measurement of NADH. The results of the two methods are given in Table 1. As can be seen, they are coincident and related by
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the following expression:
[G]GOx ) 0.49 + 1.03[G]HK, r ) 0.994 Significance tests applied on these results indicate that no systematic error is present. CONCLUSIONS The results obtained in this work suggest that monitoring the molecular fluorescence of the enzyme itself in enzymatic reactions is a useful means for establishing reaction mechanisms and determining some of their constants. Analytically, the proposed method is very interesting when the reactants or products exhibit poor spectroscopic properties and require coupled reactions. This methodology could be applied in other enzymatic reactions based on FAD-containing enzymes (i.e., xanthine oxidase, aldehyde oxidase, D-amino acid oxidase, ...) and could be tested for FMNcontanining enzymes (NADH-dehydrogenase, etc.). The use of time as the analytical parameter can partly avoid the reproducibility problems associated with fluorescence measurements, as well as the effect of the sample matrix on them. ACKNOWLEDGMENT This work has been supported by DGICYT of Spain (project 95/0793). Received for review November 4, 1996. February 6, 1997.X
Accepted
AC9611327 X
Abstract published in Advance ACS Abstracts, March 15, 1997.
