512
Anal. chem. 1981, 53, 512-515
deviation is i0.5%. Titration of binary or tertiary metallic mixtures in 75% ethanol and use of the graphite-(copper diethyldithiocarbamate-silver sulfide) electrode do not significantly affect the shape of the titration curves or the accuracy of the results. S i m u l t a n e o u s Determination of Metals i n CopperBase Alloys. The major elements of some copper-base alloys were simultaneously determined by direct potentiometric titration with NaDDC by use of the graphite-(copper diethyldithiocarbamate-silver sulfide) electrode after acid decomposition of the alloy sample and pH adjustment at 4-6. The results obtained (Table IV) for the analyses of five different alloys compare favorably with the certified values. A standard known addition "spiking" technique was also used for the analyses of these alloys. This involved addition of a standard synthetic mixture of Cu-Pb-Zn or Cu-Ni-Zn (0.5 mg of each metal) to a known volume of the alloy solution before titration. The difference between the titer of each of these metals in the standard and the standard plus the alloy sample is equivalent to the alloy metal. Good agreement within i0.370was obtained between direct and standard known addition titration procedures. The latter can be used for alloys where the constituent elements are not exactly known. Advantages a n d Limitations. The present method offers clear6advantages over many of the available procedures used for the determination of metals in mixtures and alloys (17-19). It involves little manipulation and no prior separation steps. The method can be used for titration of as low as 1 % of the metals Cu, Cd, Ni, Pb, and Zn in tertiary and quaternary mixtures and alloys. The time of assay is 25 min, and the accuracy and precision compare favorably with those obtained
by the standard techniques. The method cannot, however, be used for the determination of samples containing Fe, Mn, Ag, and Hg in concentrations >15%. The first pair of ions affects the sharpness of the other metal breaks while the latter two displace copper from CU(DDC)~ in the graphite rod, affecting the life span, response time, and sensitivity of the electrode. LITERATURE CITED (1) Bruno, P.; Caselll, M.; Fanco, A.; Fragale, C. Analyst (London) 1978, 103, 868-671. (2) Stock, J. Anal. Chem. 1980, 52, 1R-9R. (3) Borelb, A.; GuMottI, G. Anal. Chem. 1971, 43, 807-608. (4) Jagner, D. Anal. Chem. 1979, 57, 342-345. (5) Oehme, M.; Lund, W.; Jensen, J. Anal. Chlm. Acta 1978, 700, 389-398. (6) Ross, J.; Frant, M. Anal. Chem. 1989, 47, 1900-1902. (7) Meer, J.; Boef, G.; Linden, W. Anal. Chlm. Acta 1975, 76, 261-268. (8) Kalbus, L.; Kalbus, 0. Anal. Chlm. Acta 1971. 53, 225-231. (9) Toren, E.; Buck, R. Anal. Chem. 1970, 42. 284R-304R. (10) Elliott, C.; Murray, R. Anal. Chem. 1978, 48, 1247-1254. (11) Campbell, M.; Demetriou, 8.; Jones, R. Analyst(London) 1980, 705, 605-611. (12) Cosofret, V.; Zugravescu, P.; Balulescu, G. Talsnta 1877, 24, 46 1-463. (13) Pwone, S. Anal. Chem. 1863, 35, 2091-2094. (14) Ruzlcka, J.; Lamm, C. Anal. Chim. Acta 1871, 53, 206-208. (15) Hulanlckl, A. Talanta 1967, 74, 1371-1392. (16) Stary, J.; Kratzer, K. Anal. Chlm. Acta 1968, 40, 93-100. (17) Vogel, A. "A Text Book of Quantitative Inorganic Analysis", 3rd ed.; Longmans: London, 1961; Chapter IV. (18) Morelll, B. Analyst(London) 1980, 705, 398-400. (19) Elwell, W.; Schdes, I."Analysis of Copper and its Alloys"; Pwgamon Press: London, 1987; Chapter 3.
RECEIVED for review October 27,1980. Accepted December 17, 1980. This work was presented at the Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Atlantic City, NJ, March 1980.
Enzyme Amplification for Trace Level Determination of Pyridoxal !%Phosphate with a pC02 Electrode Saad S. M. Hassan' and G. A. Rechnitz" Department of Chemistry, University of Dela ware, Newark, Deb ware 197 7 7
A hlghiy sensitive and selective potentiometric method for the determination of the enzymatically active form of vttamln B6, pyridoxal I'-phosphate (PLP), has been devised. The method is based upon the decarboxylation of L-tyrosine by the apoenzyme tyrodne decarboxylase In the presence of PLP, using a pC0, electrode to monltor the reaction rate. Under optimized conditions, an amplification factor of up to 10' Is achleved, and PLP levels as low as lo-' M can be measured wHh good precision and an analysis tlme of only 20 min. Other members of the vitamin B6 group and their phosphate derlvatives do not interfere with the proposed method.
Pyridoxal 5'-phosphate (PLP), also known as codecarboxylase or cotransaminase,is the only enzymatically active form among the various members of t h e vitamin Bs group. It is involved in at least 50 enzymatic systems associated with
On leave f r o m Ain Shams University, Cairo,
Egypt.
0003-2700/8110353-0512$01.00/0
nitrogen metabolism. Urinary excretion of oxalate (oxaluria), change of free amino acid and lipid components of plasma, impairment in erythrocyte formation, and red cell transaminase activity have been used as indirect semiquantitative memum of PLP stores (1). Studies of vitamin B6deficiencies have been hampered by the lack of convenient measurement methods. The quantitative spectrophotometricdetermination of PLP by reaction with phenylhydrazine (2),and the fluorometric methods involving reaction with cyanide (3), semicarbazide (4), and methylanthranilate-sodium cyanoborohydride (5) have been previously described. These methods suffer from a lack of specificity and, moreover, require chromatographic separation steps. Enzymatic methods, based on the ability of PLP to restore the activity of tyrosine decarboxylase or tryptophanase apoenzymes with measurement of the reaction products, are reported as being more specific and sensitive. In this connection, the rate of decarboxylation of tyrosine by PLP-dependent tyrosine decarboxylase has been measured manometrically (6). The sensitivity of this method was increased by using I4C labeled tyrosine and measuring the 0 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 3, MARCH 1981
tyramine-14C produced after separation by paper chromatography (7). Tyrosine-1-14C has also been used in a method where the decrease in radioactivity was measured (8) or the 14C02released was captured and counted (9). L-Dopa was also proposed as a substrate for this reaction and the dopamine formed was measured by HPLC using amperometric detection (10). These methods, however, require several time-consuming manipulation and separation steps, sophisticated instruments, and special licensing and precautions required for the use and disposal of the radioactive reagents. Catalytic degradation of tryptophan by PLP-dependent tryptophanase, followed by spectrophotometric measurement of the indole or pyruvate products as derivatives of N-dimethylaminobenzaldehyde (11, 12) and dinitrophenylhydrazine (13), respectively, has also been reported. S-oNitrophenyl-L-cysteine has been utilized as a chromogenic substrate instead of tryptophan to permit direct color measurement of the reaction product (14). The radioactivity of pyruvate-14C product formed by using tryptophan-14C substrate has also been measured after chromatographic separation of the excess substrate and extraction of the pyruvate as hydrazone (15). These methods also involve several reaction and extraction steps resulting in low PLP recovery and poor precision. The present investigation was undertaken to develop a simple, selective, and sensitive method for the determination of PLP to levels as low as lo4 M by use of the pC02 electrode. We have recently shown (16) that the dynamic response properties of this electrode are highly favorable for rate methods of analysis. EXPERIMENTAL SECTION Apparatus. All measurements were performed by using a Corning Model 12 Research pH meter in conjunction with Heath-Schlumberger Model SR-255B strip-chart recorder. The pC02gas electrode (Model 95-02) was obtained from Orion Research Inc. (Cambridge, MA). Measurements were conducted in a 20-mL double-jacketed cell, and constant temperature was maintained at 37 i 0.1 "C by a Haake Model FM magnibath. Reagents. All the reagents used were of reagent grade unless otherwise stated and deionized water was used throughout. LTyrosine decarboxylase apoenzyme (1.1U/mg) E.C. 4.1.1.25 from Streptococcus faecalis, L-tyrosine, pyridoxal 5'-phosphate (PLP) pyridoxine monohydrochloride, pyridoxal hydrochloride, pyridoxamine 5'-phosphate hydrochloride, and pyridoxamine dihydrochloride were obtained from Sigma Chemical Co. (St.Louis, MO). L-Tyrosine solution, lo-' M, is prepared by dissolving 0.18 g of L-tyrosine in 100 mL of 0.2 M hydrochloric acid. Pyridoxal 5'-phosphate stock solution, lo4 M, is prepared by dissolving 2.74 mg of PLP in 100 mL of deionized water. The solution is refrigerated in the dark and renewed every 2 days. Working solutions are freshly prepared by 100- and 1000-fold dilution. LTyrosine decarboxylase apoenzyme, 10 U/mL, is prepared by dissolving 18 mg of the enzyme (1.1 U/mg) in 2 mL of 0.1 M sodium citrate-citric acid buffer of pH 5.8. Procedure. A 100-pL sample of the enzyme solution is transferred to a 20-mL double-jacketed reaction cell thermostated at 37 k 0.1 "C containing a small Teflon-covered spinbar. A series of aliquots ranging from 2 to 100 pL of lo4 M pyridoxal 5'phosphate (PLP) solution (equivalent to 0.55-27 ng) is added and made up to 2.0 mL with 0.1 M citrate-citric acid buffer of pH 5.8. The solution is stirred, the pC02 electrode immersed in the solution, and the potential allowed to reach a stable reading (-15 min). The speed of the chart recorder is adjusted at 0.5 in./min and 10 pL of lo-' M L-tyrosine solution is added. The rate curves are recorded and the maximum initial rate of potential change expressed in millivolts per minute is graphically determined from the recorder by using the rate portion of the curve (Figure 1). A blank experiment is carried out under identical conditions without using PLP. The initial rate is plotted as a function of PLP concentration and the graph is used for subsequent measurement of unknown PLP samples within the concentration range of 0.510 ng/mL. Lower PLP concentrations are measured by prior
>
513
95
74.8
115
Tyrosine I
I
0
.
,
,
2
,
e
4
8
Time?min
Flgure 1. Typical time vs. carbon dioxide electrode response curves for the reaction of pyridoxal 5'phosphate (1 ng/ml equals 0.37 X lo-' M) with 8 X lo-' M tyrosine and 1 U of tyrosine decarboxylase a p e enzyme at pH 5.8 and 37 OC.
preparation of another calibration graph following the above procedure but using 300 pL of the enzyme, a series of aliquots ranging from 2 to 100 pL of IO-' M PLP (equivalent to 55 pg to 2.7 ng), and 50 pL of lo-' M L-tyrosine. The initial rate is plotted as a function of PLP concentration in this range. RESULTS AND DISCUSSION The rate of COz liberation from the reaction of L-tyrosine and excess tyrosine decarboxylase apoenzyme in the presence of PLP was followed by using the pC02 electrode (eq 1). A PLP
H O ~ c ~ 2 ~ H c 0 0 TYROSINE H N H2
DECARBOXYLASE APOENZYME
HOa-CH2CH2NH2
+
CO2
> (11
preliminary investigation of the response of the pCOz electrode toward sodium bicarbonate solutions a t different pHs and various temperatures showed a Nernstian response of the M concentration levels in citrate-citric electrode to 5 X acid buffer solutions of pH 5-6 and temperatures in the range of 30-40 "C. Since the lower level of P L P in biological materials is in the range of 1O4--1O-@ M (Le., >1 ng/mL of fluid and 1pg/g tissue) (5,8,9) an amplification factor of the order of at least lo3is required to bring the amount of COz released by the above enzymatic reaction within the detection range of the pCOz electrode. Consequently, the experimental factors affecting the enzymatic reaction were carefully varied and optimized in order to achieve a 104-105-fold amplification in production of COz. Effect of Substrate and Enzyme Concentrations. The optimal L-tyrosine concentration initially present in the reaction mixture required to saturate 1 U of enzyme in the presence of 5 X lo-@M P L P in a total volume of 2 mL of citrate-citric acid buffer of pH 5.8 at 37 "C is 6.5 X M (Figure 2). Slightly higher concentrations (8 X lo4 M) were used for rate measurements throughout this investigation. A Lineweaver-Burk plot of the reciprocals of initial reaction rate vs. substrate concentrations, reveals a K , value of 5 X M which can be compared with values of 1.66 X M (17) and 6 X M (9) obtained by other workers. With an enzyme activity in the range of 0.5-1.5 U/mL in the presence of a constant PLP concentration (5 X M) and excess tyrosine ( M), a linear relationship between the rate of decarboxylation and enzyme activity was obtained. On the other hand, by changing the enzyme activity at constant concentrations of both tyrosine substrate (8 X lo-' M) and P L P (5 x M), a maximum decarboxylation rate was observed at 0.5 U/mL of the enzyme (Figure 3). This activity level is sufficient to permit the accurate and precise measurement of as little as 1 ng/mL of PLP. Lower concentra-
514
ANALYTICAL CHEMISTRY, VOL. 53, NO. 3,MARCH 1981
Table I. Comparison of Methods for the Determination of Pyridoxal 5'-Phosphate (PLP) useful
lower limit, analysis ng/mL precision, % time, min
method class
type of
measurement
chemical
spectrophotometric
10
fluorometric enzymatic
1
spectrophotometric manometric radiometric
3 10 20 5 1
2 0.5
pC0, electrode
separation step interferences
required
not reported
>30
not reported 7 10
> 150
chromatography
>70
extraction extraction none chromatography and extraction
10
not reported 15 6 2
none
ref
pyridoxal, glyoxylate,
2
5-deoxy pyridoxal,
> 90 > 30 > 180
> 30 > 100 < 20
p-nitrosalicyldehyde pyridoxal
5 11
12 6 15
none
8 9, 17
capture of CO, none
present work
I
2
6
8
4M
10x10
Flgue 2. Effect of L-tyrosine concentration on the decarboxyhtion rate with 1 U of tyrosine decarboxylase apoenzyme and 5 X lo-' M pyridoxyl 5'-phosphate at pH 5.8 and 37 O C .
tions of PLP, however, can be measured with less precision, down to 50 pg/mL, using 1.5 U/mL of the enzyme and 2.5 X M tyrosine. Under these conditions, the observed potential of the electrode system shows that the COzliberated and sensed by using 10-8-10-9 M P L P is in the range of 10-4-10-5M, which means that an amplification factor of the order 104-105 is obtained. Effect of T e m p e r a t u r e and pH. The effect of temperature on the initial rate of decarboxylation was studied over the range of 30-40 "C. The solutions used were 5 X lo-* M PLP, 8 X M tyrosine, and 1 U of tyrosine decarboxylase apoenzyme in a total volume of 2 mL of citrate-citric acid buffer of pH 5.8. The results showed a linear increase in the initial rate with increasing temperature up to 40 "C without any noticeable denaturation or decrease in the enzyme activity. The rate doubles with a 10 "C increase in temperature. A temperature of 37 OC was chosen for subsequent measurements. Figure 4 shows the pH-decarboxylation rate profile for the reaction of 8 x M tyrosine, 1 U of apoenzyme, and 5 x lo4 M PLP at 37 OC in a total volume of 2 mL of citrate-citric acid buffer solutions over the pH range from 5 to 6. The optimal activity is found at a pH 5.7-6, slightly higher than the optimum reported by using acetate-acetic acid buffer (6, 17).
Effect of Preincubation. A study of the effect of preincubation on the initial rate of decarboxylation was carried out in two parallel series of experiments using identical concentrations of reagents a t pH 5.8 and 37 "C. The first series
1 .o
0.5
LL- Ty r o s I ne]
1.5
2.0 ~ / 2 m
Enzyme a c t i v i t y
Flgure 3. Effect of enzyme activity on the decarboxylation rate of 8 X lo4 M L-tyrosine: with 5 X lo-' M pyridoxal 5'-phosphate at pH 5.8 and 37 O C , 0;blank, 0 .
l-L-l.-i-
5.2
5.4
5.6
5.8
6.0
PH
~-pk
Flgure 4. Effect of pH on the decarboxylation of 8 X lo4 M with 1 U of tyrosine decarboxylase apoenzyme and 5 X 10- M pyridoxal S'phosphate by use of citrate-citric acid buffers at 37 OC.
involved incubation of the apoenzyme and substrate, to form the enzyme-substrate complex, in the reaction cell containing the pCOz electrode. After attainment of a steady potential reading (- 15 min), PLP was added, and the rate curves were measured. The second set of experiments was performed by preincubation of the apoenzyme and P L P to permit reassociation of the two to form the holoenzyme. Tyrosine was then added, after establishment of a steady pCOz electrode po-
515
Anal. Chem. 1981, 53,515-518
tential, to initiate the reaction. The results obtained indicate that the initial rate of decarboxylation obtained by the latter technique is almost 2.5 times that obtained by the former one. Further increase in incubation time to 30 min does not influence the decarboxylation rate. Accuracy and Precision of the Method. A linear relationship between the P L P concentration in the range of 0-5 X M and the initial rate of carbon dioxide liberation was obtained. Least-squares statistical treatment of the data yielded a slope of 2.0 (standard deviation 0.05) with an intercept of 0.1 (standard deviation 0.04). The precision and accuracy of the method were evaluated in 20 repetitive analyses made on 1.85 X M pure P L P (equivalent to 5 ng/mL) under the optimum conditions he., 1U of apoenzyme, 8x M tyrosine in a totalvolume of 2 mL of citrakcitric acid buffer of pH 5.8 a t 37 "C). At this concentration level, a relative precision of *2% with an accuracy *3% (from recovery experiments) was obtained. At the lowest possible P L P concentration levels (50 pg/mL or 1.85 X M) measured using 3 U of the enzyme and 2.5 X M tyrosine in a total volume of 2 mL of citrate-citric acid buffer of pH 5.8, the precision declined to f10% and the recovery fluctuated within 80-120% owing to the invariably high blank. Selectivity. The selectivity of the method was demonstrated by measuring the decarboxylation rate at levels of 5 ng/mL (1.85 X M) of PLP in the presence of a 100-fold excess of the possible interferences pyridoxal hydrochloride, pyridoxamine 5'-phosphate hydrochloride, pyridoxine hydrochloride, and pyridoxamine dihydrochloride. None of these caused any observable interference and the method is, thus, highly selective for PLP.
Comparison with Other Methods. Table I shows a comparison of the results obtained for the proposed method with the reported analytical features of the earlier methods. I t can be seen that the use of the present technique, which features enzyme amplification with electrode detection, offers a particularly favorable combination of selectivity, sensitivity, and measurement convenience. LITERATURE CITED (1) Sauberlich, H. I n "The Vitamins Chemistry, Physldogy, Pathology Methods", 2nd ed.; Sebrell, W., Harris, R., Eds.; Academic Press: New York, 1968; Vol. 11, pp 45-51. (2) Wada, H.; Snell, E. J. Blol. Chem. 1081, 236, 2069-2095. (3) Adams, E. Anal. Bbchem. 1080, 3 1 , 118-122. (4) Srivastava, S.; Beutler, E. Biochlm. Blophys. Acts 1073, 304, 765-773. (5) Chauhan, M.; Dakshinamurti, K. Anal. Blochem. 1079, 96, 426-432. (6) Gunsaius, I.; Smith, R. I n "Methodsin Enzymology"; Colowick, S.,Kaplan, N., Eds.; Academlc Press: New York, 1957; Vol. 111, pp 963-967. (7) Hamfelt, A. Clln. Chlm. Acta 1062, 7 , 746-748. (8) Maruyama, H.; Coursin, D. Anal. Biochem. 1068, 26, 420-429. (9) Chabner, B.; Livingston, D. Anal. Blochem. 1070, 3 4 , 413-423. (10) Allenmark, S.; Hjelm, E.;Larsson-Cohn, U. J. Chromatogr. Blamed. Appl. 1078, 146, 485-469. (11) Haskeli, B.;Snell, E. Anal. Blochem. 1072, 45, 567-576. (12) Gailani, S. Ana/. Biochem. 1065, 13, 19-27. Morino, Y.; Morisue, T.; Sakamoto, Y. J. Blochem. (To(13) Nakahara, I.; kyo) 1061, 49, 339. (14) Suelter, C.; Snell, E. Methods Enzymol. 1070, 62, 561-568. (15) Okuda, K.; Fujii, S.; Wada, M. Methods Enzymol. 1070, 43. 505-509. (16) Jensen, M.; Rechnitz, G. Anal. Chem. 1070, 51, 1972-1977. (17) Sundaresan, P.; Coursln, D. Methods Enzymol. 1070, 43, 509-512.
RECEIVED for review October 9,1980, Accepted December 15, 1980. We gratefully acknowledge support of this work by Grant GM-25308 from the National Institutes of Health.
Selectivity Enhancement of a Tissue-Based Adenosine-Sensing Membrane Electrode M. A. Arnold and G. A. Rechnitz' Department of Chemistry, University of Delaware, Newark, Delaware 7977 7
A tlssue-based membrane electrode condsting of mouse small intestlne mucosal cells coupled to a potentlometric ammonia gas sensor is used as a model system to study the possibility of selectlvlty enhancement for such biocatalytlc membrane electrodes. It is shown that the electrode selectivity for adenosine over several interfering nucleotides can be greatly enhanced through elucidation of the interfering metabollc pathway and its suppression by appropriate biocklng agents.
The employment of tissue slices as catalytic layers in the construction of bioselective membrane electrodes has initiated (1) a new direction for such electrodes with a particular attractiveness for situations where the isolation of enzymes is unsatisfactory or where the sterilization requirements of bacterial electrodes are cumbersome (2). On the other hand, it must be recognized that tissue-based electrodes will rarely be highly selective for a given substrate since most tissue materials contain numerous enzymes and are capable of sustaining multiple metabolic pathways. The purpose of this brief communication is to propose a possible strategy for selectivity enhancement via selective inhibition of undesired metabolic pathways. It will be seen that some knowledge and
experimental testing of possible metabolic alternatives are required for this purpose. We selected mouse small intestine as our catalytic phase for the preparation, using the NHB gas electrode, of an adenosine-responsive membrane electrode because the enzyme adenosine deaminase (E.C. 3.5.4.4) is frequently isolated from intestinal mucosa (3). As shown below, this tissue-based electrode also exhibits response to certain adenosine-containing nucleotides. This interfering response could be the result of pathways involving either other deaminating enzymes or the enzymatic formation of intermediates which can be further degraded by the primary enzyme. Since an a priori choice between these alternatives is not possible, the successful elimination of nucleotide interference requires experimental verification of the undesired metabolic pathway and its suppression by the use of inhibitors or stereospecific blocking agents to achieve an enhanced electrode selectivity for adenosine. EXPERIMENTAL SECTION Apparatus. All potentiometric measurements were made with a Corning Model 12 pH/mV meter in conjunction with a Heath-Schlumberger SR-210 strip chart recorder. All measurements were made in thermostated cells at 26 "C controlled by a Haake Model FS bath. Orion Model 95-10 ammonia gas-sensing
0003-2700/81/0353-0515$01.00/00 1981 American Chemical Society
