ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978
in the open Erlenmeyer flasks due to the adsorption of S b onto the glass surface of the flasks. T h e quantitative recovery of this element was impossible even with repetitive washing with 1 M " O B . No losses of antimony were observed, however, when the samples were dissolved in open Teflon vessels or in t h e Teflon-lined bomb. A previous study by L. T. McClendon ( 2 4 ) cited apparent losses of organometallic chromium compounds during initial acid digestion. Because these volatile compounds were not characterized, all Bovine Liver samples analyzed in this work were digested in a Teflon-lined bomb. However, when the digested samples from the Teflon-lined bomb were boiled with HC104-HN03,no appreciable losses were observed. These observations give support to the theory that the first digestion in the Teflon bomb converts the organochromium compounds to inorganic chromium, which is not volatile in nonchloride systems. Furthermore, standards for analysis that were digested in HC104-HN03, and those pipetted directly were always in total agreement. T h e retention of arsenic, selenium, chromium, and antimony on HMD in 1 M "OB is highly selective and quantitative, while the elution of Cd and Cu is complete. T h e decontamination of the elements of interest from sodium-24 and other radioactive matrix interferences can be estimated to be a factor of 108.
1481
CONCLUSION T h e simplicity of the method permits a rapid isolation of the radioisotopes of interest without many chemical steps. T h e quantitative recovery avoids the calculation of the chemical yield. The sensitivity and accuracy obtained in this work demonstrate the validity and the precision of this radiochemical procedure. LITERATURE CITED (1) T. E. Gills, M. Galiorini, and R. R. Greenberg, Proceedings of Third International Conference on Methods in Environmental and Energy Research, University of Missouri-Columbia, Mo., October 1977. (2) K. Heydorn and E. Damsgaad, Talanfa, 20, 1 (1973). (3) A. Gaudy, B. Maziere. and D. Comar, J. Radioam/. Chem., 29, 77 (1976). (4) H. M. N. H. Irving, J . Radioanal. Chem., 33, 287 (1976). (5) K. Kudo, T. Shigematsu, and K. Kobayashi, J . Radioanal. Chem., 36, 65 (1977). (6) M. Gallorini, M. DiCasa, R. Stella, N. Genova, and E. Owini, J . Radioanal. Chem., 32, 17 (1977). (7) F. Girardi, R. Pietra, and E. Sabbioni, J , Radioanal. Chem., 5, 141 (1970). (8) J. Cuypers, F. Girardi, and F. Monsty, J. Radbaml. Chem., 17, 115 (1973). (9) A. Wyttenbach and S. Bajo, Anal. Chem., 47, 1813 (1975). (10) S. Bajo and A . Wyttenbach, Anal. Chem., 48, 902 (1976). (1 1) E. C. Kuchner, R. Abarez, P. J. Paulson, and T. J. Murphy, Anal. Chem., 44, 2050 (1972). (12) D. A. Becker and P. D. LaFleur, J . Radioanal. Chem., 19, 149 (1974). (13) H. P. Yule and H. L. Rook, J . Radioanal. Chem., 39, 255-261 (1977). (14) EPA Technical Report EPA-600/1-77-020, April 1977, p 44.
RECEIVED for review April 18, 1978. Accepted June 13, 1978.
Lysine Specific Enzyme Electrode for Determination of Lysine in Grains and Foodstuffs W. Claude White and George G. Guilbault Department of Chemistty, University of New Orleans, New Orleans, Louisiana 70 122
A totally specific enzyme electrode, useful for the assay of i-lysine in grains and foodstuffs, is described. No response is noted with any c-amino acid or any other L-amino acid. The electrode can be used for assay of this amino acid In mixtures, without the necessity for extensive separations and expensive instrumentation(i.e., the amino acid analyzer). The electrodes are quite stable, with a linear range of L-lysine concentration of 5 X 10-5-10-1 M. The only limitation is the long response time (5-10 min). After preparation, buffer is the only reagent needed.
T h e determination of amino acids is important in several applications, and often it is the determination of a single amino acid which is desired. In the clinical laboratory, determination of certain amino acids in physiological fluids or tissue may be a useful indicator of certain diseases or disorders (1-3). In nutrition, the content of certain amino acids in a foodstuff essentially controls t h e nutritional quality of the food ( 4 , 5 ) . One such situation is the relationship of L-lysine content and the protein quality of a foodstuff. Grains are the staple food in most countries, and comprise about one half of t h e world's protein supply (6). In most grains, L-lysine is the most deficient amino acid (the limiting amino acid) and controls the protein quality. For this reason, grains are routinely screened for L-lysine content and programs which are directed toward improving the L-lysine content are 0003-2700/78/0350-148 1$0 1.OO/O
underway. In such studies, a rapid simple method for L-lysine determination is valuable. In 1945, Gale (7) proposed using amino acid decarboxylases for determining amino acids, and since then a few methods based on his concept have been developed (8-12). Guilbault and Shu (23)proposed an enzyme electrode based on an amino acid decarboxylase and C 0 2 electrodes, and other similar reports have appeared (14-26). This paper describes the preparation and evaluation of immobilized enzyme L-lysine electrodes prepared by covalent coupling directly onto the C 0 2 sensor by a procedure similar to that in Ref. 17. T h e procedure is both rapid and simple. The electrode response characteristics were determined and analyses of grain samples were performed with the electrodes. The L-lysine electrodes proved to be effective in determining the L-lysine content of grains after hydrolysis, and also for the assay of L-lysine added to grains and flours. EXPERIMENTAL Apparatus. The COz electrode used was a Radiometer E-5036 fitted with a Teflon membrane (Radiometer, DG02). The electrode response was measured with a Radiometer PHM72 Mk 2 Digital Acid-Base Analyzer, and recorded with a Radiometer REC61 servograph recorder. PCOz standardization was performed with 4 % and 8% C 0 2 standards (Radiometer LG-302 and LG-303), using the cell shown in Figure 1. All measurements were carried out at room temperature in 5-mL beakers with magnetic stirring. Reagents and Materials. The L-lysine decarboxylase (E.C. 4.1.1.18) was isolated in our laboratory from E. coli B (provided 0 1978 American Chemical Society
1482
ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978 l m r n o b ~ l i z e d Enzyme Layer To M e t e ,
‘1I 1
r&
1t 1
Rubber
Inner
Electrode
\Teflon
~~
“0’
Ring
membrane
Electrode
jacket
Schematic diagram of an immobilized amino acid decarboxylase layer on the surface of a CO, electrode Figure 2.
T y q o n Tube t o S t a n d o r d Goses
Rubber Stopper
Five mi beaker ( w i t h l i p removed t
Flgure 1. Schematic diagram of the cell used for electrode Pcol
standardization and closed system measurements by Dr. E. E. Snell) or obtained from Sigma Chemical Co. (Type 11, partially purified from B. cadaueris). The amino acids were obtained from Sigma and were dissolved in buffer or distilled water. Bovine serum albumin (BSA) was from Sigma, and was prepared as a 15% solution in distilled water. Glutaraldehyde (25% aqueous solution) and pyridoxal 5’-phosphate were from Sigma. DEAE cellulose was from General Biochemicals. Flour was obtained from a local grocery, and grain samples analyzed for L-lysine were a gift from Analytical Biochemistry Laboratories, Inc. (Columbia, Mo. 65201). The remainder of the chemicals were analytical grade reagents and were not further purified. Internal electrolyte for the COz electrode was 0.005 M NaHC03/0.02 M NaCl, prepared fresh periodically. The 0.5 M acetate buffer was prepared by dissolving the appropriate amount of sodium acetate in distilled water and adjusting the pH to 5.8 with hydrochloric acid. Procedures. Preparation of L-Lysine Decarboxylase from E. coli B. The procedure used to grow the bacteria and isolate the enzyme was modified from that of Sabo et al. (18). The purification procedure was altered, in that DEAE chromatography was used in place of the pH precipitations to remove interfering L-arginine decarboxylase. The enzyme preparation was placed on a DEAE cellulose column and the L-lysine decarboxylase eluted with 0.01 M potassium phosphate, pH 6.2 (containing M pyridoxal 5’-phosphate, 0.01 70 2-mercaptoethanol, and 0.2 M NaC1). The contaminant, L-arginine decarboxylase, can then be eluted with the same buffer, which is 1 M NaC1. The purified L-lysine decarboxylase was freeze dried and stored at 0 “C. Electrode Preparation. The outer jacket of a disassembled COPelectrode was inverted and 30 pL of a 15% BSA solution was placed on the gas permeable membrane. The BSA solution was spread over the surface with a small stirring rod and over the sides until it contacted the rubber “0” ring which retained the membrane. Then 3-5 mg of the freeze dried enzyme preparation was slowly added, allowed to dissolve, and mixed with the stirring rod to blend and help dissolution, if needed. Then 2-5 WLof 25% glutaraldehyde was added and the solution mixed quickly and briefly with the stirring rod. The solution was allowed to cross-link for 10 min (complete solidification was obtained in this time). The enzyme layer was then rinsed by immersion in the following stirred solutions, in the following order: buffer, buffer containing 0.1 M glycine, and buffer. The electrode was placed in buffer and stored overnight at 4 “C. When not in use, the electrodes M were stored at 4 “C in the appropriate buffer containing pyridoxal phosphate (PLP). Electrode Measurements. Electrode measurements were made by immersing the electrode in buffered sample or by allowing the electrode to equilibrate in buffer then injecting the sample. Typically, 2-mL samples were used. The electrode response was noted as p H equilibrium (pH,), Pco, or the rate of Pcoz response. Enzyme activity and the effective activity of the enzyme electrodes were determined from the rate of PCO,response. The
electrode was allowed to equilibrate in 2 mL of buffer, then 0.5 mL of 0.5 M L-lysine in buffer was injected. The response, dPco,/dt, was determined from the recorder output and the activity was calculated as described by Berjuneau et al. (19). Hydrolysis of Grain Samples. Samples, 0.25 to 0.5 g, were placed in a hydrolysis tube and 25 mL of 6 N HC1 was added. The tube was flushed with N2, sealed by flame, and placed in a 110 “ C approximately 2 1 h. The hydrolyzate was filtered in a sintered glass crucible. The filtrate was placed in shallow tubes which were placed in a 40 “C water bath and compressed air blown over the surface to reduce the volume and remove the HC1. When the volume had been reduced to - 5 mL, 5-10 mL of distilled water was added and the volume reduced to 1-3 mL. The sample was rinsed into a 25-mL flask with buffer or acetate solution. The pH was checked and adjusted as necessary to pH 5.8. Buffer was then added to the mark.
RESULTS AND DISCUSSION Theory. T h e basis for t h e enzymatic L-lysine electrodes is the specific enzymatic decarboxylation of L-lysine followed by detection of the C 0 2 liberated with a C 0 2 electrode (Equation 1): L-lysine
L-lysine decarboxylase’
co*
(1)
T h e response of the electrode is due to the change in p H generated in a thin electrolyte layer, which is trapped between an almost flat p H electrode and the gas permeable membrane, when the C 0 2 diffuses into the layer. This is described by Equation 2:
where pH, is the p H of the thin electrolyte layer, pK, is the first dissociation constant for HzC03, (HC03J is the bicarbonate concentration of the internal electrolyte, S is the solubility of COP,and PCO,is the partial pressure of COP. From this the following relations can be developed. pH, = constant - log (CO,) = constant - log (L-lysine)
(3) log (Pco,) = constant
+ log (CO,) = constant + log (L-lysine) (4)
E l e c t r o d e P r e p a r a t i o n . The immobilization procedure utilized in this study results in a thin layer of immobilized enzyme which is attached directly to the C 0 2electrode sensor surface. The immobilized enzyme shows some adherence to the Teflon membranes but, over a period of time, can pull loose. T h e immobilized enzyme layer does, however, firmly adhere to the rubber “0” ring, probably due to increased porosity of the “0” ring. Thus, by having the protein solution contact t h e “0” ring, upon cross-linking, t h e layer is firmly attached to the surface of the COPelectrode. Figure 2 shows an immobilized enzyme layer on the surface of a C02 electrode. The effect of variations of the amounts of enzyme, albumin, and glutaraldehyde used for the enzyme layer was investigated. A series of electrodes were prepared according to Table I in which the amount of one component was varied while the other two remained essentially constant, and the effective activity of each of the electrodes was determined. T h e results
ANALYTICAL CHEMISTRY, VOL. 50, NO. ‘I 1, SEPTEMBER 1978
Table I. Effects of Variations of Amounts of Enzyme, Albumin, and Glutaraldehyde (1)varying enzyme-the
25t
1 20
following electrodes
L
were prepared electrode 1 2 3 4 5
I
mg enzyme
@L
BSA (15%)
0.8
W L glutaralde-
3.0 4.0 5.0
UNITS
hyde (25%)
30
P L
electrode
mg enzyme
BSA (15%)
1 2 3 4 5
3.2 3.3 2.9 2.9 3.1
0 5 10
mg enzyme
1 2 3
2.9
5t ~
5
6
25
glutaraldehyde (25%)
pL
I
0
2
I
3
,
1
4
5
6
1-11 GLUTARALDEHYDE ( 25 % 1 Figure 4. Effect of the amount of glutaraldehyde used in electrode preparation on the activity of L-lysine electrodes
5 O
5 5
15
20
0
k
5
following
P L
BSA
pL
(15%)
glutaraldehyde
2 (25%) 4 (25%) 1 (25%) 6 (25%) 5 (2.5%) 2.5 (2.5%)
30 30 30 30 30 30
2.9 3.0 3.0 3.1 3.0
~
5
(3)varying glutaraldehyde-the electrodes were prepared
electrode
t
‘7
5 5 5 5 5
30 30 30 30
2.0
15
( 2 ) varying BSA-the following electrodes were prepared
4
1483
6001
1
t
I
-
5
4
3
2
I
201
-LOG[-LYS] Figure 5. Response curve for a L-lysine electrode (Curve A). For comparison, electrode response using soluble enzyme is given (Curve
151
8)
UNITS
lot
0
L 5
I
I
I
IC
I5
20
% BSA Figure 3. Effect of the amount of Bovine Serum Albumin (BSA)used in electrode preparation on the activity of L-lysine electrode
are given in Figures 3 and 4, where the electrode activity is plotted against the varied component. A plot of the “effective” activity of the electrodes in units (Le., the resulting activity of each electrode) increased with t h e amount of enzyme used. This is predicted by the Michaelis-Menten equation. This type behavior was noted by Guilbault (20) who reported an increase in electrode performance which eventually levels off with increasing enzyme Concentration. There is a practical limit for those electrodes imposed by the solubility of the protein in the small amount of solution used. With the volumes used here, that limit is around 4-6 mg of enzyme powder. If larger amounts could be used, a leveling off of effective activity would probably be noted. Figure 3, which shows the relationship of “effective units” and bovine serum albumin concentration, has a small maximum a t 15% BSA. At low BSA concentrations, the enzyme
is more extensively cross-linked. This could cause obstruction of the active sites, thus lowering the effective activity by reducing the amount of reactive enzyme. At high BSA concentrations, a more complex matrix may be creating diffusional problems, thus limiting the activity. T h e combination of the two effects may result in an optimal BSA concentration of 15%. Figure 4 shows the effect of the amount of glutaraldehyde added to cross-link the protein solution. T h e amount or concentration of glutaraldehyde has the most pronounced effect of the components. This is probably entirely due to diffusional considerations. At very low glutaraldehyde concentration, the enzyme is not effectively cross-linked and may be rinsed away. Electrodes prepared using small amounts (511L) of glutaraldehyde had layers which were very soft and easily damaged or detached. At higher glutaraldehyde concentrations, the more extensive cross-linking produced diffusional limitations and, probably, obstruction of the active sites which effectively limited the activity of the layer. Above 2 1 L of glutaraldehyde, the immobilized enzyme layers were slightly yellow, transparent, and mechanically very tough. Thus, it appeared that the optimum amount of glutaraldehyde was 2-3 wL, in order to achieve a balance between good activity and a mechanically stable enzyme layer. An advantage of having the decarboxylase immobilized directly on the surface of the COz electrode is that there is an apparent increase in activity. As seen in Figures 4 and 5 , in some cases, the activity of the immobilized enzyme is as
1484
*
ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978
c
1 15Ck
t
1
1
i3ct
PCO,
pH BUFFER Figure 7.
5cL
MIN. Figure 6. Response of the L-lysine electrode, Pco2, with time
much as 20 times as high as the equivalent amount of soluble enzyme. This is probably due to an initial buildup of product in the enzyme layer before it starts to diffuse out of the layer. Thus a n increase in the C 0 2 concentration is noted for the enzyme layer over what would be observed in bulk solution. As a result of this effect, electrodes of good activity can be prepared from less active enzyme preparations. Of course, the more active the enzyme employed in electrode construction, the longer the lifetime one can expect for the electrode. Also, with higher activity enzyme preparations, the quantities of reagents used could be reduced, producing thinner enzyme membranes. With thinner membranes, the limitations of the immobilized layer should be reduced and the activity and response of the electrode improved. Electrode Response. A typical calibration curve for a L-lysine electrode is given in Figure 5 . These electrodes show a linear response range of approximately 3 x lo-*M to M in the amino acid. The lower limit of detection for these electrodes, -6 X M, which is very near the detection limit of the C 0 2 electrode itself, is imposed by the inner electrolyte and atmospheric COz. The upper limit for these electrodes is generally between 10-1M and lo-* M, which is the range of the upper practical limit of the COSelectrode. limited by the solubility of C02. A small extension of the limit to near lo-' M is noted when highly active enzyme is used and is probably due to a concentrating of the C 0 2 in the enzyme layer. T h e slope of the linear portions of those response curves approaches the theoretical 1 pH/decade when more active enzyme is used in the electrode preparation. The less active the enzyme used, the less Nernstian will be the slope. With the enzyme preparations utilized in this work, one could generally expect slopes of a t least 0.80-0.90 pH/decade. Response curves for Pco2 response and the rate of PCo2 response for L-lysine electrodes correspond to the pH, response curve in Figure 5. Response Times. Typical Pco2 response curves for a L-lysine electrode have been reproduced in Figure 6. Full Pco2 response is achieved after 6-7 min with 93% full response achieved after 3-5 min. Figure 6 also shows that if the rate is followed, sufficient data is obtained after 2 min. Generally, these response times are quite representative for this type electrode, with only slight variations in response time being noted from electrode to electrode. The increase in response time over the basic C 0 2 electrode is due to the added time of diffusion of the amino acid into
Effect of buffer pH on the response of a L-lysine electrode
the immobilized enzyme layer and to the active site, the enzymic reaction, and diffusion of the C 0 2 produced to the electrode surface. All of these processes require a finite time which must be considered. As explained by Carr @ I ) , the only way to improve response time is to make the enzyme layer infinitely thin while maintaining high enzyme activity. This is practically accomplished by using higher enzyme activities so that the quantities of reagents and, thus, the enzyme layer thickness, are reduced. Another closely related factor is the recovery time or the time necessary for return to baseline. Ross et al. (22) reported that times needed for going from high concentrations to low concentrations can be up to 13 times as long as the reverse process. In these studies, the time needed for recovery was 1 to 3 times the corresponding response time. Generally, a recovery time of twice the preceding response time is observed. The problem of these fairly long response and recovery times is probably the most serious limitation associated with these electrodes. T h e total time needed for one assay from start until ready for next sample varies from 10 to 30 min depending on the concentration of the amino acid. For electrode measurements, this is somewhat long but, considering other methods for the same analysis, especially in the light of simplicity, total specificity for the amino acid, and economy, the analysis time is not a severe handicap. Stability of Response. These electrodes show very little drift once the equilibrium response is reached. For pH, measurements, the drift is kO.001p H unit or less over several minutes. For Pco2 equilibrium measurements, a drift of less than 0.2 Pco2 over 2-3 min is noted. This type of stability is expected since the basic sensor is the extremely stable glass electrode. The reproducibility of response or precision associated with these electrodes is also quite good. When four to six replicate samples are run a t several concentrations over the dynamic range for L-lysine, the average standard deviation obtained is 10.024 p H unit for the pH, observed a t 10 min. This is within the precision values generally associated with ionselective electrodes, 1-2 mV or 0.017-0.033 p H unit. The stability of these electrodes, the drift, and the precision both are determined by the limitations of the basic sensor itself. pH Optimum. The p H a t which these electrodes exhibit maximum response was determined by testing the electrode response in a series of buffers at a constant L-lysine concentration. The results are given in Figure 7 . T h e peak denotes the pH for optimum response for the complete amino acid electrode. This includes the effect of pH on the enzymic reaction and also the C 0 2 equilibria. This approach was taken since this will reflect the true operating pH optima. T h e p H optimum for the lysine electrode appears to be shifted very slightly toward higher pH's than the soluble enzyme. T h e literature values for the soluble enzyme have been given as p H 6.0 (23) and p H 5.7 (18). In this study, the
ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978
Table 11. Standard Mixtures-Lysine L-Lysine Electrode SamPle
SLOPE
1
C 6-1
PLYScalcd ([L-LYSI1 3.537 (2.90 X M) 2.838 (1.45 x i o - 3 ~ 3.060 (8.70 x 10-4 M) 2.935 (1.16 x 10-3 M)
PH
2
DECADE -
3 02r I
,
,
4
-
_I
80
40
120
value was closer to 6.0; for later work. p H 5.8 was selected as the p H of operation. Selectivity/Specificity. Both L-lysine decarboxylase prepared from E. coli B and that obtained from Sigma yield electrodes which do not show response to any of the other common amino acids. T h e L isomers of Ala, Arg, Asn, Asp, Cys, Glu, Gln, His, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val were tested and showed no response. The electrode does not respond to D-amino acids. Because of the possibility that other decarboxylases might be present in t h e enzyme preparations, each different preparation should be tested. This is easily and economically accomplished, once t h e enzyme has been immobilized onto t h e C 0 2 electrode. L o n g T e r m Stability. T h e long-term stability of these amino acid electrodes can be illustrated by considering the effect on the response curves of age. As the electrodes begin to age, the first effect noted in an increased curvature in the upper limit regions. This results in a decrease in the linear range in the higher concentration region. Generally, the linear M. The lower region decreases to no less than around 3 X limit does not show any variation with age. After the upper limit of the response curve begins to decay, the slope of the response curve begins to decrease, that is, deviates more from the theoretical or Nernstian value. These effects are the same as one sees when less active enzyme preparations are used. This indicates t h a t the decrease in electrode performance is due to a decrease in the effective concentration of the enzyme on the electrode. Figure 8 shows the variation of slope with time for a L-lysine electrode, and illustrates the long term stability of the electrode. As seen in Figure 8, the L-lysine electrode shows a pronounced improvement in response (slope) over the first 2-3 weeks. This is often noted with enzyme electrodes and is generally attributed to the establishment of diffusion channels in the immobilized enzyme layer. From the maximum slope, there is a decrease in slope with time which seems to slow and begins to level off after 3@40 days. The L-lysine electrode was tested for 50 days; the response for the electrode was still good. T h e reduction of t h e amount of active enzyme in the enzyme layer, as noted by decreased electrode performance, is possibly due to several causes. Some of this reduction is undoubtedly due to denaturation of the enzyme. Other effects can be destruction or obstruction of the active site. This may be due to irreversible inhibition or, more probably, removal of the cofactor, which could be dialyzed away when the electrode was stored in buffer. T o test this effect, a L-lysine electrode which had been stored in buffer for several weeks was tested and then retested after storage for 2 days in approximately 10 m L of buffer containing 3.5 mg pyridoxal 5’ phosphate ( P L P ) . T h e upper limit of the linear range was increased from -3 X M to -6 X M and the slope
Determination by
Plysfounda ( [ L - L ~ s) ]
electrode
3.530 i 0.020 (2.95 x 1 0 -M~) 2.870 I0.082 (1.3’7 x 10-3 M ) 3.097 i 0.010 (8.00 X M) 2.946 t 0.036 ( i . i ‘ ix 1 0 . ~ M)
LS1 LS 1 LS2
LS2
average standard deviation = i- 0.037
DAYS
Figure 8. Long term stability of a L-lysine electrode
)
1485
a
Average of 3-5 determinations.
Table 111. l lysine Spiked Flour-L-Lysine Determined by L-Lysine Electrode sample F 1 2 3
4 1
mg flour 201.5 204.0 222.0 247.5 206.3 204.0
mg Lys (added) 0 4.8
7.9 12.1 24.6 4.8
mg LYS (found)a l3.53=
4.84 + 0.40b 8.04 I O . 1 Z b 12.Q4 r 0.35b 25.!55 i 0.60b 5.28 i 0.30b
% re-
covery
101
102 103 104 110 104
Average of a minimum of three determinations. Blank value. Corrected for blank.
a
was increased from 0.79 pH/decade to 0.84 pH/decade. After this, the lysine electrodes were generally stored in buffer containing approximately M F’LP. The stability of the electrodes given above is usually referred to as storage stability since the electrodes were periodically removed from storage and the slope was checked. However, the electrodes were also used periodically for analysis of standards, standard mixtures, and interference studies. While the use was not enough to classifyy the stability study as operational stability, the electrodes were subjected to more use than normally associated with storage stability studies. D e t e r m i n a t i o n of L-Lysine i n S t a n d a r d M i x t u r e s of Amino Acids. T h e L-lysine concentrations in synthetically prepared mixtures of the common L-amino acids were determined with L-lysine electrodes. Table I1 gives the results of the L-lysine determinations in the same standard mixture. The correlation plot yields, s. = 0 . 9 5 1 ~ 0.170, and a correlation coefficient of 0,999. T h e accuracy and precision of both electrodes were good. Recovery Studies. Table I11 shows the recovery of L-lysine added to flour samples. The L-lysine content was determined by comparing the PcoZ response to standard curves and subtracting the blank of pure flour. A correlation plot yields 3 = 1.036~- 0.006, and a correlation coefficient of 0.9998. The average recovery was 104%. Table IV shows the results of a study in which L-lysine was added to corn meal. T h e L-lysine content was determined utilizing Pco, and Pcoz/min (Rate) response curves and blank subtraction. The correlation by both methods was good. The Pco, equilibrium measurements gave a correlation plot of y = 0 . 9 0 0 ~+ 1.800 with a correlation coefficient of 0.998, and in the rate measurements gave 3 = 1 . 0 5 8 ~+ 0.512 with a correlation coefficient of 0.997. While the results by both methods are quite good, the Pco, equilibrium determinations had slightly better correlation and better precision, noted as standard deviations for samples 4-8 Also the recovery was better.
+
1486
*
ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978
Table IV. L-Lysine Spiked Corn Meal (Sample 19196-2 lysine Determined by L-Lysine Electrode sample Blank 1 2
3 4 5 6 7 8
mg L-LYS (added)
g, corn meal 1.0365 1.0438 1.0282 1.0385 1.0411 1.0175 0.9943 1.0759 1.0537
0
6.5 11.1 14.9 22.1 25.8 29.5 33.7 63.9
pc 0,
mg L-LYS(found), rate (Pco2/min)
(6.0)' 7.0 (108)b 10.8 (97) 15.1 (101) 22.5 i 0 . V (102) 27.5 t 0.6 (106) 27.5 0.9 ( 9 3 ) 32.3 i- 0.8 (96) 58.7 i 0.8 ( 9 2 )
(3.47 T 0.4)' 7.8 (119)b 12.5 (113) 1 6 . 5 (111) 21.6 t 2 . l C( 9 8 ) 30.2 t 5.6 ( 1 1 7 ) 30.2 L 0.9 (102) 36.6 * 0.7 (109) 68.3 r 3.6 (107)
_+
average recovery = (99.4) (109.5) a
Blank value.
Recovery (%).
Average of three determinations.
Table V. Determination of L -Lysine in Grain Samples By L-Lysine Electrode %
sample (grain) 19196-1 (corn) 19196-2 (corn) 19392-2 (feed) 19360 (soy residue) 19022-1 (barley) 19076 (soybean) 19196-3 (corn) 19392-1 (corn) average relative error = a
% lysine, found Pco2
rate (Pc0,)/ min)
0.94 t 0.035 ( 6.8%)'
0.96 i 0.095 (9.1%) 0.80 * 0.00 0.68 i 0.00 (9.1%) (22.7%) 0.52 ?- 0.023 0.467 i 0.046 (14.8%) (23.4%) 1.86 i 0.048 1.94 i 0.132 (9.7%) (5.8%) 0.52 i 0.017 0.36 * 0.036 (5.5%) (34.5%) 2.53 I 0.030 2.63 c 0.065 (1.2%) (5.2%) 0.89 i 0.055 0.89 * 0.096 (1.1%) (1.1%) 0.41 * 0.029 0.41 5 0.029 (5.1%) (5.1%) (6.7%)
lysine, ABC Labs 0.88
0.88 0.61 2.06 0.55 2.50 0.88
0.39
(13.4%)
Relative error.
When these samples were made up in buffer, some of the material did not dissolve. It was not necessary to remove the insoluble material before analysis. This is another advantage the electrode method has over the other methods of amino acid analysis, such as spectrophotometry, fluorometry, or chromatography. Determination of L-Lysine in Grains. Table V shows the results for determinations of L-lysine in grain samples using a L-lysine electrode. These results are compared to those obtained with a n amino acid analyzer by ABC Labs, who generously supplied the samples. The grain samples were acid hydrolyzed by a procedure similar to that recommended by ABC in order to reduce effects due to hydrolysis. L-Lysine samples subjected to the hydrolysis showed no losses of amino acid content. Both Pco, and Rate (Pco,/min) determinations show fairly good correlation with the ABC values. The Pco2 correlation plot is y = 0 . 9 7 5 ~- 0.007 with a correlation coefficient of 0.994, and the rate correlation plot is y = 1 . 0 6 1 ~ - 0.118 with a correlation coefficient of 0.989. The precision of the Pco, method is slightly better than the rate method.
T h e equilibrium Pco2method is also more accurate, having an average relative error of 6.6670, while the rate method gives 13.36%. Overall, the determinations using the L-lysine electrode compare very well with the results reported by the amino acid analyzer.
ACKNOWLEDGMENT The authors thank E. E. Snell, University of Texas, Austin, for the strain of E . coli B, and the Analytical Biochemistry Laboratories for the analyzed grain samples supplied.
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RECEIVED for review February 6, 1978. Accepted J u n e 13, 1978. T h e financial assistance of the National Science Foundation, Grant No. AER-76-23271, is gratefully acknowledged.
