ANALYTICAL CHEMISTRY, VOL. 50, NO. 1'1, SEPTEMBER 1978
is adequate for routine water analysis. Volatile acids or bases that would diffuse through the silicone fibers will interfere with carbon dioxide or ammonium determinations. Nitrate, chloride, and sulfate salts produced no response when the instrument was set up for carbon dioxide determinations. Acetate did produce a response and would interfere. Volatile amines are the most significant interferences in ammonium determinations. A test of interference from methylamine and dimethylamine showed a greater instrument response to these compounds than to ammonium solutions of the same concentration. Sample color and turbidity have no effect on performance of the instrument. The prototype instrument has required little maintenance beyond periodic replacement of the pump tubes. The hollow fiber unit shows no signs of deterioration after 24 months of use. The electrical conductivity detection system requires much less electrical shielding than potentiometric devices. There is no detectable drift in the baseline after a 5- to 10-min warm-up period. The maximum drift in response to standards observed was about 1%over a 3-h period. Segmenting the sample stream with air shortens the response time but the instrument can be operated without air injection if slightly longer sampling times are used. There is no need for debubbling the sample stream. The instrument is readily adapted to different concentration ranges by changing the sensitivity of the conductivity meter or by changing the size of the sample and reagent pump tubes. Preliminary observations indicate additional applications of the instrument. Adaptation t o continuous sampling should present no problem. I t responds to atmospheric carbon dioxide when air is pumped in place of sample and reagent so it may have use in gas analysis. It may be possible to use it
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for nitrate determinations by passing the sample stream over a bed of Devarda alloy to convert nitrate to ammonia in a manner similar to that described by Mertens et al. (11). The method described here has some advantages over methods currently in use for ammonia determination. Compared with the distillation-titration procedure, it is faster, easier to automate, and can be used with smaller samples. The reagents are simpler than those used in colorimetric procedures and the method will tolerate colored or turbid samples which could not be run colorimetrically without pretreatment. Samples with high concentrations of solutes can be analyzed if standards contain approximately the same concentration of solutes. The gas sensing ammonia electrode will not tolerate high solute concentrations. Electronic drift is much less that that encountered with the ammonia electrode. The advantages of this new method should make it an attractive alternative to the methods currently in use.
LITERATURE CITED (1) R. H.Herdricks,M. D.Thomas, M. Stout, and B. Tolman, I&. Eng. Chem., Anal. Ed., 5, 23-26 (1942). (2) L. Appieton, Chemist-Analyst,42, 4-7 (1953). (3) J. Shaw and B . W. Staddon, J . Exp. Biol., 35, 85-95 (1958) (4) F. E. Friedl, Anal. Biochem., 48, 300-306 (1972). (5) J. Keay, and P. M. A. Menage, Analyst(London), 94, 895-899 (1969). (6) Technicon Corporation, Tarrytown, N.Y., Industrial Method No. 330-74A. (7) H.P. Kollig, J. W. Falco. and F. E. Stancil. Jr.. Environ. Sci. Techno/., 9, 957-960 (1975). (8) E. Scarano and C. Calcagno, Anal. Chem., 47, 1055-1065 (1975). (9) L. B. Westover, J. C. Tou, and J. H. Mark, Anal. Chem.. 46, 568-571 (1974). (10) R. E. Gugger and S. M. Mozersky, Anal. Chem., 45, 1575-1576 (1973). (1 1) J. M e n s , P. Vandenwinkel, and D.L. Massart, AM/. Chem.,47, 522-526 (1975).
RECEIVEDfor review April 11,1798. Accepted June 23,1978.
Membrane Electrode Measurement of Lysozyme Enzyme Using Living Bacterial Cells Paul D'Orazio, M. E. Meyerhoff, and G. A. Rechnitz' Department of Chemistry, University of Delaware, Newark, Delaware
Llvlng bacterial cells of the strain Micrococcus lysodeiktlcus are used as substrate for the determination of lysozyme actlvlty. The cells are loaded with a marker ion which Is released through the action of lysozyme upon the cell wall. The rate of ion release is monitored with a highly selective membrane electrode and is readily related to the concentration of enzyme present. The proposed method has excellent sensitlvlty and offers advantages of precision and convenience over previous turbidimetric methods.
Recently it has been shown that living bacterial cells can be used in conjunction with gas sensing electrodes to form bioselective sensors (1-3). So used, these cells are effectively serving as biocatalysts a t or near the electrode surface. Similarly, other vesicles such as sheep red blood cell ghosts loaded with electroactive marker, have been employed as analytical reagents for the determination of complement and antibodies by coupling the resultant lysing action to an appropriate ion selective electrode ( 4 ) . We now have extended
1971 1
this approach by loading living bacterial cells with marker and using these cells as a reagent for the measurement of an antimicrobial enzyme such as lysozyme. Lysozyme (EC 3.2.1.17) catalyzes the hydrolysis of cell walls of several Gram-positive bacteria, with maximum activity toward Micrococcus lysodeikticus. The enzyme acts upon mucopolymers of these walls by breaking p-( 1-4)-glycosidic linkages between alternating N-acetylmuramic acid and N-acetylglucosamine residues ( 5 ) . Action of the enzyme in an osmotically protective medium results in formation of protoplasts which are intact living cells having a cell membrane but no cell wall (6). However, enzyme action in a hypotonic medium results in osmotic rupture of the membrane and release of the cytoplasmic material. Assays of lysozyme in clinical and research laboratories are most often carried out by a turbidimetric method, where decreases in absorbance of a M . lysodeihticus cell suspension are measured with time at 450 or 540 nm (7). The method suffers from a lack of precision and other problems arise from turbidity in the unknown sample. More recently immunochemical techniques have been suggested but results, thus far,
0003-2700/78/0350-1531$01.00/00 1978 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 11, SEPTEMBER 1978
rh membrane e l e c t r d? senses free trnpa?
c
., I
suspension of marker loaded M lysodeikticus
Figure 1. Schematic representation of ion electrode measuring system for lysozyme
conflict with those obtained by the turbidimetric methods (7). We now have been able to load living M.lysodeikticus cells with the marker trimethylphenylammonium ion (TMPA+) and subsequently measure its release with a TMPA+ selective membrane electrode as those cells undergo lysis due to lysozyme activity. Figure 1schematically illustrates this process where free TMPA+ released from the ruptured cells is sensed by the membrane electrode. Under appropriate conditions, the initial rate of potential change is a function of the rate of lysis and is, therefore, directly proportional to the amount of lysozyme present. The method appears to be sufficiently sensitive, reproducible, and selective to measure lysozyme in physiological fluids.
EXPERIMENTAL Equipment. Potentiometric measurements were made with a Corning model 1 2 research pH meter in conjunction with a Heath-Schlumberger model SR-204 strip chart recorder. The indicator electrode, highly selective for TMPA’ over other monovalent cations is a slightly modified version of that described previously (4,8). The organic exchanger, tetraphenylboron, was heat polymerized in poly(vinylch1oride) forming a 1-mm thick “plug” in the end of a 2-mm i.d. glass barrel. The internal element consisted of an Ag/AgCl wire dipped into a 0.01 M trimethylphenylammonium chloride solution. The selectivity coefficients ~ +were determined to of this electrode, measured as K T W ~ + ,(9) be: for M+ = K+, 6.9 X lo2; Na+, 5.2 X lo3; Li+, 1.4 X lo4. Potential measurements were made with respect to a miniature saturated calomel reference electrode. Temperature during the course of all experiments was controlled at 25 “C by means of a Haake model FS circulator and a thermostated sample cell. Reagents. Lysozyme, grade I from egg white was obtained from Sigma Chemical Co., St. Louis, Mo. The preparation was reported to contain 0.88 g protein/g solid with an activity of 46 360 units/mg protein by the standard method (10). Stock lysozyme solutions were prepared in phosphate buffer (pH 6.5). Trimethylphenylammonium chloride was obtained from Eastman Kodak Co., Rochester, N.Y. and was used without further purification. The bacterium Micrococcus Lysodeikticus, No. 4698, was obtained from the American Type Culture Collection, Rockville, Md., and was grown in medium 265 as recommended by ATCC (11). The medium components were nutrient broth and yeast extract from Difco Laboratories, Detroit, Mich., and infusion broth from Baltimore Biological Laboratories, Cockeysville, Md. Normal human control serum (Validatelot 4k055) was obtained from General Diagnostic, Morris Plains, N.J. All potentiometric measurements were made in phosphate buffer (pH 6.5) with an ionic strength of 0.05 M. Procedure. The bacterial cells were loaded with TMPA+ by a modification of previous stepwise methods outlined for inserting
i ,T
e m
3
‘ 3 :
I
Figure 2. Typical potential vs. time plot obtained after addition of lysozyme to a suspension of loaded bacteria
substances into microorganisms (12, 13): 1. Approximately 0.25 cm3of packed cells were separated from the growth medium and washed 3 or 4 times with ice cold 0.09 M NaC1. 2. The cells were resuspended in 3.5 mL of NaCl solution which had been stored at 42 “C. 3. Solid trimethylphenylammonium chloride was added to achieve a desired final concentration of TMPA+. 4. The cells were incubated at 42 “C for 30 min, followed by an incubation in an ice bath for 30 min. 5. Separation of background marker from the cell suspension by extensive dialysis at 4 “C against 0.05 M or 0.15 M LiCl depending upon the desired ionic strength of the final suspension. Sodium chloride (0.09 M)was used as an equilibrating solution during the loading steps based on reports of its ability to enhance uptake of various solutes by M.lysodeikticus (14). LiCl was chosen as the dialysis medium because of the electrode’s high selectivity over lithium. All measurements were made in a total volume of 0.6 mL. An appropriate volume of bacteria was diluted in the sample cell with 0.05 M phosphate buffer to yield a final ionic strength of 0.05 or 0.1 M. The electrodes were then immersed into the mixture and a baseline potential was obtained. This potential is indicative of the low background or untrapped level of TMPA’ in the cell suspension. Addition of high concentrations of lysozyme (-0.1 mg/mL) produced rapid and complete cell lysis and the total potential changes ( S ’ s ) reflect the amount of marker trapped. Additions of varying lower concentrations of lysozyme generated an electrode potential vs. time response typified in Figure 2. Rates were determined from the line segment labeled “rate portion of curve” and were expressed in mV/min. Rates plotted as a function of lysozyme concentration in kg/mL were used to construct calibration curves. The validity in using a potentiometric sensor in such a manner for enzyme assays has recently been discussed (15).
Calibration of the system for the determination of lysozyme in control serum was done by two methods. First, the baseline potential was obtained in buffered solutions of the lysozyme standard or control serum. Addition of known aliquots of the cell suspension (substrate) produced a negligible initial potential change owing to efficient removal of background marker. Consequently, any further change is a result of enzymatic lysis. t\lternatively, known additions of the enzyme standard or control serum could be made t o a buffered cell suspension. Because of the high selectivity of the indicator electrode toward TMPA’, baseline potential shifts produced by cationic constituents in the serum or standard solutions were insignificant (
