A Kinetic Method for Analysis of Mixtures Employing Enzyme-Catalyzed Reactions SIR: Enzymes have two properties which have made them excellent analytical reagents: they catalyze certain organic reactions, and this catalytic act'ion is very specific wit,h regard to the nat'ure of the organic reactant (substrate). Thlis, enzymes are useful because they permit ordinarily slow reactions to proceed a t reasonable rates under moderate conditions and permit the determination of a specific species in a mixture without interference from other components. ,4 considerable number of analytical techniques have been developed for the determination of a single substrate ( 1 ) . Also, techniques have been developed for the determination of a single species which either increases (activators) or decreases (inhibitors) the rate of an enzyme reaction and for the actual determination of an enzyme itself ( 1 ) . No applications for the simultaneous determination of mixtures have been reported, however. Investigators have found that, although most enzymes are specific for the reaction of a certain functional group of a molecule, the rates within a single class of substrate molecules can vary considerably (9). Also, some enzymes will catalyze the reaction of different functional groups (9). Thus, the analytical applications of enzyme reactions could be extended to allow the simultaneous determination of two or more components of a mixture by employing techniques based on differential reaction rates. Such techniques have been successfully employed (using uncatalyzed reactions) for the simultaneous determination of mixtures of closely related compounds, such as isomers, homologs, and different functional groups of polymers (10). Differential reaction rate techniques have proved to be useful because no separation of reactants is necessary. The different'ial rate relationships that enable enzyme-catalyzed reactions to be utilized for simultaneously determining the init'ial concentration of components of a mixt'ure are readily derived, as shown below. The rate expression for t,he initial rate, R,, of an enzyme-catalyzed reaction derived assuming the Michaelis-Menton mechanism ( 7 ) for enzyme cat'alyzed reactions, has the form (4, 7 ) :
strate ( I ) , [SI0 is the initial concentration of substrate, and K u is the Michaelis constant which is the substrate concentration which results in an initial rate equal to R m s x / 2 . When [ S I 0 is small compared to K , ([SI, the oxidation of both ethanol and n-propanol at different rates ( R W X IR,r,-,,ro,,snol0H=21!2) under t'he
condit’ions employed below. This system was chosen to best t,his method. The reaction involves the oxidation of the alcohols in the presence of the coenzyme. nicotinamide adenine dinucleotide (S.\D) : R--CHAOH
I) + NAD e R-CHO + S.iDH2 h
13
(7)
The reactions were run in a pyrophosphate-phosphate buffer, p H = 8.8, and the enzyme solutions were made up and their activity determined by the method of 1-allee and Hoch ( 1 1 ) . The courhe of the reaction was followed by recording the rate of increase of adsorbcnce of S;\DH2 a t 340 mp with a Berkman Xodel 1113 recording spectropho;ometer which was thermostated a t 26.1” C. A-lllsolutions were thermostated a t this temperature prior to mixing. The enzyme solution was rapidly added to the alcohol mixture with a microliter pipet which was forceemptied with a syringe. *is a binary mixture was determined, it was necessary t o measure the initial rates of reaction under two different conditions: solution of 1.25 X 10-b.ll K h D and 8.8 X mg.!ml. of A D H ; and 0.75 x ~ O - ~S.411 J I and 4.4 x mg./ml. of .IDH. Some typical results of the determinations are shown in Table I. Ailthough the accuracy is not, exceptionally good as t,he experimental
procedure was crude [automated methods are generally required to yield accurate results when enzyme-catalyzed reactions are employed ( I ) ] , it is sufficient to demonstrate that the method is valid. The application of this method for the determination of other alcohol mixtures, a-hydroxy acid mixtures [using lactic dehydrogenase (9) 1, and amide-ester mixtures [using trypsin (9)], is being investigated. Also, the optimum conditions for accurate analysis, such as optimum ratios of initial rates of the individual components and optimum values of G and G’ for each component is also being studied and will be reported in detail in the near future. LITERATURE CITED
( 1 ) Blaedel, W. J., Hicks, G. P., “Ad-
vances in Analytical Chemistry and Instrumentation,” C. S . Reilley, ed., Yol. 3, Interscience, New York, in press. ( 2 ) Briggs, G. E., Haldane, J. B., Biochem. J . 19, 338 (1925). ( 3 ) Devlin, T. hl., ANAL.CHEM.31, 977 (1959). ( 4 j Dixon, M., Webb, E. C., “Enzymes,” Academic Press, Kew York, 1960. ( 5 ) Garmon, R. G., Reilley, C. N., ASAL. CHEM.34,600 (1962). ( 6 ) Mark. H. B. Jr., Papa, L. J., Abstracts of 144th Meeting, ACS, Los Angeles, Calif., April 1963, p. 24B. ( 7 ) Michaelis, L., Menton, M. L., Biochem. 2. 49, 333 (1913).
Table I. Determination of Ethanol” in Eth anol-n-Propanol Mixtures
(Alcohol dehydrogenase catalyzed reaction with nicotinamide adenine dinucleotide. Temperature, 26.1” C. Total alcohol concn., 5 X I O - 3 M ) NO. Ethanol in mixtures, % detns. Present Found 2 2 5 2
16 36 50 80
24, 22 31, 35 57-49 73, 77
% n-Propanol can be determined by subtracting yc ethanol found from 1 0 0 ~ o . ( 8 ) Neilands, J. B., “Organic Analysis,” J. Mitchell, ed., Vo1. 4, p. 65, Interscience, Sew York, 1960. ( 9 ) Xeilands, J. B., Stanier, R. Y.,
“Enzyme Chemistry,” 2nd ed., pp. 185-7, Wiley, K e a York, 1958. (10) Papa, L. J., Mark, H. B., Jr., Reilley, C. N., ANAL.CHEM.34, 1443 (1962). ( 1 1 ) Yallee, B. L., Hoch, F. L., Proc. Natl. Acad. Scz. 41, 327 (1955).
H ~ R RB. Y MARK,JR. Department of Chemistry The University of Michigan Ann Arbor, Mich. RECEIVEDfor review March 10, 1964. Accepted April 13, 1964. Acknowledgment is made to the donors of the Petroleum Fund, administered by the ACS, for partial support of this work. Analytical Division, 147th Meeting, ACS, Philadelphia, Pa., April 1964.
Determination of Argon and Oxygen by Gas Chro matogrcphy SIR: To date, there are no rapid and simple methods for the determination of en and argon by gas chromatography. Similar retention times are exhibited by these two gases on all adsorbents at room temperature or above, and t,heir separation can be effwted only under extreme conditions. Vizard and Wynne ( 7 ) reported a n and argon on a 10-meter column of IIolecular Sieve 5A\ oprrated a t room t,emperature, using hyclrogcn carrier; with such a long colunin, 1 owever, retention times were excessively long and sensitivity was Ion.. Lard and Horn ( 3 ) were able to separate oxygen and argon on a 6-foot column of hIolecular Sieve 5 h operated a t loiv temperature ( - i 2 ” C.), using helium carrier gas; for air analyses, however, a second run a t room temperature was necessary to determine nitrogen, since this gas is irreversibly absorbed a t loiv temperatures. Other methods ( I , 6) have been reported in
which oxygen was used as carrier for argon, and argon as carrier for oxygen; sensitivity was very 1)oor, however, due to the similar thermal conductivities of carrier gas and unknown. Krejci, Tesarik, and Janak ( 2 ) have described a procedure employing hydrogen carrier, in which oxygen was converted to water by passage over a palladium catalyst and subsequently removed. This procedure requires two separate runs, one for the purpose of determining the combined 02-Ar peak before separation, the second for the measurement of Xr alone after the oxygen has been removed. An application of this technique has been briefly described in a paper concerned with the determination of dissolved gases in aqueous solutions ( 4 ) . The method described in this report for the separation and determination of oxygen and argon is a modification of the procedure of Krejci. Hydrogen carrier gas, in the presence of a palladium catalyst a t
room temperature, is used to quantitatively convert oxygen to water vapor. The gas mixture is then separated by the usual chromatographic techniques, and each component is independently measured. Only one run is thus required, and the necessity for determining one of the components by difference is eliminated. A block diagram of the system used is shown in Figure 1. The DynatronicChrom-Alyzer-100 employs two hotwire thermal conductivity detectors, the second detector normally being used as a reference detector. I n this work, however, the reference detector was employed as a second measuring detector. A constant current poner supply was used to provide detector cell currents of 350 ma. Hydrogen was used as carrier gas a t a flow rate of 65 cc. per minute. The gas mixture was first passed through a catalyzer, consisting of a 1-inch length of ’/,-inch aluminum tubing filled with 30-60 mesh palladium VOL. 36, NO. 8, JULY 1964
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