Use of Enzymes in Analytical Chemistry ... - ACS Publications

May 1, 2002 - Fluorometric substrate for sulfatase and lipase. George G. Guilbault and James. Hieserman. Analytical Chemistry 1969 41 (14), 2006-2009...
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Use of Enzymes in Analytical Chemistry G. G. Guilbault, Research laboratories,

T

surveys the literature and developments from January 1960 through March 1966 with emphasis on the past few years. HIS REVIEW

GENERAL

Enzymes are biological catalysts n hich enable the many complex chemical reactions, upon which depends the very existence of life as we know it, to take place at ordinary temperatures. Because enzymes work in complex living systems, one of their outstanding properties is specificity. An enzyme is capable of catalyzing a particular reaction of a particular substrate, even though other isomers of that substrate or similar substrates may be present. .An example of the specificity of enzymes with respect to a particular substrate is found in luriferase, which catalyzes the oxidation of luciferin to oxyluciferin (250). A rather complete study of many compounds similar in structure to luciferin, showed that the catalytic oxidation resulting in the production of the green fluorescence occurs only with luciferin. Substitution of an amino group for a hydroxyl group or addition of another hydroxyl group to the luciferin molecule alters the enzymic action, and the green fluorescence is not produced. Another example of the specificity of enzymes is glucose oxidase, which catalyzes the oxidation of p-Dglucose to gluconic acid. A rather complete study of about 60 oxidizable sugars and their derivatives showed that only 2-deoxy-~-glucoseis catalyzed a t a rate comparable to that of p-D-glucose. The anomer a-D-glucose is oxidized catalytically less than 1% as rapidly as the p-anomer (37). ‘IJrease, which catalyzes the hydrolysis of urea, is even more specific. Enzymes exhibit specificity with respect to a particular reaction. If one attempted to determine glucose by oxidation in an uncatalyzed way, for example by heating a solution of glucose and an oxidizing agent like ceric perchlorate, other side reactions would occur uncontrollably to yield products in addition to gluconic acid. With glucose oxidase, on the other hand, catalysis is so effective a t room temperature and a neutral pH that the rates of the other thermodynamically possible reactions are negligible. This specificity of enzymes, and their ability to catalyze reactions of substrates a t low concentrations is of great use in chemical analysis. Enzyme catalyzed reactions have been used for

U. S.

Army Edgewood Arsenal, Edgewood Arsenal, Md.

analytical purposes for a long time for the determination of substrates, activators, inhibitors, and also of enzymes themselves. Until recently, however, the disadvantages associated with the use of enzymes have seriously limited their usefulness. Frequently cited objections to the use of enzymes for analytical purposes have been their unavailability, instability, poor precision, and the labor of performing the analyses. While these objections were valid earlier, numerous enzymes are now available in purified form, with high specific activity, at reasonable prices. The instability of enzymes is, of course, always a potential hazard; yet, if this instability is recognized and reasonable precautions are taken, the difficulty may be minimized. Again, the poor precision, slowness, and labor that have made enzyme-catalyzed reactions unappealing as a means of analysis may be more a consequence of the methods and techniques than the fault of the enzymes. With the advent of new techniques, fluorometric and electrochemical, many of the previous difficulties have been resolved. I n addition the automation of enzymic reactions has increased the speed, ease, and reproducibility of assayutilizirig enzymes. Such automation will be discussed later in this review. Another problem in the use of enzymes in analytical chemistry, lies in the cost of using large amounts of these materials, especially in routine analysis. This problem has been solved to some extent by the development of an immobilized (insolubilized) enzyme technique which allows continuous use of the enzyme for up to one day. This aspect will likewise be discussed below. The following is a Michaelis-Menten equation for an enzyme kinetics:

+ S ekiE S + E + P ki

E

k-’

In this mechanism, the substrate, S, combines with the enzyme, E , to form an intermediate complex, E S , which subsequently breaks down into products, P , and liberates the enzyme. The equilibrium constant for the formation of the complex, K,, the Michaelis constant, is defined as (kz k.-l)/kl; the rate of reaction, oOJis then some function of the enzyme and substrate (see Equation l), as well as of activator and inhibitor concentration, if the latter two are present. At a fixed enzyme concentration,

+

uo

=

Vmx [SI,/ (Km

+ [SI,)

(1)

2 IO I 0

the initial rate increases with substrate until a nonlimiting excess of substrate is reached, after which additional substrate causes no increase in rate. The concentrations of material participating in an enzyme reaction can be calculated in one of two ways: by measuring the total change that occurs by chemical, physical, or enzymatic analysis of the product or unreacted starting material; or from the rate of the enzyme reaction. In the first method, large amounts of enzyme and small amountr of substrate are used to ensure a relatively complete reaction. The reaction is allowed to reach equilibrium, and the amount of substrate S in the sample can be calculated from the E amount of P formed ( S --+P. P is chemically and physically distinguishable from S; e.g. Ethanol

E

+ K;hD+ ~ehydrogenas~ Acetaldehyde + NADH Alcohol

of N;1DH = 6.22 X lo6 sq. cm./mole at 340 mH).

Alternatively, a coupled reaction can be used to indicate how much substrate has been decomposed. Enzyme reaction: Glucose

+ HzO + 02 ~xidase. Glucose

Gluconic Acid

+ H202

(2)

Indicator reaction:

H202

+ leuco-dye Peroxidas: H20

+ dye

(3)

The intensity of the dye produced is a measure of the concentration of glucose present. In the second method, the kinetic method, the initial rate of reaction, v o , is measured in one of many conventional ways, by following either the production of product or the disappearance of the substrate. The rate is a function of the concentration of substrate (S), enzyme ( E ) , inhibitor ( I ) , and activator ( A ) . For example, the concentration of glucose can be determined by measuring the initial rate of production of dye in the example given above (Equations 2 and 3 ) . Because it is more reliable, the total change method is generally favored over the rate method. However the former technique can only be used for substrate analysis, and not for E , A , and VOL. 38, NO. 5, APRIL 1966

527 R

I which are catalytic in nature and affect only the rate and not the equilibrium. Also, the rate method is faster, because the rate can be measured initially without having to wait for the reaction to go to completion. The conditions that affect the rate (pH, temperature, ionic strength) must be carefully controlled in the kinetic method for maximum sensitivity. The temperature coefficient of the enzyme reaction rate is roughly 10% per degree (173), and a 10’ C. rise in temperature causes a 100% increase in the reaction rate. Hence constant temperature is essential in the assay of enzyme activity. If normal precautions are taken, the error in measurement of enzyme activity is usually less than *5%. BOOKS A N D REVIEWS

Several excellent books are available on enzymic methods for the determination of enzymes, substrates, activators, and inhibitors ( I S , 29, 36, 64, 126, 230), Purdy (192) has authored a book on “Electroanalytical Methods in Biochemistry” that contains material on enzymes, and fluorometric methods of enzymic assay may be found in Udenfriend’s book (236). Dixon and Webb (44) and Long (137‘) have compiled tabulations on several hundred enzymes and their assay methods. Blaedel and Hicks (19) have written a chapter on the “Analytical AApplicationsof Enzyme Catalyzed Reactions” in Reilley’s book. Reviews on enzymic analysis have been published by Bergmeyer (14), Guilbault (7’3),and Devlin (42). A review in which about 80 enzymatically determinable substrates (C, to Clo)are tabulated has been compiled by Keilands (17‘2),and techniques of enzymic assay in clinical chemistry and in food chemistry have been written by Kingsley (114) and Sloman and Borker (228), respectively. DETERMINATION OF ENZYMES

Since the enzyme is a catalyst, theoretically one moleclue of this material would eventually produce a sufficient change in the substrate to be measured. Hence, high sensitivities may be realized in enzyme analysis. Because the concentration of enzyme is so small, it always limits the rate of reaction, and the rate can be taken as a measure of the enzyme concentration. In Equation 1, the oxidation of glucose by oxygen to give peroxide and gluconic acid is catalyzed by glucose oxidase. The rate of production of peroxide is measured by a second coupled reaction, the oxidation of a leuco dye, such as o-dianisidine, to yield a highly colored dye. When glucose, leuco dye, and oxygen are non-rate limiting, the overall rate of reaction, as indicated by the rate of production of the dye, is proportional to the glucose oxidase activity. 528 R

0

ANALYTICAL CHEMISTRY

Common techniques (spectrophotometric, change in pH, manometric) have been described for the assay of almost all enzymes (13, 44, 137), and these will not be discussed in the present review. Characteristics and assay procedures for most of the commercially available enzymes are likewise given in some manufacturer’s catalogs. Rather, some of the newer, more sensitive techniques for enzymic assay will be discussed. As was mentioned above, the use of enzymes as analytical reagents has received notoriety, mainly due to the poor precision and lengthiness of previous procedures. The recent trends in enzymic analysis have been in two main directions: to develop more sensitive procedures and to replace the long, tedious methods previously used for assay with rapid, easily instrumentable techniques. Because of the simplicity of electrochemical techniques, and their susceptibility to automation, such techniques have been used by a number of analysts to follow enzyme activity. Guilbault, Kramer, and Cannon (79) have developed a kinetic method for cholinesterase and thiocholine esters based on the electrochemical measurement of the rate of hydrolysis of the ester by the enzyme sample. Rates were measured by recording the difference in potential between two platinum electrodes polarized with a small, constant current. The thiol produced upon enzymic hydrolysis is more electroactive than the substrate, so a reduction in potential results. The complete theory of this method has been worked out and may be found in references (82) and (83). Organophosphorous compounds (Sarin, Systox, parathion, malathion) inhibit the enzyme and may be determined a t 10-9-g concentrations by this technique, with a deviation of about 1% (80). The general applicability of this potentiometric method in following any enzymic B reaction of the type A +C D where the substate A , undergoes enzymolysis by B to form products C and D,has been demonstrated in procedures developed for glucose and glucose oxidase (84), xanthine oxidase (81) and peroxidase and catalase (72). In all cases, the change in potential with time, hE/min, was found to be proportional to the concentration of the enzyme analyzed. Pardue and Malmstadt have developed automatic electrochemical methods for the determination of glucose oxidase, and glucose based on the oxidation of glucose to peroxide, followed by the oxidation of iodide to iodine by peroxide in the presence of molybdate as catalyst. The iodine produced, whose rate is proportional to the rate of oxidation of glucose, is detected either potentiometri-

+

cally (151,152,186)or amperometrically (180, 185). In either case, automatic control equipment provides a direct readout of the time required for a predetermined amount of iodine to be produced. The reciprocal of the time interval is proportional to the glucose oxidase activity or glucose concentration with relative standard deviations of about 2%. Blaedel and Olson (22) developed a method for the assay of glucose and glucose oxidase by an amperometric procedure similar to the one described, except that the peroxide produced oxidizes ferrocyanide to ferricyanide, which is measured with a tubular platinum electrode. Pardue has extended the electrochemical techniques described to the assay of galactose and galactose oxidase (183). The peroxide produced again reacts with iodide to form iodine, which is detected amperometrically. The reciprocal of the time interval required for a certain current to be produced is proportional to the materials analyzed with a deviation of about 2%. Purdy and coworkers have recently described amperometric and coulometric methods for the determination of enzymes and substrates. In one application, an amperometric method is used to follow the reaction of uric acid with the enzyme uricase (191). The reaction forms hydrogen peroxide which reacts with iodide ion t o form iodine. The formation of iodine is followed amperometrically, and the concentration of reacted uric acid is calculated from the amount of iodine formed. In another application, Purdy, Christian, and Knoblock described a method for the analysis of urease (193) based on the urease hydrolysis of urea to form ammonia. The resulting ammonia is then titrated with coulometrically generated hypobromite using a direct amperometric end point. In urine the ammonia could be titrated directly; but in blood samples, the ammonia is first separated by the microdiffusion technique. Katz (109) has described a potentiometric method for urease. A Beckman cationic sensitive glass electrode that responds to [“I+] is used to follow the course of the reaction. Polarographic techniques have been described for the determination of cholinesterase (52, 200) , 3-hydroxyanthranilic oxidase (59), and catalase in micro organisms (104). Rusznak et al. (208) determined starch by enzymic destruction using polarographic:, chromatographic, and oscillopolarographic methods. Curtain (40) assayed the activity of cholinesterase using a silver thiol electrode and a thiocholine ester as substrate. Lipner et al. (135) have developed a galvanic cell for the measurement of oxygen-consuming enzyme systems using a bucking potential circuit, and Malmstadt and Piepmeier (163) have designed an inexpensive pH

stat with digital readout for quantitative enzyme determinations. A stability of 10.002 p H units is reported. Because of limitations in molar absorptivities, measurement of gas volume, or of changes in pH, most procedures of enzymic assay are limited to substrate concentrations greater than 10-6hf. Since fluorogenic substrates are generally several orders of magnitude more sensitive than chromogenic ones, a large increase in the sensitivity of enzymic assay should result from the use of esters of fluorogenic materials. These compounds, themselves nonfluorescent, could be hydrolyzed by enzymes to form easily measured fluorescent products. Thus, much less material would be required, which is particularly important in biochemical work; and, because of the remarkable sensitivity, an easier localization of enzymes, related substrates, and coenzymes within organs and even within individual cells should result. In a recent publication, Kramer and Guilbault described a simple, rapid procedure for the assay of lipase activity in the presence of other esterases, based on the hydrolysis of fluorescein esters catalyzed by lipase (74, 121). By following the rate of production of the fluoresence of fluorescein with time, a series of curves are obtained, the slopes of which, A.F/At, are proportional to the lipase concentration over the range to 10-1 units. The method is rapid and eliminates the necessity of assay by heating the enzyme with olive oil a t high temperatures for 2 hours. Concentrations of fluorescein as low as lOW9M may be detected a t excitation and emission wavelengths of 495 and 570 mp. Four fluorogenic substrates were discovered for cholinesterase: resorufin butyrate (122),indoxyl acetate (76),and a- and p-naphthyl acetate (78). Indoxyl acetate is nonfluorescent, but is hydrolyzed by cholinesterase to first indoxyl, then indigo white, both of which are highly fluorescent(excitation and emission wavelengths 395 and 470 mp, respectively). Resorufin esters are likewise nonfluorescent, but are hydrolyzed to resorufin which is highly fluorescent. a- and @-Naphthyl acetate are nonfluorescent compounds, which are hydrolyzed by cholinesterase to the highly fluorescent 0- and @-naphthol. Using indoxyl acetate or P-napthyl acetate as substates, as little as 10-4 unit of cholinesterase can be assayed. Greenberg (68) has described the determination of alkaline phosphatase and aminopeptidase, and Rotman, Zderic, and Edelstein (207) have determined b-D-galactosidase, based on the amount of fluorescence produced from fluorogenic substrates. Keston and Brandt (llS), and Perschke and Broda (188) have developed methods for peroxidase using diacetylfluorescein and 6-methoxy-7-hydroxy 1,a-benzo-

pyrone(scopoletin), respectively, as substrates. The enzymatic hydrolysis by a-chymotrypsin of the substrates N-acetyl L-tryptophan ethyl ester and N-acetyl N-tyrosine ethyl ester was studied by Bielski and Freed ( 1 7 ) . The rate of production of the fluorescence of tryptophan or tyrosine indicated the amount of enzyme present. Moss has investigated the naphthyl phosphates as substrates for acid and alkaline phosphatase (171),and Mead et al. have prepared the glucuronides of umbelliferone and of 4-methyl umbelliferone for the assay of p-glucuronidase (161). A method for the fluorometric assay of trypsin was proposed by Roth (206),and Udenfriend, Weissbach, and Smith (237) have described the fluorometric assay of monoamine oxidase based on the oxidation of tryptamine to indoleacetic acid. A fluorometric method for p-glucuronidase using naphthyl-@-D-glucuronidewas described by Veritz, Caper, and Brown (240), and Roth (205) developed a method for leucine aminopeptidase using L-leucyl-2-naphthyl amine as substrate. An important class of enzymes are the dehydrogenases, which in the presence of a hydogen acceptor such as nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate(NADP) effect the dehydrogenation of hydroxy compounds. It has been stated that almost every material of biological interest can be assayed with the aid of auxiliary enzymes and the coenzymes NAD and NADP (140). Since NADH (the reduced form of NAD) has a high native fluorescence, and NAD yields a fluorescent derivative upon heating in alkali, many fluorometric procedures for enzyme assay have been described using the NADNADH and NADP-NADPH systems. Lowry and coworkers have described procedures for lactic dehydrogenase (141) and 6-phosphogluconate dehydrogenase (140) and for glutamic dehydrogenase (139, 1 4 1 ) . Maitra and Estabrook have developed fluorometric methods for the enzymatic determination of glycolytic intermediates by measuring the fluorescence of NADH and NADPH (145). Methods were described using the enzymes glucose-6-phosphate dehydrogenase, hexokinase, phosphoglucoisomerase, phosphoglucomutase, a-glycerophosphate dehydrogenase, triosephosphate isomerase, aldolase, glyceraldehyde 3-phosphate dehydrogenase, 3-phosphoglycerate kinase, phosphoglycerate mutase, and pyruvate kinase. Laursen and Hansen (128) and Laursen and Espersen (127) have developed methods for transaminases by coupling the following two reactions: transaminase

L-alanine

+ ketoglutarate e pyruvate + glutamate (4)

pyruvate

+ lactic acid dehydrogenase

NADH ---t lactate

>

+ NAD+

(5)

The increase in NAD+ is a measure of the transaminase activity. A simple, rapid fluorometric method was described by Guilbault and Kramer (75, 77) for measuring the activity of dehydrogenases. The method is based upon the conversion of the nonfluorescent material, resazurin, to the highly fluorescent resorufin in conjunction with the NAD-KADH or NADPS A D P H system. As little as l o w 4unit per ml. of the enzymes lactic acid dehydrogenase, alcohol dehydrogenase, malic acid dehydrogenase, glutamic acid dehydrogenase, glucose-&phosphate dehydrogenase, L-a-glycerophosphate dehydrogenase and glycerol dehydrogenase could be determined with standard deviations of less than one per cent. Likewise diaphorase (0.0004 to 0.080 unit per ml) and NADH (10-7 to 10-6M) may be determined with a standard deviation of about 0.5%.

+

+

Lactate NAD lactic acid dehydrogenase + pyruvate 2NADH

+ (6) 2 KADH + resazurin + diaphorase resorufin + 2NAD + H 2 0 (7)

+

Because of the intense fluorescence of resorufin (as little as lO-91M can be detected), an increase in sensitivity of two orders of magnitude over conventional procedures is realized. Recently Lowry has described an enzymatic cycling method for the measurement of pyridine nucleotides (158). The nucleotide to be determined is made to catalyze an enzymatic reaction between two substrates, which are transformed in amounts far greater (lo3- to lo4-fold) than the nucleotide. NAD is determined by its catalysis of the dismutation of a-ketoglutarate, NH4+, and lactate to glutamate and pyruvate. The pyruvate produced is measured in a second step with lactic dehydrogenase and an added excess of NADH. In the determination of NADP, glucose-&phosphate and 6phosphogluconate dehydrogenase are used instead of lactate and lactic dehydrogenase. As little as 10-15 mole of NAD or NADP can be determined by repeating the cycling process with an overall yield of lo7 to 108. This theoretically is sufficient to measure 1 molecule of any enzyme which forms a product that can lead to a pyridine nucleotide system. Sensitive techniques for enzyme assay have been recently developed, based on radiometric methods. Winteringham and Disney (252) assayed acetylcholinesterase using carbon-14 labeled acetylVOL. 38, NO. 5 , APRIL 1966

* 529 R

choline. The concentration of liberated acetic acid is calculated from the difference between the counts for the residual substrate and a control. Only 1-5 pl. of solution are required. Shatalova and Meerov (221) determined urease using carbon-14 labeled urea, and Sherman (223) followed phosphorylations catalyzed by galactokinase using radioactive substrate, ion exchange paper, and liquid scintillation counting. DETERMINATION OF SUBSTRATES

General. At a fixed enzyme concentration, the initial rate of an enzymatic reaction increases with increasing substrate concentration until a nonlimiting excess of substrate is reached, after which additional substrate causes no increase in rate. The region in which linearity is achieved, and in which an analytical determination of substrate concentration can be made based on the rate of reaction, lies The most important below 0.1 K,. advantage of an enzymatic assay is its specificity. Frequently only one member of a homologous series is active in the enzyme catalyzed reaction; other members are totally inactive or react a t much slower rates. Most enzymes are also specific for one optical isomer of the substrate. Thus, in the enzymatic assay of amino acids, bacterial amino acid decarboxylase is specific for Lamino acid only (60). Another advantage in the use of enzymes for substrate analysis lies in the great sensitivity obtained. Glucose, for example, is oxidized a t the rate of a few per cent per minute, regardless of concentration. Thus a lO-7.M solution can be analyzed as easily as one 10-4M. Carbohydrates. Glucose has undoubtedly received the most attention from analysts during the past few years. Hiljer ( 9 4 , Watson (244), and Hjelm and deVerdier (96) have described enzymatic methods for glucose in blood, Scott and Hammer (216) for glucose in the presence of galactose, Blecher and Glassman (23) for glucose in the presence of sucrose, Mansford and Opie (164) for glucose in starch conversion products, Fleming and Pegler (63) for glucose in maltose and isomaltose mixtures, Molne and Johanneson (166) for glucose in urine. Smith et al. (229) described an accurate glucose procedure employing Lactobacillus casei, Free (66) reviewed the enzymatic methods for the determination of glucose, and Fales (48) presented standardized procedures for glucose using glucose oxidase-peroxidase. Keston and Brandt found that the oxidation of the fi-isomer of glucose was much faster than the a-isomer, and used this to determine mixtures of the two isomers (112). Hansen (89) investigated the specificity of glucose methods, and Watson and Stevenson (245) compared four microblood glucose methods, 530 R

ANALYTICAL CHEMISTRY

and found the glucose oxidase-tolidine method the most satisfactory. Guilbault, Tyson, Kramer, and Cannon have described a sensitive electrochemical method for glucose (84). The rate of change in the potential of the system with time, due to the enzymatic production of the electroactive species, hydrogen peroxide, was found to be proportional to the glucose Concentration with a standard deviation of about 1%. Ogawa, Moriwaki, and Kishigami described a procedure for glucose in urine (179) and a test paper for small amounts of glucose using a glucose oxidase preparation has been patented by Scott (616). Specific enzymatic methods for glucose, fructose, glycerol, and malic acid in wine (61), and for glucose, fructose, and sucrose in plant material (106) have been described. Automated colorimetric methods for glucose analysis have been described by Getchell, Kingsley, and Schaffert (62) using the Auto Analyzer; and by Malmstadt and Hicks (149), Malmstadt and Hadjiioannou (146) and Blaedel and Hicks (20, 21) using a continuous analysis based on the measurement of initial reaction rates. The time required for a fixed change in the absorbance to occur or the absorbance change in a fixed time period was a measure of the concentration of glucose present. Automatic electrochemical methods for glucose by Pardue and Malmstadt (161, 162, 180, 186, 186), and by Blaedel and Olson (22) have already been discussed. Pardue has further automated the determination of glucose using the glucose-glucose oxidase-iodide system. The formation of iodine is detected potentiometrically, the slope of the response curve being measured at a preset voltage (181). Under controlled conditions, the measured slope is proportional to the glucose concentration with a deviation of 1.5%, and a response time of 10 to 100 seconds per sample. In a recent paper, Pardue, Frings and Delaney (184, described an automatic method for measuring the concentration of glucose and galactose that provides direct readout of concentration data. An electrical divider for the continuous computation of reciprocal times was used in combination with automatic control equipment to provide digital readout of concentration data. Malmstadt and Piepmeier used an automatic pH stat with digital readout for the determination of glucose and urea (163). Methods for the determination of blood galactose were reported by Ford and Haworth (64,deVerdier and Hjelm (41) and Roth, Segal, and Bertoli (S04) who used a galactose oxidase-peroxidase system; by Bowden (28) who employed a glucose oxidase-peroxidase reagent; and by Waldstein, Dublin, Newcomer, and McKenna (942) who used specially purified “glucose oxidase.” Pardue and

Frings have described automatic amperometric (183) and colorimetric (68) methods for galactose in aqueous solution and blood, respectively. Enzymatic methods for the determination of glycogen were reported by Bueding and Hawkins (32), Topi and Zane (236) and by Johnson et al. (107, 108). Maitra and Estabrook (146) have described fluorometric procedures for the determination of glycolytic intermediates, such as glucose-6-phosphate, glucose-1-phosphate, fructose diphosphate, glyceraldehyde-3-phosphate, dihydroxy acetone phosphate, and 1,3-diphospho-, 2-phospho-, and 3-phosphoglyceric acid. Inouye, Scheider and Hsia (103) developed a method for galactose-6-phosphate, and Arese has described a procedure for ~-ghconate-6-phosphate(6). Methods for glucose-6-phosphate were suggested by Greengard (69) and by Borrebaek, Abraham, and Chaikoff (27). Sempere, Gancedo, and Asensio (219) determined galactosamine and N-acetyl galactosamine in the presence of other hexosamines with galactose oxidase, and Seraydarian, Mommaerts and Wallner estimated hexose and triosephosphates using the NAD-NADH system (220). The difference between acid and enzyme hydrolysis was used to determine dextrine in beer (212),and an enzymatic method for inulin in blood or urine was described by Renschler (198). Hydrolysis to fructose was effected with perchloric acid, and the fructose was then determined using glucosed-phosphate dehydrogenase and NADP. An enzymatic procedure for lactose in meat products containing maize syrup solids was described by Hankin and Wickroski (88) and Rusznak, Gergely and Komiszar (208) have described polarographic, chromatographic, and oscillopolarographic methods for starch based on enzymatic destruction. Amino Acids. Enzymatic methods for serum phenylalanine and tyrosine were described by Knapp et al. (117) and LaDu et al. (lag). Lipner, Witherspoon, and Wahlborg (135) proposed the use of a galvanic cell technique for measuring tyrosine and cleucine. Methods for individual amino acids were described by Nederman, Reichelt, and Wolin for r,-asparagine using Streptococcus bovis Jfutant (174);by Ramadan and Greenberg (196) who determined glutamine and asparagine using a purified enzyme from a strain of Pseudomonas; by Pearson and Tubbs (187) for carnitine; by Lack and Smith (123) who described a highly specific method for a8 little as pmole of reduced glutathione using maleyl-pyruvic acid isomerase; Sowerby and Ottaway (231) and Woodward (266) who determined glutamic acid; by Yoshida (268) who developed a method for calanine (0.020.5 pmole) with calanine dehydrogenase purified from Bacillus subtilis and NAD;

and by hlalmstadt and Hadjiioannou who suggested a specific enzymatic method for the determination of as little as 10-7 equivalents of L-a-amino acids using a-amino acid oxidase, peroxidase, and o-dianisidine (148). Baldridge and Greenberg (8) determined histidine in blood by its reaction with L-amino acid oxidase in the presence of borate ions to yield an enolborate complex of imidazolepyruvic acid. Organic Acids. Steinbrecht and Hofman (232) determined low3 to lo-5M acetic acid using acetate kinase, and ascorbic acid was determined electrochemically by Kovacs (119) using catechol oxidase. Bergmeyer and Bernt (16) determined acetoacetate and D(-) 3-hydroxy butyrate in blood, and Selleck, Cohen, and Randall (218)proposed an enzymic method for the assay of aketo and a-oxo-glutarate in dog blood, plasma, urine, and tissue using glutamic dehydrogenase and NADH. Methods for lactic acid were described by Schoen @ I S ) , and by Friedland and Dietrich ( 5 7 ) ; by Blaedel and Hicks (20) using lactic acid dehydrogenase, IC'AD, and 2,6-dichloroindophenol; by Porsche (190) who used a dehydrogenase from yeast; by Mohme-Lundholm (164) in finger-tip blood; and by Lundholm et al. (142) who utilized a rabbit muscle dehydrogenase. Laycock, Thurman, and Boulter (129) determined L-lactic acid and L-malic acid using a dehydrogenase, NAD, and a tetrazolium dye. Holzer and Soling (99) proposed the use of a dehydrogenase and the 3-acetyl pyridine analog of NAD in the estimation of Llactate, L-malate, and L-glutamate. The change in absorbance was measured a t 366 mp, Wenzel and Gunther (248) proposed the preparation of specific tritiumlabeled L-lactic acid from tritiated water by means of a coupled enzyme reaction. The specific enzymic methods allowed the specific tritiation of appropriate stereoisomers, and [2-H3] L-lactate in 90-100~o yield was obtained. Nordmann, Arnaud, and Nordmann (178) developed a method for the enzymic determination of L-malic acid in blood and hfayer and Busch (160) determined malic acid in wine and grape juice using malic acid dehydrogenase. Rosano has described a procedure for 4-hydroxy-3methoxymandelic acid using l-mandelate dehydrogenase (201). Rosenbloom and Seegmiller (202) determined orotic acid in urine and serum using orotidylic pyrophosphorylase and decarboxylase, and Rfayer et al. (158) determined oxalic acid in urine with oxalic decarboxylase. Loeffler and Wieland (136) proposed a specific isotopic method for oxalacetate using [1-C14] acetyl coenzyme and a citrate-condensing enzyme. As little as 10-2 pg. could be detected. The simultaneous determination of phosphocreatine and adenosine triphosphate in muscle extracts was de-

scribed by Faway et al. (50). Maitra and Estabrook (145) described a procedure for pyruvic acid using lactic acid dehydrogenase and pyruvate kinase, and Landon et al. (125) measured blood pyruvate by a specific method employing the oxidation of NADH in the presence of lactic dehydrogenase. CurroDossi and Ripa (39) enzymatically determined pyruvic and lactic acids in cerebrospinal fluid, and Greengard (69) detected as little as 2.5 X 10-lo mole of pyruvate per ml. using lactic dehydrogenase. Succinic acid was determined electrochemically by Kovacs and Domjan (118) using succinic dehydrogenase and Rosenmund and Knob (205) determined uric acid with uricase. Buchanan, Isdale, and Rose (31) compared chemical and enzymatic methods for the serum uric acid determination, finding the latter more specific. Purdy determined uric acid electrochemically, using uricase ( 191). Alcohols and Esters. Leithoff (130) and Leithoff and Chan (131) determined blood alcohol automatically using alcohol dehydrogenase and NAD, and Malmstadt and Hadjiioannou (147) developed an automatic spectrophotometric reaction rate method employing the same enzymatic method. The absorbance of NADH was followed a t 320 mp. The use of enzymatic methods to analyze for ethanol and glycerol in beverages was proposed by Mayer (159). Drawert and Kupfer (45) and Boltralik and No11 (26) determined glycerol in wine and grape based on the esterification of glycerol to glycerol-1-phosphate with adenosine triphosphate in the presence of glycerol kinase. Hagen and Hagen (87) estimated the glycerol content in blood using glycerol dehydrogenase. Mark (156) has described a kinetic method for the analysis of mixtures of alcohols employing enzyme catalyzed reactions. The method of proportional equations was modified and applied to the determination of ethanol and n-propanol, both catalytically oxidized by alcohol dehydrogenase. Cooper (58) fluorometrically determined acetylcholine by mild reduction to ethanol and choline. The ethanol was then oxidized by alcohol dehydrogenase to acetaldehyde with concomitant reduction of NAD. Methods for L-a-glycerophosphate have been described by Nikkila and Ojala (176) and by Ciaccio (36) using L-a-glycerophosphate deydrogenase and KAD. Guilbault, Kramer, and Cannon determined thioesters electrochemically using cholinesterase (78), and West and Qureshi have proposed a qualitative test for esters using methyl red and lipase (249). A change in the color of the solution from colorless to orange or pink caused by the lipolytic hydrolysis of the ester indicated the presence of the compound. The test was found to be more specific than the hydroxamic acid test.

Miscellaneous Organic Compounds. Mei and Clegy (162) determined penicillin in milk using penicillinase; and Goryachenkova (67) assayed pyridoxyl phosphate and pyridoxamine phosphate using malate and glutamate dehydrogenases. The oxalacetic acid formed is converted into pyruvate by means of aniline citrate and the reaction rate is calculated from the amount of pyruvic acid dinitrophenylhydrazone formed. The procedure is specific for these two compounds a t a sensitivity of pg. per ml. An enzymatic method for ribonucleotides in foods has been reported by Shimazono (Z.24), and Bachrach and Oser (7) proposed a specific and sensitive (0.05-0.40 pmole) method for spermidine based on the formation of pyrroline from the polyamine by freeze dried cells of Serratia marcescens. Billiar and Eik-Nes (18) used cholinesterase to determine steroid acetates, and Margraf , Margraf, and Weichselbaum (166) assayed cortico-steroids in extracts from whole blood, plasma, and urine using 20-&hydroxy steroid dehydrogenase. Sih and Lava1 (225) enzymatically analyzed the functional groups on the steroid nucleus using A1 dehydrogenase from Nocardia restrictus. A4-3-oxosteroids are distinguished from A1s4-3-oxosteroids. Guilbault, Kramer, and Cannon determined the concentration of xanthine and hypoxanthine electro-chemically (81), and Wolf and Dugan used pacreatic lipase to determine the position of the fatty acids in triglycerides (253). Procedures for the assay of urea using urease were propsed by Nielsen (176) who determined urea in blood and urine using a pH meter, by Malmstadt and Piepmeier (153) who used a p H stat with digital readout, by Katz and Rechnitz (110) who measured the potential changes that occur with a Beckman cation sensitive glass electrode that responds to [NH4+],and by Purdy, Christian, and Knoblock (193) who followed the ammonia produced in the enzymic reaction coulometrically. Inorganic Compounds. The enzymatic determination of ammonia in tissue body fluids was described by Fawaz and Dah1 (51),in body fluids by Mondzac, Ehrlich, and Seegmiller (166), in blood and tissue by Reichelt, Kvamme, and Tveit (197),and in blood by Kirsten, Gerez, and Kirsten (115). All used the oxoglutarate-glutamic dehydrogenaseNADH system, measuring the change in absorbance a t 340 mp. NH4+

+ H + + NADH + dehydrogenase

oxoglutarate glutamate

+ NAD+ + HzO

The enzymatic procedure eliminates all the disadvantages of previous methods and is specific for ammonia in the presence of amines. Morrison (168-170) used carbonic anhydrase to determine VOL 38, NO. 5 , APRIL 1966

0

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carbon dioxide in lightly carbonated wines. Fluorometric methods for hydrogen peroxide were described by Keston and Brandt (113) who used peroxidase and diacetyl dichlorofluorescein and by Andreae (3) and Perschke and Broda (188) using peroxidase and scopoletin. The latter method is based on the decrease in fluorescence, and is not as good as the former. Guilbault assayed peroxide electrochemically using peroxidase and catalase (72),and Alexander ( 2 ) proposed a spectrophotometric assay of iodide using thyroid peroxidase. The triodide ion is measured a t 353 mp: 3 KI

+ HzOz

-peroxidase

13-

+ KOH

Nitrate was determined using nitrate reductase by Egami et al. (47). Nitrite doesnot interfere, and chlorate is the only other reducible anion. Block-Frankenthal (24) has described a method that is specific for inorganic pyrophosphate using pyrophosphatase. Mg(I1) ion is used as an activator:

P20,-*

-

+ H 2 0-

pyrophosphatase Mg + 2

2 HPOI-

DETERMINATION OF ACTIVATORS

An enzyme activator is a substance which is required for an enzyme to be an active catalyst: ElnaetlveActivator F! EaCt,,,.The activity of the enzyme will increase until enough activator has been used to activate the enzyme fully. The initial rate of the enzyme reaction is proportional to the activator concentration a t low concentrations, thus providing a method for its determination. Very little has been done in the analytical determination of activators. A method for magnesium in plasma is described by Baum and Czok ( I I ) , based on the activation of isocitric dehydrogenase. With constant amounts of enzyme, the rate is dependent on magnesium concentration down to 10-6M. A thorough study of this reaction was made by Adler, Gunther and Plass (1) and by Blaedel and Hicks (19), who found that only Mg+* and Mn+2 efficiently activate this enzyme. The useful analytical range extends up to about 100 parts per billion for Mn+2 and 2 x 10-4M for Mg+2. Hg+2 and Ag+ a t 10-5il.I~ and C a + 2 a tlo-4M inhibited the Mn+2 activation completely. Takagi and Isemura (234) found that Ca+2was needed for activation of taka-amylase A. A 10-4M concentration was needed for 80% regeneration, and only Sr+2 could replace Ca+2 in the activation. Dvornikova et al. (46) found that the potassium salts of fructose-6-phosphate and ATP activate unpurified phosphofructokinase from rabbit muscle considerably more than the Na+ salts. A number of enzymes require for their activity a specific coenzyme which participates in the enzymic reaction. By measuring the amount of activation of

+

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ANALYTICAL CHEMISTRY

such an enzyme by the coenzyme, a plot of initial rate of reaction us. coenzyme concentration may be constructed. At low concentrations of coenzyme, the degree of activation will be proportional to the concentration of coenzyme added. For example, flavine adenine dinucieotide (FAD), which is the coenzyme of Damino acid oxidase, can be determined by this method: acid + FAD n-amino Oxidase pyruvic acid + FADH + FADH + 02 FAD + HzOz

n-alanine

-

3"

Warburg and Christian (243)and Straub (233) were the first to describe this method. Unknown samples of FAD containing 0 to 0.3 pg. of FAD can be determined by measuring the increase in the rate of oxidation of alanine by Damino acid oxidase (101). Methods have likewise been developed for flavine mononucleotide (FMK) using NADP and cytochrome c reductase (101), for pyridoxal using tyrosine decarboxylase (86) for lipoic acid with pyruvate dehydrogenase (85), and for glutathione using the enzyme glyoxalase (195). The firefly reaction has been shown to require ATP and 1LIg+2in addition to luciferin, luciferase, and oxygen (250): luciferin

+ 02 + ATP

-luciferase

oxyluciferin

Mg +z

+ ADP + Pi

This reaction has been used as the basis for the most sensitive method for ATP that is known (241). This reaction may also be used to assay oxygen a t partial pressures below 10-3 mm. (34), when the gas is passed through a bacterial emulsion containing all requirements for the luminescent reaction except oxygen. A method for adenosine diphosphate (ADP) was proposed by Greengard (69) who detected as little as 2 X 10-10 mole of ADP using the phosphophenolpyruvate - KADH - pyruvate phosphokinase-lactic dehydrogenase system. Scott and Falconer (217 ) assayed adenosine 3',5'-monophosphate (69) using dog liver extract containing liver dephosphorylase, and Greengard (69-70) determined adenosine triphosphate in peripheral nerve fibers using either the glucose-hexokinase-glucose-6-phosphate dehydrogenase system : ATP

+ glucose

-hexokinase

+ glucose-6-phosphate glucose-6-phosphate + ADP

dehydrogenase

NADP -d 6-phosphogluconate NADPH

+

+ H+

or the phosphoglyceric acid-kinaseglyceraldehyde-3-phosphate dehydrogenase system :

+

kinase

ATP 3-phosphoglyceric acid __t ADP lJ3-diphosphoglycericacid

+

1,3-diphosphoglyceric acid

+

+

dehydrogenase

KADH H+Glyceraldehyde-3-phosphate KAD inorganic phosphate

+

+

DETERMINATION OF INHIBITORS

-in inhibitor is a compound that causes a decrease in the rate of an enzyme reaction, either by reacting with the enzyme to form an enzyme-inhibitor complex or by reacting with the enzymesubstrate intermediate t o form a complex :

E+I,EI

E + S T E S LT I E-S-I

P+ E

In general, the initial rate of an enzymic reaction will decrease with increasing inhibitor concentration, linearily a t low inhibitor concentrations, then will gradually approach zero. Several analytical methods have been proposed based on the inhibition of an enzyme reaction. Keller (111) has reported a fairly specific method for the determination of microgram concentrations of DDT, based on the inhibition of carbonic anhydrase. DDT inhibits this enzyme at concentrations a t which other inhibitors (except the sulfonamides) are inactive. The most specific and sensitive method for some organophosphorous compounds is based on the inhibition of cholinesterase, and numerous paper have been published on this system. Kitz (116) has published a review with 158 references on the chemistry of anticholinesterase compounds. Giang and Hall (63) assayed TEPP, paraoxan, and other insecticides that inhibit cholinesterase in vitro, and Kramer and Gamson (120) have developed a colorimetric procedure with compounds related to the indophenyl acetates for the determination of 1 to 10 pg. of various organophosphorous compounds. Fallscheer and Cook (49) have pointed out that the thiono- and dithiophosphates are poor in vitro inhibitors of cholinesterase, but can be easily converted into good in vitro inhibitors. Underhay (238)has described methods for eserine and DFP(diisopr0pyl phosphorofluoridate) using human red cell and plasma cholinesterase. Guilbault, Kramer, and Cannon (80) described an electrochemical method for the analysis of Sarin, Systox, parathion, and malathion. The decrease in the rate of the cholinesterase catalyzed hydrolysis of butyrylthiocholine iodide, as measured by dual polarized platinum indicator electrodes, is linearly related to concentrations of the organophos-

phorous compounds. Weiss and Galstatter detected pesticides in water by the inhibition of brain cholinesterase in fish. Bluegill sunfish were found to be most sensitive (247). Matousek and Cermon (157)have reported a highly sensitive, simple method for detecting cholinesterase inhibitors. Papers impregnated with butyrylthiocholine iodide and bromthymol blue as a p H indicator were used. The presence of an inhibitor was indicated by the inability of the cholinesterase used to effect the hydrolysis of the substrate. Archer et al. (4)reported a nonspecific cholinesterase inhibition procedure for the determination of ethion in olives. Peracetic acid was used t o oxidize the ethion because olefinic compounds present in olives interfered with the usual bromine treatment. The method worked well on a number of fruits and vegetables. Archer et al. (6) also used peracetic acid to oxidize phorate; the phorate was determined after oxidation by cholinesterase inhibition. Cholinesterase inhibition was used by Blumen (66) to determine Phosdrin in fruits and vegetables, and automated systems have been applied to inhibitor analysis with cholinesterase (261). Hobom and Zollner (96) detected as little as 0.5 pg. of heparin by the inhibition of ribonuclease. Other muco-polysaccharides do not interfere. X specific method for the determination of nanogram quantities of fluoride in the presence of phosphate was described by McGaughey and Stowell (143) and Linde (134). The liver esterase that is specifically inhibited by fluoride was assayed either by a p H titration of the butyric acid formed in the enzymic hydrolysis of ethyl butyrate (143) or colorimetrically (134). Shaw (221) has reported that Cu+2, Zn+2 and Ni+2 in 10-7 to 10-6M concentrations, and CO+~, to 10-4M Fe+2,and Mn+2 in 5 x concentrations inhibit urease and may be determined. Guilbault, Kramer, and Cannon (81) proposed analytical methods for silver (I) and mercury (11) based on the inhibition of xanthine oxidase. Concentrations as low as 2.5 X 1 0 - 7 N of these ions may be quantitatively determined. There are a number of reports in the literature of compounds that inhibit enzymes a t extremely small concentrations. Some of these, for which sensitive analytical procedures could be worked out, are listed below. Woronick (257) reported that straight chain C1to Cs and the branched chain amides isobutyramide and isovaleramide form strong complexes with, and hence inhibit, the action of, liver alcohol dehydrogenase. I n a study of the effect of sodium fluoride on Sarin-inhibited cholinesterase, Heilbronn (90) found that NaF in 10-6 to 10-4M concentrations reversed the inhibition and restored the enzyme activity of human erythrocyte

cholinesterase. Plasma cholinesterase was not reactivated. Scheie, Yanoff, and Isou (111) described the inhibition of chymotrypsin by aqueous humor from precataract surgery patients, and Golstein and Swain (66) have reported the inhibition of @-glucosidase, catalase, peroxidase, and alcohol and lactate dehydrogenases by tannic acid. Pyridine nucleotides strongly and specifically inhibit glutamate dehydrogenases from all sources (66), and rat liver glutamic dehydrogenase is inhibited by progesterone, thyroxine, fluorenylacetamide, and dicumarol (43). Qugliariello et al. (194) reported that 2,4-dinitrophenol in concentrations of to 3 X 10-4M inhibited the formation of aspartate in the transamination of glutamic-oxalacetic transaminase, and Igaue (102)has described the inhibition of the Q enzyme of rice plant by heavy metals such as Hg+2, Ag+, and Cu+l a t 10-6 t o 10-5M. IMMOBILIZED ENZYME

One of the primary objections to the use of enzymes in chemical analysis is the high cost of these materials. A continuous or semicontinuous routine analysis using enzymes would require large amounts of these materials, quantities greater than can be reasonable supplied, and quantities that would represent a prohibitive expenditure in many cases. If, however, the enzyme could be prepared in an immobilized(inso1ubilized) form without loss of activity, so that one sample could be used continuously for many hours, a considerable advantage would be realized. The immobilized enzyme can be used analytically in much the same way that the soluble enzyme is used, that is, to determine the concentration of a substrate that is acted upon by the enzyme, an inhibitor that inactivates the enzyme, or an activator that provides an acceleration in enzyme activity. Enzymes have been diazotized to cellulose particles (163) and to polyaminostyrene beads (71). McLaren and Peterson ( 1 4 4 , Nikolaev and Mardashev (177), and Barnett and Bull (10) have attempted the physical entrapment of the enzymes asparaginase, ribonuclease, and chymotrypsin by adsorption, absorption, or ion exchange. Enzymes have also been immobilized on polytyrosyl polypeptides (9), on a collodion matrix (65),and encapsulated in semipermeable micro capsules made of synthetic polymers (33). Bernfeld and Wan (16) used polyacrylamide gels for entrapping enzyme activity. Vasta and Usdin (239) have shown that cholinesterase could be insolubilized by entrapment in a starch gel. The preparation of immobilized cholinesterase for use in analytical chemistry was described by Bauman, Goodson, Guilbault, and Kramer (12). The enzyme, immobilized by the use of a starch matrix and placed on a polyurethane foam

pad, was found to be stable and active for 12 hours under continuous use. The activity of the enzyme was monitored electrochemically, using two platinum electrodes and an applied current of 2 pa. This immobilized enzyme was used to determine the substrates acetyland butyryl-thiocholine iodide, both in individual samples and continuously. A fluorometric system for the assay of anticholinesterase compounds using this immobilized cholinesterase was described by Guilbault and Kramer (7’8). As long as the enzyme is active a fluorescence is produced because of the hydrolysis of the 2-naphthyl acetate to 2-naphthol. Upon inhibition, the fluorescence drops to a value approaching zero. Hicks and Updike (93) have demonstrated the immobilization of enzyme activity in polyacrylamide gel. The preparation is stable and can be lyopholized and stored conveniently. Several enzyme systems were trapped in the gels, namely glucose oxidase, catalase, lactic dehydrogenase, amino acid oxidase, glutamic dehydrogenase, and enzyme activity in human serum. Detailed studies on glucose oxidase and lactic dehydrogenase were reported. AUTOMATED METHODS

Many of the experimental difficulties of using enzymes in analysis by reaction rate methods could be eliminated or lessened by the use of automation. Ideally, all the steps in an enzymic procedure would be automated : the addition of reagents, the measurement of the reaction rate, and the calculation of results. An excellent review on automated methods for the determination of enzyme activity was writtenby Schwartz and Bodansky (214); and Blaedel and Hicks have included an excellent discussion of automated methods of measuring reaction ratesin their chapteron enzymecatalyzed reactions in iidvances in Analytical Chemistry and Instrumentation (19). Jacobson et al. (105) have published a review on the p H stat, its theory, and application to automatic recording of rate curves. Various authors have published automatic electrochemical methods for analyzing enzyme reactions, and many of these have been discussed in the previous sections. Papers by Malmstadt and Piepmeier (153) on the automatic p H stat with digital readout, and by Pardue (184) on automatic control equipment to provide digital readout of concentration data in reaction rate methods bear repeating. An automated method for measuring the slopes of rate curves was described by Pardue (182). Hicks and Nalevac have described a programmer for the step-wise addition of solutions to chromatographic columns (91) and Weinburg (246) has designed a completely automatic instrument that stores 100 samples, adds reagents, measures the rate of change in VOL. 38, NO. 5, APRIL 1966

533 R

absorbance of each sample, records data on a paper tape, and shuts off automatically. A “robot chemist” has been designed by Research Specialties for automation of routine procedures (199), and Skeggs (226) has developed an automatic system, marketed by Technicon Instruments Corp. (Chauncey, N. Y.) and known as the AutoAnalyzer, that has found considerable use in enzymology. Automatic methods of assay using the AutoAnalyzer were reported by Hochella and Weinhouse (97, 98) who analyzed lactic acid and lactic acid dehydrogenase in body fluids and urine, respectively, by following the reduction of various tetrazolium dyes; by Morgenstern et al. (167) and by Levy, Dalmasso, and Daly (133) who studied the same reaction using Cu+-neocuproine and pyruvate - dinitrophenyl - hydrazone complexes, respectively, t o indicate the progress of the reaction; by Hoober and Bernstein (100) who continuously monitored enzymic activity in column effluents by measuring NADH and NADPH; by Scheidt, Levine, and Nelson ($10) and Levine and Hill (139) who automated the determination of serum glutamic oxalacetic transaminase colorimetrically using the dye azoene fast Violet B (210) and fluorometrically measuring NADH (132); and by Saifer and Robin (909) who determined glucose in biological fluids. Hicks and Updike (92) proposed an automatic system for the assay of LDH in urine using the lactic acid dehydrogenase-NAD-phenazine methyl sulfate system. Pitot and Pries (189) described a combination unit consisting of a Gilford Model 2000 multiple sample absorbance recorder, a DU monochromator, a Technicon large sampler, and a proportioning pump for the assay of a Large number of enzyme rates completely automatically using known spectrophotometric methods. Application to the determination of histidine threonine dehydrase and glucokinase-hexokinase was described. Wood and Gilford (254, 255) used an automatic cuvet positioning attachment for the spectrophotometer to automatically record simultaneous enzyme reactions, and a cell changer for automatic recording of fluorescence changes in enzymic assays was designed by Brown and Williams (30)for the determination of dihydrofolic reductase. The most recent development in automation is a multichannel analyzer developed by Skeggs (227), which determines 12 substances in a single 2-ml. sample of blood serum. Aspartate aminotransferase and alkaline phosphatase are determined, in addition to 10 other substances assayed nonenzymatically. The results from each sample are recorded in sequence in calculated form on a single piece of paper suitable for immediate transmission to the requesting physician. 534 R

ANALYTICAL CHEMISTRY

CONCLUSIONS

Enzymes possess a great potential usefulness in analytical chemistry. The specificity of enzymes can solve the primary problem of most analytical chemists, the analysis of one substance in the presence of many similar compounds that interfere in the analysis. The sensitivity of enzymes allows the determination of as little as 10-”J g of material. With the advent of new techniques, electrochemical and fluorometric, many of the previous difficulties of enzymic analysis have been eliminated. The advent of the immobilized enzyme has alleviated the problem of the cost and supply of enzymes. Finally, significant progress has been made in the direction of complete automation of enzyme methods for rapid, accurate analysis. LITERATURE CITED

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(28) Bowden, C. H., J . Clin. Pathol. 16,

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