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Methods of Enzymatic Analysis
Methods of Enzymatic Analysis
Methods of Enzymatic Analysis
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Methods of Enzymatic Analysis

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Methods of Enzymatic Analysis focuses on the general progress in enzymology and in the special field of enzymatic analysis. This book explores the commercial production of biochemical reagents for analysis and explains the transition from the possible use of enzymatic analysis to its various applications in pure and applied biochemistry. Organized into four sections, this book starts with an overview of the basis of enzymatic analysis and provides general experimental guidelines for the techniques of measurement and for the disintegration of cells and tissues. This text then provides detailed instructions for the determination of substrates and assay of enzyme activities. Other chapters explore the practical aspects and information necessary for the application of reagents to enzymatic analysis, including sources, stability, and purity required. The final section describes the commercially available enzymes, coenzymes, substrates, and several less common reagents. Biochemists, biophysicists, researchers, and graduate students will find this book extremely useful.
LanguageEnglish
Release dateDec 2, 2012
ISBN9780323141772
Methods of Enzymatic Analysis

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    Methods of Enzymatic Analysis - Hans-UIrich Bergmeyer

    SECTION A

    General

    Principles of Enzymatic Analysis

    Hans-Ulrich Bergmeyer

    Publisher Summary

    This chapter presents an overview of the principles of enzymatic analysis. The value of enzymes in analysis lies in their ability to react specifically with individual components of a mixture. The chapter highlights that the concentration of a substance which takes part in an enzymatic reaction can be determined in two ways: one, by physical, chemical, or enzymatic analysis of the product or unreacted starting material after completion of the reaction catalyzed by the enzyme; and two, from the rate of the enzyme reaction, which depends on the concentration of the substrate, cofactor, activator or inhibitor. The two methods are basically different. In the first case, the reaction should be completed as rapidly as possible. Relatively large amounts of enzyme and relatively small amounts of substrate are used. The measured values should be easily readable, not too small and not too large. In the second case, the substrate and enzyme concentrations are so arranged that the rate of the reaction, that is, the amount of substrate reacting per unit time, is not too fast and so can be measured accurately.

    1 Determination of concentration

    a). Methods measuring total changes

    α). Direct measurements

    β). Measurements with the aid of coupled reactions

    b). Determination of concentration by measuring the kinetics of the reaction

    α). Substrates

    β). Activators and inhibitors

    2 Assay of enzyme activity

    a). General information on kinetics

    b). Simple reactions

    c). Coupled reactions

    The use of enzymes for analysis is not new. As early as 1845 Osann¹) detected hydrogen peroxide with peroxidase; C. F. Schönbein²) gave the concentration which could be detected as 1 in 2000000. In the eighties of last century enzyme preparations were used extensively for the determination of carbohydrate in foodstuffs³). About 25 years ago Otto Warburg⁴) evolved the method of enzymatic analysis based on measurement of the light absorption of the reduced coenzymes, diphospho- and triphosphopyridine nucleotide. The latest advance in this field is the measurement of the fluorescence of these coenzymes⁵,⁶), with the result that the sensitivity of the determinations has been increased by two or three orders of magnitude.

    Today the term enzymatic analysis is generally understood to mean analysis with the aid of enzymes. Although in fact all metabolic reactions of living cells are catalysed by enzymes, the determination of a compound by living cells (e.g. micro-organisms) is not part of the field of enzymatic analysis; these are microbiological assays. Recently it has become common to also include in the term enzymatic analysis the assay of enzyme activity in organs and biological fluids. This has been the usual practice in food chemistry for several decades.

    The particular value of enzymes in analysis lies in their ability to react specifically with individual components of a mixture. This avoids lengthy separations of the components and therefore the time taken for the analysis is less. Owing to the mild conditions employed, enzymes often allow the detection and determination of labile substances, which can only be estimated very inaccurately by other methods.

    Basically, enzymes can be used for the determination of metabolic products, inorganic ions essential for metabolism and pharmacological agents which influence metabolic reactions.

    1 Determination of Concentration

    The concentration of a substance which takes part in an enzymatic reaction can be determined in two ways.

    a) By physical, chemical or enzymatic analysis of the product or unreacted starting material after completion of the reaction catalysed by the enzyme.

    b) From the rate of the enzyme reaction, which depends on the concentration of the substrate, cofactor, activator or inhibitor. The two methods are basically different. In the first case (measurement of total change method), the reaction should be completed as rapidly as possible. Relatively large amounts of enzyme and relatively small amounts of substrate are used. The measured values should be easily readable, not too small and not too large. In the second case (kinetic method), the substrate and enzyme concentrations are so arranged that the rate of the reaction, i.e. the amount of substrate reacting per unit time, is not too fast and so can be measured accurately.

    a) Methods Measuring Total Changes

    Enzyme reactions are equilibrium reactions. If the substrate is virtually completely consumed, then the enzymatic analysis is simple; the result is easily calculated by means of a known constant of the substance (e.g. by means of the extinction coefficient of light absorbing substances). If the substrate is not all consumed because of the unfavourable position of the equilibrium it is often possible to obtain quantitative utilization of the substrate by trapping the products (carbonyl compounds with semicarbazide, hydrazine, etc.; if a proton is formed in the reaction, then the reaction medium should be made as alkaline as possible), or by increasing the concentration of the other reactants. It is not always possible to displace the equilibrium sufficiently in favour of the product and therefore a standard curve must be prepared under defined conditions.

    α) Direct Measurements

    The substance A is to be determined. It is completely converted to B in the enzymatic reaction A → B. B can be distinguished chemically and physically from A.

    If A, in contrast to B, has a characteristic absorption spectrum, then A can be determined directly in the presence of other absorbing substances by means of the enzyme reaction which occurs in the spectrophotometer cuvette. The absorption decreases by an amount corresponding to the amount of A. Example: Determination of uric acid (ε290 mμ =12×10⁶cm.²/mole) with uricase (see p. 500). Or if B absorbs and A does not. Example: Determination of diphosphopyridine nucleotide (DPN): alcohol dehydrogenase reduces DPN to DPNH (ε340 mμ = 6.22 × 10⁶ cm.²/mole) in the presence of ethanol.

    Another typical case is if B is an acid. It can then be titrated or measured manometrieally by the production of CO2 from a bicarbonate buffer. Example: The older method for the determination of glucose with glucose oxidase (Keilin and Hartree⁷)); the reaction product is gluconic acid.

    More rare is the chemical determination of the reaction product. Example: The determination of L-arabinose by conversion to L-ribulose with L-arabinose isomerase and measurement of the ketopentose by means of the cysteine carbazole reaction (see p. 178).

    The enzymes most widely used for enzymatic analysis are the DPN*) and TPN-dependent dehydrogenases.

    The transfer of the hydrogen of the substrate to the pyridine ring of DPN or TPN leads to the formation of a peak in the absorption curve at 340 mμ (Fig. 1).

    Fig 1 ) ⁸). The Extinction Coefficient of the Purest Preparations of DPNH is 6.22×10⁶ cm.²/mole at 340 mμ. The Absorption at 260 mμ is Due in Both Cases to the Adenine Moiety of the Coenzyme.

    Consequently, the enzymatic conversion of substrate A can be followed directly in the spectrophotometer cuvette by the changes in the optical density at 340 mμ (or an adjacent wavelength). According to the Lambert-Beer law, with a light path of d = 1 cm.,

    **) the conversion of 1 μmole substrate/ml. is indicated by an optical density change

    It is the same if DPN accepts the substrate hydrogen, as in the example of the oxidation of ethanol given above, or if DPNH donates hydrogen, as in the reduction of pyruvate to lactate (see p. 253, for the reaction curve see Fig. 2 on p. 9).

    Fig 2 Measurement of the Activity of a Dehydrogenase

    A: Addition of DPNH

    B: Start of the Reaction with Substrate

    β) Measurements with the Aid of Coupled Reactions

    The use of DPN and TPN-dependent dehydrogenases is not limited to the estimation of their substrates. In many cases an enzymatic reaction can be taken further with a dehydrogenase: The substance to be determined, A, is converted to product B with an auxiliary enzyme (auxiliary reaction). B is the substrate for a DPN-linked dehydrogenase and can be determined as usual. The dehydrogenase reaction which has been coupled with the enzymatic conversion A → B is termed an indicator reaction. It indicates how much A is converted to B and, if the auxiliary reaction is quantitative, the original amount of A. An example is the determination of phosphoenolpyruvate (PEP). PEP is converted to pyruvate by adenosine diphosphate (ADP) in the auxiliary reaction; DPNH is oxidized in the indicator reaction and the pyruvate is determined by the corresponding decrease in optical density.

    General equation:

    Example:

    ADP can be determined in the same way; it is converted in the auxiliary reaction to ATP by PEP and the pyruvate formed is reduced with DPNH.

    Dyes can also serve as indicators in coupled reactions by reacting, enzymatically or non-enzymatically, with a product of the auxiliary reaction.

    An example is the determination of glucose with glucose oxidase and peroxidase, using o-dianisidine as the indicator (see p. 23).

    Auxiliary reaction:

    Indicator reaction

    The intensity of the dye is a measure of the reaction. Other examples can be found in the section on Assay of Enzyme Activity.

    b Determination of Concentration by Measuring the Kinetics of the Reaction

    α) Substrates

    KM (refer to⁹)).

    In the determination of substrates by measurement of the rate of enzyme reactions the experimental conditions must be strictly adhered to. In general, standard curves for defined conditions of temperature, buffer and pH must be used. Since the experimental result usually changes in direct proportion to the enzyme activity, a new standard curve should be prepared for each series of measurements, in which the substrate concentration is similar to the expected value for the unknown sample. The simple form of kinetic determination of substrate, such as the determination of alcohol by means of the rate of the alcohol dehydrogenase reaction (Theorell and Bonnichsen¹⁰)) is used much less. These methods have generally been superseded by methods measuring the total change.

    In contrast catalytic assays have become established where no quantitative method is possible. By this method is understood the determination of a compound which in the first reaction is consumed and in the second is regenerated. The amount of substance which can be determined is small. Example: The determination of coenzyme A with α-oxoglutaric oxidase and deacylase (v. Korff¹¹)):

    (1)

    (2)

    The coenzyme A to be determined is required for the oxidative decarboxylation of α-oxoglutarate by the oxidase. The rate of reaction (1) therefore depends on the concentration of CoA. This remains constant because the CoA is regenerated in reaction (2). The rate of reaction (1), which is measured by the increase in the optical density at 340 mμ with time due to the formation of DPNH, is a measure of the amount of CoA present.

    β) Activators and Inhibitors

    The rates of enzyme reactions in the living cell are controlled by different regulators. In principle, it should be possible to determine inorganic ions, SH compounds, etc. by their activation of certain enzymes. However, different activators may affect the same enzyme in the same way, so that the specificity of such analyses is often low. An example of such a method is the determination of magnesium by the activation of isocitric dehydrogenase¹²) (see p. 640). Inhibitors can be determined in a similar way. A compound usually inhibits a whole group of enzymes, but this lack of specificity is unimportant. More important is the fact that the same enzyme may be inhibited by several compounds. In this case the determination is only valid if the sample does not contain several inhibitors whose action is similar. It is usually possible to find an enzyme which is fairly specific for the inhibitor present in the sample. Example: The inhibition of carbonic anhydrase by DDT; determination of DDT according to Keller¹³) (see p. 626).

    2 Assay of Enzyme Activity

    a) General Information on Kinetics

    The activity of an enzyme is defined by the rate of the reaction catalysed by the enzyme. The concept of the chemical reaction rate, v = ±dc/dt, is especially well illustrated on the direct reading photometer. The rate at which the photometer needle moves over the scale indicates the speed of the chemical reaction, i.e. the decrease of the reacting compound with time or the increase of the reaction product.

    Substrate transformations involving light absorbing reactants (e.g. DPN and TPN-linked dehydrogenase reactions occurring in the cuvette, see p. 5) are measured in this way. Automatic recording or plotting the time against the optical density gives information on the course of the reaction. The reaction curve is not always linear.

    Allocation of enzyme reactions to one of the classical orders of uncatalysed reactions (first or second order) is usually not possible. Therefore by the rate of an enzyme reaction is always understood the initial rate vo*). This rate is obtained from the slope of the tangent to the reaction curve at zero time. It is possible to obtain this value exactly with non-linear curves by use of a mirror ruler¹⁴). An approximate value, which is usually sufficient for routine work, can be obtained by plotting several measurements made just after the start of the reaction and then extrapolating to t = 0 as illustrated in Fig. 10, p. 35.

    With non-linear reaction curves the intervals at which measurements are made should be small. It is incorrect to take only a few measurements after a relatively long incubation time. Unfortunately, the course of the reaction cannot always be followed continuously, as is possible with the changes in light absorption brought about by dehydrogenase reactions occurring in the spectrophotometer cuvette. For example, in the determination of trypsin activity according to Anson¹⁵) the measurement of the product formed at a certain time requires that the enzyme reaction be stopped before starting the measurement. Only by the continuous removal and analysis of samples, involving a great expenditure in time and effort, is it possible to obtain reaction curves by such methods. The simple method of determining the amount of substrate converted after a fixed time must lead to erroneous results (see¹⁵a)), because the shape of the reaction curve depends on several factors including the substrate concentration. Some of the older methods, such as the previously mentioned assay of trypsin activity, have been improved¹⁶).

    Like all chemical transformations reactions catalysed by enzymes are sensitive to changes in temperature. The temperature coefficient of the reaction rate is 10% per degree¹⁷) or more. In other words, a 10°C rise in temperature causes a 100% increase in the reaction rate. Constant temperature is essential in the assay of enzyme activity.

    All enzymes have an optimum pH range for their activity and often this optimum is very narrow. With increasing concentration of substrate the rate of enzyme catalysed reactions finally reaches a maximum value. According to the equation of Michaelis and Menten¹⁸) further increase in concentration should no longer change the rate. However, many enzymes are inhibited by a large excess of substrate. The optimum substrate concentration also depends on the pH and temperature (e.g. lactic dehydrogenase, p. 737, see also¹⁹)).

    If all the precautions taken with normal laboratory equipment are adhered to in the measurement of enzyme activity, then the error is usually less than ±5%. Greater accuracy requires a disproportionate expenditure on apparatus.

    b) Simple Reactions

    In contrast to substrate determinations the measurement of enzyme activity is always carried out with the optimum substrate concentration and if necessary the enzyme sample is diluted. The reaction should proceed so slowly (with a few exceptions) that at the end of the measurements only a small part of the substrate has been converted. The substrate must be pure since contamination with substrates of other enzymes leads to errors. For example, the oxaloacetate used for the measurement of malic dehydrogenase activity in serum must be free from its decarboxylation product pyruvate. Otherwise measurement of the decrease in the optical density of DPNH would include that due to the activity of the lactic dehydrogenase also contained in the serum (see p. 757).

    Generally the reaction is started by addition of the substrate. Since the sample cannot be deproteinized all the enzymes contained in it are active and can react with their substrates if these are present in the sample. This can cause considerable interference with the measurements. For example, in the spectrophotometric assay of dehydrogenases in serum a significant decrease in optical density is observed after the addition of DPNH. This is due to the reduction of pyruvate contained in the serum by serum lactic dehydrogenase. The real measurements are only started when the initial reaction has stopped (Fig. 2).

    The ideal situation in which the reaction can be followed directly by means of the light absorption should only be abandoned if a linear conversion of substrate with time is guaranteed. Such as is the case, for example, with glutamate-oxaloacetate and glutamate-pyruvate transaminase, where the reaction products can be determined chemically after a long incubation time. The estimation of the oxaloacetate or pyruvate formed as the dinitrophenylhydrazones requires very careful consideration of additional sources of error. Any carbonyl compounds present in the sample must be allowed for by a blank determination. It is also necessary to distinguish between the hydrazones of the reaction products and the hydrazone of the substrate α-oxoglutarate; therefore it is not always possible to use optimum substrate concentrations with this method (for further details, see pp. 842 and 851).

    c) Coupled reactions

    Just as coupled reactions can be used for the determination of substrates so they can be used for the measurement of reaction rates. The indicator enzyme is usually a DPN or TPN-linked dehydrogenase. For example, the activity of glutamate-pyruvate transaminase (GPT) can be determined with lactic dehydrogenase (LDH) as indicator enzyme. (For a three component assay with an auxiliary enzyme as intermediary, see p. 12).

    General equation :

    Example:

    The role of the indicator reaction in determinations of enzyme activity is to indicate the amount of reaction product, in this case pyruvate, formed with time. This is only possible if the pyruvate is reduced extremely rapidly by the DPNH. Then the rate of the transamination can be followed by means of the decrease in optical density due to the oxidation of the DPNH.

    For the practical execution of this type of assay it is of interest to know how much more rapid the indicator reaction must be than the reaction to be measured. To calculate this ratio the mathematical formulation of the two individual steps must be known, which at present only occurs with very few reactions. However, by means of a sequence of two uncatalysed first order reactions it is possible to estimate approximately how large the error of measurement will be with an insufficient excess of the indicator enzyme.

    With comparable rates for the individual steps the time course for the formation of the reaction product C in the sequence

    shows the typical S-shape characteristic of all sequence (or consecutive) reactions (Fig. 3).

    Fig 3 Course of Consecutive Reactions with Different Ratios of the Two Individual Rates k1: k2 (Diagrammatic). A: Start of the Reaction

    The slopes of the inflection tangents to these curves give the maximum rates, but these slopes are always less than the slope of the tangent at zero time with a more rapid indicator reaction.

    Only the tangent to the curve k1: k2 = 1:1000 comes near to the ideal case of curve k1: k2 = 1:∞, whose tangent through the origin has the slope v0. With the former curve the induction period of the reaction is practically zero and the S-shape of the curve has virtually disappeared.

    The calculations are as follows: For the step A → B the initial rate is

    (1)

    For the coupled reaction A → B → C the rate (see²⁰)) at any time is

    (2)

    a is the substrate concentration at time t.

    To obtain Vinf., i.e. the rate at the inflection point of the curve of the coupled reaction, it is necessary to substitute

    for t in equation (2)

    Let k1: k2 = 1:10. Even with this relatively low gradient in the reaction rates the expression in the round brackets in equation (2) can be neglected. Therefore

    Under the conditions k2 = 10 k1 the slope of the tangent at the inflection point represents approximately a 23% lower rate than the reaction step A → B at t = 0 actually has.

    In the same way when k2 = 100 k1 the error is only 4%. When k2 = 1000 k1 then vinf ≈ 0.993 and therefore vinf ≈ ak1 ≈ v0 (error is 0.7%).

    This estimation shows that more than a 100-fold excess of indicator enzyme is required. By excess is understood not the amount of enzyme, but rather the product of the amount and the specific activity or turnover number of the enzyme. With an unfavourable ratio for the turnover numbers (specific activities) the amount of indicator enzyme which is required may be very large.

    Three component assays are rare, because it is difficult to obtain a sufficiently rapid rate for the auxiliary reaction in comparison to the primary reaction and for the indicator reaction in comparison to the auxiliary reaction. Only if the ratios of the specific activities of the participating enzymes are favourable can this type of assay be carried out. The following example illustrates this:

    The activity of myokinase (MK) was measured in a three component assay²¹). The auxiliary enzyme was pyruvic kinase (PK) and the indicator enzyme lactic dehydrogenase (LDH):

    Auxiliary reaction:

    Indicator reaction:

    (abbreviations, see p. 5).

    The MK preparation had a specific activity of 169 units/mg. (according to Racker et al.²²)) and therefore consumed 169 μmoles AMP per minute (in the calculations it is necessary to allow for the fact that for each mole of AMP 2 moles of lactate are formed, i.e. 2 moles of DPNH are oxidized). The specific activity of PK and LDH was 156 and 455 units/mg. respectively.

    Amounts of enzyme were taken with the following activities:

    MK: 1.18 × 10-4 mg.; 1.18 × 10-4 × 169 = 1.99 × 10-2 μmoles/min.

    PK: 2 × 10-2 mg.; 2 × 10-2 × 156 = 3.12 μmoles/min.

    The ratio of the rates is about 1:170, therefore according to what has been said above the auxiliary reaction is rapid. The amount of LDH was varied.

    With these amounts of LDH the values for the ratio of the rate of the auxiliary reaction to the indicator reaction are 1: 0.3, 1: 3 and 1: 30. Accordingly the reaction curves in Fig. 4 have induction periods of different length and the rates (Δ/min.) increase from 0.030 (a), 0.036 (b) to 0.044 (c). Depending on the required accuracy, a 30-fold excess of activity of the indicator enzyme over the auxiliary enzyme may be satisfactory or the amount of LDH must be increased three or four-fold.

    Fig 4 Three Component Enzymatic Reaction: Myokinase (MK), Pyruvic Kinase (PK) and Lactic Dehydrogenase (LDH)

    MK: 1.99×10-2 μmoles/min.

    PK: 3.12 μmoles/min.

    LDH: a) 0.9 μmoles/min.

    b) 9 μmoles/min.

    c) 90 μmoles/min.

    Although the 3:1 ratio for the specific activity of lactic dehydrogenase and pyruvic kinase is favourable it is necessary to add about 1 mg. (!) LDH per assay in order to measure the activity of 1.18 × 10-4 mg. MK exactly.

    The example shows that three component reactions for the measurement of enzyme activity should be avoided if possible. Whether, as is the case for the measurement of MK activity, other methods²³) are more reliable, or whether the advantages outweigh the disadvantages indicated above, must be decided separately for each system.

    References

    1. Osann, G. Poggendorfs Ann. 1845; 67:372.

    2. Schönbein, C.F. J. prakt. Chem. 1851; 53(1):69.

    3. see Stetter, H.Enzymatische Analyse. Weinheim/Bergstr: Verlag Chemie, 1951.

    4. Warburg, O.Wasserstoffübertragende Fermente. Berlin: Verlag Dr. W. Saenger, 1948.

    5. Kaplan, N.O., Colowick, S.P., Barnes, C.C. J. biol. Chemistry. 1951; 191:461.

    6. Lowry, O.H., Roberts, N.R., Kapplahn, J.L., Lewis, C. Feder. Proc. 1956; 15:304.

    7. Keilin, D., Hartree, E.F. Biochem. J. 1948; 42:230.

    8. Warburg, O., Christian, W. Biochem. Z. 1936; 287:291.

    9. Moelwyn-Hughes, E.A.Nord, F.F., Weidenhagen, F., eds. Handbuch der Enzymologie; Vol. I. Akademische Verlagsgesellschaft, Berlin, 1940:259.

    10. Theoreil, H., Bonnichsen, R.K. Scand. J. clin. Lab. Invest,. 1951; 3:58.

    11. Korff, R.W.V. J. biol. Chemistry. 1953; 200:401.

    12. Baum, P., Czok, R. Biochem. Z. 1959; 332:121.

    13. Keller, H. Naturwissenschaften. 1952; 39:109.

    Chance, B. J. biol. Chemistry. 1960; 235:2440.

    14. see also e.g. Bergmeyer, H.-U. Biochem. Z. 1952; 323:163.

    15. Anson, M.L. J. gen. Physiol. 1938; 22:79.

    Bergmeyer, H.-U. Biochem. Z. 1952; 323:163.

    16. Schwert, G.W., Takenata, Y. Biochim. biophysica Acta. 1955; 16:570.

    17. see e.g. Netter, H. Theoretische Biochemie. Berlin-Göttingen-Heidelberg: Springer-Verlag, 1959; 554. [et seq.].

    18. Michaelis, L., Menten, M.L. Biochem. Z. 1913; 49:333.

    19. Winer, A.D., Schwert, G.W. J. biol. Chemistry. 1958; 231:1065.

    20. Bergmeyer, H.-U. Biochem. Z. 1953; 324:408.

    21. H. Möllering, unpublished.

    22. Cooper, J., Srere, P.A., Tabachnick, M., Racker, E. Arch. Biochem. Biophysics. 1958; 74:306.

    23. see Noda, L., Kuby, A. J. biol. Chemistry. 1957; 226:541.


    *Abbreviations:

    DPN = diphosphopyridine nucleotide

    DPNH = reduced diphosphopyridine nucleotide

    TPN = triphosphopyridine nucleotide

    TPN H = reduced triphosphopyridine nucleotide

    AMP = adenosine monophosphate

    ADP = adenosine diphosphate

    ATP = adenosine triphosphate

    PEP = phosphoenolpyruvate

    PK = pyruvic kinase

    LDH = lactic dehydrogenase

    **)I0 = intensity of the incident light

    I = intensity of the emergent light (from the cuvette)

    ε = extinction coefficient [cm.²/mole]

    c = concentration of the light absorbing substance [moles/ml.]

    d = light path of the cuvette [cm.]

    *)Each simple enzyme reaction is a combination of two separate steps, namely the formation of the enzyme-substrate complex ES from the enzyme E and the substrate S and its decomposition to yield the reaction product P and regeneration of the unchanged enzyme:

    The initial rate as indicated by normal techniques of measurement is only obtained when ES is formed and its concentration is stationary. The stage of the formation of ES can only be measured in very slow reactions and with highly sensitive instrumentsE + P, i.eES falls off rapidly.

    Experimental Techniques

    Hans-Ulrich Bergmeyer

    Publisher Summary

    This chapter discusses the basic principles of the experimental techniques in enzymatic analysis. In enzymatic analysis, the enzymes are used as analytical reagents. The chapter emphasizes the fact that before starting a large series of measurements, it is recommended that the activity of the enzymes used and the concentration of the coenzyme and substrate solutions should be checked. A test of a complete assay system in which a constant end-point is obtained can best be made on the completion of the reaction by the addition of a trace of the pure substance which is being assayed to the cuvette; a renewed reaction should occur immediately. All reagents required for assays, including stock buffer solutions and doubly distilled water, should be kept covered. All pipettes, flasks, and tubes used for enzymatic analysis should be thoroughly clean. After cleaning with chromic acid, it is important to remove traces of chromate by carefully rinsing with tap water, distilled water, and finally doubly distilled water. Before using modern surface-active detergents, a check should be made to see whether they are inhibitory to the enzymes being used. Pipettes should be lightly wrapped in filter paper and dried in a drying oven at a moderate temperature.

    1 Handling Biochemical Reagents

    α). Storage of biochemical substances and their solutions

    β). Control of reagents

    γ). Laboratory vapours

    δ). Glassware

    ε). Interference due to deproteinizing agents

    2 Methods of Measurement and Instruments Used

    a). Methods based on photometric measurements

    α). Principle of the photometric determination of concentration

    β). Types of instrument and their range of application

    γ). Measurement of light absorption

    δ). Special requirements of biochemistry and practical hints

    ε). Measurement of fluorescence

    b). Manometric methods

    α). Principle

    β). Practical hints

    γ). Manufacturers of apparatus

    c). Other methods

    α). Micromethods

    β). Thunberg technique

    γ). Polarography and polarometry

    3 Evaluation of the Experimental Results

    a). General information

    α). Enzyme units and their conversion

    β). Evaluation of reaction curves

    γ). Standard curves and standards

    b). Photometric methods

    α). Extinction coefficients and optical density as the basis of calculations

    β). Evaluation with a non-constant end-point

    β). Sensitivity and accuracy

    c). Manometric methods

    α). Calculation of the results

    β). Sensitivity and accuracy

    1 Handling Biochemical Reagents

    In enzymatic analysis the enzymes are used as analytical reagents. The commonly held view that enzymes are unstable only applies to a limited number. Numerous enzymes of high purity are at present available commercially (see p. 967). Their stability is indicated by the fact that it is possible to send these preparations overseas without loss of activity. Enzymes are proteins of high molecular weight which catalyse specific reactions and are therefore more sensitive than other analytical reagents, but with correct handling they are absolutely reliable.

    α) Storage of biochemical substances and their solutions

    Elevated temperatures and traces of heavy metals lead to a loss of enzyme activity especially with dilute enzyme solutions. Crystalline enzyme preparations suspended in ammonium sulphate solution should be stored at 2 to 4°C and under these conditions the loss of activity is usually minimal. Freezing of crystalline suspensions of enzymes frequently results in difficulty in resuspension of the material, fracture of the crystals and a 20–30% loss of activity. On the other hand, a few really unstable preparations can only be kept in a deep-freeze. The information on enzyme stability given in each chapter under Stability of the Solutions should be noted. An indication is also given there about the stability of the other solutions.

    The coenzymes DPNH, TPNH and CoA must be protected from light and stored in a desiccator in the cold. The sulphydryl group of CoA is easily oxidized by atmospheric oxygen. DPNH stored in the dark is about 10 times more stable (as measured by the change in its optical density at 340 mμ) than when stored in indirect sunlight. Storage at room temperature has hardly any effect on DPNH (as measured by the change in optical density at 340 mμ), but a considerable reduction in enzyme activity (e.g. with lactic dehydrogenase) is observed when DPNH preparations are used which have not been stored in the cold.

    Dry substances which have been stored in a desiccator in the cold will absorb moisture from the air if they are immediately removed from the desiccator at room temperature. If water uptake of hygroscopic substances is to be avoided, the desiccator must be allowed to warm to room temperature before opening. This precaution is especially important in warm weather. The dilute solutions of coenzymes and substrates are more stable than the more highly dilute enzyme solutions. In any case no solutions should be kept longer than about 10 days (stored in a refrigerator). It is convenient to freeze coenzyme solutions in small portions so as to avoid repeated freezing and thawing. Solutions of DPNH and TPNH are acid labile; even the CO2 content of old distilled water causes breakdown of the coenzymes. These substances should therefore be dissolved in ca. 1% bicarbonate solution or in. a dilute buffer (up to ca. pH 9). DPN and TPN are alkali labile. CoA is most stable around pH 4; ATP around pH9.

    Buffer and substrate solutions, especially phosphate, amino acid and sugar solutions, are ideal growth media for micro-organisms. They should therefore be stored in thoroughly clean and sterile dark bottles with ground-glass stoppers. To avoid contamination each day’s supply of reagent should be obtained by pouring it from the container and not by the use of a pipette.

    Because of the high activity of highly purified enzymes only a fraction of a milligram is required to provide excess of enzyme for substrate and coenzyme determinations. Hence the volumes of enzyme suspensions are small. Small containers should always be stored stoppered to avoid losses due to crystallization on the walls of the containers.

    For the busy laboratory it is recommended that the solutions should be stored on the bench in an ice bath during working hours so that they are readily available (Fig. 1). Insulated plastic containers holding racks to take about 20 tubes of various sizes are available commercially*).

    Fig. 1 Ice Bath For Biochemical Reagents

    β) Control of reagents

    Before starting a large series of measurements it is recommended that the activity of the enzymes used and the concentration of the coenzyme and substrate solutions be checked. A test of a complete assay system in which a constant end-point is obtained can best be made on completion of the reaction by the addition of a trace of the pure substance which is being assayed to the cuvette. A renewed reaction should occur immediately.

    γ) Laboratory vapours

    All reagents required for assays, including stock buffer solutions and doubly distilled water, should be kept covered. Enzymatic methods are very sensitive. For example, in the determination of alcohol, traces of alcohol absorbed by the reagents from the laboratory atmosphere lead to greatly increased blank values.

    δ) Glassware

    All pipettes, flasks and tubes used for enzymatic analysis should be thoroughly clean. After cleaning with chromic acid it is important to remove traces of Chromate by carefully rinsing with tap water, distilled water and finally doubly distilled water.

    Before using modern surface-active detergents a check should be made to see whether they are inhibitory to the enzymes being used.

    Pipettes should be lightly wrapped in filter paper and dried in a drying oven at a moderate temperature.

    Pipettes (0.1, 0.2 and 2 ml.) in which the graduations do not extend to the tip**) are recommended for the measurement of enzyme, coenzyme and substrate solutions and also for the preparation of the sample. All pipettes should be calibrated: a separate 0.5 and 1.0 ml. sample of ca. 0.1 M KMnO4 solution (acidified with H2SO4) is pipetted from an officially calibrated pipette into a 10 ml. volumetric flask, diluted to the mark and the optical density of this solution is measured at 546 mμ. The process is repeated with the pipette to be calibrated. Any difference in the optical densities corresponds to the difference in the calibration of the two pipettes.

    A pipette tray can be easily made in the laboratory (Fig. 2). The top of a commercial test tube rack is cut longitudinally and the two halves are glued or nailed along a board ca. 200 × 300 mm². The place for each pipette can be marked; a strip of adhesive tape suitable for writing on is stuck to the base board.

    Fig. 2 Pipette Tray

    In general small volumes of enzyme and coenzyme solutions are employed, especially with spectrophotometric assays carried out in cuvettes. To introduce these small volumes into the cuvette and at the same time to obtain rapid mixing a small glass rod is used that has been flattened and bent at one end (Figs. 3 and 4). This is made by flattening the heated end of a glass rod with pliers and then bending slightly. Owing to the ribbing on the jaws of the pliers an imprint is obtained on the surface of the glass which prevents large drops of fluid from flowing off. Volumes of up to about 0.05 ml. (50 μl.) can be easily pipetted onto the flat end of such a glass rod and mixed into the reaction solution.

    Fig. 3 A Glass or Plastic Rod Flattened at One End for Mixing Small Amounts of Solution Into Assay Mixtures.

    Fig. 4 Addition of 20 μl. of Reagent Solution to a Cuvette.

    To avoid scratching the walls of the cuvette it is better to make the rods from plastic (Polyvinylchloride, Plexiglas or Perspex).

    ε) Interference due to deproteinizing agents

    As all deproteinizing agents are enzyme inhibitors they must be removed as completely as possible after deproteinization. The most suitable deproteinizing agent (with a few exceptions) is perchloric acid; Perchlorate ions can be precipitated as the potassium salt. The precipitation is carried out at room temperature and then the solution is allowed to stand in an ice bath (for at least 10 minutes) to obtain quantitative precipitation. With lengthy centrifuging in the warmth a part of the KClO4 redissolves, therefore it is preferable to decant off from the precipitate. If heavy metal salts are used for deproteinization then these must be subsequently removed with, for example, hydrogen sulphide and the solution must then be aerated. The most appropriate methods are described in the individual chapters.

    2 Methods of Measurement and Instruments Used

    The various possibilities for the measurement of reactions catalysed by enzymes have already been mentioned on pages 3 to 13. For practical work it is important to know the advantages and disadvantages, and special details of the apparatus used*).

    a) Methods based on photometric measurements

    There are three different types of method: 1. Measurement of the light absorption of solutions. 2. Measurement of the light scattering of suspensions (turbidity measurements, nephelometry, refer to¹)). 3. Measurement of the fluorescent light emitted by dissolved substances after excitation of their fluorescence by irradiation with light of a suitable wavelength. Whereas nephelometry is little used in the field of enzymatic analysis, photometry is the most widely used method of measurement. For its theory, see, for example,²).

    α) Principle of the photometric determination of concentration

    The photometric determination of concentration depends on the fact that the absorption of the light passing through a solution has a definite relationship to the concentration of the solution. The ratio of the intensity of emergent light to incident light I/I0 (called the transmittance) decreases with increasing concentration. The absorption measured as extinction is directly proportional to the concentration. Extinction refers to the fraction of light absorbed and its magnitude depends on the light with which the measurements are made. Often instead of extinction the term optical density is used with respect to the measured compound*).

    The extinction (optical density) is related to the transmittance by the following formula:

    If the light absorption is measured with a photocell and an ammeter, then the transmittance of the solution is read on the linear graduated scale of the instrument. In addition photometers usually have an optical density scale which is graduated logarithmically as is required by the above formula. In the range of low optical densities therefore the graduations are further apart than those for higher optical densities (Fig. 5). This also usually applies to the graduated drum of a compensator.

    Fig. 5 Spectrophotometer Scale

    In the first phase of development of optical measurements the intensity of light from two light paths was simply compared by eye. The light path of the solution being measured was altered until it showed the same transmittance as a known standard solution or a standard light-attenuating device was adjusted in a second light path, so that it gave the same light absorption as the unknown solution (principle of compensation).

    This technique of measurement lost most of its importance when the photocell was perfected as a photoelectric transformer. Next the photocell was used instead of the eye as the null indicator and the optical compensation principle with mechanical light-attenuating devices (e.g. grey wedge or adjustable diaphragm) was retained.

    It then became evident that with good photocells a strict linearity existed over a wide range between the light absorbed and the electric current generated. The compensation of the output of the photocell could therefore be carried out electrically or direct reading instruments of high performance could be built. In the first method the precision obtained depends on the accuracy of the compensator, while in the second method (direct reading method) it depends on the accuracy of the indicator system. Certain types of instrument have a combination of the direct reading and compensation methods.

    β) Types of instrument and their range of application

    There are three different groups of instrument:

    Spectrophotometers

    In spectrophotometers the light is a continuous spectrum produced by a tungsten lamp and the desired wavelength is isolated with a prism or a diffraction grating (monochromatic light). Spectrophotometers are most versatile in their range of application. They can be used in qualitative analysis for the measurement of absorption spectra, which can be recorded directly by some instruments. For quantitative studies they allow any desired wavelength to be obtained and therefore measurements can always be made at the absorption maximum. Distinction can be made between instruments which are equipped with a tungsten lamp and a glass optical system, so that their range is limited to the visible part of the spectrum, and instruments which have a quartz optical system and can therefore be used in the UV-region.

    Spectrum line photometers

    Spectrum line photometers have a gas-discharge lamp (Hg, Cd) as the light source. From the discontinuous (line) spectrum of this lamp it is possible with optical filters to isolate the wavelengths which are emitted because of the nature of the gas (monochromatic light). Spectrum line photometers were developed because of the need which arose when the technique of photometric measurements was applied more and more to routine work. As they emit monochromatic light, although only at specific, fixed wavelengths, they can be employed for nearly all the methods developed for spectrophotometers. They are easier to handle, their accuracy is comparable, and they are cheaper and better value than spectrophotometers. With some instruments the optical density can also be recorded as a function of time.

    A survey of some of the best known spectrophotometers and spectrum line photometers is given in Table 1. Data on the wavelengths of some spectrum line photometers is given in Table 2.

    Table 1

    Survey of some well-known spectrophotometers and spectrum line photometers. (The list is not exhaustive.)

    Table 2

    Wavelengths of some spectrum line photometers. Hg (mercury) and Cd (cadmium) lamps produce monochromatic light, tungsten lamps polychromatic light. The filters for the Elko II and III instruments have a band-width at half maximum intensity of ca. 20 mμ and those for the Leifo E instrument of 20–30 mμ.

    *)Interference filter

    Photometers with tungsten lamp and filters

    These instruments use a tungsten lamp as the source of a continuous spectrum and the radiation is limited to a region of the spectrum by means of a filter (polychromatic light). Such instruments are used for routine work which does not require a high accuracy of measurement. As these need fewer components they are cheaper than spectrum line photometers. They sometimes can also be fitted with recorders.

    The spectrophotometer, which operates at any wavelength, is best suited to the determination of absorption spectra for qualitative analysis and to the development of new photometric methods of analysis. In quantitative analysis, especially in routine work, the accuracy with which a given concentration can be determined and the time required, are the criteria for the suitability and efficiency of a instrument.

    When a method is routinely carried out at a fixed wavelength, there is no need for a continuous change of wavelength, Rather the need is for a simple reproduction of the chosen wavelength. Metallic vapour lamps have proved especially successful in fulfilling this condition. The energy radiated by them is limited to certain fixed wavelengths because of the properties of the particular metal atoms. With a filter it is possible to separate a line of the spectrum. If it is established that adjacent lines are suppressed, then the wavelength of the light is not changed when the range of maximal transmittance of the filter differs from the λmax of the line. This is in contrast to the use of filters with spectrophotometers which emit continuous spectra. Photometers with metallic vapour lamps as the light source are therefore most frequently used in routine measurement requiring high standards of accuracy. The use of a mercury vapour lamp has the additional advantage that it produces a series of intense lines in the UV-range.

    To make the best use of the monochromatic light given by a metallic vapour lamp and filter, a correspondingly efficient device for measuring the light absorption is required. To obtain a similar efficiency with a tungsten lamp requires that only a narrow band be transmitted by the filters. It is possible to reduce this band-width to about 10 mμ. without the light intensity becoming too low. The narrower the wave band of light transmitted by the filter, the more efficient must be the device for measuring the light, because the energy decreases with the narrowing of the band-width.

    Due to the greater proportion of light absorbed at the absorption maxima, non-monochromatic light results in non-linear standard curves at high concentrations of the absorbing substance and therefore to a decrease in the accuracy of the measurements (Fig. 6).

    Fig. 6 The Influence of Monochromatic and Polychromatic Light on the Shape of the Standard Curve (Diagrammatic)

    a) Absorption curve of a red solution

    1. Monochromatic light (492 mμ)

    2. Polychromatic light (centre 520 mμ, band-width of the filter at half maximum intensity: 20 mμ)

    3. Monochromatic light (546 mμ)

    b) Standard curves obtained by use of the light indicated under a).

    Here the reproducibility of the results depends on the absorption curve of the substance being investigated. If the absorption of the compound is similar for all wavelengths of the polychromatic beam, i.e. if the width of the absorption maximum of the compound is considerably wider than the transmission range of the filter, then essentially no change in the measured values will be brought about by a change in the spectral distribution of the energy of the lamp due to ageing or to an alteration of the working voltage. On the other hand, if the absorption band is narrow in comparison to the transmission range of the filter, then it is to be expected that the standard curve will depend considerably on the factors mentioned above. It therefore depends on the type of problem and on the accuracy required, whether an instrument employing a tungsten lamp and filter is suitable or not.

    γ) Measurement of light absorption

    Although the nature of the light source and the type of light produced are probably most important for classifying photometers, the method of measurement of the absorption also has a considerable importance in determining the efficiency of an instrument.

    The measurement of absorption is either by a compensation method resulting in a null point or by a direct reading method. In the true optical compensation method the absorption of the solution is balanced by a calibrated light-attenuating device. The value for the absorption is read directly from the light-attenuating device (example: Elko II).

    Thus the measured extinctions are no more accurate than the calibration of the light-attenuating device. Since the chemist is normally concerned with the accuracy of measurements of concentration, this accuracy must be determined relative to the optical density measured. Generally it can be assumed that if the light-attenuating device is a graduated diaphragm, it has a constant uncertainty over the whole range of measurements of about 0.1%, relative to 100% transmittance. If this uncertainty is plotted against the measured optical density a curve is obtained, in which the smallest error is 0.29% when the diaphragm is half closed (optical density 0.3) and this error slowly rises with larger and smaller optical densities. In such a case the concentration range is so chosen that the measurements are made on the range of the smallest error.

    With development the photoelectric transformer (photocell) became sufficiently reliable to give a constant and linear relationship between the light intensity and the current generated. The output of the photocell in most instruments was then balanced by an electrical system. This can cause errors of measurement due to variations in the photocell characteristics, but on the other hand it permits the construction of an electric compensator and the possibility of switching to several ranges of measurement. For example, with such an arrangement, optical densities from 0–1.0 can be measured with range 1 and from 1.0–2.0 with range 2. The range of measurement of the instrument depends on the spectral purity of the light beam and the degree of amplification of the output that can be achieved.

    In the electrical compensation method the current generated by the photocell is balanced and the measured value is read off on the scale of the compensator (examples: Beckman DU, Unicam SP 500, SP 600). Considered optically, the transfer of the compensator to the electrical system is equivalent to using a direct reading instrument. For consistent results the current of the light source must be stabilized.

    In the true direct reading method the electrical balance circuit is replaced by an instrument for measuring the current output of the photocell. The output is in nearly all cases amplified before measurement. The extent to which the direct measurement of the photoelectric current compares in accuracy with the compensation method depends on the quality of the indicator system. Good instruments therefore have an indicator system incorporating a light index scale to avoid errors due to parallax, and because this type of scale is easier to read than one employing a pointer (example: Zeiss PMQ II).

    A measure of the capability of an instrument is given by the reproducibility of its measurements. In a well designed instrument the accuracy of reading the instrument is commensurate with its internal accuracy.

    The scales of direct reading instruments and of equivalent null point instruments are seldom longer than 20 cm. because of lack of space. With these types of scale the accuracy of the readings relative to the optical density is greatest in the middle of the scale, i.e. at optical densities of about 0.3. This accuracy can be increased by optical magnification of the scale (example: Zeiss PMQ II).

    Another way of reducing errors of reading is to combine the direct reading and null point methods. A series of equal electrical resistances is placed between the photocell and the indicator system. These can be shunted out when measuring high optical densities. In this way the readings with high optical densities are displaced from the condensed part of the scale to its initial part, where the readings are more accurate. The additional optical density due to the resistance shunted out of circuit is simply added. By this method the accuracy in measuring an optical density of 1.0 is increased 10-fold (e.g. Eppendorf photometer) and therefore the optimal accuracy is no longer around optical densities of 0.3. Since the accuracy of the compensating resistors is greater than the accuracy of the indicator system, and as the absolute accuracy of the latter remains the same, the accuracy relative to the optical density increases with increase in optical density (Fig. 7).

    Fig. 7 Influence of Partial Compensation on the Accuracy of the Readings.

    Curve A: without Partial Compensation

    Curve B: with Partial Compensation

    The upper limit is fixed by the properties of the photocell and the capacity of the amplifier. The indicator system has been improved to such a degree that the accuracy of the instrument is virtually dependent on the photocell. Important factors determining the efficiency of the apparatus are the performance of the photocell and the ease of controlling and replacing it.

    δ) Special requirements of biochemistry and practical hints

    General Information

    The direct reading system is especially suited to the measurement of reactions catalysed by enzymes, because the optical density changes per unit time can be read directly on the scale. Recording the optical density (with a linear optical density scale) against the wavelength and more especially against reaction time simplifies the measurements and increases the performance of the apparatus.

    An important factor in deciding the suitability of a photometer for the assay of enzyme activity is whether it can be used in the UV-region.

    Because of the sensitivity of enzymatic reactions to temperature it is important that a constant temperature cuvette chamber is available for the instrument and that the cuvettes are easily accessible.

    Measurement of optical densities

    In the application of photometry to analytical chemistry only the optical densities or optical density changes are of interest, because these are related to amounts of the substance (Lambert-Beer Law, see p. 5).

    Measurement of the optical transmittance is unsuitable for chemical analysis. Its use in the initial stages of photometry arose from the fact that it could be read off from the linear scale of a normal indicator system, since transmittance is proportional to the galvanometer deflection. With optical-attenuating devices the transmittance is proportional to the angle of rotation and thus an exact graduation of the scale, with resultant greater accuracy of the readings, is made easier. Use of a transmittance scale always leads to additional work and sources of error.

    In enzymatic analysis optical density differences are measured and therefore the absolute values are not important. With determinations in which the reaction proceeds to completion ΔE values between 0.020 and 0.200 are suitable, as long as no other directions are given in the procedure. To obtain sufficiently accurate readings, the measurements should be made on a part of the scale on which the graduations are far apart, by choosing the concentration of the absorbing compound or by partial compensation of the photoelectric current (refer to Fig. 5).

    Two wavelengths, 334 mμ (weaker) and 366 mμ (stronger), are available when using spectrum line photometers for the measurement of DPN and TPN-dependent reactions. Usually 366 mμ is chosen. The extinction coefficients for DPNH and TPNH are

    These values are for 25°C. It should be noted that both extinction coefficients, especially that at 366 mμ, are dependent on temperature (in contrast to the extinction coefficient at 340 mμ); the values decrease with increasing temperature.

    Control cuvettes and types of cuvette

    If the construction of the instrument allows it*) the measurements can be made against a cuvette containing all the reactants of an assay mixture except one (reagent blank), or against a cuvette containing water or even with the omission of a cuvette altogether (against air).

    Control cuvettes are indispensable if the optical density of the solution to be measured is relatively high before the start of the reaction. If the intensity of the light is sufficient it is possible to partially or completely eliminate this initial optical density by reading against a control cuvette in which no reaction occurs.

    For example, in the assay of serum transaminase from patients with hepatitis (see p. 848) the strongly coloured serum has a considerable optical density. The initial optical density of the reaction mixture containing DPNH is so high at 340 or 366 mμ that the relatively small optical density changes can no longer be read accurately on the condensed part of the logarithmically graduated optical density scale. A control cuvette is prepared from the strongly coloured serum or a little DPNH and the instrument is so adjusted that when the control cuvette is in the light path, full transmittance or zero optical density is given (the instrument is zeroed with the control cuvette or measurements are made against the control or blank cuvette).

    In some instruments high optical densities are compensated for electrically in steps (for example of 0.25) instead of by means of a blank, thus enabling measurements of the optical density changes to be made in the range 0–0.25. In this way readings of high accuracy are attained even with high optical densities.

    All spectrophotometers and the Eppendorf photometer (see p. 21) use cuvettes with a base of 1 × 1 cm.²,1 × 2 cm.² or 1 × 4 cm.² (internal measurements). The Elko II, Elko III and Leifo E (p. 21) instruments have special cuvettes, which can only be used for these instruments.

    Care must be taken that the cuvettes are sufficiently full; only light that has passed through the solution should be allowed to reach the photocell. If the volume of the assay mixture is insufficient to fill the cuvettes, they can be raised up with pieces of wood or cardboard (e.g. in the Elko II by 3 mm.; in the Elko III by 7 mm.).

    The amount of reagent required can be reduced if the area of the optical surface of the cuvette through which the light passes is made smaller. With efficient instruments this need only be a few mm²., so that if necessary the assay volume can be reduced by one or two orders of magnitudes. Some manufacturers supply so-called micro-cuvettes for this purpose.

    )ε) Measurement of fluorescence

    Measurement of fluorescence (fluorimetry) is being employed more and more. The native fluorescence of a compound (e.g. of FAD or DPNH) or that produced by chemical action (e.g. by making a DPN solution strongly alkaline) can be measured. In principle all TPN and DPN-dependent reactions involved in enzymatic analysis can be measured fluorimetrically. In this way the sensitivity of measurement is increased by two to three orders of magnitudes (refer to p. 551).

    Suitable instruments for routine fluorimetric measurements are the fluorescence attachments of the well-known spectrophotometers and the Eppendorf photometer, and especially the Farrand fluorimeter (Farrand Optical Co., New York, USA). For the development of fluorimetric methods with the aim of obtaining the optimum conditions with regard to the excitation and fluorescent light a spectrophotofluorimeter (e.g. Aminco-Bowman*)) is necessary. For the practical details of fluorescence measurements and the theoretical principles see⁵) and⁶).

    b) Manometric methods

    α) Principle

    In manometry, which was founded by Barcroft⁷) and further developed by O. Warburg⁸), a volume of gas is measured by means of a constant volume manometer. An efficient thermo-regulator is essential for manometric measurements. The following types of reaction can be measured:

    The last two groups of reactions have the disadvantage that they are not specific. Interference due to the presence of two gases, for example, O2 and CO2 in aerobic glycolysis can be eliminated by the adsorption of the CO2 in KOH or by use of two flasks.

    For details of the various methods and instruments, see, for example⁹), ¹⁰) and Fig. 8.

    Fig. 8 A simple (open) Manometer After Haldane-Barcroft-Warburg A. Manometer tap. —- B. Reaction Flask with Side-Arm and Centre Well (Manometer Vessel, Flask or Cup). —- C. Graduated capillary Tube (Manometer). —- D. Brodie Solution. —- E. Rubber Tubing. —- F. Adjusting Screw.

    β) Practical hints

    The Warburg bath should not stand in a room which has large fluctuations in temperature

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