P1: SFK/UKS BLBS102-c08 P2: SFK BLBS102-Simpson March 21, 2012 12:8 Trim: 276mm X 219mm Printer Name: Yet to Come Enzyme Activities 175 Figure 8.8 The effect of pH on the kcat /K m value of an enzymatic reaction that is not associated with the Henderson-Hasselbalch equation the pKa value can be determined from the point of intersection of lines on the plot, especially for an enzymatic reaction that is not associated with the Henderson-Hasselbalch equation pH = pKa + log ([A− ]/[HA]) (Fig 8.8) Thus, the number of ionizing groups involved in the reaction can be evaluated (Dixon and Webb 1979, Tipton and Webb 1979) Effects of Temperature Temperature is one of the important factors affecting enzyme activity For a reaction to occur at room temperature without the presence of an enzyme, small proportions of reactant molecules must have sufficient energy levels to participate in the reaction (Fig 8.9A) When the temperature is raised above room temperature, more reactant molecules gain enough energy to be involved in the reaction (Fig 8.9B) The EA is not changed, but the distribution of energy-sufficient reactants is shifted to a higher average energy level When an enzyme is participating in the reaction, the EA is lowered significantly, and the proportion of reactant molecules at an energy level above the activation energy is also greatly increased (Fig 8.9C) That means the reaction will proceed at a much higher rate Most enzyme-catalyzed reactions are characterized by an increase in the rate of reaction and increased thermal instability of the enzyme with increasing temperature Above the critical temperature, the activity of the enzyme will be reduced significantly; while within this critical temperature range, the enzyme activity will remain at a relatively high level, and inactivation of the enzyme will not occur Since the rate of reaction increases due to the increased temperature by lowering the activation energy EA , the relationship can be expressed by the Arrhenius equation: k = A exp(−EA /RT ), where A is a constant related to collision probability of reactant molecules, R is the ideal gas constant (1.987 cal/mol—deg), T is the temperature in degrees Kelvin (K = ◦ C + 273.15), and k represents the specific rate constant for any rate, that is, kcat or Vmax The equation can be Figure 8.9 Plots of temperature effect on the energy levels of reactant molecules involved in a reaction (A) The first plot depicts the distribution of energy levels of the reactant molecules at room temperature without the presence of an enzyme (B) The second plot depicts that at the temperature higher than room temperature but in the absence of an enzyme (C) The third plot depicts that at room temperature in the presence of an enzyme The vertical line in each plot indicates the required activation energy level for a reaction to occur The shaded portion of distribution in each plot indicates the proportion of reactant molecules that have enough energy levels to be involved in the reaction The x -axis represents the energy level of reactant molecules, while the y -axis represents the frequency of reactant molecules at an energy level transformed into: ln K = ln A − EA /RT , where the plot of ln K against 1/T usually shows a linear relationship with a slope of −EA /R, where the unit of EA is cal/mol The calculated EA of a reaction at a particular temperature is useful in predicting the EA of the reaction at another temperature And the plot is P1: SFK/UKS BLBS102-c08 P2: SFK BLBS102-Simpson March 21, 2012 12:8 176 Trim: 276mm X 219mm Printer Name: Yet to Come Part 2: Biotechnology and Enzymology useful in judging if there is a change in the rate-limiting step of a sequential reaction when the line of the plot reveals bending of different slopes at certain temperatures (Stauffer 1989) Taken together, the reaction should be performed under a constantly stable circumstance, with both temperature and pH precisely controlled from the start to the finish of the assay, for the enzyme to exhibit highly specific activity at appropriate acidbase conditions and buffer constitutions Whenever possible, the reactant molecules should be equilibrated at the required assay condition following addition of the required components and efficient mixing to provide a homogenous reaction mixture Because the enzyme to be added is usually stored at low temperature, the reaction temperature will not be significantly influenced when the enzyme volume added is at as low as 1–5% of the total volume of the reaction mixture A lag phase will be noticed in the rate measurement as a function of time when the temperature of the added enzyme stock eventually influences the reaction Significant temperature changes in the components stored in different environments and atmospheres should be avoided when the reaction mixture is mixed and the assay has started METHODS USED IN ENZYME ASSAYS General Considerations A suitable assay method is not only a prerequisite for detecting the presence of enzyme activity in the extract or the purified material: it is also an essential vehicle for kinetic study and inhibitor evaluation Selection of an assay method that is appropriate to the type of investigation and the purity of the assaying material is of particular importance It is known that the concentration of the substrate will decrease and that of product will increase when the enzyme is incubated with its substrate Therefore, an enzyme assay is intended to measure either the decrease in substrate concentration or the increase in product concentration as a function of time Usually, the latter is preferred because a significant increase of signal is much easier to monitor It is recommended that a preliminary test be performed to determine the optimal conditions for a reaction including substrate concentration, reaction temperature, cofactor requirements, and buffer constitutions, such as pH and ionic strength, to ensure the consistency of the reaction Also, an enzyme blank should be performed to determine if the nonenzymatic reaction is negligible or could be corrected When a crude extract, rather than purified enzyme, is used for determining enzyme activity, one control experiment should be performed without adding substrate to determine if there are any endogenous substrates present and to prevent overcounting of the enzyme activity in the extract Usually, the initial reaction rate is measured, and it is related to the substrate concentration Highly sufficient substrate concentration is required to prevent a decrease in concentration during the assay period of enzyme activity, thus allowing the concentration of the substrate to be regarded as constant Types of Assay Methods On the basis of measuring the decrease of the substrate or the increase of the product, one common characteristic property that is useful for distinguishing the substrate and the product is their absorbance spectra, which can be determined using one of the spectrophotometric methods Observation of the change in absorbance in the visible or near UV spectrum is the method most commonly used in assaying enzyme activity NAD(P)+ is quite often a cofactor of dehydrogenases, and NAD(P)H is the resulting product The absorbance spectrum is 340 nm for the product, NAD(P)H, but not for the cofactor, NAD (P)+ A continuous measurement at absorbance 340 nm is required to continuously monitor the disappearance of the substrate or the appearance of the product This is called a continuous assay, and it is also a direct assay because the catalyzed reaction itself produces a measurable signal The advantage of continuous assays is that the progress curve is immediately available, as is confirmation that the initial rate is linear for the measuring period They are generally preferred because they give more information during the measurement However, methods based on fluorescence, the spectrofluorometric methods, are more sensitive than those based on the changes in the absorbance spectrum The reaction is accomplished by the release or uptake of protons and is the basis of performing the assay Detailed discussions on the assaying techniques are available below Sometimes, when a serial enzymatic reaction of a metabolic pathway is being assayed, no significant products, the substrates of the next reaction, can be measured in the absorbance spectra due to rapid changes in the reaction Therefore, there may be no suitable detection machinery available for this single enzymatic study Nevertheless, there is still a measurable signal when one or several enzymatic reactions are coupled to the desired assay reaction These are coupled assays, one of the particular indirect assays whose coupled reaction is not enzymatic This means that the coupled enzymes and the substrates have to be present in excess to make sure that the rate-limiting step is always the reaction of the particular enzyme being assayed Though a lag in the appearance of the preferred product will be noticed at the beginning of the assay, before reaching a steady state, the phenomenon will only appear for a short period of time if the concentration of the coupling enzymes and substrates are kept in excess So, the Vmax of the coupling reactions will be greater than that of the preferred reaction Besides, not only the substrate concentration but also other parameters may affect the preferred reaction (see previously) To prevent this, control experiments can be performed to check if the Vmax of the coupling reactions is actually much higher than that of the preferred reaction For other indirect assays in which the desired catalyzed reaction itself does not produce a measurable signal, a suitable reagent that has no effect on enzyme activity can be added to the reaction mixture to react with one of the products to form a measurable signal Nevertheless, sometimes no easily readable differences between the spectrum of absorbance of the substrate and that of the product can be measured In such cases, it may be possible to measure the appearance of the colored product, by the P1: SFK/UKS BLBS102-c08 P2: SFK BLBS102-Simpson March 21, 2012 12:8 Trim: 276mm X 219mm Printer Name: Yet to Come Enzyme Activities chromogenic method (one of the spectrofluorometric methods) The reaction is incubated for a fixed period of time Then, because the development of color requires the inhibition of enzyme activity, the reaction is stopped and the concentration of colored product of the substrate is measured This is the discontinuous or end point assay Assays involving product separation that cannot be designed to continuously measure the signal change, such as electrophoresis, high performance liquid chromatography (HPLC), and sample quenching at time intervals, are also discontinuous assays The advantage of discontinuous assays is that they are less time consuming for monitoring a large number of samples for enzyme activity However, additional control experiments are needed to ensure that the initiation rate is linear for the measuring period of time Otherwise, the radiolabeled substrate of an enzyme assay is a highly sensitive method that allows the detection of radioactive product Separation of the substrate and the product by a variety of extraction methods may be required to accurately assay the enzyme activity Detection Methods Spectrophotometric Methods Both the spectrophotometric method and the spectrofluorometric method discussed below use measurements at specific wavelengths of light energy (in the wavelength regions of 200–400 nm (UV, ultraviolet) and 400–800 nm (visible)) to determine how much light has been absorbed by a target molecule (resulting in changes in electronic configuration) The measured value from the spectrophotometric method at a specific wavelength can be related to the molecule concentration in the solution using a cell with a fixed path length (l cm) that obeys the Beer-Lambert law: A = − log T = εcl, where A is the absorbance at certain wavelength, T is the transmittance, representing the intensity of transmitted light, ε is the extinction coefficient, and c is the molar concentration of the sample Using this law, the measured change in absorbance, A, can be converted to the change in molecule concentration, c, and the change in rate, vi , can be calculated as a function of time, t, that is, vi = A/εl t Choices of appropriate materials for spectroscopic cells (cuvettes) depend on the wavelength used; quartz cuvettes must be used at wavelengths less than 350 nm because glass and disposable plastic cuvettes absorb too much light in the UV light range However, the latter glass and disposible plastic cuvettes can be used in the wavelength range of 350–800 nm Selection of a wavelength for the measurement depends on finding the wavelength that produces the greatest difference in absorbance between the reactant and product molecules in the reaction Though the wavelength of measurement usually refers to the maximal wavelength of the reactant or product molecule, the most meaningful analytical wavelength may not be the same as the maximum wavelength because significant overlap of spectra may be found between the reactant and product molecules Thus, a different spectrum between two molecules can be calcu- 177 lated to determine the most sensitive analytical wavelengths for monitoring the increase in product and the decrease in substrate Spectrofluorometric Methods When a molecule absorbs light at an appropriate wavelength, an electronic transition occurs from the ground state to the excited state; this short-lived transition decays through various high-energy, vibrational substrates at the excited electronic state by heat dissipation, and then relaxes to the ground state with a photon emission, the fluorescence The emitted fluorescence is less energetic (longer wavelength) than the initial energy that is required to excite the molecules; this is referred to as the Stokes shift Taking advantage of this, the fluorescence instrument is designed to excite the sample and detect the emitted light at different wavelengths The ratio of quanta fluoresced over quanta absorbed offers a value of quantum yield (Q), which measures the efficiency of the reaction leading to light emission The light emission signals vary with concentration of fluorescent material by the Beer-Lambert law, where the extinction coefficient is replaced by the quantum yield, and gives: If = 2.31 I0 εclQ, where If and I0 are the intensities of light emission and of incident light that excites the sample, respectively However, conversion of light emission values into concentration units requires the preparation of a standard curve of light emission signals as a function of the fluorescent material concentration, which has to be determined independently, due to the not strictly linear relationship between If and c (Lakowicz 1983) Spectroflurometric methods provide highly sensitive capacities for detection of low concentration changes in a reaction However, quenching (diminishing) or resonance energy transfer (RET) of the measured intensity of the donor molecule will be observed if the acceptor molecule absorbs light and the donor molecule emits light at the same wavelength The acceptor molecule will either decay to its ground state if quenching occurs or fluoresce at a characteristic wavelength if energy is transferred (Lakowicz 1983) Both situations can be overcome by using an appropriate peptide sequence, up to 10 or more amino acid residues, to separate the fluorescence donor molecule from the acceptor molecule if peptide substrates are used Thus, both effects can be relieved by cleaving the intermolecular bonding when protease activity is being assayed Care should be taken in performing the spectrofluorometric method For instance, the construction of a calibration curve as the experiment is assayed is essential, and the storage conditions for the fluorescent molecules are important because of photodecomposition In addition, the quantum yield is related to the temperature, and the fluorescence signal will increase with decreasing temperature, so a temperature-constant condition is required (Bashford and Harris 1987, Gul et al 1998) Radiometric Methods The principle of the radiometric methods in enzymatic catalysis studies is the quantification of radioisotopes incorporated into the substrate and retained in the product Hence, successful radiometric methods rely on the efficient separation of the P1: SFK/UKS BLBS102-c08 P2: SFK BLBS102-Simpson March 21, 2012 12:8 178 Trim: 276mm X 219mm Printer Name: Yet to Come Part 2: Biotechnology and Enzymology radiolabled product from the residual radiolabeled substrate and on the sensitivity and specificity of the radioactivity detection method Most commonly used radioisotopes, for example, 14 C, 32 P, 35 S, and H, decay through emission of β particles; however, 125 I decays through emission of γ particles, whose loss is related to loss of radioactivity and the rate of decay (the half-life) of the isotope Radioactivity expressed in Curies (Ci) decays at a rate of 2.22 × 1012 disintegrations per minute (dpm); expressed in Becquerels (Bq), it decays at a rate of disintegrations per second (dps) The experimental units of radioactivity are counts per minute (cpm) measured by the instrument; quantification of the specific activity of the sample is given in units of radioactivity per mass or per molarity of the sample (e.g., µCi/mg or dpm/µmol) Methods of separation of radiolabeled product and residual radiolabeled substrate include chromatography, electrophoresis, centrifugation, and solvent extraction (Gul et al 1998) For the detection of radioactivity, one commonly used instrument is a scintillation counter that measures light emitted when solutions of p-terphenyl or stilbene in xylene or toluene are mixed with radioactive material designed around a photomultiplier tube Another method is the autoradiography that allows detecting radioactivity on surfaces in close contact either with X-ray film or with plates of computerized phosphor imaging devices Whenever operating the isotopescontaining experiments, care should always be taken for assuring safety Chromatographic Methods Chromatography is applied in the separation of the reactant molecules and products in enzymatic reactions and is usually used in conjunction with other detection methods The most commonly used chromatographic methods include paper chromatography, column chromatography, thin-layer chromatography (TLC), and HPLC Paper chromatography is a simple and economic method for the readily separation of large numbers of samples By contrast, column chromatography is more expensive and has poor reproducibility The TLC method has the advantage of faster separation of mixed samples, and like paper chromatography, it is disposable and can be quantified and scanned; it is not easily replaced by HPLC, especially for measuring small, radiolabeled molecules (Oldham 1992) The HPLC method featured with low compressibility resins is a versatile method for the separation of either low molecular weight molecules or small peptides Under a range of high pressures up to 5000 psi (which approximates to 3.45 × 107 Pa), the resolution is greatly enhanced with a faster flow rate and a shorter run time The solvent used for elution, referred to as the mobile phase, should be an HPLC grade that contains low contaminants; the insoluble media is usually referred to as the stationary phase Two types of mobile phase are used during elution; one is an isocratic elution whose composition is not changed, and the other is a gradient elution whose concentration is gradually increased for better resolution The three HPLC methods most commonly used in the separation steps of enzymatic assays are reverse phase, ion-exchange, and size-exclusion chromatography The basis of reverse phase HPLC is the use of a nonpolar stationary phase composed of silica covalently bonded to alkyl silane, and a polar mobile phase used to maximize hydrophobic interactions with the stationary phase Molecules are eluted in a solvent of low polarity (e.g., methanol, acetonitrile, or acetone mixed with water at different ratios) that is able to efficiently compete with molecules for the hydrophobic stationary phase The ion-exchange HPLC contains a stationary phase covalently bonded to a charged functional group; it binds the molecules through electrostatic interactions, which can be disrupted by the increasing ionic strength of the mobile phase By modifying the composition of the mobile phase, differential elution, separating multiple molecules, is achieved In the size-exclusion HPLC, also known as gel filtration, the stationary phase is composed of porous beads with a particular molecular weight range of fractionation However, this method is not recommended where molecular weight differences between substrates and products are minor, because of overlapping of the elution profiles (Oliver 1989) Selection of the HPLC detector depends on the types of signals measured, and most commonly the UV/visible light detectors are extensively used Electrophoretic Methods Agarose gel electrophoresis and polyacrylamide gel electrophoresis (PAGE) are widely used methods for separation of macromolecules; they depend, respectively, on the percentage of agarose and acrylamide in the gel matrix The most commonly used method is the sodium dodecyl sulfate (SDS)-PAGE method; under denaturing conditions, the anionic detergent SDS is coated on peptides or proteins giving them equivalently the same anionic charge densities Resolving of the samples will thus be based on molecular weight under an electric field over a period of time After electrophoresis, peptides or proteins bands can be visualized by staining the gel with Coomassie Brilliant Blue or other staining reagents, and radiolabeled materials can be detected by autoradiography Applications of electrophoresis assays are not only for detection of molecular weight and radioactivity differences, but also for detection of charge differences For instance, the enzyme-catalyzed phosphorylation reactions result in phosphoryl transfer from substrates to products, and net charge differences between two molecules form the basis for separation by electrophoresis If radioisotope 32 P-labeled phosphate is incorporated into the molecules, the reactions can be detected by autoradiography, by monitoring the radiolabel transfer after gel electrophoresis, or by immunological blotting with antibodies that specifically recognize peptides or proteins containing phosphate-modified amino acid residues Native gel electrophoresis is also useful in the above applications, where not only the molecular weight but also the charge density and overall molecule shape affect the migration of molecules in gels Though SDS-PAGE causes denaturing to peptides and proteins, renaturation in gels is possible and can be applied to several types of in situ enzymatic activity studies such as activity staining and zymography (Hames and Rickwood P1: SFK/UKS BLBS102-c08 P2: SFK BLBS102-Simpson March 21, 2012 12:8 Trim: 276mm X 219mm Printer Name: Yet to Come Enzyme Activities 1990) Both methods assay enzyme activity after electrophoresis, but zymography is especially intended for proteolytic enzyme activity staining in which gels are cast with high concentrations of proteolytic enzyme substrates, for example, casein, gelatin, bovine serum albumin, collagen, and others Samples containing proteolytic enzymes can be subjected to gel electrophoresis, but the renaturation step has to be performed if a denaturing condition is used; then the reaction is performed under conditions suitable for assaying proteolytic enzymes The gel is then subjected to staining and destaining, but the entire gel background will not be destained because the gel is polymerized with protein substrates, except in clear zones where the significant proteolysis has occurred; the amount of staining observed will be greatly diminished due to the loss of protein This process is also known as reverse staining Otherwise, reverse zymography is a method used to assay the proteolytic enzyme inhibitor activity in gel Similar to zymography, samples containing proteolytic enzyme inhibitor can be subjected to gel electrophoresis After the gel renaturation step is performed, the reaction is assayed under appropriate conditions in the presence of a specific type of proteolytic enzyme Only a specific type of proteolytic enzyme inhibitor will be resistant to the proteolysis, and after staining, the active proteolytic enzyme inhibitor will appear as protein band (Oliver et al 1999) Other Methods The most commonly used assay methods are the spectrophotometric, spectrofluorometric, radiometric, chromatographic, and electrophoretic methods described above, but a variety of other methods are utilized as well Immunological methods make use of the antibodies raised against the proteins (Harlow and Lane 1988) Polarographic methods make use of the change in current related to the change in concentration of an electroactive compound that undergoes oxidation or reduction (Vassos and Ewing 1983) Oxygen-sensing methods make use of the change in oxygen concentration monitored by an oxygen-specific electrode (Clark 1992), and pH-stat methods use measurements of the quantity of base or acid required to be added to maintain a constant pH (Jacobsen et al 1957) Selection of an Appropriate Substrate Generally, a low molecular mass, chromogenic substrate containing one susceptible bond is preferred for use in enzymatic reactions A substrate containing many susceptible bonds or different functional groups adjacent to the susceptible bond may affect the cleavage efficiency of the enzyme, and this can result in the appearance of several intermediate-sized products Thus, they may interfere with the result and make interpretation of kinetic data difficult Otherwise, the chromophore-containing substrate will readily and easily support assaying methods with absorbance measurement Moreover, substrate specificity can also be precisely determined when an enzyme has more than one recognition site on both the preferred bond and the functional groups adjacent to it Different sized chromogenic substrates 179 can then be used to determine an enzyme’s specificity and to quantify its substrate preference Unit of Enzyme Activity The unit of enzyme activity is usually expressed as either micromoles of substrate converted or product formed per unit time, or unit of activity per milliliter under a standardized set of conditions Though any unit of enzyme activity can be used, the Commission on Enzymes of the International Union of Biochemistry and Molecular Biology (IUBMB) has recommended that a unit of enzyme, Enzyme Unit or International Unit (U), be used An Enzyme Unit is defined as that amount that will catalyze the conversion of micromole of substrate per minute, U = µmol/min, under defined conditions The conditions include substrate concentration, pH, temperature, buffer composition, and other parameters that may affect the sensitivity and specificity of the reaction, and usually a continuous spectropotometric method or a pH stat method is preferred Another enzyme unit that now is not widely used is the International System of Units (SI unit) in which katal (kat) = mol/sec, so kat = 60 mol/min = × 107 U In ascertaining successful purification of a specified enzyme from an extract, it is necessary to compare the specific activity of each step to that of the original extract; a ratio of the two gives the fold purification The specific activity of an enzyme is usually expressed as units per milligram of protein when the unit of enzyme per milliliter is divided by milligrams of protein per milliliter, the protein concentration The fold purification is an index reflecting only the increase in the specific activity with respect to the extract, not the purity of the specified enzyme REFERENCES Barry MJ 1997 Emzymes and symmetrical molecules Trends Biochem Sci 22: 228–230 Bashford CL, Harris DA 1987 Spectrophotometry and Spectrofluorimetry: A Practical Approach IRL Press, Washington, DC Bell JE, Bell ET 1988 Proteins and Enzymes New Jersey: PrenticeHall Briggs GE, Haldane JBS 1925 A note on the kinetics of enzyme action Biochem J 19: 338–339 Brocklehurst K, Dixon HBF 1977 The pH dependence of secondorder rate constants of enzyme modification may provide freereactant pKa values Biochem J 167: 859–862 Brown AJ 1902 Enzyme action J Chem Soc 81: 373–386 Burbaum JJ, Schimmel P 1991 Structural relationships and the classification of aminoacyl-tRNA synthetases J Biol Chem 266: 16965–16968 Cardenas ML et al 1998 Evolution and regulatory role of the hexokinases Biochim Biophys Acta 1401: 242–264 Cherfils J et al 1990 Modelling allosteric processes in E coli aspartate transcarbamylase Biochimie 72(8): 617–624 Clark JB 1992 In: Eisenthal R, Danson MJ (eds.) Enzyme Assays, A Practical Approach New York: Oxford University Press Copeland RA 2000 Enzymes, 2nd ed New York: Wiley-VCH Inc Cornish-Bowden A 1995 Fundamentals of Enzyme Kinetics, 2nd ed London: Portland Press ... freereactant pKa values Biochem J 167: 85 9? ?86 2 Brown AJ 1902 Enzyme action J Chem Soc 81 : 373– 386 Burbaum JJ, Schimmel P 1991 Structural relationships and the classification of aminoacyl-tRNA... Washington, DC Bell JE, Bell ET 1 988 Proteins and Enzymes New Jersey: PrenticeHall Briggs GE, Haldane JBS 1925 A note on the kinetics of enzyme action Biochem J 19: 3 38? ??339 Brocklehurst K, Dixon HBF... 1 988 ) Polarographic methods make use of the change in current related to the change in concentration of an electroactive compound that undergoes oxidation or reduction (Vassos and Ewing 1 983 )