Enzyme concentration defined

Enzymes are excellent catalysts for two reasons great specificity and high turnover rates. With but few exceptions, all reac tions in biological systems are catalyzed by enzymes, and each enzyme usually catalyzes only one reaction. For most of the important enzymes and other proteins, the amino-acid sequences and three-dimensional structures have been determined. When the molecular struc ture of an enzyme is known, a precise molecular weight could be used to state concentration in molar units. However, the amount is usually expressed in terms of catalytic activity because some of the enzyme may be denatured or otherwise inactive. An international unit (lU) of an enzyme is defined as the amount capable of producing one micromole of its reaction product in one minute under its optimal (or some defined) reaction conditions. Specific activity, the activity per unit mass, is an index of enzyme purity. 

Overall enzyme balance and equilibrium constants are defined for the intermediate substrate and enzyme complex. The total enzyme concentration is the sum of free and conjugated enzymes with the substrates. 

The initial and total enzyme concentrations are defined based on measurable components given below ... 

From the equilibrium constant, the free enzyme concentration must be defined. We know the total enzyme concentration as the sum of the conjugated enzymes with substrates and the free enzymes. 

End-point methods are often not based on kinetic-ally optimum conditions. However, an end-point method is often the only convenient one available. In this case, the method should have been validated by showing that the catalysis of the substrate follows well defined kinetics, rate of reaction is proportional to enzyme concentration, blanks and interfering substances are corrected for, and that appropriate standards are available. 

Flaving minimized the enzyme concentration as described above, one can attempt to measure the IC50 of the compound at several enzyme concentrations, at and above the minimum, and then attempt to define A app by use of Equation (7.12) and the graphical methods described in Section 7.2. We have already discussed the limitations of this approach. Nevertheless, when the enzyme concentration can be varied over an appropriate range, relative to the Apapp, this approach can work well. 

The turnover number of an enzyme is defined as the maximum number of moles of substrate reacted per mole of enzyme (or molecules per molecule) per minute under optimum conditions (i.e., saturating substrate concentration, optimum pH, etc). If 2 mg/cm3 of a pure enzyme (50,000 molecular weight, Michaelis constant Km = 0.03 mole/m3) catalyzes a reaction at a rate of 2.5 jumoles/nUksec when the substrate concentration is 5 x 10 3 moles/m3, determine the turnover number corresponding to this definition and the actual number of moles of substrate reacting per minute per mole of enzyme. 

The concentrations of substrate and product are invariably in molar units (M this includes mM, iM, etc.), but enzyme concentrations may be given in molar (M), milligrams per milliliter (mg/mL), or units/mL. The amount of enzyme you have can be expressed in molecules, milligrams, nanomoles (nmol), or units. A unit of enzyme is the amount of enzyme that will catalyze the formation of 1 prnol of product per minute under specifically defined conditions. A unit is an amount, not a concentration. 

Assay of Homogenate for Aldrin Epoxidation. The following experimental sequence was designed to determine the optimum in vitro conditions for aldrin epoxidation in larval whole body homogenates 1) the effect of component chemicals generally included in an incubation mixture, 2) a pH profile, 3) a temperature profile, 4) a molarity profile, 5) a reaction time profile, 6) a larval concentration (enzyme concentration) profile, 7) a substrate concentration profile, and 8) a restudy of the effects of component chemicals in the initial incubation mixture (Step 1) upon aldrin epoxidation under optimum conditions as defined by steps 2-7 above. The effect of PBO, FMN, and FAD upon enzyme activity was also tested. 

The / -glucosidase activity was determined by measuring the release of p-nitrophenol from p-nitrophenyl-/i-D-glucopyranoside one unit of / -glucosidase activity (U) is defined as the amount of enzyme that releases 1 [mu] mol p-nitrophenol per minute. AU samples were assayed in potassium phosphate buffer (50 mM, pH 7.0) at 50 °C under conditions that activity was proportional to enzyme concentration. 

Easterby proposed a generalized theory of the transition time for sequential enzyme reactions where the steady-state production of product is preceded by a lag period or transition time during which the intermediates of the sequence are accumulating. He found that if a steady state is eventually reached, the magnitude of this lag may be calculated, even when the differentiation equations describing the process have no analytical solution. The calculation may be made for simple systems in which the enzymes obey Michaehs-Menten kinetics or for more complex pathways in which intermediates act as modifiers of the enzymes. The transition time associated with each intermediate in the sequence is given by the ratio of the appropriate steady-state intermediate concentration to the steady-state flux. The theory is also applicable to the transition between steady states produced by flux changes. Apphcation of the theory to coupled enzyme assays makes it possible to define the minimum requirements for successful operation of a coupled assay. The theory can be extended to deal with sequences in which the enzyme concentration exceeds substrate concentration. 

The surfactant mass fraction in a microemulsion defines the size of the interfacial area between the water and oil. The reaction rate of organic reactions in microemulsions can be dramatically enhanced by increasing the specific interfacial area [95]. Enzyme catalysis in microemulsions is usually not influenced by the size of the interfacial area because only a small fraction of the reverse micelles are hosting a bio-molecule. Most investigations published so far were made with low enzyme concentrations resulting in a low population of enzymes per reverse micelle. 

One unit of immobilised enzyme is defined as the amount of enzyme bound to glass beads that generates 1 pmol NADH/min under standard assay conditions . BA concentration in the serum and bile is extrapolated from the standard curve be-... 

It is usually difficult to express the enzyme concentration in molar unit because of difficulties in determining enzyme purity. Thus, the concentration is sometimes expressed as a unit, which is proportional to the catalytic activity of an enzyme. The definition of an enzyme unit is arbitrary, but one unit is generally defined as the amount of enzyme that produces 1 pmol of the product in 1 min at the optimal temperature, pH and substrate concentration. 

Storch and Segelcke (1874) proposed that the product of coagulation time (tc) and enzyme concentration (E) should be defined as a constant (k) (McMahon and Brown 1983, 1984B). 

The reverse reaction, i.e., the direct conversion of inosine to adenosine catalyzed by both the calf duodenal and the Takadiastase nonspecific adenosine aminohydrolase (Section V) and measured as a function of pH with the calf enzyme, was defined by a theoretical curve for the equilibrium, K,= ([inosine] [NH i])/([adenosine] H20]) =38, with water concentration taken as one and pKa values of 8.8 and 9.2 for inosine and ammonium ion, respectively. The calculated AF = —5400 cal/mole at pH 7.0 was in reasonable agreement with —6000 cal/mole estimated from the summation of a series of partial reactions (114). 

Enzyme concentration may be expressed in mass unit instead of molar unit. However, the amount of enzyme is not well quantified in mass unit because actual contents of an enzyme can differ widely depending on its purity. Therefore, it is common to express enzyme concentration as an arbitrarily defined unit based on its catalytic ability. For example, one unit of an enzyme, cellobiose, can be defined as the amount of enzyme required to hydrolyze cellobiose to produce 1 /imol of glucose per minute. Whatever unit is adopted for CEq, the unit for k3CEQ should be the same as r, that is, kmole/m3s. Care should be taken for the consistency of unit when enzyme concentration is not expressed in molar unit. 

In the absence of catalysis, the inhibitor concentration and the ratio of k to k u the equilibrium association constant, will define the fraction of the enzyme bound with inhibitor at a given enzyme concentration. The enzyme-inhibitor complex proceeds to transform the inhibitor to an intermediate that may decompose to form a metabolite or react with the enzyme to form an inactive complex. First-order rate constants /o, /t , and kA determine the rates of these reactions and the concentration of intermediate at a given concentration of inhibitor and enzyme. 

In this equation the subscripts on the concentration and enhancement terms denote total Ga3+ (T), free Gd3+ (F), and Gd3+ bound at site 1 (Bl) or Gd3+ bound at site 2 (B2). In this paper we will define site 1 as the site which binds Gd3+ more tightly. The data of Figure 11 are consistent with an Eg- of 9.4 and an Eg2 of 5.4. When solutions containing 10 or 50 pM Gd3+ were titrated with Ca2+-ATPase, the enhancement increased as the enzyme concentration increased. The reciprocal of the observed enhancement was plotted against the reciprocal of the total ATPase concentration (Figure 12) yielding a linear behavior, except at high levels of enzyme where a sharp increase in the observed enhancement is found. This behavior is consistent with two environments for bound Gd3+ ion on the Ca2+-... 

In practical applications, it is quite likely that quantitative description of the relationship between the dependent variable (eg., conversion yield) and the independent variable(s) (eg., reaction time, temperature, enzyme concentration, and substrate molar ratio) is impossible, and the optimum point cannot be found analytically. On such occasions, the graphical method, although less precise, is a practical and straight forward option, providing the graphical version of the description of the function without knowing how the function is mathematically defined. 

The kCM value is defined as kcat = vmax/[/i0l where /i(1l is the employed enzyme concentration. 

Because of the nonstandard units associated with the I.U. system for defining enzyme concentrations, an equivalent International system of units (Systeme International) (S.I.) unit has been defined, and is called the katal. One katal (kat) of enzyme activity is that quantity that will consume 1-mol substrate/s 1 pkat = 60 I.U. 

The international unit itself may eventually be replaced by the SI unit termed the katal, the SI derived unit for catalytic activity (see Chapter 1). It is defined as moles per second. The name katal had been used for this unit for decades, but did not become an official SI derived unit until 1999 with Resolution 12 of the 21st CGPM, on the recommendation of the International Federation of Cfinical chemistry and Laboratory Medicine. Both the International Union of Pure and Applied Chemistry and the lUB now recommend that enzyme activity be expressed in moles per second and that the enzyme concentration be expressed in terms of katals per liter (kat/L). Thus, lU = lO mol/fiOs = 16.7 X 10 mol/s, or l.Onkat/L - 0.06U/L. The formal adoption of the katal is hoped to discourage the use of a non-SI unit called unit, symbol U, defined as micromoles per minute. Units are more commonly used than the katal in practice at present, but their definition lacks coherence with the SI system. 

Three basic quantities are defined to describe membrane performance. Flux is the permeate flow rate normalized to total membrane filter area. For protein recovery in the cell separation step, instantaneous protein transmission can be measured by determining enzyme concentration simultaneously on the retentate and permeate sides of the filter during cell concentration. Percent transmission is calculated as ... 

The kinetic description of enzyme-catalyzed reactions in vitro has allowed their rates to be expressed as a function of reactant and enzyme concentrations, and a set of empirically defined kinetic parameters. Enzymologists also have sought to modify this basic relationship in systematic ways so as to reveal something about the mechanism by which the enzyme acts. Traditionally, in such studies the enzymologist has been concerned primarily with the enzyme, its proper substrates and products, and closely related chemical analogs of these proper reactants (Jencks, 1969). 

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