Dear Debaprasad,
The molar attenuation coefficient is a measurement of how strongly a chemical species such as substrate or product attenuates light at a given wavelength. It is an intrinsic property of the species. The SI unit of molar attenuation coefficient is the square metre per mole (m2/mol), but in practice, it is usually taken as the M−1⋅cm−1 or the L⋅mol−1⋅cm−1. The molar attenuation coefficient is also known as the molar extinction coefficient and molar absorptivity, but the use of these alternative terms has been discouraged by the IUPAC.
The absorbance of a material that has only one attenuating species also depends on the pathlength and the concentration of the species, according to the Beer–Lambert law
A = εcℓ
where
ε is the molar attenuation coefficient of that material;
c is the amount concentration of those species;
ℓ is the pathlength.
Enzymes are usually protein molecules that manipulate other molecules — the enzymes' substrates. These target molecules bind to an enzyme's active site and are transformed into products through a series of steps known as the enzymatic mechanism
E + S ES ES*< ——> EP E + P
In enzyme kinetics, the reaction rate is measured by monitoring the reaction by UV or Visible spectra. Generally, it is much easier to monitor the enzymatic reaction by calorimetric methods in which the reaction mixture is allowed to react with a reagent that forms a colored complex. This is because the attenuation coefficient of lamda max in the UV spectrum is relatively low, thus providing a weak absorbance. Whereas, in calorimetric method the attenuation coefficient of lamda max in the visible region is relatively high and the thus the absorbance is strong.
An attenuation coefficient of a particular wavelength that can be used is largely dependent on the enzyme, substrate(s) and/or products. There is no particular wavelength and attenuation coefficient that is applied for all enzymes. These vary depending on the enzyme's system. Generally, the methods to monitor most of the enzymes reactions are well-known and the monitoring lamda and its attenuation coefficient are known as well.
Knowing the monitoring wavelength and its extinction coefficient allows monitoring the conversion of a substrate to product/s, and basically allows the quantification of such conversion such as the product quantity formed and the reaction rate. Please note that that when using a calorimetric method with a certain extinction coefficient you should consider the stoichiometry of the reaction.
Alternatively, monitoring the enzymatic reaction can be done by measuring the disappearance of the substrate. In this case the extinction coefficient allows you to determine the remaining amount of the substrate after reaching an equilibrium.
It should be emphasized that whether the enzymatic reaction is monitored by the formation of a product or disappearance of a substrate, you have to use the extinction coefficient to convert the absorbance units to concentration.
Enzyme assays (General)
An enzyme assay consists of mixing the enzyme with a substrate in a solution of
controlled pH with any additional substance whose effect is to be tested, incubating the reaction mixture at a suitable temperature for a suitable time, stopping the reaction precisely, and then somehow measuring the amount of reaction that has occurred. In general, the amount of reaction that has taken place may be quantified in one of two ways: in terms of the disappearance of substrate or the appearance of product, depending upon which is chemically
the more advantageous.
The actual method of measurement depends on some biochemical or biophysical
property of the molecule being assayed.Frequently, some reagent is used which combines with a product of the enzyme reaction to produce a color. The intensity of the color can then be measured spectrophotometrically and compared with color produced by known amounts of product to provide a basis for expressing units of enzyme activity.
An Example of Calorimetric Assay:
Wheat germ (the wheat embryo that is removed from the wheat grain before milling so that the flour will keep better) contains a phosphatase that can be extracted readily in water. The enzyme is not as specific as some other enzymes and will act on a wide range of possible substrates. We will use p-nitrophenyl phosphate (NPP) as substrate because it allows us a simple assay procedure: wheat germ phosphatase acting on NPP produces p-nitrophenol (PNP) as the product and PNP is yellow at alkaline pH. By adding Na2CO3 to our reaction mixtures, the pH can be raised enough to stop the reaction (an important requirement in enzyme studies)
and to make the product colored for spectrophotometric quantification.
Construction and use of a standard graph
You remember that if we measure the optical density (O.D.) of a solution at all the
possible wavelengths and plot the data of wavelengths (λ) vs. O.D., we will obtain a bell-shaped curve with a single peak. The wavelength at which the peak occurs, called lambda-max (λmax) is the wavelength at which the molecules have the highest absorption of light. Every solution has its own characteristic λmax. For example, the λmax of PNP is 420 nm. According to Beer's Law, if we prepare a solution of a substance at a known concentration, dilute it to several additional known concentrations, measure the absorbance of each of these samples at the λmax of the substance and plot optical density as a function of concentration, we should obtain a straight line that passes through the origin. This line is called
the standard graph of the substance. Consequently, if someone handed us a solution of the substance of unknown concentration, we could quickly determine its concentration by measuring its absorbance. This could be done for as many unknowns as we wish.
In this example the extinction coefficient to be used is the one of the wavelength of 420 nm which can be calculated according to the following equation:
Using A = εcℓ; ε = A/cℓ
Hoping this will be helpful,
Rafik
Dear Debaprasad,
The molar attenuation coefficient is a measurement of how strongly a chemical species such as substrate or product attenuates light at a given wavelength. It is an intrinsic property of the species. The SI unit of molar attenuation coefficient is the square metre per mole (m2/mol), but in practice, it is usually taken as the M−1⋅cm−1 or the L⋅mol−1⋅cm−1. The molar attenuation coefficient is also known as the molar extinction coefficient and molar absorptivity, but the use of these alternative terms has been discouraged by the IUPAC.
The absorbance of a material that has only one attenuating species also depends on the pathlength and the concentration of the species, according to the Beer–Lambert law
A = εcℓ
where
ε is the molar attenuation coefficient of that material;
c is the amount concentration of those species;
ℓ is the pathlength.
Enzymes are usually protein molecules that manipulate other molecules — the enzymes' substrates. These target molecules bind to an enzyme's active site and are transformed into products through a series of steps known as the enzymatic mechanism
E + S ES ES*< ——> EP E + P
In enzyme kinetics, the reaction rate is measured by monitoring the reaction by UV or Visible spectra. Generally, it is much easier to monitor the enzymatic reaction by calorimetric methods in which the reaction mixture is allowed to react with a reagent that forms a colored complex. This is because the attenuation coefficient of lamda max in the UV spectrum is relatively low, thus providing a weak absorbance. Whereas, in calorimetric method the attenuation coefficient of lamda max in the visible region is relatively high and the thus the absorbance is strong.
An attenuation coefficient of a particular wavelength that can be used is largely dependent on the enzyme, substrate(s) and/or products. There is no particular wavelength and attenuation coefficient that is applied for all enzymes. These vary depending on the enzyme's system. Generally, the methods to monitor most of the enzymes reactions are well-known and the monitoring lamda and its attenuation coefficient are known as well.
Knowing the monitoring wavelength and its extinction coefficient allows monitoring the conversion of a substrate to product/s, and basically allows the quantification of such conversion such as the product quantity formed and the reaction rate. Please note that that when using a calorimetric method with a certain extinction coefficient you should consider the stoichiometry of the reaction.
Alternatively, monitoring the enzymatic reaction can be done by measuring the disappearance of the substrate. In this case the extinction coefficient allows you to determine the remaining amount of the substrate after reaching an equilibrium.
It should be emphasized that whether the enzymatic reaction is monitored by the formation of a product or disappearance of a substrate, you have to use the extinction coefficient to convert the absorbance units to concentration.
Enzyme assays (General)
An enzyme assay consists of mixing the enzyme with a substrate in a solution of
controlled pH with any additional substance whose effect is to be tested, incubating the reaction mixture at a suitable temperature for a suitable time, stopping the reaction precisely, and then somehow measuring the amount of reaction that has occurred. In general, the amount of reaction that has taken place may be quantified in one of two ways: in terms of the disappearance of substrate or the appearance of product, depending upon which is chemically
the more advantageous.
The actual method of measurement depends on some biochemical or biophysical
property of the molecule being assayed.Frequently, some reagent is used which combines with a product of the enzyme reaction to produce a color. The intensity of the color can then be measured spectrophotometrically and compared with color produced by known amounts of product to provide a basis for expressing units of enzyme activity.
An Example of Calorimetric Assay:
Wheat germ (the wheat embryo that is removed from the wheat grain before milling so that the flour will keep better) contains a phosphatase that can be extracted readily in water. The enzyme is not as specific as some other enzymes and will act on a wide range of possible substrates. We will use p-nitrophenyl phosphate (NPP) as substrate because it allows us a simple assay procedure: wheat germ phosphatase acting on NPP produces p-nitrophenol (PNP) as the product and PNP is yellow at alkaline pH. By adding Na2CO3 to our reaction mixtures, the pH can be raised enough to stop the reaction (an important requirement in enzyme studies)
and to make the product colored for spectrophotometric quantification.
Construction and use of a standard graph
You remember that if we measure the optical density (O.D.) of a solution at all the
possible wavelengths and plot the data of wavelengths (λ) vs. O.D., we will obtain a bell-shaped curve with a single peak. The wavelength at which the peak occurs, called lambda-max (λmax) is the wavelength at which the molecules have the highest absorption of light. Every solution has its own characteristic λmax. For example, the λmax of PNP is 420 nm. According to Beer's Law, if we prepare a solution of a substance at a known concentration, dilute it to several additional known concentrations, measure the absorbance of each of these samples at the λmax of the substance and plot optical density as a function of concentration, we should obtain a straight line that passes through the origin. This line is called
the standard graph of the substance. Consequently, if someone handed us a solution of the substance of unknown concentration, we could quickly determine its concentration by measuring its absorbance. This could be done for as many unknowns as we wish.
In this example the extinction coefficient to be used is the one of the wavelength of 420 nm which can be calculated according to the following equation:
Using A = εcℓ; ε = A/cℓ
Hoping this will be helpful,
Rafik
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