Study of Enzyme Activity - basics & standard curve calibration

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Department
Chemical and Biochemical Engineering
Course
Chemical and Biochemical Engineering 2290A/B
Professor
Mita Ray
Semester
Fall

Description
Lab Notes - 2011 Contents Objective.......................................................................................................................................................2 Theory...........................................................................................................................................................2 Enzymes................................................................................................................................................2 Competitive Inhibition..........................................................................................................................4 Noncompetitive Inhibition....................................................................................................................4 Absorption Spectroscopy......................................................................................................................6 Alkaline Phosphatase............................................................................................................................8 Assay Method .......................................................................................................................................9 WEEK 1....................................................................................................................................................10 Lab Notes - 2011 Objective The objective of this lab is to familiarize you with biological catalysts – enzymes and their activity. Theory Enzymes WHAT ARE ENZYMES? Enzymes are protein catalysts that carry out the chemical reactions of metabolism. All chemical reactions require activation energy to break chemical bonds and begin the reaction. The need for activation energy acts as a barrier to the chemical reaction occurring and/or to the speed at which it occurs. Enzymes lower the barriers that normally prevent chemical reactions from occurring (or slow them down) by decreasing the required activation energy. Thus, in the presence of enzymes, reactions proceed and/or proceed at a faster rate. Figure 1 Enzyme Activity Lab Notes - 2011 HOW ENZYMES LOWER ACTIVATION ENERGY Enzymes carry out their function of lowering activation energy by temporarily combining with the chemicals involved in the reaction. These chemicals are called the substrate. Enzymes are specific for their substrate: A particular substrate molecule will combine temporarily with one enzyme type, and the active site of a particular enzyme will fit only one kind of substrate. For example, the enzyme sucrase will attach only to the substrate sucrose. The combination is called the enzyme- substrate complex. When the enzyme and substrate combine, the substrate is changed to a different chemical called the product. The enzyme is not consumed or altered by the reaction. Equation 1 Enzyme Kinetics k k 1 2 E+ S ES E+ P k-1 ENZYME KINETICS Definition: The study of the rate at which an enzyme works is called enzyme kinetics. The rate at which an enzyme works is influenced by several factors, e.g., A) Substrate concentration (the more of them available, the quicker the enzyme molecules collide and bind with them). The concentration of substrate is designated [S] and is expressed in units of molarity. B) Temperature. As the temperature rises, molecular motion — and hence collisions between enzyme and substrate — speed up. But as enzymes are proteins, there is an upper limit beyond which the enzyme becomes denatured and ineffective. C) pH. The conformation of a protein is influenced by pH and as enzyme activity is crucially dependent on its conformation, its activity is likewise affected. D) Presence of inhibitors. Competitive inhibitors are molecules that bind to the same site as the substrate — preventing the substrate from binding as they do so — but are not changed by the enzyme. Non- competitive inhibitors are molecules that bind to some other site on the enzyme reducing its catalytic power. Lab Notes - 2011 ENZYME INHIBITION Figure 2 Enzyme Inhibition Enzymes can be inhibited  competitively, when the substrate and inhibitor compete for binding to the same active site or  non-competitively, when the inhibitor binds somewhere else on the enzyme molecule reducing its efficiency. Competitive Inhibition In the presence of a competitive inhibitor, it takes a higher substrate concentration to achieve the same velocities that were reached in its absence. So while V maxcan still be reached if sufficient substrate is available, one-half Vmaxrequires a higher [S] than before and thus K ism larger. Noncompetitive Inhibition With noncompetitive inhibition, enzyme molecules that have been bound by the inhibitor are taken out of the game so  enzyme rate (velocity) is reduced for all values of [S], including Vmax and one-half V max Lab Notes - 2011  K memains unchanged because the active site of those enzyme molecules that have not been inhibited is unchanged Lineweaver-Burk plot Figure 3 Lineweaver Burk Plot Enzyme activity Enzyme activity is defined as the initial rate of either product formation or substrate being consumed. The concentration of substrate or product is in moles/ litre and the time is in minutes. One unit of activity ( U ) is usually defined as 1 uM of product produced per minute Lab Notes - 2011 Absorption Spectroscopy The most widely used analytical instrument in biochemical studies is the absorption spectrophotometer. Utilizing wavelenths of light from the ultraviolet region (180 nm – 350 nm) through the visible light region (350nm – 800nm ) the technique can be used for quantifying materials, for following reactions, and for characterizing isolated substances. Many compounds absorb ultraviolet (UV) or visible (VIS) light. Figure 4 Visible Spectrum Many compounds absorb ultraviolet (UV) or visible (Vis.) light. The diagram below shows a beam of monochromatic radiation of radiant power P , directed at a sample solution. 0 Absorption takes place and the beam of radiation leaving the sample has radiant power P. Figure 5 Absorption of incident light The amount of radiation absorbed may be measured in a number of ways : Transmittance : T = P/P 0 % Transmittance : %T = 100 T Absorbance : A = log P10P0 A = log 101/T A = log1000/%T A = 2 – log 10 Lab Notes - 2011 The last equation, A = 2 - log %T10 is worth remembering because it allows you to easily calculate absorbance from percentage transmittance data. So, if all the light passes through a solution without any absorption, then absorbance is zero, and percent transmittance is 100%. If all the light is absorbed, then percent transmittance is zero, and absorption is infinite. Beer-Lambert Law Spectrophotometry is used for both qualitative and quantitative investigations of samples. The wavelength at the maximum of the absorption band will give information about the structure of the molecule or ion and the extent of the absorption is proportional with the amount of the species absorbing the light. Quantitative measurements are based on Beer’s Law :- Equation 2 Beer-Lambert Law A = εbc A: absorbance (no units, because it is calculated as A = log (P /P)10whe0e P is the inc0dent light’s intensity and P is the light intensity after it passes through the sample ) ε : molar absorbtivity with units of L mol cm -1 -1 b is the path length of the sample - that is, the path length of the cuvette in which the sample is contained. -1 c is the concentration of the compound in solution, expressed in mol L Note 1.: Instead of the molar absorbance you can also use the specific absorbance, but you have to make sure that the proper concentration unit is applied. Note 2.: Molar absorbance is a function of wavelength, so Beer’s Law is always applied at one (or several) specific wavelength values. Extra information : Detailed description of Uv-Vis spectrophotometer: http://www2.chemistry.msu.edu/faculty/reusch/VirtTxtJml/Spectrpy/UV-Vis/uvspec.htm#uv1 Lab Notes - 2011 Alkaline Phosphatase Alkaline phosphatase (ALP, ALKP) (EC 3.1.3.1) is a hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids. The process of removing the phosphate group is called dephosphorylation. As the name suggests, alkaline phosphatases are most effective in an alkaline environment. It is sometimes used synonymously as basic phosphatase. Figure 6 Structure of Alkaline Phosphatase Alkaline phosphatase reaction:- Substrate :- p-nitrophenol phosphatase (p-NPP) : colorless Enzyme : alkaline phosphatase Product : p-nitrophenol (p-NP) : yellow colored at 405nm Equation 3 Alkaline phosphatase reaction Lab Notes - 2011 Assay Method End point Assay: Sampl
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