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Lecture

# Spectrophotometry, absorption spectrum, Beers-Lambert law, enzyme kinetics, Michaelis constant, enzyme action

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School
Department
Life Science
Course
LIFE 212
Professor
Semester
Fall

Description
12 September Spectrophotometry Is the most often used theory and technique in a biochemistry lab Is a technique that measures the interaction of electromagnetic radiation with molecules, atoms, or ions Absorption Emission Fluorence Principle of spectrophotometry Each substance absorbs a unique wavelength of light (monochromatic light) from the light spectrum Substances can be analyzed qualitatively and quantitatively Substances appear a certain color because they absorb certain wavelengths of light and transmit others. The Absorption Spectrum Absorption over a range of wavelength Lmax: wavelength at which absorbance is the greatest Lmax is characteristic of each chemical substance and provides information on the electronic structure of the analyte. Action Spectrum Is the rate of a physiological activity plotted against wavelength of light Beers-Lambert Law Direct linear relationship of Absorbance (A) to concentration (C) of a solute A = ELC A = absorbance, E = extinction coefficient, C = concentration The equation follows the equation of a straight line Y = mx + b Where m is the slope of the line and b is the y intercept A C = EL A E = CL Note: Absorbance has no units Standard Curve Determining the concentration (C) of a substance in solution when E is unknown or when a substance does not obey Beer’s law Enzyme Kinetics Enzymes run all cell functions Enzyme activity is studied by enzyme kinetics Important in disease diagnosis and research Enzyme Kinetics: study of Rate or velocity of an enzyme catalyzed reaction Factors which may affect it Enzymes act as catalysts Enzyme brings the substrate(s) into the proper alignment or configuration to speed up their reaction or conversion into products Enzymes are highly selective in binding of substrates Because of their 3-dimensional structure Key-Lock mode of enzyme action Steric specificity Binding of substrate to enzyme is in a geometric arrangement Active site of an enzyme The molecular place where the substrate binds and at which catalysis takes place Catalytic groups: protein residues that directly participate in the enzymatic reaction Making and breaking of bonds Substrates are bound to enzymes by multiple weak interactions Km: Michaelis constant Units of Kmare concentrations (M) Characteristic for an enzyme-substrate interaction under defined pH and temp [E [S] K m [ES] Gives an idea about the affinity of an enzyme to its substrate, important measure of the enzyme rate of reaction K mow = high affinity of the enzyme to the substrate K migh = low affinity of the enzyme to the substrate Enzymes often have multiple substrates: a substrate with low Kmwill bind more quickly to the enzyme than those with high Km Environment affects enzyme activity Enzyme-substrate best-fit configuration – reaction maximum rate Under optimal environmental conditions Factors affecting rate of enzyme action pH Temperature Salts Cofactors and coenzymes Concentration of the substrate Concentration of enzymes pH: How do
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