Previous Class. Today. Reasons for analyzing pre-steady state conditions Methods for pre-steady state measurements

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1 Previous Class Reasons for analyzing pre-steady state conditions Methods for pre-steady state measurements Today Practical Methods for Kinetics and Equilibria Spectrophotometry Radioactive Procedures Spectrofluorimetry Plotting kinetic data (initial rates) Coupled assays

2 Spectrophotometry A spectrophotometer is employed to measure the amount of light that a sample absorbs. The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector A = log I0/I What are the factors that affect the amount of light a sample absorbs? Beer's Law (1852) : The absorbance displays a simple dependence on the concentration of the analyte and the cell path length A = εcl Where ε is the molar absorptivity, c is the concentration, and l is the length of sample that the light passes through (in cm)

3 Is a measure of the ability of an analyte to absorb light at a specified wavelength. (find λ max) Is supposed to be constant for Beer s law to be valid DETERMINE ε Molar Absorptivity Determination Measure the intensity of absorbed light for solutions with various concentrations of analyte. Construct a plot of A vs c (should be a straight line) Plot the line-of-best-fit through the experimental points. The slope of this line is the molar absorptivity (at a particular path length)

4 Molar Absorptivity Determination ε = L/mol cm

5 Spectrophotometry A classic example NADH assays NADH and NAD are coenzymes involved in oxidation-reduction reactions such as dehydrogenase reactions. Reaction rate of enzymes can be determined by the rate of formation or decomposition of NAD and NADH. Pyruvate + NADH LDH Lactate + NAD N R CONH 2 H N R H CONH 2 Extinction Coefficient (cm -1 M -1 x 10-3 ) Abs NAD + NADH NAD NADH wavelength (nm)

6 Spectrophotometry Determination of Product Concentration The application of spectrophotometry to Enzymology is to determine the concentration of the product over a period of time. Exploiting Beer's Law (linear relationship between absorbance of the solution and the concentration of the product) v (velocity) [S]

7 Plotting kinetic data Michaelis-Menten kinetics (steady state kinetics) Measure Initial rates (v 0 ) at different substrate concentrations by detecting the absorbance difference over time and obtaining the slope of the line Obtain various initial rates at different substrate concentrations and plot Use Lineweaver-Burke plots to obtain kcat, Km, kcat/km

8 Plotting kinetic data Measuring initial rates: The initial velocity is the amount of product produced per minute Assay involves adding all of the components including substrate first Once enzyme is added the absorbance is continually monitored and recorded with respect to time Abs vs time can be plotted Determine slope of tangent Absor. time

9 Plotting kinetic data Measuring initial rates: The slope of tangent = Abs/unit of time (min) Recall A = εcl, therefore A = ε cl When c occurs in a known time period then c min -1 = v 0 v 0 = A min -1 / εl = c min -1 Has M/min after multiplying by vol. of enzyme assay solution the units change to mol/min. Determine v 0 at different substrate concentrations and plot

10 Plotting kinetic data v (velocity) [S]

11 Spectrofluorimetry Fluorescent compounds absorb light (photon) to give excited state then re-emits light (photon) during decay at a different (usually longer) wavelength Fluorimetry is usually much more sensitive than spectrophotometry Common natural fluorophores are: Tryptophan, tyrosine, and phenylalanine (amino acids), NADH (340nm nm) Quenching occurs when re-emission energy is transferred to another group (enzymes with SH3 and WW domains and regulation of folding) Fluorescence can be enhanced in different solvents (tryptophan, NADH)

12 Radioactive Procedures Most sensitive assay methods involve radioisotopes (orders of magnitude more sensitive than spectrophotometry) Common isotopes include P 32, C 14, H 3, S 35 ATP all phosphate groups can be labeled (gamma most common) Radioactive decay (measured in cpm) involves the emitting of electrons that are converted to light quanta by a scintillant solution P 32 can spontaneously emit photons and can be captured by film detector Often radioactive methods are used to examine enzymes in vivo to provide qualitative data (but can be quantitative also)

13 Kinase Assay Lyse Transfected cells and remove cell debris Immunoprecipitate protein kinase of interest Incubate with γ-[ 32 P] ATP and substrate SDS-PAGE Autoradiography

14 Enzyme activity of MLK3 kinase and a MLK3 oligomerization mutant MLK3: - WT L410P MLK3 P histone P substrate

15 Mitogen Activated Protein Kinase Signaling Cascades

16 Kinase Assay Lyse Transfected cells and remove cell debris Immunoprecipitate protein kinase of interest Incubate with γ-[ 32 P] ATP and substrate SDS-PAGE Autoradiography

17 Enzyme activity of Downstream Kinase (JNK) MLK3: - WT L410P JNK IP substrate c-jun P Most Sensitive!!! Experiment from one plate of human cells [E] = fmol vs Spectrophotometry [E] = µmol

18 Next Class ph dependence of Enzyme catalysis