Post on 22-Oct-2020
Previous Class
•Reasons for analyzing pre-steady state conditions
•Methods for pre-steady state measurements
TodayPractical Methods for Kinetics and Equilibria
Spectrophotometry Radioactive Procedures
Spectrofluorimetry Plotting kinetic data (initial rates)
Coupled assays
Spectrophotometry
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 pathlength
A = εclWhere ε is the molar absorptivity, c is the concentration, and l is the length of sample that the light passes through (in cm)
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 detectorA = log I0/I
•Is a measure of the ability of an analyte to absorb light at a specified wavelength. (find find λλ maxmax)
•Is supposed to be constant for Beer’s law to be valid
DETERMINE DETERMINE εε
•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)
Molar Absorptivity Determination
Molar Absorptivity Determination
εε = L/mol cm= L/mol cm
wavelength (nm)260 300 340 380
Ext
inct
ion
Coe
ffici
ent
(cm
-1 M
-1 x
10-
3 )
20
15
10
5
NAD+
NADH
N
CONH2
R
N
CONH2
R
H H
NAD NADH
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 Lactate + NADLDH
Abs
Determination of Product ConcentrationThe 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)
Spectrophotometry
v (v
eloc
ity)
[S]
Plotting kinetic data
Michaelis-Menten kinetics (steady state kinetics)
Measure Initial rates (v0) 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
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 andrecorded with respect to time
•Abs vs time can be plotted
•Determine slope of tangentA
bsor
.
time
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 = v0
•v0 = ∆A min-1/ εl = ∆c min-1
•Has M/min after multiplying by vol. of enzyme assay solution theunits change to mol/min.
Determine v0 at different substrate concentrations and plot
v (v
eloc
ity)
[S]
Plotting kinetic data
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 ---- 460nm)
•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)
Radioactive Procedures
Most sensitive assay methods involve radioisotopes (orders of magnitude more sensitive than spectrophotometry)
Common isotopes include P32, C14, H3, S35
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
P32 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)
Kinase AssayKinase Assay
LyseLyse TransfectedTransfected cells and remove cell debriscells and remove cell debris↓↓
Immunoprecipitate protein kinase of interestImmunoprecipitate protein kinase of interest↓↓
Incubate with Incubate with γγ--[[3232PP]] ATP and substrateATP and substrate↓↓
SDSSDS--PAGEPAGE↓↓
AutoradiographyAutoradiography
histone
MLK3
MLK3:
L410
P
WT-
P
P
Enzyme activity of MLK3 kinase and a MLK3 oligomerization mutant
substrate
Mitogen Activated Protein Kinase Signaling CascadesMitogen Activated Protein Kinase Signaling Cascades
Kinase AssayKinase Assay
LyseLyse TransfectedTransfected cells and remove cell debriscells and remove cell debris↓↓
Immunoprecipitate protein kinase of interestImmunoprecipitate protein kinase of interest↓↓
Incubate with Incubate with γγ--[[3232PP]] ATP and substrateATP and substrate↓↓
SDSSDS--PAGEPAGE↓↓
AutoradiographyAutoradiography
Enzyme activity of Downstream Kinase (JNK)
MLK3:
-W
T
c-Jun
L410
P
JNK IP P
substrate
Most Sensitive!!! – Experiment from one plate of human cells
[E] = fmol vs Spectrophotometry [E] = µmol
Next Class
pH dependence of Enzyme catalysis