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•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