lab training – enzyme kinetics & photometry · calculate the blood alcohol content (bac) in...

Lab training – Enzyme Kinetics & Photometry

Qing Cheng

[email protected] Division, MBB, KI

Schedule

Lab lecture - Enzyme Kinetics and Photometry (QC HT16)

19/9, lab lecture (mandatory)

20/9, odd groups, lab work (mandatory)

21/9, even groups, lab work (mandatory)

22/9, 12:45-15:30, compilation (mandatory, teacher will sign your lab journal)

23/9, 9:00-11:30, compilation (not mandatory)

1/10, deadline for the lab report

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Lab lecture


Introduction on enzyme and kinetics Michaelis-Menten kinetics KM, Vmax and kcat Lineweaver-Burk plot Enzyme inhibition, competitive and non-competitive inhibition

Spectrophotometer and Beer-Lambert Law

Lab procedure Lab execution Lab report

Safety in the lab

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Introduction – Enzyme

ΔG‡ (with enzyme)

ΔG‡ (without enzyme)

Substrate

Product

Transition state, S‡

Free

ene

rgy

Reaction progress

ΔG for the reaction

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Enzymes are biological catalysts characterized by Catalytic efficiency Specificity Regulated activity

Enzyme-catalyzed reactions are affected by Enzyme concentration Substrate concentration Temperature pH Inhibitors Activators

SubstrateEnzyme

Product

Lab lecture - Enzyme Kinetics and Photometry (QC HT16) 20160919

Kinetics is the study of chemical reaction rate (v, stands for velocity)

Enzyme kinetics is the study of enzyme catalyzed reaction

Determination of kinetics parameter measurement of enzyme activity

Introduction – Kinetics

ΔG‡ (with enzyme)

ΔG‡ (without enzyme)

Substrate

Product

Transition state, S‡

Free

ene

rgy

Reaction progress

ΔG for the reaction


Introduction – Order of reactoin

·

S k P

Reactio

n rate, v

Substrate concentration [S]

Zero order reaction

First order reaction

k

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First order reaction (v k · S ) Reaction rate is proportional to the substrate’s concentration. This is

true when the substrate concentration is low during the reaction, and the substrate is the determine factor for the reaction rate.

Zero order reaction (v k · S ) The reaction rate is independent of the substrate concentration. This is

true when the substrate concentration is much higher than the enzyme concentration during the reaction, and the enzyme is the determine factor for the reaction rate.

Michaelis-Menten kinetics

To understand how enzyme functions, we need a kinetic description of their activity.

The reaction rate rises linearly as substrate concentration increases (first order reaction) and then begins to level off and approach a maximum at higher substrate concentration (zero order reaction)

For many enzymes, the reaction rate V0 is defined as the number of moles of product formed per unit time when [P] is low, that is at times close to zero (hence, V0)


Reactio

n rate, v


First order reaction

Zero order reaction

Reactio

n rate, v

Time

[S1]

[S2]

[S3]

[S4]V0

V0

V0

V0

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At very low substrate concentration ( ≪ → ) : The reaction rate is directly proportional to the substrate concentration

At very high substrate concentration ( ≫ → ): The reaction rate is maximal, independent of substrate concentration.

When KM is equal to the substrate concentration ( → ): KM is equal to the substrate concentration at which the reaction rate is half

its maxim value


Reaction rate


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⇌ ⇀


Michaelis constant (Km) KM has the units of concentration KM is a physical constant characterizing a certain enzyme in term of specificity, affinity, etc.

KM is independent of either [E] or [S] KM is an inverse measure of the substrate's affinity for the enzyme

A bigger KM indicates lower affinity.

Lab lecture - Enzyme Kinetics and Photometry (QC HT16) 20160919

Velo

city

..........................

..........................

Lineweaver-Burk plot

Vmax is difficult to estimate because the initial reaction rate approaching Vmax asymptotically withincreasing substrate concentration. In addition, the high concentration of substrate often inhibitsreaction rate. To solve this problem, Lineweaver and Burk (1934) had inverted the Michaelis-Menten equation, which is referred as Lineweaver-Burk plot (or Double reciprocal plot):


·

In this equation, 1/V and 1/[S] arevariables, while KM/Vmax and 1/Vmax areconstants. This can be plotted as alinear equation (y = ax + b).Specifically, 1/v is y, 1/[S] is x, KM/Vmaxis a (slope) and 1/Vmax is b (y-intercept). We can accurately calculateKM and Vmax value from a Lineweaver-Burk plot.

y = ax + b

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Nonlinear regression

Indeed, KM and Vmax values can be calculateddirectly from the Michaelis-Menten equationthrough nonlinear regression.


y = ax/(b+x)

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http://www.colby.edu/chemistry/PChem/scripts/lsfitpl.html(in short: http://bit.ly/1re1XU4)

Input the data pairs (V and [S])

Choose fit function: ax/(b+x)

Leave Parameter guesses as it is.

Choose Convergence Mode: Damped or Strongly damped

Click Fit or Fit & Plot (Java needed for plotting)

Inhibition of enzyme activity

Competitive inhibition

Noncompetitive inhibition


Enzyme Enzyme

Competitive inhibitor

Enzyme

Noncompetitive inhibitor

Substrate

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Inhibition of enzyme activity Competitive inhibition


Enzyme


No inhibitor

Competitive inhibitorNo inhibitor

+ Competitive inhibitor

Vmax is not affected KM is increased

+I

EI

E + S ES E + P

ki S

I

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Inhibition of enzyme activity Noncompetitive inhibition


No inhibitor


S IE + I ES E + P

kiS

EI EIS ×

No inhibitor

+ Noncompetitive inhibitor

Enzyme


Vmax is decreasedKM is not affected

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Inhibition of enzyme activity


No inhibitor

Mix inhibition

Vmax is decreasedKM is not affected

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No inhibitor


No inhibitor


Vmax is not affected KM is increased

Vmax is decreasedKM is increased


Photometry - Spectrophotometer

How to measure the chemical reactions rate Different molecules have different

absorption Some molecules (e.g. proteins) have

several absorbance peaks during the wave scan

Spectrophotometer

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Light source Filter Sample Detector Readout


Photometry - Beer-Lambert law

Beer-Lambert law is a mathematical means of expressing how light is absorbed bymatter. The law states that the amount of light emerging from a sample isdiminished by three physical phenomena:

The concentration of absorbing sample in its pathway (C, in unit of molarity, M)

The distance the light travels through the sample (ℓ, in units of centimeters, cm)

The probability that the light of that particular wavelength will be absorbed bythe material, also known as molar absorption (or extinction) coefficient (ε), inunits that are reciprocals of molarity and distance in centimeters, M-1cm-1)

T: 0 - 1A: ∞ - 0

Due to technical limitation, the best readingrange of spectrophotometer is normallyfrom 0.1 – 1, thus:

o If A is too high, dilute the sampleo If A is too low, concentrate the sample

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Lab training

Enzyme: Alkaline Phosphatase (ALP) Remove phosphate groups from many types of molecules. Function as a dimer, and take effect under alkaline conditions Made in liver, bone, and other tissues. It can be measured in a routine blood test. Abnormally high serum

levels of ALP may indicate bone, liver disease, etc.

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Lab training – Outline

Determine the following parameters of alkaline phosphataseusing p-nitro-phenyl-phosphate (NPP) as substrate Optimal pH KM

Vmax

Inhibition

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p-Nitro-Phenyl-Phosphate (NPP)

p-Nitrophenol


Determine the maximal absorbance (λmax) for p-nitrophenol

Set wavelength scan in the range of 350 – 500 nm

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Calculating molar absorption coefficient (ε) using Beer-Lambert law

· · ·

Lab training – Maximal absorbance


Lab training – Incubation time

The effect of incubation time on the amount of product formed

Incubation at 40⁰C water bath Incubation time (9 different time from 0.5-20 min) Measure absorbance within 15 min at the end of the reaction Plot: amount of product formed (y-axis) against time (x-axis)

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dH2ONFF

Buffer with pH 9.5

Testing tubes (× 9)Add enzyme

Blank tubes (× 9)Add dH2O


Lab training – Optimal pH

The effect of pH on the reaction rate (v)

Incubation at 40⁰C water bath Incubation time: 10 min Measure absorbance within 15 min at the end of the reaction Plot: amount of product formed (y-axis) against time (x-axis)

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dH2ONFF

Buffer with differnt pH

Testing tubes (× 9)Add enzyme

Blank tubes (× 9)Add dH2O


Lab training – KM, Vmax, Inhibition

Determine the KM and Vmax value for alkaline phosphatase, and the inhibition type of the inhibitor provided

Incubation at 40⁰C water bath Incubation time: 10 min Measure absorbance within 15 min at the end of the reaction Michaelis-Menten plot: Reaction rate (y-axis) against [S] (a-axis) Lineweaver-Burk plot: 1/V (y-axis) against 1/[S] (a-axis) Determine KM, Vmax, and inhibition type

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Each series contains 6 tubes with different substrate concentration [S]

a. With enzyme onlyb. With enzyme and 1st inhibitor (0.5 mM phosphate solution)c. With enzyme and 2nd inhibitor (1 mM phosphate solution)d. Blank (with neither enzyme nor the inhibitor)

a b c d


Lab training – Case study (ADH)

KM determination for alcohol dehydrogenase

Determine KM, Vmax, of alcohol dehydrogenase using eithermethanol or ethanol as substrate from the values in the labcompendium.

Determine inhibition type of ethanol on ADH when usingmethanol as substrate.

Calculate the blood alcohol content (BAC) in the patientundergone ethanol treatment.

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Lab journal and reports

Write journal carefully, which should be approved and countersigned by your teaching assistant.

A written lab report per group is needed Brief introduction and aim Brief description of the execution of the lab work Results including all the raw data, calculations including the

equation, and all the plots. Don’t forget to specify units in calculations and plots Brief conclusions and discussion Answer all the questions in the compendium.

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Lab safty

Lab coat, goggles, gloves No eating or drinking in the lab Prevent accidents

Be well prepared Work calmly

When you leave the lab Wash hands Remove lab coat

Use your judgment Ask teaching assistant

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Question?

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lab training – enzyme kinetics & photometry · calculate the blood alcohol content (bac) in...

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