DAFTAR ISI
DAFTAR ISI ..................................................................................................................................................... 1
DAFTAR GAMBAR ........................................................................................................................................ 6
DAFTAR TABEL …..…………………..……………………………………………………………………………………………6
1.
CLASSIFICATION AND MEASUREMENT OF ENZYME ACTIVITY ........................................ 7
A. CLASSIFICATION OF ENZYMES ......................................................................................................... 7
Organization ............................................................................................................................................. 7
Characteristics .......................................................................................................................................... 7
Presentation of an entry in Enzyme Nomenclature ................................................................................................ 7
System of classification ............................................................................................................................. 7
Classification system used in Enzyme Nomenclature ............................................................................................ 7
Isoenzymes ................................................................................................................................................ 7
Classification of common enzymes ........................................................................................................... 7
Common enzymes ................................................................................................................................................. 7
B. UNITS OF ENZYME ACTIVITY ............................................................................................................ 7
References ................................................................................................................................................. 7
2.
PURIFICATION AND ANALYSIS OF ENZYME PREPERATIONS ............................................. 8
A. DYE-LIGAND CHROMATOGRAPHY IN ENZYME PURIFICATION ............................................... 8
Introduction .............................................................................................................................................. 8
General use of immobilized dye matrices ................................................................................................. 9
Protein adsorption ................................................................................................................................................. 9
Elution of target enzyme ....................................................................................................................................... 9
Dye column regeneration and storage ...................................................................................................... 9
Common conditions used for enzyme adsorption to immobilized dye affinity matrices ....................................... 9
Common parameters used for elution to enzymes from the dye-immobilized matrix ........................................... 9
Structure and chemistry of dyes ................................................................................................................ 9
The structure of some reactive dyes ...................................................................................................................... 9
Examples of requirements for metal ions for proteins to bind the dye affinity matrices ....................................... 9
Dye immobilization ................................................................................................................................... 9
Properties of some commercial immobilized affinity matrices ............................................................................. 9
Choice of the support matrix ................................................................................................................... 10
Choosing the activation chemistry .......................................................................................................... 10
Summary of the properties of various activated gels ........................................................................................... 10
Immobilizing the dye ............................................................................................................................... 10
Absorbance properties of some reactive dyes...................................................................................................... 10
B. CRITERIA OF ENZYME PURITY ........................................................................................................ 10
Detection of non protein contaminants ................................................................................................... 10
The quantification of nucleic acid contaminants in protein samples ................................................................... 10
Detection of protein contaminants .......................................................................................................... 10
Specific activity measurement ................................................................................................................. 10
Electrophoretic methods ......................................................................................................................... 10
The choice of electrophoretic method for the determination of protein purity .................................................... 10
Some common problems encountered in electrophoretic and their remedies ...................................................... 11
Capillary electrophoresis........................................................................................................................ 11
he uses of capillary electrophoresis ..................................................................................................................... 11
Capillary electrophoresis can be used in a variety of modes ............................................................................... 11
Chromatography technique .................................................................................................................... 11
Gel filtration ........................................................................................................................................................ 11
Ion exchange ....................................................................................................................................................... 11
Affinity chromatography ..................................................................................................................................... 11
Chromatofocusing ............................................................................................................................................... 11
1
Centrifugation methods ........................................................................................................................... 11
Sediments velocity ................................................................................................................................... 11
Sedimentation equilibrium .................................................................................................................................. 11
Mass spectrometry .................................................................................................................................. 11
The basis of elctrospray in matrix assisted laserdesorption mass spectrometry .................................................. 11
The properties of electrospray and matrix-assisted laser desorption mass spectrometry ..................................... 11
Amino acids analysis and sequence analysis .......................................................................................... 11
Active-site titration ................................................................................................................................. 11
C. THE DETERMINATION OF PROTEIN CONCENTRATION ............................................................. 12
Introduction ............................................................................................................................................ 12
Why determine protein concentration? ................................................................................................... 12
The choice of method .............................................................................................................................. 12
Gravimetric determination biuret method ........................................................................................................... 12
UV absorbance .................................................................................................................................................... 12
Amino acid analysis lowry method ..................................................................................................................... 12
Bicinchoninic acid (BCA) reagent ...................................................................................................................... 12
Coomassie blue binding ...................................................................................................................................... 12
Reaction with OPA.............................................................................................................................................. 12
D. FRACTIONATION AND ANALYSIS OF ENZYME BY GEL ELECTROPHORESIS ....................... 12
Introduction ............................................................................................................................................ 12
Recipes for gel and buffers ..................................................................................................................... 12
Separation of enzymes by non denaturing gel electrophoresis ............................................................................ 12
Buffers for non denaturing discontinuous system ............................................................................................... 12
Recipe for gel preparation using non denaturing discontinuous buffer system ................................................... 12
Determination of molecular weigths of enzymes by gel electrophoresis on SDS Error! No index entries
found.denaturing gels .......................................................................................................................................... 12
Determination of isoelectric points of enzyme using isoelectric focusing ........................................................... 12
Electrolytes used in IEF .......................................................................................................................... 12
Recipes for preparation of IEF gel ......................................................................................................... 12
Electrolytes used in immobilized pH gradients ....................................................................................... 12
Two dimensional gel electrophoresis ...................................................................................................... 13
Sample preparation................................................................................................................................. 13
Standard marker proteins used in non denaturing gels ........................................................................................ 13
Standard marker proteins used in denaturing gels ............................................................................................... 13
Analysis of gels ....................................................................................................................................... 13
Staining and detection methods used in IEF........................................................................................................ 13
Staining procedures for two dimensional polyacrylamide gels ........................................................................... 13
Staining procedures used after immunoelectrophoresis ...................................................................................... 13
Detection of specific enzymes on gels ................................................................................................................ 13
Common blotting membrane ............................................................................................................................... 13
Transfer buffer used in blotting ........................................................................................................................... 14
General stains for blot transfer ............................................................................................................................ 14
Sensitivities of methods for radioisotope detection in polyacrylamide gel ......................................................... 14
E. ACTIVE SITE TITRATION ................................................................................................................... 14
Introduction ......................................................................................................................................................... 14
Principle of titration using (chromogenic analog) substrates............................................................................... 14
Development of the titration technique using chromogenic and fluorogenic substrates ...................................... 14
Titrations using specific nhibitors without chromogenic leaving groups ............................................................ 14
Use of spectroscopic labels and radiolabels ........................................................................................................ 14
Use of antibodies ................................................................................................................................................. 14
Activity loss measurements ................................................................................................................................. 14
3.
ENZYME KINETICS .......................................................................................................................... 14
A. TYPE OF ASSAY PROCEDURE .......................................................................................................... 14
Product or substrate? ............................................................................................................................. 14
Physical or chemical property to measure .............................................................................................. 15
A summary of the main enzyme assay methods .................................................................................................. 15
2
Alternative assay strategies when reactants do not offer useful properties for measurement .............................. 15
Dependence on time and enzyme concentration ..................................................................................... 15
Continuous or stopped assay? ................................................................................................................ 15
Enzyme instability ................................................................................................................................... 15
Testing for enzymes instability ........................................................................................................................... 15
Choice of reaction mixture...................................................................................................................... 15
Volumetric accuracy ............................................................................................................................... 15
B. ENZYMES KINETICS ........................................................................................................................... 15
Analysis of initial rate data .................................................................................................................................. 15
Two substrate systems ......................................................................................................................................... 15
Single substrates/pseudo single-substrate system ................................................................................................ 15
Three substrate systems ....................................................................................................................................... 15
Assignment of mechanism .................................................................................................................................. 15
C. PLOTTING METHODS ......................................................................................................................... 15
The michaelis-Menten equation .......................................................................................................................... 15
Plots of the Michaelis-Menten equation .............................................................................................................. 15
Plots of rate against substrate concentration for enzyme obeying the Michaelis-Menten equation doublereciprocal plot 1/v against 1/a.............................................................................................................................. 15
Alternative form of direct linear plot ................................................................................................................... 15
The integrated Michaelis-Menten equation ......................................................................................................... 15
Plots for linear inhibition data ............................................................................................................................. 15
Plots for two substrate data ................................................................................................................................. 15
Non Michaelis-Manten data ................................................................................................................................ 15
Plot for testing enzyme hypotheses ..................................................................................................................... 15
4.
PATTERNS OF ENZYME INHIBITION .......................................................................................... 16
SIMPLE REVERSIBLE INHIBITION ................................................................................................................... 16
Some basic differences between reversible and irreversible inhibitors ............................................................... 16
Simple Michaeilis-Menten kinetics ..................................................................................................................... 16
Kinetic mechanism or reversible inhibition ......................................................................................................... 16
Inhibitor and substrate concentration effect in reversible inhibition.................................................................... 16
Competitive inhibition ............................................................................................................................. 16
Dependence of the degree of inhibition resulting from a fixed inhibitor ............................................................. 16
Some reversible enzyme inhibitors...................................................................................................................... 16
Uncompetitive inhibitors ......................................................................................................................... 16
Mixed and noncompetitive inhibitors ...................................................................................................... 16
Determination of inhibitor constants ...................................................................................................... 17
Apparent values of the Michaelis-Menten parameters in the presence of simple reversible inhibitors ............... 17
Dixon plots for mixed inhibitors ......................................................................................................................... 17
Cornish-Bowden plots for mixed inhibition ........................................................................................................ 17
TIGHT-BINDING INHIBITORS ......................................................................................................................... 17
Analysis of tight-binding inhibitors by the Dixon methods ................................................................................. 17
Analysis of tight-binding inhibitors by the Dixon’s procedure ........................................................................... 17
Reaction kinetics of tight-binding inhibitors by the Henderson procedure.......................................................... 17
Some tight-binding enzyme inhibitors ................................................................................................................ 17
IRREVERSIBLE INHIBITION ............................................................................................................................ 17
Non specific irreversible inhibitors......................................................................................................... 17
Behavior of non specific irreversible inhibitors .................................................................................................. 17
Some commonly used non specific enzyme inhibitors ........................................................................................ 17
Irreversible reaction of amino acid residues in an enzymes that results in inhibition .......................................... 17
Specific irreversible inhibition ................................................................................................................ 17
Behavior of specific irreversible inhibitorsspesific irreversible inhibition .......................................................... 17
5.
PHYSICAL FACTORS AFFECTING ENZYME ACTIVITY ........................................................ 17
A. PH DEPENDENT KINETICS ................................................................................................................. 17
Potential pitfalls: general considerations ............................................................................................... 17
Parallel pathways ................................................................................................................................... 17
3
Kinetic models for product formation a central ionization state of an enzyme substrate complex ...................... 17
The quasi equilibrium condition ............................................................................................................. 17
Six type of pKa ........................................................................................................................................ 17
Most commonly encountered types of acid dissociation constant and their significance .................................... 17
Kinetic consequences of reaction via a minor ionization state ............................................................... 17
Protonic dissociation from a two site acid ........................................................................................................... 18
Reaction of the nucleophilic from of an enzyme (E-) with electrophilic from a time dependent inhibitor .......... 18
B. THE EFFECT OF TEMPERATURE ON ENZYME-CATALYSED REACTION................................. 18
Variations of equilibrium constant with temperature ............................................................................. 18
Variation of rate constant with temperature ........................................................................................... 18
The transition state change in energy of molecular during a chemical reaction .................................................. 18
Interpretation of Arrhenius plots ............................................................................................................ 18
6.
ANALYSIS OF LIGAND BINDING BY ENZYMES ....................................................................... 18
INTRODUCTION TO LIGAND BINDING............................................................................................................. 18
THEORETICAL BACKGROUND TO LIGAND BINDING ....................................................................................... 18
Interactions at equilibrium ..................................................................................................................... 18
The Scatchard plot............................................................................................................................................... 18
A binding curve measured by a change in the protein fluorescence intensity ..................................................... 18
Linearized plot for measurement sites concentration and dissociation constant from binding data at equilibrium
............................................................................................................................................................................ 18
Conditions for collecting Scatchard binding data and their attendant errors ...................................................... 18
Binding data collected in tight binding and weak binding conditions ................................................................. 18
The kinetics of binding ............................................................................................................................ 18
Data from a single exponential reaction .............................................................................................................. 18
Linearization of single exponential data .............................................................................................................. 18
Conformational change in an induced-fit, two step binding process ................................................................... 18
Practical techniques for studying interactions at equilibrium ................................................................ 18
Methods for Scatchard analysis ........................................................................................................................... 18
Equilibrium dialysis ....................................................................................................................................... 18
A protein ligand interaction measured by gel filtration .................................................................................. 18
A filter binding assay ..................................................................................................................................... 19
Methods for measuring saturation of binding sites .............................................................................................. 19
A protein ligand interaction measured by a change in the emission properties of the enzyme and resonance
energy transfer ............................................................................................................................................... 19
Practical techniques for studying the kinetics of ligand binding ............................................................ 19
General ................................................................................................................................................................ 19
Stopped flow mixing methods ............................................................................................................................. 19
Schematics representation of a stopped flow apparatus ................................................................................. 19
4
DAFTAR GAMBAR
GAMBAR 1 ........................................................................................................................................................... 7
GAMBAR 2 ........................................................................................................................................................... 7
GAMBAR 3 ........................................................................................................................................................... 9
GAMBAR 4 ......................................................................................................................................................... 13
GAMBAR 5 ......................................................................................................................................................... 14
5
DAFTAR TABEL
TABEL 1 ............................................................................................................................................................... 9
TABEL 2 ............................................................................................................................................................ 10
TABEL 3 ............................................................................................................................................................ 11
TABEL 4 ............................................................................................................................................................ 12
TABEL 5 ............................................................................................................................................................ 13
TABEL 6 ............................................................................................................................................................ 14
TABEL 7 ............................................................................................................................................................ 14
TABEL 8 ............................................................................................................................................................ 14
TABEL 9 ............................................................................................................................................................. 16
TABEL 10 .......................................................................................................................................................... 16
6
1.CLASSIFICATION AND MEASUREMENT OF ENZYME ACTIVITY
A. CLASSIFICATION OF ENZYMES
Organization
Characteristics
Presentation of an entry in Enzyme Nomenclature
Lihat gambar 1
gambar 1
System of classification
Classification system used in Enzyme Nomenclature
Isoenzymes
Classification of common enzymes
Common enzymes
gambar 2
B. UNITS OF ENZYME ACTIVITY
References
Lihat gambar 2
7
2. PURIFICATION AND ANALYSIS OF ENZYME PREPERATIONS
A. DYE-LIGAND CHROMATOGRAPHY IN ENZYME PURIFICATION
Introduction
Affinity chromatography is a highly efficient techniques which biological
molecules based on the specific interaction of a covalently bound ligand
with its complementary biological counterpart lihat gambar 3. The types
of immobilized ligand used can be separated into two classes: biospecific
ligand such as receptors, enzyme substrate, antibodies, nucleotides,
coenzyme, and pseudo-biospecific ligand such as dyes, hydrophobic
compounds and metals ions. From this list, dyes are rapidly becoming the
most frequently tested for use as affinity ligands in the purification of a
wide range of enzyme and other proteins. Lihat tabel 1
8
General use of immobilized dye matrices
Lihat Tabel 2
Protein adsorption
gambar 3
Elution of target enzyme
Lihat gambar 3
Dye column regeneration and storage
Common conditions used for enzyme adsorption to immobilized dye affinity
matrices
Lihat gambar 4
Common parameters used for elution to enzymes from the dye-immobilized
matrix
Structure and chemistry of dyes
The structure of some reactive dyes
Examples of requirements for metal ions for proteins to bind the dye affinity
matrices
Lihat Tabel 3
Dye immobilization
Properties of some commercial immobilized affinity matrices
tabel 1
9
Choice of the support matrix
Choosing the activation chemistry
Summary of the properties of various activated gels
Immobilizing the dye
Absorbance properties of some reactive dyes
B. CRITERIA OF ENZYME PURITY
Detection of non protein contaminants
The quantification of nucleic acid contaminants in protein samples
Detection of protein contaminants
gambar 4
Specific activity measurement
Electrophoretic methods
Lihat Tabel 2
The choice of electrophoretic method for the determination of protein purity
Tabel 2
10
Some common problems encountered in electrophoretic and their remedies
Capillary electrophoresis
The uses of capillary electrophoresis
Capillary electrophoresis can be used in a variety of modes
Chromatography technique
Gel filtration
Ion exchange
Affinity chromatography
Chromatofocusing
Centrifugation methods
Sediments velocity
Sedimentation equilibrium
Mass spectrometry
The basis of elctrospray in matrix assisted laserdesorption mass
spectrometry
The properties of electrospray and matrix-assisted laser desorption mass
spectrometry
Lihat Tabel 3
Amino acids analysis and sequence analysis
Active-site titration
Tabel 3
11
C. THE DETERMINATION OF PROTEIN CONCENTRATION
Introduction
Why determine protein concentration?
The choice of method
Gravimetric determination biuret method
UV absorbance
Amino acid analysis lowry method
Bicinchoninic acid (BCA) reagent
Coomassie blue binding
Reaction with OPA
Lihat Tabel 4
D. FRACTIONATION AND ANALYSIS OF ENZYME BY GEL
ELECTROPHORESIS
Introduction
Tabel 4
Recipes for gel and buffers
Separation of enzymes by non denaturing gel electrophoresis
Buffers for non denaturing discontinuous system
Recipe for gel preparation using non denaturing discontinuous buffer system
Determination of molecular weigths of enzymes by gel electrophoresis on
SDS denaturing gels
Determination of isoelectric points of enzyme using isoelectric focusing
Electrolytes used in IEF
Recipes for preparation of IEF gel
Electrolytes used in immobilized pH gradients
Lihat gambar 5
12
Two dimensional gel electrophoresis
gambar 5
Sample preparation
Standard marker proteins used in non denaturing gels
Standard marker proteins used in denaturing gels
Analysis of gels
Staining and detection methods used in IEF
Lihat Tabel 5
Staining procedures for two dimensional polyacrylamide gels
Staining procedures used after immunoelectrophoresis
Detection of specific enzymes on gels
Common blotting membrane
Tabel 5
13
Transfer buffer used in blotting
General stains for blot transfer
Sensitivities of methods for radioisotope detection in polyacrylamide gel
E. ACTIVE SITE TITRATION
Introduction
Lihat Tabel 6
Principle of titration using (chromogenic analog) substrates
Tabel 6
Development of the titration technique using chromogenic and fluorogenic
substrates
Titrations using specific nhibitors without chromogenic leaving groups
Lihat Tabel 7
Use of spectroscopic labels and radiolabels
Tabel 7
Use of antibodies
Tabel 8
Activity loss measurements
Lihat Tabel 8
3. ENZYME KINETICS
A. TYPE OF ASSAY PROCEDURE
Product or substrate?
14
Physical or chemical property to measure
A summary of the main enzyme assay methods
Alternative assay strategies when reactants do not offer useful properties for
measurement
Dependence on time and enzyme concentration
Continuous or stopped assay?
Enzyme instability
Testing for enzymes instability
Choice of reaction mixture
Volumetric accuracy
B. ENZYMES KINETICS
Analysis of initial rate data
Two substrate systems
Single substrates/pseudo single-substrate system
Three substrate systems
Assignment of mechanism
C. PLOTTING METHODS
The michaelis-Menten equation
Plots of the Michaelis-Menten equation
Plots of rate against substrate concentration for enzyme obeying the
Michaelis-Menten equation double-reciprocal plot 1/v against 1/a
Alternative form of direct linear plot
The integrated Michaelis-Menten equation
Plots for linear inhibition data
Plots for two substrate data
Non Michaelis-Manten data
Plot for testing enzyme hypotheses
15
4. PATTERNS OF ENZYME INHIBITION
Simple reversible inhibition
Some basic differences between reversible and irreversible inhibitors
Simple Michaeilis-Menten kinetics
Lihat tabel 9
tabel 9
Kinetic mechanism or reversible inhibition
Inhibitor and substrate concentration effect in reversible inhibition
Competitive inhibition
Dependence of the degree of inhibition resulting from a fixed inhibitor
Some reversible enzyme inhibitors
Uncompetitive inhibitors
Lihat Tabel 10
Mixed and noncompetitive inhibitors
Tabel 10
16
Determination of inhibitor constants
Apparent values of the Michaelis-Menten parameters in the presence of
simple reversible inhibitors
Dixon plots for mixed inhibitors
Cornish-Bowden plots for mixed inhibition
Tight-binding inhibitors
Analysis of tight-binding inhibitors by the Dixon methods
Analysis of tight-binding inhibitors by the Dixon’s procedure
Reaction kinetics of tight-binding inhibitors by the Henderson procedure
Some tight-binding enzyme inhibitors
Irreversible inhibition
Non specific irreversible inhibitors
Behavior of non specific irreversible inhibitors
Some commonly used non specific enzyme inhibitors
Irreversible reaction of amino acid residues in an enzymes that results in
inhibition
Specific irreversible inhibition
Behavior of specific irreversible inhibitorsspesific irreversible inhibition
5. PHYSICAL FACTORS AFFECTING ENZYME ACTIVITY
A. Ph DEPENDENT KINETICS
Potential pitfalls: general considerations
Parallel pathways
Kinetic models for product formation a central ionization state of an enzyme
substrate complex
The quasi equilibrium condition
Six type of pKa
Most commonly encountered types of acid dissociation constant and their
significance
Kinetic consequences of reaction via a minor ionization state
17
Protonic dissociation from a two site acid
Reaction of the nucleophilic from of an enzyme (E-) with electrophilic from a
time dependent inhibitor
B. THE EFFECT OF TEMPERATURE ON ENZYME-CATALYSED
REACTION
Variations of equilibrium constant with temperature
Variation of rate constant with temperature
The transition state change in energy of molecular during a chemical reaction
Interpretation of Arrhenius plots
6. ANALYSIS OF LIGAND BINDING BY ENZYMES
Introduction to ligand binding
Theoretical background to ligand binding
Interactions at equilibrium
The Scatchard plot
A binding curve measured by a change in the protein fluorescence intensity
Linearized plot for measurement sites concentration and dissociation
constant from binding data at equilibrium
Conditions for collecting Scatchard binding data and their attendant errors
Binding data collected in tight binding and weak binding conditions
The kinetics of binding
Data from a single exponential reaction
Linearization of single exponential data
Conformational change in an induced-fit, two step binding process
Practical techniques for studying interactions at equilibrium
Methods for Scatchard analysis
Equilibrium dialysis
A protein ligand interaction measured by gel filtration
18
A filter binding assay
Methods for measuring saturation of binding sites
A protein ligand interaction measured by a change in the emission properties of
the enzyme and resonance energy transfer
Practical techniques for studying the kinetics of ligand binding
General
Stopped flow mixing methods
Schematics representation of a stopped flow apparatus
19