Analytical techniques

UNIT 1

Self Study
Principle of the following instruments/techniques and identify specific analytes that are measured by each instrument: Flourometry Turbidimetry Nephelometry Chemiluminescence Chromotography (HPLC; GLC & TLC) Elisa Prof T. Matsha

Lecture Outline
Photometry & Spectrophotometry
Mass spectrophotometry

Electrochemistry (Nersnt equation) Electrophoresis Osmometry Enzyme kinetics

Prof T. Matsha

Photometry & Spectrophotometry (1)

Measurement of light intensity Light as other forms of electromagnetic radiation make characteristic patterns (waves) as they travel through space Wavelength distance between two peaks (high points) as the light travels in a wave like manner

Prof T. Matsha

Photometry & Spectrophotometry (2)

Each wave has a certain shape and length depending on the frequency of the waves.
Frequency of a wave is inversely proportional to the wavelength

Prof T. Matsha

Photometry & Spectrophotometry (3)

Previously, photometric instruments measured light intensity independently of wavelength Sunlight mixture of spectrum of radiant energy at different wavelengths (rainbow) human eye recognizes it as white

Modern Instruments can isolate a narrow wavelength range of the spectrum for measurements Filters filter photometers Prisms or gratings - spectrophotometers
Prof T. Matsha

Photometry & Spectrophotometry (4)

Determinations in the Clinical Laboratory are based on measurement of radiant energy
Emitted Partially reflected Transmitted Absorbed

Photometers light emitted Spectrophotometers - absorbed

Prof T. Matsha 7

SPECTROPHOTOMETER

Used to measure to concentrations of substances

Prof T. Matsha 8

SPECTROPHOTOMETER cont.

A combination of a spectrometer & a photometer Spectrometer produces light of any selected Photometer measures light intensity A cuvette with liquid is placed between the 2.
Prof T. Matsha 9

SPECTROPHOTOMETER cont.

The amount of light passing through the cuvette is measured by the photometer. The photometer delivers a voltage signal to a display device. The signal changes as the amount of light absorbed by the liquid changes
Prof T. Matsha 10

SPECTROPHOTOMETER cont.
Transmitted light

Light absorbed
Prof T. Matsha 11

SPECTROPHOTOMETER cont.

Units: Absorbance (A) or Optical density (OD) (logarithmic scale) Light absorbed Transmitted light - % transmission (Arithmetic scale) Prof T. Matsha

12

SPECTROPHOTOMETER cont.
Obeys Beers law:

If a solute absorbs light of a particular , the absorbance is directly proportional to the concentration of substance in solution.
I0 I

A = log

= cl

Prof T. Matsha

13

Beers Law (1)

Beers law The intensity of a solution when viewed through monochromatic light (single wavelength, e.g. 600nm) is directly proportional to the concentration of the substance through which the light passes. Lamberts law - When monochromatic light passes through a transparent medium, the rate decrease in intensity with the thickness of the medium is proportional to the intensity of light Beer-Lambert law (also known as Beers law) - When monochromatic light passes through a coloured solution the amount of light transmitted decreases exponentially with the increase in concentration of the solution through which the light passes or simply; absorbance is directly related to concentration if the light path stay constant

Prof T. Matsha

14

Beers Law (2)

Absorbance Absorption is a process in which incident radiated energy is retained without reflection or transmission on passing through a medium Therefore for ray to be absorbed it must have the same frequency as a rotational or vibrational frequency in the atom or molecule it strikes

Transmittance The ratio of transmitted energy to the amount of incident energy is called transmittance.
Prof T. Matsha 15

USE OF A SPECTROPHOTOMETER

Blank

Prof T. Matsha

16

Beers Law (3)

Some of Io is either reflected by surface of the cell or absorbed by solvent or cell wall , Io < Is Percent transmittance %T = Io / Is X 100 For some applications in optics it might be useful to see transmittance values as percent transmittance values. All intensities will be scaled to fit an interval between 0 and 100 percent transmittance. Focus interest eliminate factors use blank Blank absence of compound of interest but same solvent. No light absorbed %T = 100% Add compound of interest serially %T varies inversely and logarithmically with concentration
Prof T. Matsha 17

Clinical Example

1.

Blank Reading reagent buffer without serum Read the blank Most light transmitted & small amount absored by cuvette, solvent or reflected from detector Set instrument abitrarily at 100%T (A = 0) 2. Sample Reading Reagent buffer + serum Difference amount light passed blank vs. sample due to presence of compound measured %T Sample beam signal X 100 Prof T. Matsha 18 Blank beam signal

USE OF A SPECTROPHOTOMETER

1. Switch on 2. Set wavelength 3. Use after 30 min

Prof T. Matsha

19

USE OF A SPECTROPHOTOMETER

4. Calibrate a. Insert blank

Prof T. Matsha

20

SPECTROPHOTOMETER cont.

Prof T. Matsha

21

USE OF A SPECTROPHOTOMETER

5. Set to 0
Prof T. Matsha 22

USE OF A SPECTROPHOTOMETER

4. Insert sample & read

Prof T. Matsha

23

Beers Law (4)

Absorbance is more convenient to use because it is directly proportional to concentration. Amount of light absorbed particular wavelength depends: 1. Molecules and ions present 2. [ ] 3. pH 4. Temperature
Prof T. Matsha 24

Standard Curve
Unknown [ ] is determined from a calibration curve or standard curve Standards of known concentration Plot on graph linear curve
Prof T. Matsha

Absorbance (450nm)

B2MG Concentration (g/ml) 0 0.625 1.25 2.5 5 10 0.046 0.385 0.723 1.241 2.199 3.094

25

Spectrophotometic Instruments
Measure light transmitted by solution Mathematically converted absorbance Determine [ ] light absorbing substance

Prof T. Matsha

26

Spectrophotometer (2)
Light Source Provides radiant energy Visible light (350 - 700nm) tungsten light bulb To increase lifetime iodine or bromide is added tungsten-iodide lamp UV region (165 -360nm) low pressure mercury-vapor lamp, emits discontinuous spectrum Hydrogen & deuterium lamps low. Deuteriumdischarge more stable than hydrogen Mercury & xenon high pressure

Prof T. Matsha

27

Monochromator
Monochromator - isolate radiant energy of desired wavelength but excludes others

Prof T. Matsha

28

Filters
To obtain monochromatic light use Types 1. coloured-glass filter Transmistts energy over a wide range of wavelength Not precise Simple & inexpensive 2. Interference filters Pass very narrow range wavelength Efficient
Prof T. Matsha 29

Prisms
Type of monochromator Separates white light to continuous spectrum by refraction, i.e. shorter wavelengths are refracted or bent. Consequently nonlinear spectrum with longer wavelengths closer together, but Suitable narrow-bandwith portion of the spectrum

Prof T. Matsha

30

Diffraction gratings
Separation of light to different wavelengths Most commonly used monochromators Consists of parallel grooves etched polished surface

Prof T. Matsha

31

Cuvettes
Cuvette/Sample/Absorption cell: Holds sample & provides constant light path Round / square Light path must be kept constant Otherwise A not C

Prof T. Matsha

32

Cuvette types
Round Difficult constant light path Not uniform Etched for constant position Square/rectangular Plane-parallel optical surface, constant light path Less error Most common Plastic cells inexpensive, but designed for single use application Good clarity for both UV and visible light Problems etching by solvents, temp. deformations, cleaning Quartz cuvettes expensive, UV range
Prof T. Matsha 33

QC: Cuvettes
Scratched optical surface scatter light Do not touch on optical surface Wipe optical surface tissue before use Insert correct orientation spectrophotometer Clean immediately after use (DO NOT soak) (mild detergent & rinse deionised water) Drain upside down to dry

Prof T. Matsha

34

Photodetector
Convert transmitted radiant energy equivalent amount electrical energy

Prof T. Matsha

35

Photodetector types
Photocell / barrier-layer cell => least expensive => film light-sensitive material (eg. selenium / iron / silver) => require no external voltage => rely internal e- transfer produce current => wide bandpass instruments

Prof T. Matsha

36

Phototube
=> also photosensitive material => e- generated from light energy => outside voltage required

Prof T. Matsha

37

photomultiplier tube
=> detects & amplifies radiant energy => e- attracted series anodes / dynodes => each - (+) voltage => generate 2ndary e=> multiple cascade => amplification => thus 200x more sensitive than phototube => narrow bandpass instruments wavelength scanner instruments double-beam spectrophotometers

Prof T. Matsha

38

SINGLE-BEAM SPECTROPHOTOMETER

A - reading blanked appropriate reference solution

ie. solution without compound measured

Followed by test measure

Prof T. Matsha 39

DOUBLE-BEAM SPECTROPHOTOMETER
Automatic correction sample A & reference A

Prof T. Matsha

40

QA/QC Spec.
Wavelength accuracy Stray light scratched and dust particles Linearity

Prof T. Matsha

41

Spec in Clin lab

General chemistry analytes, e.g. glucose

Prof T. Matsha

42

Mass spectrophotometer
MS used to: Identify unknown compounds [ ] of known substances Molecular structure Chemical composition of both in-& organic materia In clinical chemistry: Drug metabolism Drug abuse (steroids use in sport) Damage to DNA Metabolic disorders in infants Research, e.g. search for unique proteins in specimen for use as diagnostic or therapeutic targets
Prof T. Matsha 43

Mass spec basic components

Sample converted into ions or molecules by thermal or electrical energy The ions in a gaseous medium are accelerated into the mass analyzer where they are separated into species, such that different species of ions strike the detector at
Prof T. Matsha 44

 Measure [ ]. by detecting A of electromagnetic radiation by atoms (not molecules)

ATOMIC ABSORPTION SPECTROPHOTOMETER

Prof T. Matsha

45

Components

Prof T. Matsha

46

Light Source
Hallow-cathode lamp Consists of evacuated gas-tight chamber +
Cathode Inert gas (argon / helium) => voltage applied => filler gas ionised => ions excite metal atoms => light energy emitted

anode

Prof T. Matsha

47

Sample Cell
Flame Why? Sample must contain reduced metal in atomic vaporised state Done via heat of flame break chemical bonds unexcited atoms

Prof T. Matsha

48

AAS vs SPEC
Light source passes through sample Note, atoms though bonds are broken ground state (unexcited) Light source excites atoms returns to ground state emits energy = absorbed light

Prof T. Matsha

49

Chopper
Aim: measure light absorbed by atoms Need to distinguish between light emitted by light source and excited atoms Hence, the chopper

Prof T. Matsha

50

Monochromator
Also used to protect the photodetector from excessive light from flame emissions

Prof T. Matsha

51

Adv:Disadvantages
ADV: => very sensitive & precise DISADV: only measure elements that exist atomic state eg. Mg2+, Mn2+, Cu2+, Pb2+ Flame not dissociate samples into free atoms, ie. PO42- interfere Ca2+
analysis (forms CaPO complex) 4 Overcome adding cations compete with Ca for P

Ionisation atoms upon dissociation by flame, overcome reducing flame temp. Matrix interference, eg. atoms in organic solvents enhanced light
absorption.

Overcome

pretreat sample (extraction)

Prof T. Matsha

52

Fluorometry
Measure concentration of solution that contain fluorescing molecules Principle: is based on an energy exchange process that occurs when valence shell electrons absorb EMR, become excited and return to an energy level lower than their original level. The lifetime of an excited state is about 10-9 to 10-6 seconds and the light emitted fro a single excited state is called fluorescence
Prof T. Matsha 53

Monochromator
Primary filter selects the wavelength Secondary filter as in AAS protects the photodetector from radiant energy emitted by flourescing molecules in sample Spectrofluorometer filters are replaced with prisms or gratings

Prof T. Matsha

54

Advantages
Specificity - because selection optimal

wavelength for both absorption & fluorescence spectrophotometry

Sensitivity - 1000x more sensitive than

Prof T. Matsha

55

Disadvantages
Sensitive to environmental changes pH changes => affects availability e Temperature changes => affects probability loss energy via collision (rather than fluorescence) Contaminating chemicals / change solvent => change structure molecules

Prof T. Matsha 56

QC Fluorometry
Any fluorescence as result of environmental changes Quenching QC: extreme care mandatory => analytical technique => instrument maintenance

Prof T. Matsha

57

Chemiluminescence
Chemiluminescence differs from flourescence in that emission of light is created from a chemical or electrochemical reaction (rxn) and not from EMR stimulation of electrons. It involves the oxidation of an organic compound such as luminol, in the presence of a catalyst such as an enzyme, metal ions (though not always) The excited products formed during the oxidation rxn produce chemiluminescence on return to the singlet state that can be measured by a luminometer.

Prof T. Matsha

58

ADVANTAGES CHEMILUMINESCENCE
Subpicomolar detection limits Speed rapid Ease of use, ie. one-step procedure Simple instrumentation Increased sensitivity over flourescence

Prof T. Matsha

59

TURBIDITY & NEPHELOMETRY

Techniques used to measuring the [ ] of a solution that contains particles too large for absorption spectoscopy. Nephelometry measurement of light scattered by a particulate solution. Commonly used for antibodyantigen rxns. TURBIDIMETRY measurement of the reduction in light transmission caused by particle formation. Applications: microbiology bacterial growth in broth cultures; hematology clot formation in coagulation analysers.
Prof T. Matsha 60

QC
Reagents free particles Cuvettes no scratches

Sample handling critical because particles tend aggregate & settle out solution

Prof T. Matsha

61

Electrochemistry
Measurement of current of voltage generated by the activity of specific ions Clinical chemistry: potentiometry; coulometry; voltammetry; and amperometry All use eithergalvanic electrochemical cell OR electrolytic cell
Prof T. Matsha 62

GALVANIC & ELECTROLYTIC CELLS

Electrochemical cell consists of: Two half-cells and a salt bridge (textbook figure) Electrodes (cathode & anode) immersed 2 beakers salt solution If only 1 beaker then solution = salt bridge
Prof T. Matsha 63

ION-SELECTIVE ELECTRODES (ISE)

Potentiometric method pH electrodes
Sensitive to individuals ions measure direct electrical potential due to activity of free ions

Prof T. Matsha

64

pH Electrodes
Components Indicator Electrode consists of a silver wire coated with AgCl in 0.1mmol/L HCl

All above place in into a tube containing special glass membrane tip sensitive to H+ only.
Prof T. Matsha 65

pH METER
Measures the acidity of a solution.

pH = - log aH+

pH = - log [H+]
AH+ = hydrogen ion activity [H+] in moles / Prof T. Matsha of solution
66

pH METER

2 electrodes measure voltage 1 electrode is in a liquid with fixed acidity reference electrode Other electrode responds to acidity of the solution sensing electrode Prof T. Matsha 67

pH METER

A voltmeter measures the difference between the voltage of the electrodes A meter converts this into pH
Prof T. Matsha 68

Indicator Electrode
pH electrode into test solution => movement H+ near tip electrode => produce potential difference => between internal & test solution => measured as pH by voltmeter

Prof T. Matsha

69

Reference electrode
Most commonly used - calomel electrode Calomel is a paste of mercurous chloride & potassium chloride In electrolyte solution KCl it is in direct contact with metallic mercury All reference electrodes must generate a stable electrical potential [ ] of electrolyte must constant & temperature stable voltage
Prof T. Matsha 70

NERNST EQUATION
Electromotive force generated because H+ at glass tip => described by Nernst equation (self study) THUS - temperature - H+ activity Set temperature-compensation knob

Prof T. Matsha

71

pH COMBINATION ELECTRODE
Indicator + reference electrode combine in one small probe Consists of: internal reference
=> Ag/AgCl OR => Hg/Hg2Cl2 Sealed into narrow glass cylinder with pH-sensitive glass tip
Prof T. Matsha 72

electrode

pH METER
Combination probe contains both electrodes

Prof T. Matsha

73

QC pH electrode
Balance the system with the electrodes in a buffer whose pH is 7.0 Replace buffer with one of different pH, usually 4.0 or 10
DONT touch glass bulb with your fingers. Rinse with distilled water Keep within ambient temperature range Avoid air bubbles Keep clean of deposit
Prof T. Matsha 74

GAS-SENSING ELECTRODES
Similar pH electrodes but separated from solution by gas-permeable hydrophobic
BUT designed detect specific gases in solutions eg. CO2 (PCO2 electrode) eg. O2 (Clark electrode) eg. NH3 (NH3 gas electrode)

membrane

Prof T. Matsha

75

Electrophoresis
Migration charged solutes in electrical

field In clin lab protein serum, urine, CSF Lately, Nucleic acids

Prof T. Matsha

76

Components
Electrical power => driving force Support medium Buffer Sample Detecting system

Prof T. Matsha

77

SUPPORT MATERIAL
All gels transparent Scanned densitometer Dried permanent record

Cellulose acetate Agarose gel Polyacrylamide gel Starch gel

Prof T. Matsha

78

PRINCIPLE
Charged particles migrate to opposite charged electrode
Velocity of migration controlled by => particle net charge (directly ) => particle size & shape (inversely ) => strength electrical field => chemical & physical properties supporting

medium => temperature

Prof T. Matsha

79

Procedure
Support with gel placed in

electrophoresis chamber Chamber filled buffer - contact both

ends support/gel Samples applied to gel Apply constant voltage / current specific time - Electrophoresis
Prof T. Matsha

80

Detection
Support/gel in fixative / dried F: prevent diffusion sample Stain with appropriate dye aid visualisation & quantitation NOTE: dye uptake sample conc. Excess dye washed away Dry gel (permanent record)
Prof T. Matsha 81

QC electrophoresis
Operate either constant current / constant voltage - constant current preferred Why? Current flows through medium heat is produced Results increased agitation of dissolved solutes increased current increased heat & buffer evaporation ionic strength of buffer increased current

Prof T. Matsha

82

QC buffer
Affects charge ampholytes (e.g Protein) 1 => pH & 2 => ionic strength of buffer Ampholyte net charge either (+) / (-) if buffer more acidic than pI ampholyte

=> => => if => => =>

ampholyte binds H+ ie. (+) charge migrate cathode (-) buffer more basic than pI ampholyte ampholyte loses H+ ie. (-) charge migrate anode (+)
Prof T. Matsha 83

 Serum may need dilution urine, CSF - concentrate before electrophoresis Application: 2-5 l sample (eg. serum) loaded / applied through thin plastic template slots Serum allowed diffuse into gel (5 min) Template blotted to remove excess serum Remove template

TREATMENT & APPLICATION OF SAMPLE

Prof T. Matsha

84

DETECTION & QUANTITATION

Separated protein fractions stained visualise - UV light (nucleic acids) Quantitation:densitometer => each band = peak => surface area of peak = % of total

Prof T. Matsha

85

Prof T. Matsha

86

Prof T. Matsha

87

Chromatography
Refers to a group of techniques used to separate complex mixtures on the basis of different physical interactions between the individual compound and stationary phase of the system

Prof T. Matsha

88

Components
Mobile phase => gas / liquid F: carry sample Stationary phase => solid/liquid F: mobile phase flows Column: F: hold stationary phase & separated components

Prof T. Matsha

89

MODES OF SEPARATION
Adsorption chromatography Partition chromatography Steric exclusion chromatography Ion-exchange chromatography

Prof T. Matsha

90

Adsorption chromatography
Liquid-solid chromatography Competition sample & mobile phase for adsorptive sites solid stationary phase High affinity molecules retained longer Stationary phase:(a) acidic polar (eg. silica

gel) (b) basic polar (eg. alumina) (c) nonpolar (eg. charcoal)

Prof T. Matsha

91

Partition chromotography
Liquid-liquid chromatography Separation solute basis relative solubility

Molecules polar & nonpolar groups in aqueous solution added to immiscible organic solvent Vigorous shaking -two phases separate Chloroform method DNA extraction
Prof T. Matsha

nonpolar (organic) solvent & polar (aqueous) solvent

92

Steric Exclusion
Liquid-solid chromatography Separate solute molecules basis of size

& shape

Prof T. Matsha

93

Method
Column packed porous material Packing material=> silica / glass Pore size controlled ie. choose according to molecules separated Sample => molecules different sizes move down column dissolved in mobile solvent Small molecules enter pores & momentarily trapped Large molecules excluded from small pores thus move past particles faster rate Intermediate-sized molecules partially restricted thus move intermediate rate
Prof T. Matsha 94

 Solute mixtures separated by charge Stationary phase is a resin consisting

Ion-exchange chromatography

of large polymers with charged functional groups Cation exchange resin,anion exchange resin or mixed bed exchange resin Resin is insoluble in water

Prof T. Matsha

95

USES OF ION-EXCHANGE CHROMATOGRAPHY

Remove interfering substances from solution Concentrate dilute ion solutions Separate mixtures charged molecules (eg. amino acids)

Prof T. Matsha

96

Chromotographic procedures
1. THIN-LAYER CHROMATOGRAPHY (TLC) Variant column chromatography Thin layer sorbent eg. alumina, silica gel, cellulose, cross-linked dextran => uniformly coated glass / plastic plate Sample applied near bottom edge plate Mobile phase / solvent in closed container until atmosphere saturated solvent vapour Bottom edge plate in solvent NOTE: samples NOT immersed solvent solvent migrates up thin layer => capillary action => sample molecules dissolved => ie. carries sample molecules
Prof T. Matsha 97

TLC
Separation depends sorbent & solvent

Solvent close top => plate removed & dried Sample Rf compared standards Rf - Rf => Rf = distance sample component total distance solvent front Method semiquantitative screening test
Prof T. Matsha 98

(1) (2) (3) (4)

adsorption partition steric exclusion ion-exchange

retention factor

HPLC
Improved TLC Components: Pump forces mobile phase through column much greater velocity than gravity-flow Column: stationary phase packed into long stainless steel column Fine & uniform packing => high resolution separation => requires pressure to force mobile phase through
Prof T. Matsha 99

Components HPLC
Sample injector - small syringe introduce sample into path Mobile phase carries sample through column Detector - monitor eluate as leaves column produce electronic signal conc. each component Spectrophotometers most common Recorder - record detector signal versus time mobile phase ie. from time sample injection Graph => chromatogram

Prof T. Matsha

100

Chromatograph
Identify compounds => compared retention time to standard retention times (BUT only identical conditions) Determine conc. each compound => peak area conc. compound

Prof T. Matsha

101

GAS CHROMATOGRAPHY
Separate mixtures volatile compounds / made volatile Similar HPLC except mobile phase = gas Thus samples partitioned between gaseous mobile phase & liquid stationary phase Carrier gas => nitrogen / helium / argon, selection depends on type of detector used Sample must be injected as gas OR Temperature of the injection port must be above boiling point of the components to vaporise sample upon injection
Prof T. Matsha 102

SCINTILLATION COUNTER
Radioimmunoassay used to measure trace concentrations of hormones or drugs Detect radioactive signals Development of non-isotopic immunoassay diminished use

Prof T. Matsha

103

Osmometry
Measure conc. solute particles in solution Refers to measurement of osmolality of an aqueous solution such as serum, plasma or urine 4 physical properties solution change as number dissolved particles in solvent: collectively they are called colligative properties of the solution because they can be related to each other and to the osmolality Thus, osmometry is based on measuring changes in the colligative properties and the freezing-point depression is the most commonly used in clin Prof T. Matsha

(1) osmotic pressure; (2) vapour pressure; (3) boiling point; (4) freezing point

104

FREEZING-POINT OSMOMETER
Consists of a sample refrigerated chamber containing a stirrer and a thermistor (temperature sensing device) Sample is supercooled below its freezing point in a chamber usually containing ethylene glycol The stirrer is used to agitate the sample in order to initiate freezing As the ice crystals form, heat is released from the solution, which at some point reaches an equilibrium with the rate of heat removed by the colder temp. of the sample chamber The equilibrium temp is known as the freezing point and is detected by the thermistor osmolality of the sample and is expressed as milliosmoles per kilogram of water (mOsm/kg)

Prof T. Matsha

105

Enzyme Action: Lock and Key Model

An enzyme binds a substrate in a region called the active site Only certain substrates can fit the active site Amino acid R groups in the active site help substrate bind Enzyme-substrate complex forms Substrate reacts to form product Product is released

Lock and Key Model

P
+ S S + P

E E

ES complex

Enzyme Action: Induced Fit Model

Enzyme structure flexible, not rigid
Enzyme and active site adjust shape to bind substrate Increases range of substrate specificity Shape changes also improve catalysis during reaction

Enzyme Action: Induced Fit Model

P
S S P

E E

ES complex

Learning Check E1
A. The active site is (1) the enzyme (2) a section of the enzyme (3) the substrate B. In the induced fit model, the shape of the enzyme when substrate binds (1) Stays the same (2) adapts to the shape of the substrate

Factors Affecting Enzyme Action: Substrate

Increasing substrate concentration increases the rate of reaction (enzyme concentration is constant) Maximum activity reached when all of enzyme combines with substrate First order kinetics rxn rate a [substrate] Zero order kinetics rxn depends on [enzyme]

Michaelis-Menten constant (Km)

1913 Michaelis & Menten hypothesised role [S] S binds free E at low [S] (ie. more E than S) reaction rate - steadily as more S added thus reaction rate [S] => first-order kinetics eventually E saturated with S thus maximum reaction velocity as P formed free E immediately combines excess free S => zero-order kinetics thus reaction rate depends [E]

Factors Affecting Enzyme Action: Temperature

Little activity at low temperature Rate increases with temperature
- Movement of molecules - Rate of intermolecular collusion - Energy for rxn

Most active at optimum temperatures (usually 37C in humans) Activity lost with denaturation at high temperatures

Factors Affecting Enzyme Action: Temperature

low temp. (eg. refrigeration / freezing) => enzymes reversible inactive (specimens for enzyme => some enzymes NOT frozen (activity lost) => avoid repeated freeze-thaw (denature) control temp. lab => accurate 0.1C labs choose enzyme analysis => 25C => 30C => 37C (most common) NOTE: reference ranges vary...

analysis frozen or refrig.)

Factors Affecting Enzyme Action

Optimum temperature

Reaction Rate

Low

High

Temperature

Factors Affecting Enzyme Action: pH

Maximum activity at optimum pH R groups of amino acids have proper charge Tertiary structure of enzyme is correct Narrow range of activity Most lose activity in low or high pH

Factors Affecting Enzyme Action

Reaction Rate Optimum pH

5 pH

11

Enzyme Inhibition
Inhibitors cause a loss of catalytic activity Change the protein structure of an enzyme May be competitive or noncompetitive Some effects are irreversible

Competitive Inhibition
A competitive inhibitor Has a structure similar to substrate Occupies active site Competes with substrate for active site Has effect reversed by increasing substrate concentration

Noncompetitive Inhibition
A noncompetitive inhibitor Does not have a structure like substrate Binds to the enzyme but not active site Changes the shape of enzyme and active site Substrate cannot fit altered active site No reaction occurs Effect is not reversed by adding substrate

Learning Check E2
Identify each statement as describing an inhibitor that is (1) Competitive (2) Noncompetitive A. B. C. D. Increasing substrate reverses inhibition Binds to enzyme, not active site Structure is similar to substrate Inhibition is not reversed with substrate

Solution E2
Identify each statement as describing an inhibitor that is (1) Competitive (2) Noncompetitive A. B. C. D. 1 2 1 2 Increasing substrate reverses inhibition Binds to enzyme, not active site Structure is similar to substrate Inhibition is not reversed with substrate