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Part 1: pH, Buffers & Amino Acids


Part 1: pH, Buffers & Amino Acids Introduction Cells made up of many organic cpds: nucleic acids polysacharrides - chains of sugars lipids - chains of fatty acids – PowerPoint PPT presentation

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Title: Part 1: pH, Buffers & Amino Acids

Part 1 pH, Buffers Amino Acids
  • Introduction
  • Cells made up of many organic cpds
  • nucleic acids
  • polysacharrides - chains of sugars
  • lipids - chains of fatty acids
  • proteins
  • gt contain weak acidic basic groups
  • gt change in pH --gt change in ionisation state of
    the groups
  • Solution? Use buffers to resist changes in pH


  • What is pH?
  • What is strong/weak acid base?
  • What are buffers?
  • What are amino acids?

1. pH, Acids Bases
  • Introduction (pg.2)
  • Bronsted Lowry
  • Acid is a molecule that can ionise to release a
    hydrogen ion
  • HA lt--gt H A-
  • In aqueous solution, hydrogen ion exists as
    hydronium ion
  • HA H2O lt--gt H3O A-
  • (conjugate acid) (conjugate

1.1 What is pH Kw?
  • Definition of pH (pg2)
  • Negative logarithm of the concentration of free
    hydrogen/hydronium (H / H3O) ions in a solution
  • pH -log10H
  • If H 10-3M gt log10 H -3 gt pH
    -log10 H 3
  • Ionisation of Water, Kw (pg3)
  • H2O H2O lt--gt H3O OH-
  • Dissociation constant of a solution
  • Kw H3O OH- 1 x 10-14 M
  • For Water H3O OH- 1 x 10-7 M gt neutral
  • Table 1 the pH scale (pg4)

1.3 Acid Dissociation Constant, Ka
  • A measure of the extent an acid dissociates in
    aqueous solution (pg3)
  • HA H2O --gt H3O A-
  • Ka H3O A-
  • HA
  • gt greater dissociation gt larger Ka gt
  • pKa -log10 Ka

1.4 pH and pKa
  • Henderson-Hasselbalch Equation (pg4)
  • pH pKa log10(base/acid) pKa
  • (i) RCOOH ltgt H RCOO- ---gt pH
  • (ii) RNH3 ltgt H RNH2 --gt pH
  • For weak acid, pH pKa when base acid
  • gt acid is
    50 dissociated

2.1 Strong Acid Base
  • HA H2O --gt H3O A- (pg4)
  • Completely dissociates in water gt H3O HA
  • gt pH -log10 H3O -log10 HA
  • (i) pH of 0.1M HCl
  • (ii) pH of 5 x 10-2 M H
  • (iii) pH of 0.01M NaOH
  • (iv) pH of 0.030M OH-

2.2 Weak Acid Base
  • Ionisation not complete (50) (pg5)
  • pH (of weak acid) 1/2(pKa - log10HA)
  • pH (of weak base) 7 1/2(pKa - log10base)
  • pH of 0.1M acetic acid (Ka 1.73 x 10-5 M)
  • pH of 0.1M NH4 (Ka 1.8 x 10-5 M)

3. Buffers
  • Weak acid-base pairs (pg6)
  • To maintain pH of a solution
  • Biological rxns --gt acidic or basic products
  • gt inhibits enz rxn (pg5)
  • Buffers mops up free H or OH- ionic products
  • Buffers cannot withstand large amounts of acid or
  • Effective buffer is one that can buffer between
    pKa 1 and pKa - 1 (pg6)
  • eg. acetic acid-acetate buffer at pH
  • phosphoric acid-phospate at pH
  • ammonia-ammonium at pH

3.1 Acetic acid-Acetate Buffer
  • Acetic acid solution has mixture of undissociated
    acetic acid, acetate ions hydronium ions (pg6)
  • CH3COOH H2O ltgt CH3COO- H3O

  • OH- --gt 2H2O
  • Each OH- ion neutralise one H3O ion
  • Acetic acid ionise to produce more H3O ions to
    neutralise OH- gt pH of solution remains
    relatively constant
  • End point is when there is no more acetic acid
  • --gt pH increase
  • The reverse happens when when a small quantity of
    H is added

4. Amino Acids
  • Free amino acids consist of (pg9)
  • an a-amino group(NH2)
  • an a-carboxyl group (COOH)
  • a side chain group (R)
  • The NH2 COOH group ( sometimes the R group)
    can gain or lose protons - dependg on the
    concentration of protons (pH) of the solution
  • gt AA can be used

4.1 Ionisable forms of Amino Acids
  • AA with non ionisable R group can exist as (pg9,
  • cation (overall ve charge) - at low pH
  • anion (overall -ve charge) - at high pH
  • zwitterion (ve -ve charge) - at
    isoionic/isoelectric point
  • AA with ionisable R group can exist as (pg10)
  • cation
  • zwitterion
  • intermediate ion (overall ve or -ve dependg on
    R group)
  • anion

4.2 Fractional charge of ionisable group
  • Tells the percent of the group that carries a
    charge at any one time for a given pH (pg12)
  • eg.
  • (i) FC of -0.5 for carboxyl group for a given pH
  • gt 50 of the carboxyl grps carries a -1
  • 50 carry no charge
  • (ii) FC of 0.3 for an amino group for a given pH
  • gt 30 carry 1 charge (RNH3) 70 carry no
  • (iii) If at a given pH an AA has 50 carboxyl
    grps charged -1
  • and 30 charged 1 gt overall charge

cont. Fractional charge
  • Can calculate FC on any ionisable group in an AA
    for any pH if you know the pKa (pg12)
  • FC charged species x magnitude sign (/-)
  • total species
  • Tables (a) (b) pg 12 13

4.3 Free AAs Bound AAs
  • pKas for free AA side chains differ by up to 1
    pH from pKas of Aas in a protein (pg14)
  • Acid-base properties of an AA residue in a
    polypeptide/protein depends upon
  • neighbourg residues can influence the properties
    of a side chain
  • a residue buried within the interior of a protein
    molecule and the same type of residue located on
    the surface of the protein may have a different

4.4 Effects from changes in pH
  • pH determines the types of ionic forms their
    proportions in an environment (pg14)
  • The different ionic forms determine other
    physical chemical properties of AAs eg. optical
    rotation, uv absorbance, metal chelating
  • These properties are also affected by changes
  • gt pH influences the charge of amino acids
  • hence also biomolecules --gt thus affectg
    its function

Part 2 Proteins
  • Introduction
  • Proteins are components of all living things
  • Contain the elements C, H, O, N S
  • Polymer of amino acids linked by peptide bonds
  • Polypeptides of more than 50 AA
  • Simpleprotein - made up entirely of AA residues
  • Conjugated protein - has another non-AA
    component (prosthetic grp) incorporated
  • Fibrous protein eg. collagen or
  • Globular protein eg.

1 Molecular Properties of Proteins
  • Protein Structure (pg55)
  • 1o structure - long, continuous,
  • 2o structure - linear chain folds turns on
  • 3o structure - chain aggregates to form a 3-D
  • 4o structure - aggregation of 1 or more chains
  • Folding Aas with hydrophilic R grps on the
    outside Aas with hydrophobic R grps are inside
    the molecule
  • Shape size - covalent peptide, disulfide bonds
    hydrogen bonds,
  • Changes in physical, chemical bioogical
    properties mainly due to changes in non covalent
  • Prots have different types proportions of Aas
    gt differ in shape, size, charge, solubility,

cont. Molecular Properties
  • Protein denaturation (pg56)
  • reversible or irreversible change in 2o, 3o, 4o
  • by a chemical eg. SDS, urea, high acid/alkaline
  • by physical process eg. rapid stirring, heat
  • Absorption of light by Proteins
  • absorbs uv light at 250nm to 300nm
  • measure protein concn
  • Chemical assays of Proteins by spectrophotometry
  • Biuret method (540nm)
  • Lowry method (750nm)
  • Coomassie Brilliant Blue or Bromocrsol Green

2. Effect of pH on Structure Function
  • Aas bound to each other by peptide bonds -
    a-amino group of 1 AA linked to the a-carboxyl
    group of another AA (pg57)
  • Ionisable groups
  • the a-amino the a-carboxyl grps at the ends of
    the chain
  • the side grps
  • Changes in pH (pg58)
  • proteins can act as buffers
  • can alter proportion distribution of ve -ve
  • gt may cause changes in shape, orientation
  • gt may affect structure, stability, function

2.1 Isoelectric Point (pI)
  • the pH at which the protein has no net charge
  • acidic protein low pI negatively charged
  • basic protein high pI positively charged
  • protein in a solution wh pH gt pI gt will bear a
    net -ve
  • protein in a solution wh pH lt pI gt will bear a
    net ve

2.2 Effect of pH Ionic Strength on
Protein Solubility
  • Adjust pH or ionic strength -gt alter distribution
    of ve -ve charges on the molecule -gt affect
    proteins solubiliy (affinity with water) (pg59)
  • Isoelectric point of a protein gt pH where
    protein is least soluble (pg59 Fig2, pg60)
  • Ionic strength - measure of the total charge of
    ions in solution
  • increase in IS of solution
  • low salt concn gt increase solubility of protein
  • very high salt concns gt solubility decreases
    (see pg 59)

3. Separation Methods
  • Chromatography (pg60)
  • separates a mixture into its components or
  • isolates one component fr a complex mixture
  • Stationary phase (SP) - solid, liquid or
    solid/liqd mixture
  • -
  • Mobile phase (MP) - liquid or gas wh flows over
    or through
  • the SP
  • Elution movement of the MP over or through the
  • Components of a mixture distribute themselves
    bewtn the MP
  • Kd (distribution coeffcnt) concn of component

  • concn of component in MP

cont. Chromatography
  • Classification based on method used in separation
  • Physical system - column
  • Phase - liquid or gas
  • Chemistry - adsorption, filtration, ion exchange,
  • Single step (batch)
  • Multi-step (column)

3.1 Ion Exchange Chromatography
  • separates molecules based on their net charge
    (pgs62, 63)
  • stationary phase -vely or vely charged
    functional groups covalently bound to a
  • Cation (acidic) exchanger SP -ve functnl grp,
    binds mobile ve ions
  • Anion (basic) exchanger SP ve functnl grp
    binds mobile -ve ions
  • Matrix--C- A X lt--gt
    Matrix--C- X A
  • ion exchange resin counterion sample ion
  • Molecules of opposite charge interacts with the
    SP neutral ions ions of same charge are eluted
  • Molecules with large charge will interact
    strongly small charge interact moderately

3.2 Gel Filtration Chromatography
  • Gel permeation or Size exclusion chromatography
    (pgs 64, 65)
  • Separates molecules based on molecular size
  • SP polymers of organic cpds cross-linked to
    create a 3-D porous matrix
  • Size of pores in gel determined by degree of
  • Large molecules cannot enter (excluded) the gel
  • -gt first to elute
  • Small molecules permeate pores -gt last to elute
  • Elute molecules in decreasing size

3.3 Electrophoresis
  • Proteins are charged at a pH other than their pI
  • gt exist with net -ve or ve charge in solution
  • Proteins move thru a solution under an electric
  • cations travel toward cathode
  • anions travel toward anode
  • gt Separate proteins based on electrophoretic
  • Solid matrix to support molecules during
  • gt GE

cont. Electrophoresis
  • Buffers used (pg69)
  • Non-dissociating buffer separates proteins by
    charge, molecular wt size - prots retain 3-D
  • Dissociating buffer (SDS) separates proteins by
    molr wt size (not charge) gt can estimate molr
    wt of unknown when compared with prots of known
    mol wt
  • Continuous system uses the same buffer ions in
    sample, gel electrophoresis
  • Discontinuous buffer system uses difft buffer
    ions for the gel electrophoresis

4. Protein Purification
  • Preparative Purification Techniques (physical
    chemical) (pg70)
  • isolate proteins in relatively large amounts
  • purify protein
  • evaluate purity (using analytical techns) at each
    step eg. enzyme activity
  • estimate total amount of protein by standard
    protein assay

cont. Protein Purification
  • Measurement of Enzyme Activity (pg71)
  • A B --gt C D
  • gt C absorbs light at nm but not Ab, B D
  • gt measure production of C at nm
  • a buffered rxn mixt containg substrate(s) a
    known volume
  • measure (spectrophotometrically) the production
    of a product
  • calculate the rate of change
  • enzyme activity in International Units (I.U.) -
    amt of enzyme wh catalyses conversion of 1umol
    substrate/min under defined rxn conditions

cont. Protein Purification
  • Specific Activity (SA) (pg72)
  • by assayg for enzymic activity absolute prot
  • SA (IUmg-1) enz activity per ml sample(IUml-1)
  • total prot concn
  • Purification Factor
  • proportion of the total prot wh is made up of the
    prot of interest
  • Purification specific activity of sample
    (IU per mg)
  • specific actvty of
    startg material (IU per mg)
  • Yield Total units of enz actvty in sample
    x 100
  • Total units of enz actvty in
    startg material

cont. Protein Purification
  • Factors Causing Loss of Enzyme Activity (pg73)
  • Physical loss (eg. not all precipitated)
  • Inactivation or denaturation
  • frothing of solution by vigorous agitation
  • inappropriate pH, temp.
  • heavy metal ions wh inhibit
  • breakdown of enz
  • Analysis of purity (pg74)
  • indirectly, by determining specific activity
  • directly by separating prots

Part 3. Enzyme Activity Kinetics
  • Enzyme assays
  • estimate amount of active enz present in cell
  • monitor purification of enzymes
  • provide info on catalytic mechanisms
    physiological role of enz
  • Enzymes (pg98, 99)
  • catalysts to a rxn gt affect only rxn rate
  • 3-D struc controlled by many factors pH, salts,
  • changes in temp - alter rxn rates, activity,
  • salts cause denaturation (maybe reversible)
  • heavy metals alter struc

cont. Enzymes
  • E S ES ltgt EP E P (pg98)
  • v1 k1ES formtn of enz-substr
  • v2 k2ES reformtn of free enz substr
  • v3 k3ES formtn of product v4 k4EP
    reformtn of enz-prodt complex
  • In steady state equilibrium, v1-v2 v3-v4
  • if all product is removed/does not recombine
    with enz
  • gt k1ES - k2ES k3ES gt (k2 - k3)/k1
  • where (k2 - k3)/k1 is Km (rate constant or
    Michaelis constant
  • measure
    of enz actvty)
  • Enzs fr difft sources (but same function) may
    have difft Km

1. Active Sites
  • The particular part of the enzyme structure which
    specifically binds to a substrate (pg99)
  • Enzyme does not react
  • gt it brings substrate into proper
    alignment/configuration for
  • spontaneous rxn or rxn with another
  • Rxn proceeds by random kinetic action of
    molecules bumping into eah other gt enzyme align
    substrate to facilitate rxn
  • When enzyme is in ideal configuration
  • -gt rxn proceed
  • -gt overall rate of activity dependent on
    substrate concn.
  • Maximum rxn rates at controlled conditions
    optimal pH, salt envmnt, temp, presence of
    cofactors or co-enzymes, sufficient substrate to
    saturate all enzyme

1.1 Michaelis-Menten Plots
  • Plot enz activity /velocity of rxn (y-axis) vs
    substrate concn
  • Velocity of rxn Initial velocity measured at
    each substrate concn.
  • From plot, as concn of substrate increases,
    velocity increases approaches the maximum rate
  • At Vm, all enzyme molecules are complexed with
    substrate gt any additional substrate has no
    effect on rate
  • Value of Vm dependent on enzyme concn substrate

1.2 Michaelis-Menten Equation
  • Michaelis-Menten equation v VmS
  • Km S
  • Km (Michaelis constant) rate constant
  • concn of substrate wh will give exactly 1/2 Vm
  • reacted with an enz with optimum pH, temp
  • However, difficult to obtain Vm accurately fr M-M
  • gt use Lineweaver/Burke

1.3 Lineweaver/Burke equation
  • 1/v Km S Km S
  • Vm S Vm S Vm S
  • 1/v Km 1
  • Vm S Vm
  • gt straight line eqn
  • y 1/v x 1/S slope (m) Km/Vm intercept
    (b) 1/Vm
  • easily obtain Km Vm

2. Specific Activity
  • Enzyme units per mg enzyme protein (pg103)
  • An enzyme unit catalyse transformation of 1umole
    substrate per minute under specific rxn
    conditions (pH, temp substrate concn)
  • Specific activity (SA) relates the enz units to
    the amt of prot
  • To obtain SA, measure amount of prot
    kinetically measure

3. Enzyme Inhibition
  • If a molecule interferes with the binding of enz
    to substrate gt inhibit the activity of the enz
  • Competitive Inhibition inhibitor molecule binds
    to same active site
  • gt No change in Vm, Km changes (req more
    substrate to

  • compete)
  • Non-competitive Inhibition Inhibitor binds to
    another site on enz alters struc of enz or
    blocks access
  • gt Vm change (enz removed fr rxn) but Km no
  • Uncompetitive Inhibition has effects on both
    active site allosteric site
  • gt Vm Km change