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pH Buffers Amino Acids

VIEWS: 36 PAGES: 40

  • pg 1
									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

 => contain weak acidic & basic groups
 => change in pH --> change in ionisation state of the groups


 Solution? Use buffers to resist changes in pH (pg.1)
Objectives


   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 <--> H+ + A-

    In aqueous solution, hydrogen ion   exists as hydronium ion


            HA + H2O <--> H3O+ + A-
    (conjugate acid)         (conjugate base)
             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 = -log10[H+ ]

   If [H+] = 10-3M => log10 [H+ ] = -3 => pH = -log10 [H+ ] = 3


    Ionisation of Water, Kw (pg3)
         H2O + H2O <--> H3O+ + OH-
   Dissociation constant of a solution
    Kw = [H3O+] [OH-] = 1 x 10-14 M
   For Water: [H3O+] = [OH-] = 1 x 10-7 M => neutral pH
   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 --> H3O+ + A-

        Ka = [H3O+] [A-]
               [HA]

=> greater dissociation => larger Ka =>

   pKa = -log10 Ka
    1.4 pH and pKa


   Henderson-Hasselbalch Equation (pg4):
    pH = pKa + log10([base]/[acid]) = pKa + log10([A-]/[HA])

    (i) RCOOH <=> H+ + RCOO- ---> pH =

    (ii) RNH3+ <=> H+ + RNH2 --> pH =
   For weak acid, pH = pKa when [base] = [acid]
                            => acid is 50% dissociated
2.1 Strong Acid & Base


      HA + H2O --> H3O+ + A-         (pg4)

     Completely dissociates in water => [H3O+ ] = [HA]
      => 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 - log10[HA])
   pH (of weak base) = 7 + 1/2(pKa - log10[base])

    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 --> acidic or basic products
                         => inhibits enz rxn (pg5)
   Buffers ‘mops’ up free H+ or OH- ionic products
   Buffers cannot withstand large amounts of acid or alkalis
   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 <=> CH3COO- + H3O+
                                 + OH- --> 2H2O

    Each OH- ion neutralise one H3O+ ion
    Acetic acid ionise to produce more H3O+ ions to
     neutralise OH- => pH of solution remains relatively
     constant:
    End point is when there is no more acetic acid
     --> 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 - depend’g on the concentration of
    protons (pH) of the solution
                  => AA can be used
4.1 Ionisable forms of Amino Acids

     AA with non ionisable R group can exist as (pg9, 10)
        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 depend’g 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
      => 50% of the carboxyl grps carries a -1 charge
         & 50% carry no charge
(ii) FC of +0.3 for an amino group for a given pH
     => 30% carry +1 charge (RNH3+) & 70% carry no charge



(iii) If at a given pH an AA has 50% carboxyl grps charged -1
      and 30% charged +1 => 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
      neighbour’g 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 pKa
        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 activity,
   These properties are also affected by changes

=> pH influences the charge of amino acids &
     hence also biomolecules --> thus affect’g 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 or
    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 itself
      3o structure - chain aggregates to form a 3-D structure:
      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 bonds
      Prots have different types & proportions of Aas =>
       differ in shape, size, charge, solubility, stability
          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 charges
     => may cause changes in shape, orientation
     => 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 > pI => will bear a net -ve

   protein in a solution wh pH < pI => will bear a net +ve
     2.2 Effect of pH & Ionic Strength on
          Protein Solubility

   Adjust pH or ionic strength -> alter distribution of +ve & -ve
    charges on the molecule -> affect protein’s solubiliy (affinity
    with water) (pg59)

   Isoelectric point of a protein => 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 => increase solubility of protein but
       very high salt concns => 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
                           - immobilised
   Mobile phase (MP) - liquid or gas wh flows over or through
                          the SP
   Elution: movement of the MP over or through the SP
   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, affinity

   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 water-insoluble
    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+ <--> 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 cross-linking
   Large molecules cannot enter (excluded) the gel matrix
    -> first to elute
   Small molecules permeate pores -> last to elute
   Elute molecules in decreasing size
     3.3 Electrophoresis


   Proteins are charged at a pH other than their pI
    => exist with net -ve or +ve charge in solution (pg68)
   Proteins move thru’ a solution under an electric field:
    cations travel toward cathode
    anions travel toward anode
    => Separate proteins based on electrophoretic ability
   Solid matrix to support molecules during separation
    => GE
             cont. Electrophoresis

   Buffers used (pg69):
      Non-dissociating buffer separates proteins by charge,
       molecular wt & size - prots retain 3-D struc

       Dissociating buffer (SDS) separates proteins by molr wt &
        size (not charge) => 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 --> C + D
    => C absorbs light at nm but not Ab, B & D
    => measure production of C at nm
      a buffered rxn mixt contain’g 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 assay’g for enzymic activity & absolute prot
      SA (IUmg-1) = enz activity per ml sample(IUml-1)
                        total prot concn (mgml-1)

   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 start’g material (IU per mg)

        Yield = Total units of enz actvty in sample          x 100
                Total units of enz actvty in start’g 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 => affect only rxn rate
      3-D struc controlled by many factors: pH, salts,
      changes in temp - alter rxn rates, activity, struc
      salts cause denaturation (maybe reversible)
      heavy metals alter struc
    cont. Enzymes

    E + S == ES <==> EP == E + P          (pg98)

     v1 = k1[E][S] formtn of enz-substr
     v2 = k2[ES]   reformtn of free enz & substr
     v3 = k3[ES]    formtn of product v4 = k4[E][P]   reformtn of
     enz-prodt complex

In steady state equilibrium, v1-v2 = v3-v4
& if all product is removed/does not recombine with enz

=> k1[E][S] - k2[ES] = k3[ES] => (k2 - k3)/k1 = [E][S]/[ES]

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
    => it brings substrate into proper alignment/configuration for
        spontaneous rxn or rxn with another substance
   Rxn proceeds by random kinetic action of molecules bumping
    into eah other => enzyme align substrate to facilitate rxn
   When enzyme is in ideal configuration
    -> rxn proceed
    -> 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 => any additional substrate has no effect on
    rate
   Value of Vm dependent on enzyme concn & substrate
    concn
1.2 Michaelis-Menten Equation

   Michaelis-Menten equation: v = Vm[S]        (pg102)
                                  Km + [S]

   Km (Michaelis constant) = rate constant
    = concn of substrate wh will give exactly 1/2 Vm when
      reacted with an enz with optimum pH, temp

   However, difficult to obtain Vm accurately fr M-M plots

    => 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

=> 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
    => inhibit the activity of the enz (pg104)
   Competitive Inhibition: inhibitor molecule binds to same
    active site
    => 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
    => Vm change (enz removed fr rxn) but Km no change
   Uncompetitive Inhibition: has effects on both active site
    & allosteric site
    => Vm & Km change

								
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