Amino acid electrophoresis PDF

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Briony yorke...

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Amino Acid electrophoresis pH = -log10[H+] pH of water = -log10 (1x10^-7) = 7 Acids and bases = strong and weak – strong = dissociates completely, weak = dissociate partially Ka = acid dissociation constant tells us how strong an acid is – equilibrium towards products - pKa = strong acids = lower pKa and higher Ka than weak pKa = -log10[Ka] = -log10 [H+][A-]/[HA] pH = pKa + log10 [A-]/[HA] = Henderson Hasselbalch – able to use it to calculate pH of a solution and total charge of an amino acid or protein at particular pH pH=pKa = acid/base = 50% Dissociated Henderson- hasselbalch – pH of a buffer = mixture of weak acid and its conjugate base – pH of it resists changes when strong acid or base added Calculating pH of a buffer: - pH = pKa + log10 [A-]/[HA] - E.g: Benzoic acid = pKa of 4.2, what’s pH of a solution made up of 20mM benzoic acid and 500mM benzoate? pH = 4.2 = log10[500]/[20] Amino acids contain weak acid and weak base Zwitterions = molecule with both pos and neg charged functional groups – so net charge is 0 and that is the isoelectric point Calculating amino acid charge at particular pH: - E.g. calculate charge for each group separate at pH 1 for COOH and NH3 pKa COOH = 2.4 pKa NH2 = 9.9

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pH = pKa = log10 [A-]/[HA] 1 = 2.4 = log10[COO-]/[COOH] Log10 [COO-]/[COOH] = 1 – 2.4 = -1.4 ANTILOG of -1.4 = 0.0398 = 0.04 Assume [COOH] = 1 COO-:COOH = 0.04:1 %COO- = 0.04/1.04 X 100 = 3.8% Neg charge is 3.8% of 1 = -0.038

NH2 = 1 = 9.9 + log10[B]/[BH+] Log10[B]/[BH] = 1 – 9.9 = -8.9 Antilog -8.9 = 1.26x10^-9 [NH3+] = 1 so 1.26x10^-9/1.00000000126 x 100 = 1.25 x10^-7 = neutral (%NH2) 1/1.00000000126 x 100 = 99.9999% pos (NH3) – pos charge is 99.9% of 1 =+0.999

So overall charge of Alanine at pH 1: - COO- = -0.038 - NH3+ = +0.999 - = 0.961 Determination of protonation: - pH bigger than pKa – group deprotonated - pH smaller than pKa – group is protonated - pH = pKa – half protonated Isoelectric point: - pH greater than isoelectric point – charge = negative - pH less than isoelectric point – charge = positive - can be determined by pKa – where R group is non ionisable isoelectric point (pI) = pKa1 + pKa2...