University:
Boston University
Course:
MET HI 215 | Special Topics in History
Academic year:

2022
Views:

401

Pages:

17
Author:

Mike Williamson

Buffers and Titrations Purpose: 1) Get to know your pH meter 2) Make a common buffer used in biochemistry and perform titrations of that buffer with acid or base to find the pKa values for the buffer 3) Hydrolyze BSA with trypsin and calculate the number of Lys and Arg residues that BSA contains pH Meter ● ● ● Glass-electrode sensitive to hydrogen ions Electrode somewhat sensitive to other alkali metals Complete system contains: ● Electrometer – 5 ● Reference Electrode – 6 ● Solution to be measured – 1,4 ● Glass Electrode – 2,3 Titration Curves in Non-buffered Solutions Weak Acid = 0.1 M Acetic Acid Strong Acid = 0.1 M Hydrochloric Acid ● Equivalence Point ● ● Point at which reaction is neutralized Inflection point in titration curve ● Strong Acid – pH 7.0 ● Weak Acid – pH 8.8 ● ● Buffered solutions behave as weak acids Table of pKa values – Lab Manual p. 36 pH Changes in Buffered Solutions pKa of Buffer More H+ present More -OH present Begin deprotonating buffer Titration begins with an acidic pH Acidic Add -OH Fully deprotonated buffer pH of the Solution Titration ends at basic pH Basic Buffered Titration Curve Titration of a Buffer ● 8 7 6 5 pH 4 3 2 1 0 0 20 40 60 Quantities of -OH Added 80 100 120 Modeled on HendersonHasselbach equation Buffered Titration Curve Titration of a Buffer 8 ● 7 6 5 ● pH 4 3 Empirically, H-H equation useful for buffering range Buffers most effective near pKa 2 1 0 0 20 40 60 Quantities of -OH Added 80 100 pH = pKa when 120 [A ] = [HA] Buffering Capacity ● ● Ability of buffer to resist changes in pH with addition of acid or base Highest buffering capacity obtained when [A-] = [HA] Procedure: Titration ● Make His Buffer ● ● ● Starting pH? Four Titrations ● Titrate Acid Group of His ● Titrate the Two Basic Groups of His ● Titrate Water with Acid ● Titrate Water with Base Subtract Water Values from His to Get Pure His Curve Procedure: Titration ● Make His Buffer – 0.4 M His-HCl = 0.4 M HA ● Deprotonated His (His0) = [A-] = [H+] -log[H+] = -log[Ka] + log[H+] - log [HA] 2(-log[H+]) = -log[Ka] - log [HA] 2pH = pKa - log[HA] • Substituting pKa2 of His = 6.04 pH = (6.04 - log [0.4])/2 = 3.22 Derivation p. 54 of Lab Manual Procedure: Titration ● Make His Buffer ● ● ● Starting pH = 3.22 Four Titrations ● Titrate Acid Group of His ● Titrate the Two Basic Groups of His ● Titrate Water with Acid ● Titrate Water with Base Subtract Water Values from His to Get Pure His Curve Digestion of BSA with Trypsin Proteolytic Cleavage of Proteins Trypsin Cleaves C-terminal of (+) charged side chains Trypsin Procedure: Determining the Number of Lys and Arg (combined) in BSA ● Denature BSA at 80-90 °C until cloudy ● Digest BSA with Trypsin ● ● Titrate during reaction to maintain pH value 8.5 ● Indicate volume KOH added and the time elapsed Calculate the Number of Peptide Bonds Cleaved When Reaction is Complete ● Calculate mmols KOH added at endpoint ● Calculate number of Arg + Lys per molecule BSA Relating the Titration to Arg + Lys Residues Denatured BSA, Mr = 66,000 g/mol Trypsin Cleavage H+ H+ H+ H+ H+ H+ H+ New N-Termini Add to Buffer Capacity H+ Relating the Titration to Arg + Lys Residues ● Since pH is only slightly greater than the pKa of Nterminus ● Each new N-terminus will buffer the new H+ released from the reaction – ● Not every amino group will gain a proton How much H+ is actually produced? ● Depends on ratio of [A-]/[HA] ● If pH is constant, [A-]/[HA] must remain constant Relating the Titration to Arg + Lys Residues ● What is ratio of [A-]/[HA] for the protonation of an amine with a pKa = 8.2, at pH 8.5? 8.5 = 8.2 + log [R-NH2]/[R-NH3] = 8.2 + log [A-]/[HA] pH of reaction Amino group pKa 0.3 = log [A-]/[HA] [A-]/[HA] = 100.3 = 2/1 2/3 depronated [A-], 1/3 protonated [HA] Relating the Titration to Arg + Lys Residues ● ● ● The trypsin digestion alters the buffer capacity of the solution ● As more amino groups are formed, some accept a proton ● Other protons are neutralized by KOH titration Total # of peptide bonds cleaved = (mmol of KOH added)(3 peptide bonds cleaved/2 mmol KOH added) Total # of Lys + Arg per molecule of BSA = (# of peptide bonds cleaved)/(mmol of BSA used) ● Calculate mmol of BSA using MW (66,000 g/mol)