Announcements. Chapter 1 post lab write-ups are due at the end of your Chapter 2 lab. Complete your Chapter 2 pre-lab beforehand

Transcription

1 Announcements Chapter 2 labs begin Tuesday (9/16) Chapter 1 post lab write-ups are due at the end of your Chapter 2 lab Complete your Chapter 2 pre-lab beforehand Quiz at the end of discussion today (no longer take home)

2 Chapter 2: Buffers and Titrations Procedures Calibrate meter and measure ph of different solutions Titrate histidine solution and deionized water to obtain histidine titration curve Perform a ph stat experiment (hydrolyze BSA with trypsin) to calculate the number of Lys and Arg residues in BSA

3 ph Meter Glass- electrode sensi2ve to hydrogen ions Electrode somewhat sensi2ve to other alkali metals Complete system contains: Electrometer 5 Reference Electrode 6 Solu2on to be measured 1,4 Glass Electrode 2,3

4 Titra2on Curves & Key Points Weak Acid = Ace2c Acid Strong Acid = Hydrochloric Acid Equivalence Point Point at which reac2on is neutralized Inflec2on point in 2tra2on curve Weak acids are good buffers Table of pk a values Lab Manual p. 36

5 Buffered Titra2on Curve Empirically, H- H equa2on useful for buffering range Buffers most effec2ve near pk a ph = pk a when [A - ] = [HA]

6 Buffering Capacity Ability of buffer to resist changes in ph with addi2on of acid or base Highest buffering capacity obtained when [A - ] = [HA]

7 Procedure: Titra2on Make His Buffer Star2ng ph? Titra2on of a tripro2c acid Titrate Acid Group of His Titrate the Two Basic Groups of His Titrate imidazole Titrate amino group Controls Titrate Water with Acid Titrate Water with Base Histidine Subtract Water Values from His Values to Get Corrected His Curve

8 Procedure: Titra2on Make His Buffer! 0.4 M His- HCl = 0.4 M HA Deprotonated His! [A - ] = [H + ] - log[h + ] = -log[k a ] + log[h + ] - log [HA] 2(-log[H + ]) = -log[k a ] - log [HA] 2pH = pk a - log[ha] Subs2tu2ng pk a2 of His = 6.04 ph = ( log [0.4])/2 = 3.22 Problem #9 in Lab Manual

9 Make His Buffer Procedure: Titra2on Star2ng ph = 3.22 (calculated) Four Titra2ons Titrate Acid Group of His Titrate the Two Basic Groups of His Titrate Water with Acid Titrate Water with Base Histidine Subtract Water Values from His Values to Get Corrected His Curve

10 Diges2on of BSA with Trypsin Proteoly2c Cleavage of Proteins Trypsin Cleaves C- terminal of (+) charged side chains

11 Trypsin

12 Procedure Denature BSA at C un2l cloudy Digest BSA with Trypsin Titrate during reac2on to maintain ph value 8.5 Do not overshoot +tra+on! Record volume KOH added and the 2me elapsed Calculate the Number of Pep2de Bonds Cleaved When Reac2on is Complete Calculate mmols KOH added at endpoint Calculate number of Arg + Lys per molecule BSA

13 Rela2ng the Titra2on to Arg + Lys Residues How much H + is actually produced? Since ph is only slightly greater than the pk a of N- terminus Not every amino group will gain a proton

14 Rela2ng the Titra2on to Arg + Lys Residues Problem 10, p. 43: What is ra2o of [A - ]/[HA] for the protona2on of an amine with a pk a = 8.2, at ph 8.5? 8.5 = log [R-NH 2 ]/[R-NH 3 ] = log [A - ]/[HA] ph of reaction Amino group pk a 0.3 = log [A - ]/[HA] [A - ]/[HA] = = 2/1 2/3 depronated [A - ], 1/3 protonated [HA]

15 Rela2ng the Titra2on to Arg + Lys Residues The trypsin diges2on alters the buffer capacity of the solu2on As more amino groups are formed, some accept a proton Other protons are neutralized by KOH 2tra2on Total # of pep+de bonds cleaved = (mmol of KOH added)(3 pep2de bonds cleaved/2 mmol KOH added) Total # of Lys + Arg per molecule of BSA = (# of pep2de bonds cleaved)/(mmol of BSA used) Calculate mmol of BSA using MW (66,000 g/mol)

16 Chapter 2 laboratory checklist At the end of lab, you should have: " ph recordings for tap water, deionized water, 0.01 N HCl, 0.01 N KOH, and 0.01 N NaOH " Titrated histidine solution to ph 1.0 with 0.5 N HCl, and recorded ph readings and volumes. " Titrated a fresh aliquot of histidine solution to ph 12.5 with 0.5 N KOH, and recorded ph readings and volumes " Repeated titration method with deionized water between ph 2 and Recorded ph readings and volumes. " Completed proteolytic hydrolysis with BSA & Trypsin. Recorded volumes and corresponding timepoints. " Cleaned out burets and leave filled with dih 2 O. Properly stored ph meter. Cleaned bench. " TURNED IN YOUR CHAPTER 1 POST-LAB!

17 Questions?