Announcements• Chapter 2 labs begin Wednesday (9/18)

• Get to lab on time with tool-kits checked out, pre-labs submitted, etc

• You should know who your partner is, meet with them ahead of time!

• Chapter 2 pre-lab write-ups are due at the beginning and end of lab

• Chapter 1 post-lab write-ups are also due at the beginning of lab

• Quiz at the end of discussion today

• Biochem Staff Office Hours posted on blackboard• Some TFs have phone extensions to get past security doors

Chapter 2: Buffers and Titrations

Procedures• Calibrate pH meter and measure pH of different solutions• Titrate histidine solution and deionized water to obtain a

corrected histidine titration curve• Perform a pH stat experiment (hydrolyze BSA with

trypsin) to calculate the number of Lys and Arg residues in BSA

pH Meter● Measures the difference in electric 

potential between a pH electrode and a reference electrode

● Difference in electric potential related to acidity (pH) of the solution.

● Use standards to set the pH values in meter

● Glass‐electrode sensitive to hydrogen ions

● Electrode somewhat sensitive to other alkali metals (Na+ vs K+)

● Complete system contains:● Electrometer – 5● Reference electrode – 6● Solution to be measured – 1,4● pH glass electrode – 2,3

● Always protect your pH meter!

Titration Curves in Non‐buffered Solutions

● Equivalence Point 

● Point at which reaction is neutralized

● Vertical‐like inflection point in titration curve

● Weak acids are good for buffering solutions

● Strong acids are good for titrating solutions

● Table of pKa values – Lab Manual page 40

Weak Acids: mediocre H+ donors(e.g. acetic acid)[HA] = CH3COOH[A‐] = CH3COO‐

Strong acids:excellent H+ donors (e.g. hydrochloric acid)[HA] = HCl[A‐] = Cl‐

Buffered Titration Curve

● Empirically, Henderson‐Hasselbalch equation is useful for buffering range

● Buffers most effective near pKa

pH = pKa when [A‐] = [HA]

← Henderson‐Hasselbalch equation

Buffering Capacity

● Ability of buffer to resist changes in pH with addition of acid or base

● Highest buffering capacity obtained when [A‐] = [HA] (when pH = pKa)

● At any given pH, the buffer capacity increases proportionally as the concentration of the buffer increases

Evidence that Buffer Capacity is highest at pH near pKa

𝑏𝑢𝑓𝑓𝑒𝑟 𝑐𝑎𝑝𝑎𝑐𝑡𝑖𝑦 2.303𝐻𝐴 𝐴

𝐻𝐴 𝐴 2.303𝐻𝐴 1 𝐻𝐴

𝐻𝐴 1 𝐻𝐴

2.303𝐻𝐴 𝐻𝐴 2

1

Procedure A: Calibration and Measurements

● Calibrate your pH meters● YouTube video uploaded tonight!● Immediately approach your TF is you encounter problems (don’t waste precious lab time)

● Measure the following solutions● Tap water● Deionized water (take measurement in first 10 seconds, pH fluctuates, why?)

● 0.01 N HCl● 0.01 N KOH● 0.01 N NaOH

● Pay attention to all the solutions and concentrations throughout the lab

Procedure B: Titration● Make His Buffer

● Starting pH?

● Titration of a triprotic acid● Titrate Acid Group of His (1.82)

● Titrate the Two Basic Groups of His● Titrate imidazole (6.00)

● Titrate amino group (9.17)

● Water corrections● Titrate Water with Acid

● Titrate Water with Base

● Subtract water titration values from histidine titration values to obtain corrected histidine curve

Histidine

Procedure B: Titration● Make His Buffer – 0.4 M His‐HCl = 0.4 M [HA]

● Deprotonated His‐HCl = [A‐] = [H+]

‐log[H+] = -log[Ka] + log[H+] - log [HA]

2pH = pKa - log[HA]

pH = (6.04 ‐ log [0.4])/2 = 3.22Problem #9 in Lab Manual

• Substituting pKa of His‐HCl = 6.04

2(-log[H+]) = -log[Ka] - log [HA]

Procedure B: Titration● Make His Buffer

● Starting pH = 3.22(show your work for pre‐lab)

● Four Titrations● Titrate acid group of His

● Titrate the two basic groups of His

● Titrate di‐water with acid (count drops of titrant instead of adding certain volumes of titrant)

● Titrate di‐water with base (count drops of titrant instead of adding certain volumes of titrant)

● Subtract water titration values from histidine titration values to obtain corrected histidine curve in post‐lab write‐up

Histidine

Part C: Digestion of BSA with Trypsin

Proteolytic Hydrolysis (Cleavage) of Proteins

Trypsin (enzyme):

Cleaves c‐terminus of the amino acids lysine or arginine, except when either is followed by proline

Trypsin

Procedure C● Denature BSA at 80‐90 °C until cloudy‐> Use a 50 mL beaker (not 25 mL)

● Digest BSA with Trypsin● Titrate during reaction to maintain pH value 8.5

● Do not overshoot titration! You’ll ruin your trypsin enzyme

● Record 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

● How much H+ is actually produced?

● pH of reaction is only slightly greater than the pKa of N‐terminus

● Not every amino group will gain a proton

● Problem 10, p. 47:  What is ratio of [A‐]/[HA] for the protonation of an amine with a pKa = 8.2, at pH 8.5?

Relating the Titration to Arg + Lys Residues

8.5 = 8.2 + log [R-NH2]/[R-NH3] = 8.2 + log [A-]/[HA]Amino group pKa

pH of reaction

0.3 = log [A-]/[HA]

[A-]/[HA] = 100.3 = 2/1

2/3 depronated [A‐], 1/3 protonated [HA]

But what does this 2:1 ratio actually mean?(pages 44-45 in lab manual)

N’-Gly-Lys-Ala-Arg-Val-Lys-Leu-C’

Nterm-Gly-Lys-COOH

HHN-Val-Lys-COOHHHN-Ala-Arg-COOH

HHN-Leu-Cterm

Nterm-Gly-Lys-COO- H+

HHN-Val-Lys-COO-HHN-Ala-Arg-COO- H+

HHHN-Cterm

> Water molecules are incorporated into peptide hydrolysis

> At pH 8.5, carboxyl is completely deprotonated, and one out of every three amino groups accepts these protons

> You are titrating the other two hydrogen ions ( H+ and H+)

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)

● This 3/2 correction ratio is highly dependent on the starting and ending pH of your experiment. If your pH did not begin exactly at 8.5, then you need to recalculate this ratio for your experiment.

● 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)

Chapter 2 pre-lab checklist

In your data collection sheets, you should have:

5 self-prompts for the pHs of your various solutions

4 tables for the pH titrations (you will record a lot of points!)

A table for time, volume of KOH added, and corresponding pH for proteolysis experiment

- Don’t forget to record the starting pH of your experiment

Chapter 2 laboratory plan

To maximize in-lab efficiency:

One person calibrates the meter and measures pH of solutions; another person prepares the histidine buffer and prep the burets

During histidine titrations, one person controls the buret and calls out volumes/drops, another person records the data and checks the pH

One person does the histidine titrations, another person prepares and denatures the BSA solution

Work together on the BSA proteolysis experiment (~35 min)

A few lab notes moving forwardAt 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 12-13. Recorded pH readings and volumes.

Completed proteolytic hydrolysis with BSA & Trypsin. Recorded volumes and corresponding timepoints.

Cleaned out burets and leave filled with diH2O. Properly stored pH meter. Cleaned bench.

Lab notes moving forwardMeet with your lab partners ahead of time to be better organized in lab

No need to rinse tubes or plastic tip/liquid waste buckets with extra water. Washing/rinsing the plastic soup cups at the sink is okay if you dumped as much as possible in the appropriate waste! (Trace amounts is okay down the drain!)

Be conscious of all the reagents, and put them back where you found them!