Announcements• UV/VIS printer issues during Chapter 1 – TFs should

have emailed a PDF of UV/VIS data

• Chapter 2 labs begin Wednesday (9/20)

• 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

Example of a proper figure legend

Absorbance Spectrum

Figure 3. Absorbance spectra of different macromolecules. Absorbance spectra for gelatin (red), BSA (blue), and yeast RNA (green) are ploted between 230 and 360 nm. Between this wavelength range, BSA displays an absorbance peak at 277 nm…

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.

● 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

● Patented by Arnold Beckman in Oct 1934

Titration Curves in Non‐buffered Solutions

● Equivalence Point 

● Point at which reaction is neutralized

● Inflection point in titration curve

● Weak acids are good buffers

● Table of pKa values – Lab Manual p. 38

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)

Procedure A: Calibration and Measurements

● Calibrate your pH meters● TF’s will demo this● 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 fluxuates, 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

● Titrate the Two Basic Groups of His● Titrate imidazole

● Titrate amino group

● Controls● 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 pKa2 of His = 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 water with acid

● Titrate water with base

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

Histidine

Digestion of BSA with Trypsin

Proteolytic Hydrolysis (Cleavage) of Proteins

Trypsin:

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● Digest BSA with Trypsin

● Titrate during reaction to maintain pH value 8.5● Do not overshoot titration!

● 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. 45:  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?(page 42-43 in lab manual)

N’-Gly-Lys-Ala-Arg-Val-Lys-Leu-C’

N’-Gly-Lys-COOH

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

HHN-Leu-C’

N’-Gly-Lys-COO- H+

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

H2HN-Leu-C’

> 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)

● 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 laboratory checklistAt 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.