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
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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
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!