TOPHAT SETUP
• Navigate to www.tophat.com
• In the top right corner, click “sign up”
- Sign-up for a student account
- When prompted to input a “Course Join Code”
input number 740959
• Ensure that “Biochemistry I- Lab” with Professor
Didem Vardar-Ulu listed as instructor
• Click “Yes, that’s right”
• Fill out your personal info (make sure to enter
your Student ID staring with U), agree with the
terms and conditions, and click “Next”
Please note there is TopHat App for many types of mobile
devices (phone, tablets, etc. that work really well. Make sure
to have that app downloaded on your device.
PRELAB DISCUSSION #2
•
•
ANNOUNCEMENTS
CH1 Short Reports due at the beginning of your
next lab (Electronic submission on blackboard
and hardcopy to your TFs
Please look over the entire week of OHs and try
to plan in advance to get help for your
questions. Emails can not substitute a good
discussion in terms of the desired learning gain.
In the fields of observation,
chance favors only the
prepared mind.
Louis Pasteur, University of Lille, Dec 7, 1854
TOP HAT QUESTION 1
PART I- CHAPTER 2 LAB
Buffers and Titrations
Will be completed in three parts:
PART A: Calibrating and using pH meters
PART B: Titration of Histidine
PART C: Monitoring Proteolytic Hydrolysis of BSA by
trypsin via titration
pH Meters
How a pH electrode works
Strong & Weak Electrolytes
substances that can generate ions in solution and
increase its electrical conductivity
Strong electrolytes completely dissociate to form ions in
solution. (Ex. NaCl, K2SO4, HCl, NaOH)
Weak electrolytes have limited tendency to dissociate into ions
in solution. (Ex. Acetic acid –CH3COOH)
The acid dissociation constant of a weak acid (HA) is:
HA
H+ + AKa =
[ H+ ] [ A- ]
----------------[HA]
Henderson-Hasselbalch (HH) Equation
pH = pKa +
log10
[A-]
------[HA]
For any weak acid (HA) in the presence of its conjugate base
(A-), there is a relationship between the pKa, the
concentrations existing at equilibrium, and the solution pH.
pH = pKa
when [A-]=[HA]
TOP HAT QUESTION 2
Part A- Procedure
1. Calibrate the pH meter with two standard
solutions (pH 7 and pH 4).
2. Measure the pH of the following solutions:
a. Tap water
b. De-ionized water
c. 0.01 N HCl
d. 0.01 N KOH
e. 0.01 N NaOH
Note: For this part you are expected to create your own
empty data table for the prelab work.
What are buffers?
• Buffers are solutions that resist changes in pH as acid and base are
added and consist of a weak acid and its conjugate base
• Buffers can only be used reliably within a
pH unit of their pKa The plot of pH versus
base added is flat only near the pKa
Common buffers in biochemistry labs
Table of pKa values on p/38 of the manual
BUFFERING CAPACITY
Ability of buffer to resist changes in pH when
an acid or base is added
Highest buffering capacity is achieved when
[A-]=[HA]
[A-] & [HA] can either be calculated theoretically
using the HH Equation, or can be determined
experimentally from a titration curve.
Part B- Procedure
1. Make 75 mls of 0.4 M Histidine solution.
2. Calculate the expected pH for this solution (see
problem #9 in the manual)
3. Measure the pH of this solution.
4. Transfer 25 mls of this solution to a new beaker, add
25 ml of water and set-up a titration with 0.5 N HCl.
5. Repeat the titration using KOH.
- Why not NaOH?
6. Skip the titrations using water as the starting
material (a sample data set for this titration will be
given to you for data analysis)
7. Plot a His titration curve and a corrected curve.
TITRATION OF HISTIDINE BUFFER
IONIZATION STATES
OF HISTIDINE
To calculate starting pH remember:
Deprotonated His (His0) = [A-] = [H+]
[HA]=0.4 M Why?
-log[H+] = -log[Ka] + log[H+] - log [HA]
Rearrange and solve p/57
Titration Curve for Pure Histidine
STEP 1: Titrate Histidine Buffer
●
●
●
●
Titrate Acid Group of His with KOH
Titrate the Two Basic Groups of His with HCl
Make table of pH versus volume and mmoles
Plot this by connecting dots or fitting
STEP 2: Titrate pure water
(You will NOT be performing this titration in the lab, but will be given a sample
dataset for analysis)
●
Titrate Water with HCl
●
Titrate Water with NaOH
●
Make table of pH versus volume and mmoles
●
Plot this by connecting dots or fitting
STEP 3: Construct the titration curve for pure His
●
●
Subtract Water Values in mmoles from mmoles for His titration at each value
of pH in first table to create a table with values for a pure His curve
Plot this curve
TOP HAT QUESTION 3
Part C: Digestion of BSA with Trypsin
Hydrolysis of Peptide Bond
THESE TWO SPECIES SET THEIR OWN EQUILIBRIA
TOP HAT QUESTION 4
Trypsin
Part C- Procedure
1. Denature BSA at 80-90C until cloudy
2. Digest BSA with trypsin
- Titrate during reaction to maintain pH 8.5
- Be careful not to overshoot the pH.
- Record the amount KOH is added and
time elapsed. (data Table)
3. Calculate the number of peptide bonds
cleaved when reaction is complete.
- Calculate mmols KOH added at endpoint
- Calculate number of Arg+Lys in BSA
WITHOUT DOING ANY
CALCULATIONS:
DO YOU EXPECT THE pH OF YOUR
REACTION SOLUTION TO INCREASE OR
DECREASE AS THE TIME GOES ON? WHY?
See problem #10 p/45
for calculations.
TOPHAT Q5
pKa?
pKa?
pH = pKa + log10
[A-]
------[HA]
Relating the Titration to Arg + Lys
Residues
Denatured BSA, Mr = 66,000 g/mol
Trypsin
Cleavage
H
H
H
H
H
+
+
+
+
+
H
H
H
+
+
+
New N-Termini Add to Buffer Capacity
Relating Titration to the # of Arg/Lys
Residues
CH2 Specific Cautions &Clean-up
• Make sure to have the probe guard piece attached
to your pH probe BEFORE you insert it in your
sample and start stirring.
•
At the end of the lab, clean out burets thoroughly
and leave them filled with water.
• Properly store away the pH meter.
• Clean up your bench
PART II
REFLECTING ON CH1
DIRECT UV-VIS READINGS
Spectroscopic method
Direct UV absorption data cannot tell you
[Protein] without ε
The absorption maxima is determined by the electronic
structure of the functional groups.
The absorption intensity is determined by the #of absorbing
functional groups.
DYE-BINDING (BRADFORD)
Colorimetric Methods
Protein amount in your sample:
• Theoretical (expected)
• Experimental (measured/ determined)
Colorimetric data cannot tell you [Protein] without a standard curve
Standard
Curve
.
A
.
. .
µg/mL Protein
To measure [Protein]
Find A
.
.
.
A
.
. .
µg/mL Protein
Error in determined [Protein]
% Error by which a protein concentration determined
from a standard curve differs from the expected value.
% Error= [(measured value-expected value)/expected value]x100
What do we do with interfering
substances? Can we always get by?
Observing Interference While
Quantifying [Protein]
●
●
To observe and quantify interference you should
●
Run assay without protein but all other components
●
Run assay with protein and other components
This will give you correction values to
compensate for IS
Actual
Actual