Discussion Problems
BT 3016
Protein Structure
• How do the amino acids differ from one
another structurally?
• What properties differ?
• What are the kinds of secondary
structure? Describe them.
• What is quaternary structure?
• What causes proteins to fold?
Structure - continued
• What is a Ramachandran diagram?
• What are the four classifications of
protein folds?
• How can you identify a transmembrane
protein?
pH, pKa, HH equation
• What is the charge on an amino acid in
a solution that is 0.1 unit below its pI?
• How do you make a buffer of a
particular pH and concentration?
50mM glycine buffer, pH 8.8
• What is its ionic strength?
Solubility
• Describe three ways to cause a protein
to precipitate?
• Describe three ways to cause an
insoluble protein to dissolve?
Separations
• What techniques separate according to
the size of a protein?
• What techniques separate according to
charge on a protein?
• What techniques separate based on a
combination of both charge and size?
Separation by Size
• What fundamental difference is there
between determination of molecular
weight by SDS PAGE and by Size
Exclusion chromatography?
Which will separate?
Mr
(subunit) subunits
pI
Charge
@ pH 8
A
14,200
Monomer
9.5
+14
B
13,900
monomer
5.6
-4
C
67,000
monomer
5.2
-8
D
66,950
dimer
4.9
-20
By the following techniques?
Technique
gel filtration
chromatography
ion exchange chromatog. on
DEAE cellulose, pH 8.1
ion exchange chromatog. on
CM cellulose, pH 6.2
Native Polyacrylamide gel
electrophoresis
SDS Polyacrylamide gel
electrophoresis
Isoelectric focusing
A from B
C from D
B from D
Activity
•
•
•
•
•
•
What is an activity unit?
How is it different from a rate?
What is total activity?
What is specific activity?
What is purification fold?
What is recovery?
Reaction Order
•
•
•
•
Define the order of the reaction
Define the order in a component
What does pseudo zero order mean?
How could a reaction be pseudo zero order
in a component?
• What does the order of reaction imply about
a mechanism?
Determine the order of the
reaction
• Initial rates
– Change one [reactant]
– Measure initial rate
– See how the rate changes
• Integrated Rate Equation
– Follow the reaction until significant changes in
concentrations occur
– See which integrated rate equation describe
the data
How do you find KM and Vmax
[P] vs. time
Measure initial rates at several values of [S]0
300
[S]0=
250
[P] (mM)
200
0.005
0.01
0.02
0.04
0.08
150
100
50
0
0
10
20
30
time (s)
40
50
Find Initial Rate for each [S]0
[S]= 5mM
60
50
[P] (mM)
40
30
Slope
(convert to mM/min)
20
10
0
0
5
10
15
20
time (s)
25
30
35
40
45
Make table for all [S]0,V0
[S]0
V0
1/[S]0
1/V0
5
83
200
0.012
10
143
100
0.007
20
222
50 0.0045
40
308
25 0.0033
80
380
12.5 0.0026
Plot 1/V0 vs. 1/[S]0
Double Reciprocal Plot
0.014
1/Vo
0.012
0.01
0.008
-1/KM 0.006
0.004
0.002
1/Vmax
0
-50
0
50
100
1/[S]o
150
200
250
Cooperativity?
Vmax
.9Vmax
[S].9Vmax
[S].1Vmax
V0
.1Vmax
[S].1Vmax
[S].9Vmax
[S]0
Reversible Inhibition
[I]
[I]
1/Vo
1/Vo
? competitive
Inhibition
1/So
1/Vmax
?competitive
Inhibition
1/So
-1/KM
[I]
1/Vo
1/Vmax
?competitive
Inhibition
-1/KM
1/So
XI = X (1 + [I]/KI)
Effect of pH?
EH2 + S
+H+
EH- + S
+H+
E-2 + S
EH2S
EH2 + P
+H+
EHS-
EH- + P
+H+
ES-2
E-2 + P
Effect of T?
•Choose a good pH
•Measure V0 over a pH range
•Plot what?
•V0 vs T
•ln V0 vs. 1/T
•ln (V0/T) vs. 1/T