Lecture'3'Practice'Problem'Answers'Fall'2015'
BIOSE10
- Lecture 3 Practice Problems - Answers
19'
(a) Ion9exchange'chromatography'separates'proteins'on'the'basis'of'their'charges.''(b)'Size9
exclusion'or'gel'filtration'chromatography'separates'on'the'basis'of'size''(c)'Affinity'
chromatography'separates'proteins'with'specific,'high'affinity'for'some'ligand'(attached'
to'an'inert'support)'from'other'proteins'with'no'such'affinity.''
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29''
(1) Use'Edman'degradation'to'determine'the'sequence'of'each'peptide.''(2)'Create'a'
second'set'of'peptides'by'treatment'of'the'protein'with'a'specific'protease'(e.g.,'
trypsin),'and'determine'the'sequence'of'each'of'these.''(3)''Place'the'peptides'in'order'
by'their'overlaps.'''
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39''
From'the'percentage'recovery'of'activity'(units),'we'calculate'percentage'yield'and'specific'
activity'(units/mg).'
(b)$Step'4,'ion9exchange'chromatography;'this'gives'the'greatest'increase'in'specific'activity'
(an'index'of'purity'and'degree'of'increase'in'purification).'
(c)$Step'3,'pH'precipitation;'two9thirds'of'the'total'activity'from'the'previous'step'was'lost'
here.'
(d)$Yes.'The'specific'activity'did'not'increase'further'after'step'5.'SDS'polyacrylamide'gel'
electrophoresis'is'an'excellent,'standard'way'of'checking'homogeneity'and'purity.'
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Lecture'3'Practice'Problem'Answers'Fall'2015'
BIOSE10
- Lecture 3 Practice Problems - Answers
59'Myoglobin'is'all'three.'The'folded'structure,'the'“globin'fold,”'is'a'motif'found'in'all'
globins.'The'polypeptide'folds'into'a'single'domain,'which'for'this'protein'represents'the'
entire'three9dimensional'structure.'
'
69'The'affinity'of'hemoglobin'for'O2'is'regulated'by'the'binding'of'the'ligands'H+,'CO2,'
and'BPG.'The'binding'of'each'ligand'shifts'the'O29saturation'curve'to'the'right—that'is,'the'
O2'affinity'of'hemoglobin'is'reduced'in'the'presence'of'ligand.'(a)$decreases'the'affinity;'
(b)$increases'the'affinity;'(c)$decreases'the'affinity'
'
7.'These'observations'indicate'that'the'cooperative'behavior—the'sigmoid'O29binding'curve'
and'the'positive'cooperativity'in'ligand'binding—of'hemoglobin'arises'from'interaction'
between'subunits.'
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BIOSE10 – Lecture 3 Practice Problems
Multiple choice questions:
1. Which of the following technique/s do NOT utilize a primary antibody specific for a
protein of interest?
A. 2-D gel electrophoresis
B. affinity chromatography
C. western blot
D. Edman degradation
E. A and D
F. B and C
2. Which of the following statements is false with regards to protein purification
A. In size-exclusion chromatography larger proteins elute first
B. Affinity chromatography involves specific interaction of the protein with a ligand
C. Specific activity may be calculated by dividing the enzyme’s activity by total protein
D. “Salting out” is a means of purifying proteins based on their electronegative/charge properties
E. None of the above
3. Myoglobin and a single chain of hemoglobin have similar _______?
A. prosthetic groups
B. tertiary structures
C. BPG-binding sites
D. A and B
E. A, B and C