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Ch. 5 - Protein TechniquesWorksheetSee all chapters
All Chapters
Ch. 1 - Introduction to Biochemistry
Ch. 2 - Water
Ch. 3 - Amino Acids
Ch. 4 - Protein Structure
Ch. 5 - Protein Techniques
Ch. 6 - Enzymes and Enzyme Kinetics
Ch. 7 - Enzyme Inhibition and Regulation
Ch. 8 - Protein Function
Ch. 9 - Carbohydrates
Ch. 10 - Lipids
Ch. 11 - Biological Membranes and Transport
Ch. 12 - Biosignaling
Clutch Review 1: Nucleic Acids, Lipids, & Membranes
Clutch Review 2: Biosignaling, Glycolysis, Gluconeogenesis, & PP-Pathway
Clutch Review 3: Pyruvate & Fatty Acid Oxidation, Citric Acid Cycle, & Glycogen Metabolism
Clutch Review 4: Amino Acid Oxidation, Oxidative Phosphorylation, & Photophosphorylation
Protein Purification
Protein Extraction
Differential Centrifugation
Salting Out
Column Chromatography
Ion-Exchange Chromatography
Anion-Exchange Chromatography
Size Exclusion Chromatography
Affinity Chromatography
Specific Activity
Native Gel Electrophoresis
SDS-PAGE Strategies
Isoelectric Focusing
Diagonal Electrophoresis
Mass Spectrometry
Mass Spectrum
Tandem Mass Spectrometry
Peptide Mass Fingerprinting
Overview of Direct Protein Sequencing
Amino Acid Hydrolysis
Chemical Cleavage of Bonds
Edman Degradation
Edman Degradation Sequenator and Sequencing Data Analysis
Edman Degradation Reaction Efficiency
Ordering Cleaved Fragments
Strategy for Ordering Cleaved Fragments
Indirect Protein Sequencing Via Geneomic Analyses

Concept #1: Trypsin

Practice: Draw out each of the peptide fragments that would be generated if the peptide is treated with trypsin.

Concept #2: Chymotrypsin

Practice: Draw out the resulting peptide fragments that would be generated if the peptide is treated with chymotrypsin.

Concept #3: Other Relevant Peptidases

Practice: What would the resulting peptide fragments be if the following peptide were treated with excess Pepsin? 


Practice: Which is the expected result of chymotrypsin cleavage of the following peptide? 


Practice: You perform multiple tests to derive the amino acid sequence of a purified peptide (see results below). Which of the following peptides listed best represents the sequence of the unknown peptide?

Practice: A) The octapeptide AVGWRVKS was digested with the enzyme trypsin. Would ion exchange or size-exclusion chromatography be most appropriate for separating the fragments? Explain. 

a. Ion-Exchange chromatography.

b. Size-exclusion chromatography.

B) Suppose the same peptide was digested with chymotrypsin. Which would be the optimal separation technique? Explain.

a. Ion-Exchange chromatography.

b. Size-exclusion chromatography.

Practice: A nonapeptide was determined to have the following amino acid composition: (Lys)2, (Gly)2, (Phe)2, His, Thr, Met.  The native peptide was incubated with 1-fluoro-2,4-dinitrobenzene (FDNB) and then hydrolyzed; 2,4-dinitrophenylhistidine was identified by HPLC.  When the native peptide was exposed to cyanogen bromide (CNBr), an octapeptide and free glycine were recovered.  Incubation of the native peptide with trypsin gave a pentapeptide, a tripeptide, and free Lys.  2,4-Dinitrophenyl-histidine was recovered from the pentapeptide, and 2,4-dinitrophenylphenylalanine was recovered from the tripeptide.  Digestion with the enzyme pepsin produced a dipeptide, a tripeptide, and a tetrapeptide.  The tetrapeptide was composed of (Lys)2, Phe, and Gly.  The native sequence was determined to be:

*Recall: Pepsin cleaves N-terminal peptide bond of F, Y, W & L residues.*

Practice: The following reagents are often used in protein chemistry. Match the reagent with the purpose for which it is best suited. Some answers may be used more than once or not at all and more than one reagent may be suitable for a given purpose.