Sections
Protein Purification
Protein Extraction
Differential Centrifugation
Salting Out
Dialysis
Column Chromatography
Ion-Exchange Chromatography
Anion-Exchange Chromatography
Size Exclusion Chromatography
Affinity Chromatography
Specific Activity
HPLC
Spectrophotometry
Native Gel Electrophoresis
SDS-PAGE
SDS-PAGE Strategies
Isoelectric Focusing
2D-Electrophoresis
Diagonal Electrophoresis
Mass Spectrometry
Mass Spectrum
Tandem Mass Spectrometry
Peptide Mass Fingerprinting
Overview of Direct Protein Sequencing
Amino Acid Hydrolysis
FDNB
Chemical Cleavage of Bonds
Peptidases
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 #2: FDNB + Amino Acid Hydrolysis

Practice: The peptide Lys-Leu-Leu-Arg-Phe is incubated with FDNB. After exposure to 6M HCl, the solution contains:

Practice: After purifying a protein, you react the protein with 1-fluoro-2,4-dinitrobenzene (FDNB or Sanger’s reagent) then with 6M HCl. You obtain DNP-Arg and DNP-Asp. Which of the following is an appropriate conclusion of the results?

Practice: A tetrapeptide of unknown sequence, containing 2 moles of glycine (Gly) and 1 mole each of leucine (Leu) & alanine (Ala), gives a dinitrophenyl derivative of Gly (DNP-Gly) after treatment with 1-fluoro-2,4-dinitrobenzene (FDNB) and then 6M HCl for complete hydrolysis. Partial hydrolysis of the tetrapeptide gives Gly-Ala and Gly-Gly fragments. What is the amino acid sequence of the original tetrapeptide?

Practice: A 660 mg sample of a protein with quaternary structure (MW=132,000 g/mol) was treated with excess 1-fluoro-2,4-dinitrobenzene (FDNB) under slightly alkaline conditions until the reaction was complete. After FDNB treatment, the peptide bonds of the protein were then completely hydrolyzed by heating it with concentrated 6M HCl. The resulting solution was found to contain 5.5 mg of DNP-Val (shown in the figure below):

2,4-Dinitrophenyl derivatives of the α-amino groups of the other amino acids were not detected. Assuming 100% yield for the reaction of FDNB with the protein, determine the number of subunits in this protein.