Chapter7-PrimarySequence

Chapter7-PrimarySequence - Chemistry 233 Fundamentals of...

Info iconThis preview shows pages 1–12. Sign up to view the full content.

View Full Document Right Arrow Icon
Chemistry 233 Fundamentals of Biochemistry Primary Sequence Determination
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Covalent Structures of Proteins Sequence determination techniques: Composition (how many Asp, Ala, etc) N-terminal and C-terminal reagents cleavage of peptides (smaller fragments are easier to sequence) sequencing: Edman method or mass spectrometry
Background image of page 2
We are here
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Proteins come in all sorts of sizes and shapes What methods can we use on all proteins for sequence determination? Primary structure (sequence) of bovine insulin
Background image of page 4
Composition of a Protein Gives pretty much all the amino acids in the protein, but no sequence information! The steps in the procedure: i)hydrolyze all amide bondswith 6 N HClat 100-110 o C for 12-36 hrs in sealed tube ii) run the mixture on an ion exchange column (usually cation exchange at low pH) or reverse-phase (C18-hydrophobic) HPLC (high performance liquid chromatography)
Background image of page 5

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
amino acids are derivatized for visualization integrate peaks to get relative numbers of each amino acid separation of an amino acid mixture on HPLC.
Background image of page 6
Cautions: all glutamine and asparagines residues are converted to the acids, glutamate and aspartate (why?). How could we be able to tell how many asparagines/glutamines are present? serine, threonine and tyrosine are degraded under the hydrolysis conditions; acid conditions also degrade Trp. Must do a time course hydrolysis (we will assume that we CAN measure Ser, Thr, Tyr and Trp in this manner)
Background image of page 7

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Acid hydrolysis time course
Background image of page 8
Cautions, continued Amino acid analysis only gives the empirical composition need the MW of peptide (or protein) to determine the real composition. Or add known amount of standards to the experiment to be able to quantitate the amino acids exactly by integrating the peaks.
Background image of page 9

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Example: both the peptides Asp-Ser-Gly and (Asp-Ser-Gly) n will give the same composition, but clearly are different peptides Solution:the MW of the tripeptideis 259. Suppose the MW of your sample is 1036; 1036/259 = 4. So the composition must be 4 Asp, 4 Ser, 4 Gly the MW of the sample does not need to be an integral multiple of the empirical MW
Background image of page 10
For example: composition: 7 Gly + 10 Leu + 13 Asp MW =
Background image of page 11

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Image of page 12
This is the end of the preview. Sign up to access the rest of the document.

This note was uploaded on 03/12/2009 for the course CHEM 233 taught by Professor Daub during the Winter '09 term at Waterloo.

Page1 / 52

Chapter7-PrimarySequence - Chemistry 233 Fundamentals of...

This preview shows document pages 1 - 12. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online