SC BIOL 541 - Extract Crystallography (5 pages)

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BIOL 541 1st Edition Lecture 6 Outline of Last Lecture I Characterization of Protein Outline of Current Lecture II Edmund Degradation III Extract Crystallography IV Electron Microscope Current Lecture Biochemistry Lecture 6 Carboxy peptidase enzyme technique Extract crystallography Sequencing from COOH terminus The most common technique is quick and dirty Carboxy peptidase cleaves amino acid from COOH terminus Carboxy peptidase A cleaves all amino acids except basic ones or proline Carboxy peptidase B cleaves lysine and proline Carboxy peptidase C cleaves proline Method The enzyme continues to nibble away on first and then second amino acid Therefore reaction condition has to be chosen in order to continue slowly The sample is freeze dried followed by adding trichloroacetic acid to form precipitate Then the amino acid is added on the analyzer The graph shows that at 15 minutes phenyl alanine is picked up followed by serine coming out thus this process is very messy These notes represent a detailed interpretation of the professor s lecture GradeBuddy is best used as a supplement to your own notes not as a substitute First carboxy peptidase A is added and lysine is cleaved Then carboxy peptidase B is added to cleave 4 5 amino acids Schlack Kemp degradation This is the chemical means of Edmund degradation The reaction used does not allow for standards and hence not used frequently The organic extraction of amino acids following Edmund degradation has increasing loss of product as the peptide gets smaller and smaller as the product is partially extracted and washed out of the vessel Mass spectroscopy This method is used to determine molecular weight and sequence A The peptide bonds are ruptured using helium B Standard mass spectroscopy is done to determine the molecular weight Disadvantage This method can not distinguish identical amino acid molecular weight such as leucine and isoleucine appear the same Small peptides that are 25 amino acids long can be determined but