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Chemistry 3B Lecture 28

Chemistry 3B Lecture 28 - Chemistry 3B Lecture 28 Thursday...

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Chemistry 3B Lecture 28 Thursday May 7 th 2009 N-terminal analysis and C-terminal analysis: this is another way to do these kinds of analyses is called an Edman Degredation. It is different because the N/C terminal analyses both destroy the sample completely. You always need to figure out a primary sequence and break it up into several fractions so that you don’t blow it up all at once. This method does allow you to take a small peptide and figure out it’s sequence; it’s an iterative process and has been automated. If you have something on the order of 10-20 amino acids, it makes use of a peptide and the reagent shown. It is a cousin of CO2 because it has a similar structure. It has sulfur and a phenyl group on each side. It reacts with the N terminus as a nucleophile through an addition reaction and then you end up protonating the negative charge on the nitrogen. we have labeled the N terminus but the point of the exercise : if you treat this with mild acid, it will rearrange to the Thihydantoin. We’ve literally chopped off the N terminal group and made it a discrete species but the rest of our peptide is intact. With ansilation or stanglers reagent we label the N terminus and hit it with 6M HCl and 24 hours 100 degrees C and the whole thing falls apart. You don’t know what the N terminus or rest of the sequence was. In this case though, we chopped off the N terminus but the rest of the peptide is intact. It is carboxylic acid chemistry mechanism wise. It becomes a thiydanotin even though it goes through a cyclic intermediate. The Sulfur is part of the ring and is in the ring itself. Kinetic to thermodynamic product is witnessed. You can’t do this for 1000 amino acids in a row because each of these is a reaction and reactions don’t typically go to 100% yield especially if you are reacting thousands of molecules. There is 5% of something in this reaction left behind like unreacted starting materials or impurities and if you have an interative sequence like we are doing, the impurities start to build up after steps 15, 20 etc… which is the problem with this method. Other methods for sequencing: if you have a large protein, it will be hard to sequence using techniques like this one. People will try to take the larger peptide and break it up into smaller pieces and then put it back
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together. There are enzymes that do this in selective fashions and there is a chemical reagent that will selectively cleave that one particular amino acid residue.
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