DETERMINATION and treated with aqueous acid phenylthiohydantoin Steps: (PTH) AA derivative c. PTH-AA identified by HPLC 1. Hydrolyze peptide bond - Up to 50 AA - Acid hydrolysis - For shorter peptides, only step 3 - 6 M HCL at 110 C for 16-72 is done hrs If protein contains 1 cysteine 2. Analyze hydrosylate using residues, disulfide bonds must be chromatographic procedure cleaved by: (amino acid analysis) a. Treatment with performic acid a. Treatment with oxidizes cysteine to 2 phenylisothiocyanate (PITC) cysteic acid residues, OR
b. Reduction by dithiothreitol to phenylcarbamoyl (PTC)- form cystein residues amino acid derivatives treated with iodoacetate b. PTC-AA subjected to HPLC (prevents disulfide bond c. Eluted PTC-AA detected and reformation) concentration is determined by measuring absorbance at If protein is large selective 240nm (peak absorbance) cleaving a. Use proteases or reagents to - timing of peaks used to form shorter peptides identify AA Treatment Cleavage - amount of AA proportional Site to area under peak Trypsin Lys, Arg (C)* Submaxillus Arg (C) protease AMINO ACID SEQUENCING Staphylococcus Asp, Glu (C) aureus V8 Steps protease Asp-N-protease Asp, Glu 1. Determine AA composition 2. Identify N-terminal residue (N)** - Sangers (1-fluoro-2,4- Pepsin Phe, Trp, Tyr dinitrobenzene) (N) - Dabsyl chloride (highly Cyanogen Met (C) fluorescent derivatives) bromide - Dansyl chloride intensely * C-terminal side colored and greater sensitivity) ** N-terminal side These destroy the b. Use Edman degradation polypeptide c. Infer AA sequence by 3. Use Edman degradation procedure locating overlapping - Labels and removes N-terminal sequences residue only; leaves the rest of The nucleotide sequence of the the chain intact gene that codes fsor a protein a. Polypeptide reacts with PITC can also be used to determine PTC-AA AA sequence. However, this cannot determine the disulfide bonds that occur and whether Horton, H.R., Moran, L.A., Ochs, R.S., modification of AA residues Rawn, J.D., Scrimgeour, K.G. 1993. occurred after synthesis. Principles of Biochemistry. USA: Neil Patterson Publishers/Prentice-Hall, Inc.
Lehninger, A.L. Nelsonm, D.L., Cox,
References: M.M. 1993. Principles of Biochemistry. 2nd ed. New York: Worth.