Enzymatic Methods C-terminal analysis by carboxypeptidase A,B,C,Y: Carboxypeptidase A cleaves all except for P,R,K; Carboxypeptidase B cleaves only R,and K; .Carboxypeptidase Y cleaves all; .Carboxypeptidase C cleaves all; Trypsin-cleaves on the C-side of Lys or Arg; Chymotrypsin-C-side of Phe,Tyr,Trp Clostripain-like trypsin,but attacks Arg more than Lys; Endopeptidase Lys-C cleaves at C-side of Lys; Staphylococcal protease C-side of Glu or Asp in phosphate buffer specific for Glu in acetate or bicarbonate buffer
Enzymatic Methods C-terminal analysis by carboxypeptidase A, B, C, Y: • Carboxypeptidase A cleaves all except for P, R, K; • Carboxypeptidase B cleaves only R, and K; • Carboxypeptidase Y cleaves all; • Carboxypeptidase C cleaves all; Trypsin - cleaves on the C-side of Lys or Arg; Chymotrypsin - C-side of Phe, Tyr, Trp Clostripain - like trypsin, but attacks Arg more than Lys; Endopeptidase Lys-C cleaves at C-side of Lys; Staphylococcal protease C-side of Glu or Asp in phosphate buffer specific for Glu in acetate or bicarbonate buffer
CNBr Cleavage CNBr(cyanogen bromize)acts only on methionine residues CNBr is useful because proteins usually have only a few Met residues HgC CS:一CN H2C H2C CH, Br displacement of Br CH2 CH CH CH H2 Methyl thiocyanate C H3C C-terminal H2C S一CEN Peptide 十， 二0 Homoserine lactone
CNBr Cleavage CNBr(cyanogen bromize) acts only on methionine residues CNBr is useful because proteins usually have only a few Met residues N H C H C C H2 H2C S H3C H N O O R' Br C N N H C H C C H2 H2C S H3C H N O O R' C N displacement of Br N H C H C H2C H2 C S H3C N O C N + + Methyl thiocyanate N H C H C H2C H2 C O O H2O C-terminal Peptide + Homoserine lactone
An example: Trypsin cleavage: A-E-F-S-G-I-T-P-K L-V-G-K Chymotrypsin Cleavage: L-V-G-K-A-E-F S S-G-I-T-P-K Edman degradation:L Correct sequence: L-V-G-K-A-E-F-S-G-I-T-P-K
An example: Trypsin cleavage: A-E-F-S-G-I-T-P-K L-V-G-K Chymotrypsin Cleavage: L-V-G-K-A-E-F S-G-I-T-P-K Edman degradation: L Correct sequence: L-V-G-K-A-E-F-S-G-I-T-P-K
An example: Amino Acid Analysis yielded:Asn,Gin,Leu,Lys,Met, Tyr,Trp Trypsin had no effect; Edman degradation yielded PTH-Tyr; CNBr treatment yielded a tetrapeptide of positive charge and a tripeptide of zero charge at pH 7.0; Brief chymotrypsin(cleaved at Trp)yielded a dipeptide and a tetrapeptide which contains GIn,Leu, Lys,and Met; What is the sequence of the peptide?
An example: Amino Acid Analysis yielded: Asn, Gln, Leu, Lys, Met, Tyr, Trp Trypsin had no effect; Edman degradation yielded PTH-Tyr; CNBr treatment yielded a tetrapeptide of positive charge and a tripeptide of zero charge at pH 7.0; Brief chymotrypsin (cleaved at Trp) yielded a dipeptide and a tetrapeptide which contains Gln, Leu, Lys, and Met; What is the sequence of the peptide?
Cleavage of Disulfide Bonds Oxidation of a disulfide by performic acid: 0 -CH- CHC-0- CH2 SO3 H-C-0-O-H performic Acid SO3 CH2 H-c40-02 -Ht- o Reduction of S-S bridges by sulfhydryl compound: -CHC-0- N-CHe-o. CH2 CH2 SH 2HSCH2CH2OH 2SCH2CH2OH SH CH2 CH2 月-Hc-0- -CHC-O
Cleavage of Disulfide Bonds Oxidation of a disulfide by performic acid: Reduction of S-S bridges by sulfhydryl compound: N H CH C CH2 O O S H N CH C CH2 O O S N H CH C CH2 O O SO3 H N CH C CH2 O O SO3 C O O H O H performic Acid N H C H C C H2 O O S H N C H C C H2 O O S 2HSCH2C H2OH N H C H C C H2 O O SH H N C H C C H2 O O SH + + 2SCH2C H2OH