Rapid and sensitive techniques for identification and analysis of 'reactive-centre' mutants of C1-inhibitor proteins contained in type II hereditary angio-oedema plasmas.
Novel procedures for structural analysis of the 'reactive-centre' residues, particularly the P1 residue, of the dysfunctional C1-inhibitor proteins found in the plasmas of type II hereditary angio-oedema (HAE) patients are described. C1-inhibitor is adsorbed directly from plasma on to Sepharose-anti-(C1 inhibitor) beads. The P1 residue of C1 inhibitor is arginine and hence a potential cleavage site for trypsin. Thus trypsin digestion of the immobilized protein, followed by SDS/PAGE of the released fragments, identifies P1 residue mutations. Pseudomonas aeruginosa elastase digestion of the immobilized protein, followed by purification of the released C-terminal peptide (by h.p.l.c.) and N-terminal sequence analysis defines the new P1 residue (or other mutations in the reactive-centre region). The techniques are both rapid and highly sensitive, requiring only 400 microliters of plasma. In addition, they permit accurate assessment of the level of normal (functional) inhibitor in a subclass of type II HAE plasmas, those containing P1-residue mutant proteins.
Aulak
K S
KS
MRC Molecular Immunopathology Unit, MRC Centre, Cambridge, U.K.
Harrison
R S
RS
D000799:Angioedema / Q000097:blood* / Q000145:classification / Q000235:genetics; D003062:Codon / Q000235:genetics; D003174:Complement C1 Inactivator Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006801:Humans; D009154:Mutation; D010446:Peptide Fragments / Q000097:blood / Q000302:isolation & purification; D012685:Sepharose / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D003062:Codon; D003174:Complement C1 Inactivator Proteins; D010446:Peptide Fragments; D006639:Histidine; D012685:Sepharose; D014357:Trypsin; D003545:Cysteine