Studies on the Active Center of Human Plasmin

Abstract

Abstract A new method is described for the isolation of the reduced and S-carboxymethylated light chain derivative of human plasmin. Complete reduction and carboxymethylation of urokinase-activated human plasmin followed by dialysis against 0.002 m NH4HCO3 results in the formation of a precipitate which was shown to be the S-carboxymethylated light chain derivative of human plasmin by gel filtration through Sephadex G-200, carboxyl-terminal amino acid analysis, and diisopropyl phosphorofluoridate-32P and l-1-chloro-3-tosylamido-7-amino-2-heptanone-14C incorporation. A complete amino acid composition of the S-carboxymethylated light chain derivative is reported. Reaction of human plasmin with l-1-chloro-3-tosylamido-7-amino-2-heptanone-14C resulted in the incorporation of 0.96 ± 0.13 mole of l-1-chloro-3-tosylamido-7-amino-2-heptanone-14C per mole of plasmin. Amino acid analysis showed the loss of a single histidine residue and analysis following performic acid oxidation yielded 0.74 residue of 3-carboxymethylhistidine, thus confirming the involvement of a histidine residue. A similar analysis of the S-carboxymethylated light and heavy chain preparations of l-1-chloro-3-tosylamido-7-amino-2-heptanone-14C plasmin showed that the single histidine residue in question was located on the light chain of plasmin. The light chain derivative prepared in this manner from diisopropyl phosphorofluoridate-32P-treated human plasmin contains 1 mole of diisopropylphosphoryl-32P per mole of light chain. A peptide map of the tryptic digest of the diisopropyl phosphorofluoridate-32P-treated S-carboxymethylated light chain gave a single radioactive peptide containing histidine but devoid of both tyrosine and tryptophan. The isolated peptide had the composition Lys1, His1, Cys3, Asp2, Thr2, Ser3, diisopropylphosphoryl-32P1, Glu4, Pro1, Gly6, Ala2, Val2, Leu3, Phe1.