On the Primary Structure of Human Plasminogen and Plasmin, Cyanogen Bromide Fragments

Abstract

Human plasminogen is a single chain molecule with a molecular weight of 93,000. This protein contains 8 methionine residues per mole. Cleavage with cyanogen bromide and subsequent reduction and S-carboxymethylation resulted in about 15 fragments ranging in size from 11 residues to about 310 residues. Most of these fragments have been purified by combinations of gel filtration and ion exchange chromatography. Characterization by molecular weight determination, amino acid composition, carbohydrate analysis and direct NH2-terminal amino acid sequence determination were thereafter performed. Some of the fragments were found to be the result of incompletely cleaved methionyl bonds which in a very convenient way allowed us to elucidate almost the complete organization of the cyanogen bromide fragments. — Plasmin is a two-chain molecule with a molecular weight of 85,000 formed from plasminogen by activation with urokinase involving proteolytic cleavage in two steps. The first step is release of the NH2-terminal 68 residues and the second step cleavage of an arginyl-valyl bond at about residue number 600 during formation of the two-chain structure. The cyanogen bromide fragment which contains that arginyl-valyl bond and thus forms the linkage between the heavy (A) and light (B) chains, consists of 117 amino acid residues. Its primary structure is almost completely determined. The NH2-terminal 93 residues form the carboxyterminal part of the heavy (A) chain and the carboxyterminal 24 residues form the NH2-terminal part of light chain. Furthermore this fragment and another fragment, which forms the major part of the light (B) chain, show a microheterogeneity which is very similar to that found in the intact plasminogen molecule. Neither of these two fragments seems to contain any sialic acid, which instead seems to be concentrated together with most of the hexoses to another heavy (A) chain fragment, which is very difficult to purify.