Structural changes of subunit polypeptides of bovine fibrinogen during proteolysis

Abstract

Native fibrinogen consisting of three pairs of subunit polypeptides, Aα, Bβ and γ, was degraded by various bacterial proteinases, using a weight ratio of substrate to enzyme of 1:2000. After various periods of digestion, each digest was reduced and subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis. The protein band on the gel corresponding to the Aα chain disappeared after proteolytic digestion for 3 min and two new fragments appeared with molecular weights of 46 000 (±- 2000) and 30 000 (± 2000). After this period of digestion, the ability to form α-multimer in a fibrin clot derived from degraded fibrinogen was completely lost. The protein bands of the Bβ and γ chains on the gel appeared unchanged after this period of digestion, and the γ chain was readily converted to the γ-dimer, having a molecular weight of 90 000 (± 2000). After digestion for 6 min, some degradation of the Bβ chain was seen but the γ chain remained essentially intact. On digestion for between 6 and 15 min some degradation of the γ chain occurred, since the intensity of the γ-dimer band formed from degraded fibrinogen clearly decreased. Titration of fibrinogen with alkali during the proteolysis showed that an average of five peptide bonds per 340 000 unit weight of molecule were cleaved within 3 min. The thrombin clotting time of fibrinogen seemed to be prolonged greatly on degradation of the Aα chain and no coagulable protein was produced when degradation of the γ chain was initiated. Similar sequences of degradation of fibrinogen subunits were observed with all of the crystalline proteinases tested, including semi-alkaline protease, alkaline proteases I and II, subtilisin BPN' and thermolysin of bacterial origin. However, the degradation sequences of denatured fibrinogen with the proteinases were essentially different from those of the native molecule. These results indicate that in general the three peptide chains of the native fibrinogen molecule are degraded in the order, A α → B β → γ by bacterial proteinases. The Aα chain is the most susceptible of the chains to all proteinases suggesting its external location on the protein molecule.