PubMed · 6169293
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J M Thomas, M E Hodes. 1981-05-15. Improved method for ribonuclease zymogram.. https://doi.org/10.1016/0003-2697(81)90087-7
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We used a perfused clot system to study the degradation of cross-linked fibrin. Multiangle laser light scattering showed that plasmin-mediated cleavage caused the release of noncovalently associated fibrin degradation products (FDPs) with a weight-averaged molar mass (Mw) of approximately 6 x 10(6) g/mol. The Mw of FDPs is dependent on ionic strength, and the Mw observed at 0.15 M NaCl resulted from the self-association of FDPs having Mw of approximately 3.8 x 10(6) g/mol. Complete solubilization required the cleavage of approximately 25% of fragment D/fragment E connections, with 48% alpha-, 62% beta-, and 42% gamma-chains cleaved. These results showed that D-E cleavage cannot be explained by a random mechanism, implying cooperativity. Gel filtration and multiangle laser light scattering showed that FDPs range from 2.5 x 10(5) to 1 x 10(7) g/mol. In addition to fragment E, FDPs are composed of fragments ranging from 2 x 10(5) Da (D-dimer, or DD) to at least 2.3 x 10(6) Da (DX8D). FDP mass distribution is consistent with a model whereby FDPs bind to fibrin with affinities proportional to fragment mass. Root mean square radius analysis showed that small FDPs approximate rigid rods, but this relationship breaks down as FDPs size increases, suggesting that large FDPs possess significant flexibility.
The photosystem II (PSII) reaction center (RC) complex was isolated from spinach and characterized by gel electrophoresis, gel filtration and analytical ultracentrifugation. The purified complex contained the PsbA, PsbD, PsbE, PsbF and PsbI subunits. Gel filtration and analytical ultracentrifugation indicated the presence of a homogeneous complex. The mass of the RC complexes was found to be 107 kDa by analytical ultracentrifugation and 132 kDa by scanning transmission electron microscopy (STEM). The mass obtained showed the isolated complex to exist as a monomer and only one cytochrome b559 (cyt b559) to be associated with the RC complex. Digital images of negatively stained RC complexes were recorded by STEM and analyzed by single-particle averaging. The complex was 9 nm long and 5 nm wide, and exhibited a pronounced quasi-twofold symmetry. This supports the symmetric organization of the PSII complex, with the PsbA and the PsbD proteins in the center and symmetrically arranged PsbB and PsbC proteins at the periphery of the monomeric complex.
Fibrin is degraded by the fibrinolytic system in which a plasminogen activator converts plasminogen to plasmin, a serine protease that cleaves specific bonds in fibrin leading to solubilization. To elucidate further the biophysical processes involved in conversion of insoluble fibers to soluble fragments, fibrin was treated with either plasmin or the combination of plasminogen and plasminogen activator, and morphologic changes were observed using scanning electron microscopy. These changes were correlated with biochemical analysis and with characterization of released, soluble fragments by transmission electron microscopy. Initial changes in the fibrin matrix included creation of many free fiber ends and gaps in the continuity of fibers. With more extensive digestion, free fiber segments associated laterally, resulting in formation of thick fiber bundles. Supernatants of digesting clots, containing soluble derivatives, were negatively contrasted and examined by transmission electron microscopy. Large, complex fragments containing portions of multiple fibers were observed, as were pieces of individual fibers and smaller fragments previously identified. Some large fragments had sharply defined ends, indicating that they had been cleaved perpendicularly to the fiber direction. Other fibers showed splayed ends or a lacy meshwork of surrounding protofibrils. Longer times generated more small fragments whose molecular composition could be inferred from their appearance. These results indicate that fibrinolytic degradation results in larger pieces than previously identified and that plasmin digestion proceeds locally by transverse cutting across fibers rather than by progressive cleavage uniformly around the fiber.