Search

A peptide corresponding to a 13-residue segment of the human protein semenogelin I has been shown to generate a hydrogel consisting of amyloid-like fibrils. The relative chemical diversity (compared to synthetic de novosequences) with 11 distinct amino acids makes this peptide (P0) an ideal candidate for investigating the role of individual residues in gelation. Herein, the N-terminal residues have been sequentially removed to furnish a series of truncated peptides, P1–P10, ranging from 12 to 3 residues in length. FTIR spectroscopy investigations reveal that P0–P6 forms a β-sheet secondary structure while shorter sequences do not self-assemble. Site-specific isotope labeling of the amide backbone of P0–P2 with the IR-sensitive vibrational probe 13C═O yields FTIR spectra indicative of the initial formation of a kinetic product that slowly transforms into a structurally different thermodynamic product. The effects of the isotopic labels on the IR spectra facilitate the assignment of parallel and antiparallel structures, which are sometimes coexistent. Additional IR studies of three PheCN-labeled P0 sequences are consistent with an H-bonded β-sheet amide core, spanning the 7 central residues. The macromolecular assembly of peptides that form β-sheets was assessed by cryo-TEM, SAXS/WAXS, and rheology. Cryo-TEM images of peptides P1–P6 display μm-long nanofibrils. Peptides P0–P3 generate homogeneous hydrogels composed of colloidally stable nanofibrils, and P4–P6 undergo phase separation due to the accumulation of attractive interfibrillar interactions. Three amino acid residues, Ser39, Phe40, and Gln43, were identified to be of particular interest in the truncated peptide series as the removal of any one of them, as the sequence shortens, leads to a major change in material properties.

In semen, the gel proteins SgI and SgII (semenogelins I and II) are digested by PSA (prostate-specific antigen), resulting in liquefaction and release of motile spermatozoa. Semen contains a high concentration of Zn2+, which is known to inhibit the protease activity of PSA. We characterized the binding of Zn2+ to SgI and SgII and found evidence that these proteins are involved in regulating the activity of PSA. Intact SgI and SgII and synthetic semenogelin peptides were used in the experiments. Binding of Zn2+ was studied by radioligand blotting, titration with a zinc (II) fluorophore chelator and NMR analysis. A chromogenic substrate was used to measure the enzymatic activity of PSA. SgI and SgII bound Zn2+ with a stoichiometry of at least 10 mol (mol of protein)−1 and with an average dissociation constant of approx. 5 μM per site. Moreover, Zn2+-inhibited PSA was activated by exposure to SgI or SgII. Since both proteins have high affinity for Zn2+ and are the dominating proteins in semen, they probably represent the major Zn2+ binders in semen, one function of which may be to regulate the activity of PSA. The system is self-regulating, and PSA is maintained in an active state by its substrate.

The catabolism of purified radiolabelled antithrombin III (AT III) concentrate was studied in two normals and two patients with congenital AT III deficiency both alone and combined with warfarin. The radiolabelling with iodine mono-chloride did not change the quality of the concentrate. The half-life varied between 3.4 and 4.8 days. No difference between normals and patients with congenital deficiency in non-acute stage could be observed in the catabolic parameters; nor was there any influence with warfarin.

Lupus anticoagulants are spontaneously occurring antibodies with specificity for negatively charged phospholipids. The plasma of a patient with such a polyclonal antibody of IgM type demonstrated low levels of factor VIII coagulant activity (VIII:C) and factors IX, XI and XII when analyzed by biologic clotting assays, whereas in immunochemical assays, normal levels of VIII coagulant antigen and factor IX were obtained. After immunoadsorption of patient plasma with anti-IgM Sepharose, normal biologic activities were demonstrated in clotting assays for VIII:C, factors IX, XI, and XII. The addition of the patient's isolated IgM to normal plasma resulted in grossly abnormal results in these coagulation assays, and a pattern similar to that of the patient's plasma was obtained. The inhibitory effect of the patient's lupus anticoagulant on blood coagulation was demonstrated also in platelet-rich plasma. The results of the clotting assays indicated that the anticoagulant inhibited several of the reactions in the blood coagulation cascade. The availability of purified components made it possible to demonstrate an inhibiting effect on the activation of prothrombin by factor Xa in the presence of isolated platelets, as well as in a system where purified factor V and well defined phospholipid vesicles were substituted for the platelets.