The thirteenth type III domain of fibronectin binds heparin almost as well as fibronectin itself and contains a so-called heparin-binding consensus sequence, Arg6-Arg7-Ala8-Arg9 (residues 1697-1700 in plasma fibronectin). Barkalow and Schwarzbauer (Barkalow, F.J., and Schwarzbauer, J.E. (1991) J. Biol. Chem. 266, 7812-7818) showed that mutation of Arg6-Arg7 in domain III-13 of recombinant truncated fibronectins abolished their ability to bind heparin-Sepharose. However, synthetic peptides containing this sequence have negligible affinity for heparin (Ingham, K.C., Brew, S.A., Migliorini, M. M., and Busby, T.F. (1993) Biochemistry 32, 12548-12553). We generated a three-dimensional model of fibronectin type III-13 based on the structure of a homologous domain from tenascin. The model places Arg23, Lys25, and Arg54 parallel to and in close proximity to the Arg6-Arg7-Ala8-Arg9 motif, suggesting that these residues may also contribute to the heparin-binding site. Domain III-13 and six single-site mutants containing Ser in place of each of the above-mentioned basic residues were expressed in Escherichia coli. All of the purified mutant domains melted reversibly with a Tm near that of the wild type indicating that they were correctly folded. When fluorescein-labeled heparin was titrated at physiological ionic strength, the wild type domain increased the anisotropy in a hyperbolic fashion with a Kd of 5-7 microM, close to that of the natural domain obtained by proteolysis of fibronectin. The R54S mutant bound 3-fold weaker and the remaining mutants bound at least 10-fold weaker than wild type. The results point out that the Arg6-Arg7-Ala8-Arg9 consensus sequence by itself has little affinity for heparin under physiological conditions, even when presented in the context of a folded domain. Thus, the heparin-binding site in fibronectin is more complex than previously realized. It is formed by a cluster of 6 positively charged residues that are remote in the sequence but brought together on one side of domain III-13 to form a "cationic cradle" into which the anionic heparin molecule could fit.

Pasteurization of Antithrombin III (AT III) for 10 hours at 60°C is necessary to reduce the risk of transfusion hepatitis. Addition of appropriate stabilizers can largely prevent the loss of antithrombin activity which otherwise occurs during pasteurization. Studies of the mechanism of denaturation and stabilization have been facilitated by the use of 1,8-anilinonaphthalene sulfonate (ANS) which binds weakly to the inhibitor and whose fluorescence undergoes a sigmoidal response to increasing temperature as the protein unfolds. The extent of the increase in ANS fluorescence correlated roughly with the loss of antithrombin activity and with the extent of protein aggregation as determined by high pressure exclusion chromatography. The midpoint, Td, of the thermal denaturation curve increased by 13 and 19°C in the presence of 0.5 M and 1.0 M sodium citrate respectively. Phosphate, sulfate, and EDTA were also strong stabilizers while the chaotropic anions, iodidé and thiocyanate were potent destabilizers. Heparin, at 10 mg/ml, increased Td by 7°, presumbly through a direct binding mechanism. Reducing agents increased ANS fluorescence by an amount similar to that seen with thermally denatured samples, an effect which was inhibited by heparin but not by citrate. Furthermore, incorporation of 14C-iodoacetamide into AT III during thermal titration was coincident with the increase in ANS fluorescence suggesting that disulfide cleavage is the event which triggers the unfolding of the protein. Samples pasteurized for 10 hours at 60°C in the presence of 0.5 M and 1.0 M citrate retained full antithrombin activity but exhibited evidence of minor alterations in the ability to bind heparin.

The major sites of heparin binding by fibronectin are located in fragments of 30 or 40 kDa that contain type III modules 12 through 14 or 15. Various proteolytic or recombinant subfragments and several synthetic peptides derived from this region have been compared with respect to their binding to fluorescein-labeled heparin in solution. Binding was monitored by the change in fluorescence anisotropy at 25 degrees C and pH 7.4 in 0.02 M Tris buffer, alone (TB) or with 0.15M NaCl (TBS). A 23-kDa fragment containing III13 and III14 but lacking III12 had Kd values of 0.3 and 1.8 microM in TB, and TBS, respectively, indistinguishable from the 30-kDa parent. Fragments containing only module III13 bound 2-3-fold weaker than the parent while those containing only III14 bound 6-50-fold weaker depending on the ionic strength. Fragments containing only III12 or III15 failed to bind at all in TBS. A cationic peptide derived from the amino terminus of III13 and containing the Arg-Arg-Ala-Arg consensus sequence, whose integrity was shown by Barkalow and Schwarzbauer [Barkalow, F. J., & Schwarzbauer, J. E. (1991) J. Biol. Chem. 266, 7812-7818] to be critical, failed to bind in TBS but bound weakly in TB. Two additional cationic peptides derived from the middle and C-terminal regions of III14 showed similar behavior. Thus while the major determinant(s) of heparin binding are located in III13, those determinants are only active when part of a properly folded structure. Furthermore, module III13 when isolated had a slightly lower affinity than fragments containing both III13 and III14.(ABSTRACT TRUNCATED AT 250 WORDS)

A better understanding of the structure and function of C1 requires knowledge of the regions (domains) of the subcomponents that are responsible for Ca2+-dependent assembly. Toward this end, C 1s was digested with trypsin in the presence of Ca2+, a treatment that rapidly degraded the B chain, leaving a 56-kDa fragment comprised of a complete A chain disulfide linked to a small (<4-kDa) residual piece

These findings suggest that level of residual moisture of lyophilized fibrinogen with B19 spike correlated with a different resistance of B19 to dry-heat treatment, and that low moisture may stabilize virus against heat.