Some Characteristics of the Clottable Protein of Limulus Polyphemus Blood Cells

Summary

1. The endotoxin-clottable protein of Limulus blood cell extracts has been studied. The concept of the clottable protein as a true cell protein was confirmed.

2. 35–55% of total extractable protein was removed from the extracts by clotting. Polyacrylamide disc electrophoresis of the extracts showed 6 to 9 protein bands one of which was reduced in intensity by the clotting. Dimethylf ormamide could be used for fractionated precipitation of the proteins.

3. The gel protein was easily soluble in HCl or NaOH and reprecipitated by neutralization. It was also soluble in 0.05 M formate/6.7 M urea pH 4.3 but not in neutral solutions of urea.

4. Light absorption spectra of the gel protein in 0.188 N NaOH showed maxima at 283 mμ and 290 mμ, whereas one maximum, at 276 mμ, was observed in 0.189 N HCl. E_{1 %} ^{1 cm} in 0.188 N NaOH was 10.4 and 11.1 at 283 mμ and 290 mμ, respectively, and 9.0 at 276 mμ in 0.189 N HCl.

5. Data on the total amino acid composition of the gel protein are given. A mean minimal molecular weight of about 20,000 is calculated from these.

6. In starch gel electrophoresis with a discontinuous acid buffer system the gel protein separated into two main zones. Possible relationships between these are discussed in terms of clotting mechanism.

7. The data show that Limulus clottable protein differs markedly in its molecular characteristics from those of mammalian fibrinogens.

• References

• 1 Loeb L. Über die Blutgerinnung bei Wirbellosen. Biochem. Z 24: 478 1910;
• 2 Levin J, Bang F. B. Clottable protein in Limulus: Its localization and kinetics of its coagulation by endotoxin. Thrombos. Diathes. haemorrh. (Stuttg.) 19: 186 1968;
• 3 Loeb L. On the blood lymph cells and inflammatory processes of Limulus . J. med. Res 7: 145 1902;
• 4 Rustum Maluf N. S. The blood of arthropods. Quart. Rev. Biol 14: 149 1939;
• 5 Copley A. L. The clotting of Limulus blood. Fed. Proc 6: 90 1947;
• 6 Levin J, Bang F. B. The role of endotoxin in the extracellular coagulation of Limulus blood. Bull. Johns Hopk. Hosp 115: 265 1964;
• 7 Levin L, Bang F. B. A description of cellular coagulation in the Limulus . Bull. Johns Hopk. Hosp 115: 337 1964;
• 8 Levin J. Blood coagulation and endotoxin in invertebrates. Fed. Proc 26: 1707 1967;
• 9 Dumont J. N, Anderson E, Winner G. Some cytologic characteristics of the hemocytes of Limulus during clotting. J. Morph 119: 181 1966;
• 10 Alsberg C. L, Clark E. D. The blood clot of Limulus polyphemus . J. biol. Chem 5: 523 1908;
• 11 Lopaciuk S, Solum N. O. Bovine platelet proteins. I. Soluble proteins studied by Immunoelectrophoresis and Polyacrylamide disc electrophoresis. Thrombos. Diathes. haemorrh. (Stuttg.) 21: 409 1969;
• 12 Solum N. O, Lopaciuk S. Bovine platelet proteins. II. Purification of platelet fibrinogen. Thrombos. Diathes. haemorrh. (Stuttg.) 21: 419 1969;
• 13 Solum N. O, Lopaciuk S. Bovine platelet proteins. III. Some properties of platelet fibrinogen. Thrombos. Diathes. haemorrh. (Stuttg.) 21: 428 1969;
• 14 Blombäck B, Blombäck M. Purification of human and bovine fibrinogen. Ark. Kemi 10: 415 1956;
• 15 Vogel A. I. A text-book of macro and semimicro qualitative inorganic analysis. Fourth edition. 319 Longmans, Green and Co.; London: 1957
• 16 Abildgaard U. Purification of two progressive antithrombins of human plasma. Scand. J. clin. Lab. Invest 19: 190 1967;
• 17 Miller G. L. Protein determinations for large number of samples. Analyt. Chem 31: 964 1959;
• 18 Lowry O. H, Rosebrough N. J, Farr A. L, Randall R. J. Protein measurement with the Folin phenol reagent. J. biol. Chem 193: 265 1951;
• 19 Łipiński B, Budzyński A. Z, Latałło Z. S, Kowalski E. Isolation and characterization of cold-insoluble fibrinogen complex from bovine plasma. Acta biochim. pol 11: 527 1964;
• 20 Laki K, Gladner J. A. Chemistry and physiology of the fibrinogen-fibrin transition. Physiol. Rev 44: 127 1964;
• 21 Lorand L, Rule N. G, Ong H. H, Furlanetto R, Jacobsen A, Downey J, Oner N, Bruner-Lorand J. Amine specificity in transpeptidation. Inhibition of fibrin cross-linking. Biochemistry 7: 1214 1968;