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A simple reproducible procedure has been developed for the large scale isolation of bovine factor X and prothrombin in highly purified states. Both proteins isolated by this method show a single component in four different electrophoretic systems. The specific activity of prothrombin obtained by this procedure is 1300 ± 100 NIH thrombin units per mg of prothrombin, whereas the specific activity of factor X is 90 ± 10 units of activated factor X (factor Xa) per mg of factor X, when assayed by the standard technique (Bachmann, F., Duckert, F., andKoller, F. (1958) Thromb. Diath. Haemorrh. 2, 24). The over-all yields of bovine prothrombin and factor X are 70 mg and 4.5 mg, respectively, per liter of starting plasma. The isolation procedure involves adsorption of factor X and prothrombin on to barium citrate, their elution from the barium citrate complex with EDTA, extensive dialysis against 0.02 mtrisodium citrate and 0.9% sodium chloride, ammonium sulfate fractionation, diisopropylfluorophosphate treatment, and DEAE-cellulose chromatography. DEAE-cellulose chromatography in (sodium) citrate buffer at pH 6.0 effectively and completely separates prothrombin from factor X. Factor X is then further purified by DEAE-Sephadex chromatography. Both proteins are completely stable in 50% glycerol-H2O (v/v) at -20° for at least 18 months.

Bovine prothrombin with tritium incorporated into its sialic acid residues has been prepared by the method of Van Lenten and Ashwell ((1970) J. Biol. Chem.246, 1889–1894). The resulting radiolabeled protein ([sialyl-3H]prothrombin) was electrophoretically indistinguishable from the untreated protein. The thrombin activity generated by factor Xa activation of [sialyl-3H]prothrombin was about 90 % of that obtained from nonlabeled prothrombin.

Guinea pig eosinophil granules are characterized by the presence of a basic protein of low molecular weight which accounts for greater than 50% of granule protein. This protein, termed the major basic protein (MBP), readily aggregates and becomes insoluble, and the formation of aggregates is dependent on the establishment of disulfide bonds. Analysis of concentrated preparations of MBP often revealed a series of bands which were multiples of a monomeric unit with a mol wt of approximately 11,000. Analysis of reduced and alkylated MBP on a 10% agarose column equilibrated with 6 M guanidinium chloride revealed a single polypeptide chain with a mol wt of 10,800. Amino acid analysis of the protein revealed the presence of 13% arginine, consistent with the basic character of the molecule. Four residues of tryptophan, were present, indicating that MBP is not a histone. The MBP did not increase vascular permeability when injected into the skin of guinea pigs, nor did it antagonize the effect of histamine and bradykinin in the skin. MBP also did not contract the isolated guinea pig ileum and when mixed with histamine or bradykinin did not inhibit their activity on the gut. MBP had only weak, if any, antihistaminic activity. MBP possessed weak bactericidal activity when compared to histone and then only with one strain of E. coli. MBP precipitated DNA, neutralized heparin, and activated papain. On a molar basis MBP was more active than cysteine in activating papain. These results do not point to any unique biological activity associated with MBP other than those expected of a protein as basic as it is and one which possesses reactive sulfhydryl groups. Possible functions of eosinophils based on the properties of the MBP are discussed.

Bovine lactoferrin has a molecular weight of 77,100 ± 1500 in dilute aqueous solution as determined by the method of sedimentation equilibrium in the ultracentrifuge. In 6 mguanidine hydrochloride in the presence or absence of mercaptoethanol, bovine lactoferrin has a molecular weight of 72,500 to 77,200 ±1300 as measured by sedimentation equilibrium in the ultracentrifuge or by gel filtration on agarose columns. The molecular weight obtained by gel electrophoresis in the presence of sodium dodecyl sulfate is 76,000 ± 2400. Tryptic peptide maps reveal a number of peptides approximately equal to the content of lysine plus arginine in the molecule. Quantitative end group analysis shows 1 residue of alanine at the amino terminus of the molecule. These data demonstrate that bovine lactoferrin is composed of a single polypeptide chain. Analysis of the carbohydrate content revealed 1 residue of terminal sialic acid, 10 to 11 residues of N-acetyl glucosamine, 5 to 6 residues of galactose, and 15 to 16 residues of mannose per molecule of lactoferrin. Another protein, isolated as a major contaminant in commercial preparations of bovine α-lactalbumin showed identical properties to bovine lactoferrin except that it appeared, based upon molecular weight measurements, to be an aggregating system.

Bovine prothrombin can be labeled with fluorescein isothiocyanate to produce a fluorescent proenzyme which maintains up to 85% of native prothrombin-specific activity. Other proteins labeled similarly were analyzed by sodium dodecyl sulfate gel electrophoresis, and it was found that the chromophores did not affect the molecular weights as determined by the electrophoretic system. Sodium citrate activation of both labeled and unlabeled prothrombin produced molecular weight species consistent with our previously proposed model (Mann, K. G., Heldebrant, C. M., andFass, D. N. (1971) J. Biol. Chem.246, 6106–6114): prothrombin (72,000), Intermediate 1 (65,000), Intermediate 2 (39,000), and Intermediate 3 (25,000). The thrombins are produced from Intermediate 2 and consist of multichain disulfide-linked structures; α-thrombin (33,000 + 6,000), β-thrombin (18,000 + 10,000), and γ-thrombin (14,000 + 10,000 + 4,000). An analog of the physiological coagulation system was constructed using serum and a phospholipid source. Labeled prothrombin when activated in this mixture yielded products which, by virtue of their fluorescence, could be distinguished from the serum proteins. All precursors of thrombin which are produced during citrate activation are also produced during serum activation. The large thrombin, α-thrombin, is composed of 33,000- and 6,000-dalton chains in both activating systems. These results indicate that prothrombin activation in biological systems produces intermediates and products identical with those formed in highly purified synthetic activation systems. Our studies also indicate that the kinetics of the activation process in serum is demonstrably different from that previously described in highly purified systems.

The intermediates formed during the activation of prothrombin in 25% sodium citrate solutions have been isolated and partially characterized. Their properties are in agreement with those predicted by a previously proposed model for prothrombin activation in 25% sodium citrate: prothrombin (72,000 daltons) → intermediate 1 (65,000 daltons) → Intermediate 2 (39,000 daltons) and Intermediate 3 (25,000 daltons); Intermediate 2 (39,000 daltons) → thrombin (α-thrombin, 39,000 daltons) (Mann, K. G., Heldebrant, C. M., andFass, D. N. (1971) J. Biol Chem.246, 6106–6114).