Complement factor B (fB) is purified from normal human serum. Complement factor B is a glycosylated protein composed of a single 93,000 Da polypeptide chain. It is an essential component of the alternative pathway of complement activation and is found in plasma at approximately 200 µg/mL. In the presence of Mg++ factor B binds to C3b and the C3b,B complex can be activated by factor D, a serine protease that circulates as an active trypsin-like serine protease. Cleavage of factor B by factor D causes the release of the Ba fragment (33,000 Da) and leaves the 60,000 Bb fragment bound to C3b. This Bb subunit is a serine protease. C3b,Bb is called a C3 and a C5 convertase because it converts both of these proteins to their active forms by cleaving off the small peptides C3a and C5a, respectively (Morikis, D. and Lambris, J.D. (2005); Morley, B.J. and Walport, M.J. (2000)).

Another role for factor B is in the initiation of the alternative pathway. Continuous conversion of native C3 to a C3b-like form is a result of spontaneous hydrolysis of the thioester in C3. This C3(H2O) binds factor B in a Mg++ stabilized complex. Factor B in the C3(H2O),B complex can be activated by factor D releasing Ba. During alternative pathway initiation, fluid phase C3(H2O),Bb cleaves C3 producing metastable C3b which can attach to carbohydrates on cell surfaces and on plasma proteins. If this C3b attaches to a host cell or protein it is rapidly inactivated by a variety of mechanisms due to the actions of factor H, CR1, MCP, and factor I. C3b that attaches to a foreign target lacking these regulators remains active long enough to bind factor B and form C3b,Bb as described above. This is the cell surface-bound C3/C5 convertase of the alternative pathway of complement. C3b,Bb is an unstable trypsin-like serine protease with a half-life of approximately 90 seconds in the absence of factors that accelerate decay (factor H, DAF, and CR1). The proteolytic site is in the C-terminal domain of the Bb subunit (Morley, B.J. and Walport, M.J. (2000)).

A unique feature of the alternative pathway is the ability of C3b,Bb to amplify itself on the surface of a complement-activating target particle. This enzyme cleaves C3 producing metastable C3b which can attach to the cell near the initial C3b. Each C3b deposited can bind factor B and form another C3/C5 convertase and deposit more C3b in an expanding ring of attached proteins. C3b,Bb with a second C3b nearby becomes a more efficient C5 convertase and it cleaves C5 releasing C5a and depositing C5b-9 complexes in the bilipid layer of the target cell. This amplification mechanism of the alternative pathway can deposit 2,000,000 C3b molecules on a yeast cell or 30,000 C3b on a bacterium 10-15 min after they come in contact with blood. These numbers represent a monolayer of covalently attached opsonins (C3b, iC3b and C3d) which are ligands for phagocytic immune cells. The numbers of C3b and C5b-9 deposited far exceed those produced by the classical or lectin pathway due to the factor B-containing convertase and its ability to amplify itself and spread across the surface of a target.

The major protein of cobra venom is able the bind factor B from many species of animals. The protein, known as cobra venom factor (CVF), once combined with factor B and activated by factor D forms the C3 convertase CVF,Bb which circulates as a stable complex (half-life ~7 hrs) and is able to cleave C3 and C5. This complex is not decayed by any of the decay accelerating factors that shorten the half-life of C3b,Bb to a few seconds in blood. Thus, the cobra can generate an enzyme that releases C3a and tahe advantage of its vasodilatating effect to speed dissemination of the other toxins in the venom. CVF from some species of cobras (naja naja Kaouthia) form enzymes that can also bind C5 with high affinity. The resulting CVF,Bb enzyme can cleave C5 thus releasing the anaphylatoxin C5a (Rawal, N. and Pangburn, M.K. (2001)).

The fragments of factor B (Ba and Bb) have been proposed to elicit several biological responses. The smaller fragment Ba has been reported to have chemotactic activity with neutrophils and macrophages, but this effect is so much lower than that of C5a or even C5adesArg that its effect in vivo may be negligible (Morgan BP, (1990)). Fragment Bb has been reported to stimulate macrophage spreading, to enhance monocyte-mediated cytotoxicity and to promote B lymphocyte proliferation. The larger fragment Bb possesses the proteolytic site, but once Bb is released from C3b it no longer expresses proteolytic activity toward C3 or C5. Reports of low level proteolytic activity towards synthetic substrates have been shown to be due to contaminating thrombin in some Bb preparations. Reported activities toward clotting factors probably have a similar explanation.

Sizes Available: 250 µg/vial

Concentration: 1.0 mg/mL (see Certificate of Analysis for actual concentration)

Form: Frozen liquid

Activity: >90% versus normal human serum standard.

Purity: >95% by SDS-PAGE Buffer: 10 mM Sodium phosphate, 145 mM NaCl, pH 7.2

Molecular weight: 93,000 Da (single chain)

Extinction Coeff.: A280 nm = 1.27 at 1.0 mg/mL

Preservative: None, 0.22 µm filtered

Source: Normal human serum (shown by certified tests to be negative for HBsAg and for antibodies to HCV, HIV-1 and HIV-II).

Precautions: Use normal precautions for handling human blood products.

Molecular weight: 93,000 daltons, single chain protein containing 7.3-8.6% carbohydrate (Rother (1998)). The protein is negatively charged at serum pH and exhibits a heterogeneous pI = 5.6-6.1. Factor B is one of the heat labile proteins of the complement system. It binds divalent metal ions such as Mg++ or Ni++, which stabilize its interaction with C3b and C3b-like molecules. In the electron microscope factor B exhibits a three domain structure one domain of which is Ba. Bb contains two domains one of which is a von Willebrand factor-like A domain that binds to Mg++ and to C3b and the other, the C-terminal domain, contains the active serine protease site. The Ba domain actually contains of three SCR or CCP domains which interact with C3b. Crystal structures for the serine protease domain at 2.1 angstrom resolution (Jing, H. (2000)), the A domain (Milder, F.J. (2007)) and the whole protein (Bhattacharya, A.A. (2004)) at 2.3 A resolution have been published.

This protein is purified from human serum and therefore precautions appropriate for handling any blood-derived product must be used even though the source was shown by certified tests to be negative for HBsAg, HTLV-I/II, STS, and for antibodies to HCV, HIV-1 and HIV-II.

Hazard Code: B

MSDS available upon request.

-70℃ or below. Avoid repeated freeze/thaw.

Factor B is a zymogen of a serine protease. It must be bound to C3b, C3(H2O) or CVF to be activated by the serine protease factor D which cleaves the Arg233-Lys234 bond in factor B. Even in its active form while bound to C3b, the Bb enzyme is a very inefficient protease. It cleaves C3 at about 1/1000 the rate of trypsin. But unlike trypsin it is highly specific for the single bond in C3 or C5. The turnover number for C3 is 107 C3/min/enzyme and cleavage of C5 has a turnover number of 0.3 C5/min/enzyme. The activity toward C5 is slower than some RNA-based enzymes (ribozymes), but it is apparently sufficient to support the biological effects of complement including immune system stimulation by C5a and target killing by C5b-9.

Split products of factor B in plasma are indicative of activation of the alternative pathway in vivo. ELISA kits for measurement of Ba and Bb are commercially available.