Electrophoresis 1986. 7.379-386 Irnmunoblotting analysis zyxw of complement factor 3 379 Jsrgen Folkersen' B~rge Teisner' Gosta Eggertsed Robert B. Sim3 'Institute of Medical Microbiology, Odense University *Department of Biochemistry, Uppsala University 3MRC Immunochemistry Unit, Department of Biochemistry, Oxford University Immunoblottinganalysis of the peptide chain structure of the physiological breakdown products of the third component of human complement Immunoblotting after sodium dodecyl sulfate polyacrylamide gel electrophoresis of the purified third component of human complement (C3) and its fragments and of un- purified C3 and its degradation fragments in serum or plasma has been examined. The pattern of C3 degradation observed corresponded well with previous observa- tions made with purified proteins. The immunoblotting technique demonstrated a high resolution, and proved to be capable of providing afull analysisofmixtures of C3 fragments in serum with the use of chain-fragment-specific antibody preparations. Additional heterogeneity in C3 and its breakdown products were observed, and covalent adducts between activated C3 and other proteins were detected directly in serum and plasma without any purification of the components. The results further in- dicated that antibodies raised against denatured C3 reacted quantitatively and qualitatively better with the nitrocellulose-bound C 3 than those antibodies raised to native C3, probably reflecting the partially denatured state of molecules after separa- tion by polyacrylamide gel electrophoresis and blotting onto the nitrocellulose. zy 1 Introduction The third component of human complement (C3) constitutes a key molecule in the complement system taking part in both the classical and alternative pathway of complement activa- tion. C3 is synthesized as a singlepolypeptide chain and subse- quently cleaved to form a molecule composed oftwo disulfide- linked peptide chains: the a-chain of 120 kDa and the @-chain of 75 kDa zyxwvutsrq [ 1,21. During complement activation by either the classical (C4b2a) or alternative pathway C3 convertase (C3bBb), the C3 molecule is cleaved in a position close to the N-terminus of the a-chain. A small anaphylatoxin C3a (8 kDa) is then detached from the rest of the molecule, now called C3b (178 kDa) [31, which undergoes a conformational change exposing a labile thiolester capable of forming a covalent bond with nearby acceptor structures possessing amino or hydroxylgroups [4,51.C3bisinactivated by another two cleavages in the alpha-chain, mediated by factor I with factor H as cofactor [6, 71. This gives rise to an N-terminal fragment of 68 kDa and a C-terminal fragment of 43 kDa (formed through an intermediate 46 kDa fragment). The product formed by this cleavage, termed iC3b is still held together by disulfide bridges, and is further degraded zyxwvut in vivo by unidentified proteases to form two separate molecules, C3dg or C3d and C3c. There has been some controversy in the interpretation of the later steps in the breakdown pattern of C3, mainly due to the fact that various artificially introduced enzymes have been employed to study the breakdown of purified C 3 in vitro [ 1, 8-101. This controversy can be resolved to some extent by direct study of complement conversion in vivo. The reason why this has not been attempted in detail earlier, arose from the methodological difficulty in the discrimination and detec- tion of a singlemolecule in a complex biological fluid. One ear- ly trial was published by Spitzer in 1971 [l l ] using immu- nochemical techniques in combination with polyacrylamide zyxwv Correspondence: Dr. J. Folkersen, Institut for Medicinsk Mikrobiologi, OdenseUniversitet, J. B. WinsI~fwsVej 19, DK-5000OdenseC, Denmark Abbreviations: C3, third component of human complement; SDS, sodium dodecyl sulfate; CVF, cobra venon factor zyxwvutsrq 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheirn, 1986 gel electrophoresis. Later methods have employed a study of radiolabelled purified C3b in whole serum by means of auto- radiography of polyacrylamide gels [ 71. Davis et al. [ 121have isolated specific C3 fragments from serum using affinity chromatography with monoclonal antbodies. The present work was undertaken to apply the immunoblotting technique [ 131 and modifications thereof [ 141 to clarify the physiological breakdown of C3 in human serum. 2 Materials and methods 2.1 Plasma and serum samples Whole blood was drawn by venepuncture into either dry glass tubes (for serum) or plastic tubes containing a concentrated solution of EDTA (for plasma) to give a final concentration of 10 mM EDTA. The serum or plasma was separated from the blood cells within 2 h of collection and stored at -20 "C until use. In the study of the molecular pattern of C3 conversion, one single healthy donor was used for the in vitro experiments (lanes 2-5, Fig. 2A, B, C). This pattern was compared with that of a patient with an acute anaphylactoid reaction to blood transfusion 1151 (lane 1, Fig. 2A, B, C). In a separate experi- ment the uncleavedC3 patternof 22 individual healthy donors was compared and found to show no significant variation between individuals. In all other experiments plasma or serum pools from healthy donors were used both for purification of complement factors and for application in the immunoblot- ting analysis. 2.2 zyxwv In zyxwv vitro conversion of C3 in serum samples The degree of C 3 conversion was estimated by crossed im- munoelectrophoresis with an intermediate gel [ 161 using monospecific antibodies to C3c and C3dg(produced at Oden- se University). Complement activation in serum with E. coli was performed by addition of 1 g wet pellet of phenol-killed and washed E. coli per 100 mL of serum, followed by incuba- tion for 1 h at 37 OC. The reaction was stopped by addition of EDTA (10 mM) and transferred to 4 "C. 0173-0835/86/0808-0379 %02.50/0