Localization of a hydrophobic domain in human C5

The fifth component of human complement (C5), under physiological salt conditions, bound firmly to phenyl-Sepharose. This indicated the presence of hydrophobic sites on C5. These sites may play an important role in C5 haemolytic activity since this activity was inhibited by chaotropic ions (Mg2+, SCN-, I-) at relatively low concns (0.6-1 M). Charge shift experiments performed on purified C5 provided further evidence for the presence of hydrophobic sites within C5. Bidirectional charge shifts of C5 mobility were observed with the two detergent systems TX-100 + DOC and TX-100 + CTAB. In order to localize these sites, C5 was subjected to trypsin digestion. Three major fragments were evidenced by two-dimensional electrophoresis (agarose/SDS-PAGE and SDS-PAGE/SDS-PAGE), and were isolated by hydrophobic affinity chromatography. The first fragment, C5FI, was composed probably of a mixture of six alpha chain fragments (alpha 1-alpha 6) linked to the beta chain by disulphide bridges. The second fragment, C5FII, was composed of the beta chain linked to two alpha chain fragments: alpha 2 (Mr = 58 K) and alpha 4 (Mr = 32 K). The third fragment, C5FIII, contained two covalently linked chains (beta chain and alpha 4). The rank order for mol. wt, agarose gel electrophoretic mobility and hydrophobicity was respectively: C5 greater than or equal to C5FI greater than C5FII greater than C5FIII; C5 less than or equal to C5FI less than or equal to C5FII much less than C5FIII; and C5FII greater than or equal to C5 greater than C5FI much greater than C5FIII. From the relative hydrophobicity, the hydrophobic sites of C5 can be localized to the alpha 2 fragment (Mr = 58 K) since C5FIII lacked the alpha 2 fragment and was not hydrophobic. Cleavage and the liberation of the N-terminal part of C5 induced an increase in hydrophobicity of the remaining part of C5 (C5b and C5FII), suggesting a possible role of the hydrophobic sites in association of C5b with membranes.