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The rst component of the complement pathway, C1, consists of three interacting subcomponents, C1q, C1r and C1s. The two latter components are homologous serine protease zymogens which form a Ca2+ -dependent tetramer, C1s-C1r2 -C1s which associates with C1q to form C1. The zymogens are activated to functional serine proteases C1s and C1r in a process mediated by Ca2+ which triggers the rst step in complement activation. C1s forms a dimer when Ca2+ binds at an N-terminal domain and the stabilization of the C1s-C1r2-C1s tetramer is also facilitated by Ca2+ coordination to homologous N-terminal domains. Interaction with Ca2+ is therefore a major feature of both complement structure and activation. Several binding sites of varying af nity have been identi ed by methods such as equilibrium dialysis (Section 7.3.2). Detailed analysis of Ca2+ -binding requires a technique suitable for studying a wide range of Ca2+ and protein concentrations at equilibrium. Sedimentation equilibrium lends itself in particular to this type of analysis. Protein was equilibrated by dialysis with either 5 mM or 1 nM Ca2+ . It was then centrifuged at 6500 7000 rpm in swinging bucket rotors (90 l volume) in a preparative centrifuge for 24 hours. Note that, in contrast to the previous example, this centrifugation experiment was not carried out in an analytical centrifuge. Sample was collected as individual fractions and the equilibrium distribution of protein under these conditions was stabilized by inclusion of dextran-70 with the protein sample. A280 was plotted as a function of radial distance for the two samples as shown below. The molecular mass, Mr, was estimated using essentially the equations described in Section 7.2.8 and was found to be 79 (1 nM Ca2+ ) and 143 kDa (5 mM Ca2+ ), respectively. This suggests that Ca2+ promotes dimer formation between C1s monomers. c# print windows form to pdf Convert a Windows Form to PDF - CodeProject
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