The ability of C1 to bind to (Fc)5µ-fragment of a monoclonal IgM was determined by means of a hemolytic C1-inhibition assay. (Fc)5µ-fragments were produced by trypsin digestion at five different temperatures ranging from 54 to 62°C (2°C increments) and were purified by immunoadsorption through a column of monospecific anti-Fabµ and by molecular exclusion chromatography. Analytical ultracentrifugation showed the final preparations to be free of aggregates. A plot of µg (Fc)5µ required to inhibit

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... 50% of available C1 (Z = 1 to 1.5) vs temperature of production of the fragment, yielded a curve with a minimum (5 µg) at 58 to 60°C. Upon mild reduction and alkylation of these (Fc)5µ's their C1-fixing capacity reverted to being approximately the same (60 µg) irrespective of temperature of production. (Fc)5µ was also prepared at 25°C in the presence of 5 M urea, purified by immunoadsorption as before and aliquots then exposed to temperatures ranging from 40 to 70°C (5°C increments) for 15 min.