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+, theor 686.2365).
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note
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The I107C gene was cloned and the protein purified as previously described for other gp45 mutants (ref 7f). I107C (150 μM in monomers) was dialyzed into NR buffer (20 mM HEPES, pH 7.0, 50 mM NaCl, 1 mM EDTA, and 10% glycerol) and 3 added in 0.1 vol DMF (1.5 mM final). This mixture was nutated in the dark at 4 °C for 12 h and then purified on a MonoQ (Pharmacia, Piscataway, NJ) anion-exchange FPLC column. Although we have excluded light in both the synthesis of 3 and the formation of the I107C-3 conjugate, we have found that the aryl azide of 3 is stable enough to produce satisfactory results without the rigorous exclusion of light. Exclusion of free thiols in the conjugation and photo-cross-linking reactions is essential.
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