and CtrHb cautions that the PTM may occur in additional heme proteins, not only wild-type but also unwittingly in His-tagged versions, (histidine) variants, or designed proteins. (2) In preparing the histidine Nε2–heme vinyl Cα crosslink, care must be exercised in the choice of a reducing agent to avoid heme and protein damage, as occasionally caused by the oxidative by-products of aerobic DT reduction. Anaerobic DT treatment or reduction with alternative agents such as a ferredoxin system [56] if sufficiently powerful may be preferable. (3) It is possible to engineer the crosslink in a b heme protein using relatively mild reaction conditions. Although detailed kinetic analysis was not performed on CtrHb variants, the products are consistent with the mechanism described previously [21], in which case the pKa of the reactive histidine, in addition to its ability to adopt the geometry of the product, is an important factor
and CtrHb cautions that the PTM may occur in additional heme proteins, not only wild-type but also unwittingly in His-tagged versions, (histidine) variants, or designed proteins. (2) In preparing the histidine Nε2–heme vinyl Cα crosslink, care must be exercised in the choice of a reducing agent to avoid heme and protein damage, as occasionally caused by the oxidative by-products of aerobic DT reduction. Anaerobic DT treatment or reduction with alternative agents such as a ferredoxin system [56] if sufficiently powerful may be preferable. (3) It is possible to engineer the crosslink in a b heme protein using relatively mild reaction conditions. Although detailed kinetic analysis was not performed on CtrHb variants, the products are consistent with the mechanism described previously [21], in which case the pKa of the reactive histidine, in addition to its ability to adopt the geometry of the product, is an important factor
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