Proteins biotin-BMCC

Biotin-BMCC is insoluble in water and must be dissolved in an organic solvent prior to addition to an aqueous reaction mixture. Preparing a concentrated stock solution in DMF or DMSO allows transfer of a small aliquot to a buffer reaction. The upper limit of biotin-BMCC solubility in DMSO is approximately 33 mM or 17 mg/ml. In DMF, it is only soluble to a level of about 7 mM (4 mg/ml). Upon addition of an organic solution of the reagent to an aqueous environment (do not exceed 10 percent organic solvent in the aqueous medium to prevent protein precipitation), biotin-BMCC may form a micro-emulsion. This is normal and during the course of the reaction, the remainder of the compound will be driven into solution as it couples or hydrolyzes. 

The following protocol is a suggested method for modifying sulfhydryl-containing proteins with biotin-BMCC. Some optimization of biotinylation levels may have to be done for particular applications. 

Add 100 pi of the biotin-BMCC solution to each ml of the protein solution. Mix well. 

Additional reaction components can be used that may enhance or be essential for catalytic activity in a particular system. These components may serve functions analogous to the cofactors used by protein catalysts (e. g., ATP, NADH, or metal ions). These components can be supplied free in solution or incorporated into the RNA library as previously described. As an example, Figure 8.5 oudines the RNA-catalyzed carbon-carbon bond-forming [4 + 2] cycloadclition reaction between a tethered diene substrate, (2E.4E)-hexa-2,4-clien-l-0-PEG (1), and 1-biotinamido-4-[A -(maleimidomethyl)cyclohexanecarboxamido] butane (BMCC-biotin, 2), a clienophile that is free in solution. The RNA catalyzes the formation of (3), which contains biotin for partitioning purposes. 

FIGURE 20.2 Adduction of proteins with the biotin-labeled reactive electrophiles 1 -biotinamido-4-(4 -[maleimidoethylcyclohexane]-carboxamido)-butane (BMCC) and A-iodoacetyl-A-biotinylhexylenediamine (lAB). Adduction is followed by SDS PAGE gel separation followed by gel digestion and peptide extraction. The protein-electrophile adducts are then captured by biotin-avidin chromatography and analyzed by LC-MS/MS. 

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