Biotin sulfhydryl group

Figure 2.7. Identification ofphosphoproteins by site-specific chemical modification. A. Method of Zhou et al. (2001) involves trypsin digest of complex protein mixture followed by addition of sulfhydryl groups specifically to phosphopeptides. The sulfhydryl group allows capture of the peptide on a bead. Elution of the peptides restores the phosphate and the resulting phosphopeptide is analyzed by tandem mass spectrometry. B. Method of creates a biotin tag in place of the phosphate group. The biotin tag is used for subsequent affinity purification. The purified proteins are proteolyzed and identified by mass spectrometry.

Conjugates of (strept)avidin with these fluorescent probes may be prepared by activation of the phycobiliprotein with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) to create a sulf-hydryl-reactive derivative, followed by modification of (strept)avidin with 2-iminothiolane or SATA (Chapter 1, Section 4.1) to create the free sulfhydryl groups necessary for conjugation. The protocol for SATA modification of (strept)avidin can be found in Section 3.1, this chapter. The procedure for SPDP activation of phycobiliproteins can be found in Chapter 9, Section 7. Reacting the SPDP-activated phycobiliprotein with thiol-labeled (strept)avidin at a molar ratio of 2 1 will result in highly fluorescent biotin binding probes. 

The required sulfhydryl groups for biotin-BMCC modification may be indigenous... 

CoA to form malonyl CoA using C02 in the form of bicarbonate HC03 (Fig. 2). This reaction is catalyzed by the enzyme acetyl CoA carboxylase which has biotin as a prosthetic group, a common feature in C02-binding enzymes. One molecule of ATP is hydrolyzed in the reaction, which is irreversible. The elongation steps of fatty acid synthesis all involve intermediates linked to the terminal sulfhydryl group of the phosphopantetheine reactive unit in ACP phosphopantetheine is also the reactive unit in CoA. Therefore, the next steps are the formation of acetyl-ACP and malonyl-ACP by the enzymes acetyl transacylase and malonyl transacylase, respectively (Fig. 2). (For the synthesis of fatty acids with an odd number of carbon atoms the three-carbon propionyl-ACP is the starting point instead of malonyl-ACP.)... 

Reagents for biotinylation of proteins and peptides are commercially available and allow the fast and efficient derivatization of antibodies and enzymes without the loss of enzyme or antibody activity. Several reagents are also available for the biotinylation of sulfhydryl groups aldehydes, nucleic acids and carbohydrates. Hence, in addition to proteins and peptides, a variety of antigens can be labeled with biotin. 

Coenzyme A (CoA), biotin, and pyridoxal phosphate are also activation-transfer coenzymes synthesized from vitamins. CoA (CoASH), which is synthesized from the vitamin pantothenate, contains an adenosine 3, 5 -bisphosphate which binds reversibly, but tightly, to a site on an enzyme (Fig. 8.12A). Its functional group, a sulfhydryl group at the other end of the molecule, is a nucleophile that always... 

Fig. 8.12. CoA and biotin, activation-transfer coenzymes. A. Coenzyme A (CoA or CoASH) and phosphopantetheine are synthesized from the vitamin pantothenate (pantothenic acid). The active sulfhydryl group, shown in blue, binds to acyl groups (e.g., acetyl, succinyl, or fatty acyl) to form thioesters. B. Biotin activates and transfers CO2 to compounds in car-boxylation reactions. The reactive N is shown in blue. Biotin is covalently attached to a lysine residue in the carboxylase enzyme.

Figure 11.8 Biotin-BMCC provides sulfhydryl reactivity through its terminal maleimide group. The reaction creates a stable thioether linkage.

Figure 11.9 Biotin-HPDP reacts with sulfhydryl-containing molecules through its pyridyl disulfide group, forming reversible disulfide bonds. The biotin group may be released from modified molecules by reduction with DTT.

Purify the SH-labeled oligo by gel filtration on a desalting resin using 10 mM sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2. The probe now may be used to conjugate with an activated enzyme, biotin, fluorescent tag, or other molecules containing a sulfhydryl-reactive group. 

Figure 28.15 Two similar label transfer reagents containing a thiol-reactive methanethiolsulfonate group to label bait protein through available sulfhydryls, a tetrafluorophenyl azide group for high-efficiency photoreac-tive conjugation with interacting prey proteins, and a long biotin affinity tag.

Chemistry of sulfhydryl-specific modification. Reaction of MTS (CH3SO2SCH2CH2X) with a cysteine will lead to specific modification of the SH group by MTS. Note that X = NH+ (MTSEA), SO3- (MTSES), N(CH3)+ (MTSET), NH-biotin (MTSEA-biotin) or NHCO(CH2)5 (MTSEA-biotincap)... 

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