Reaction with biotin

Time Succinimidyl ester derivatives will react with a protein within 1 h. Periodate oxidation will require 2 h at pH 6.0. Reaction with biotin hydrazide can be performed in a few hours. Stabilization with cyanoboro-hydride... 

Percentage of amino groups substituted after reaction with biotin -hydroxysuccini-mide (BNHS) . From Guesdon et al. (1979)... 

Sauers et al. proposed that carboxy phosphate may decompose to give carbon dioxide and inorganic phosphate prior to reaction with biotin (57). These workers suggest that this provides a tamed form of carbon dioxide that is low in entropy in that it is localized in the vicinity of biotin and thus is a much better reactant than bicarbonate. 

Fig. 2. Detection of myristoylated and palmitoylated proteins by metabolic labeling with co-alkynyl fatty acids. Cellular extracts were prepared from MDCK cells treated with the different co-alkynyl fatty acid probes (100 jiM) for 24 h. The proteome was subjected to click reaction with biotin-azide, separated by SDS-PAGE and detected by streptavidin-linked horseradish peroxidase. The blot in (b) represents an experiment done at the same time as in (a) except that it has been treated with hydroxylamine. Lanes 1,Alk-C10 (/r=6) 2,Alk-C11 (/ =7) 3,Alk-C13 (n=9) 4,Alk-C14 (n=10) 5,Alk-C16 (/ = 12) 6, Alk-C18 (/7=14). The DMSO lane serves as a background control for labeling with alkyne fatly acids.

The mechanism of the C02 transfer reaction with acetyl CoA to give mal-onyl CoA is thought to involve C02 as the reactive species. One proposal is that loss of C02 is favored by hydrogen-bond formation between the A -carboxy-biotin carbonyl group and a nearby acidic site in the enzyme. Simultaneous deprotonation of acetyl CoA by a basic site in the enzyme gives a thioester eno-late ion that can react with C02 as it is formed (Figure 29.6). 

Addition of amines to a,/J-unsaturated sulfones has been used in synthesis of key intermediates of biotin. In this reaction, benzylamine serves first as a base in the reaction with 60 to afford thiophene 1,1-dioxide (61) and also as a nucleophile to introduce two amino groups (equation 57)49. 

Reaction with avidin-labeled alkaline phosphatase (AP)-biotin conjugate... 

Figure 11.4 NHS-LC-biotin provides an extended spacer arm to allow greater distance between the biotin rings and a modified molecule. Reaction with amines forms amide linkages.

The increased length of this spacer (24.7 A) provides more efficient interaction potential with avidin or streptavidin probes, possibly increasing the sensitivity of assay systems. The reactions of biotin-LC-hydrazide are identical to those of biotin-hydrazide. 

Figure 11.22 Azido-sialic acid-containing glycans can be labeled in vivo with biotin-PEG-phosphine using the Staudinger ligation reaction, which forms an amide bond.

The methods used for in vivo incorporation of azido-monomers and performing a labeling reaction with live cells are relatively simple. The following protocol is based on the methods of Saxon and Bertozzi (2000), which uses acetylated azidoacetylmannosamine as the azido-monomer source and a biotin-PEG-phosphine compound to biotinylate cell surface glycoproteins at the specific azide-sialic acid incorporation sites (Figure 17.19). 

With mixing, add an aliquot of the biotinylation stock solution to the protein solution to provide at least a 10-fold molar excess over the concentration of protein present. Doing a series of reactions with different molar amounts of the NHS-PEG -biotin compound may be done to optimize the modification level. 

Reaction of Biotin-BMCC with Sulfhydryl-Modified DNA... 

Reisfeld, A., Rothenberg, J.M., Bayer, E.A., and Wilchek, M. (1987) Nonradioactive hybridization probes prepared by the reaction of biotin hydrazide with DNA. Biochem. Biophys. Res. Comm. 142, 519-526. 

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