Biotinylation determining level

For instance, a dendrimer easily can be coupled with a large number of fluorescent dyes and still provide additional coupling sites for biotinylation. The only limitation to the number of fluorescent modifications is if fluorescence quenching starts to take place, in which case no further modifications will result in increased signal. A series of such conjugates using different levels of fluorophore modification should be done to determine the optimal level of dye-to-dendrimer before quenching occurs. 

Proteins biotinylated with this reagent will have a characteristic absorbance band at 354 nm, which can be used to determine accurately the number of biotin groups per molecule. No other biotinylation compound has such built-in quantification capability. This feature eliminates the need to consume conjugate by doing a HABA assay to test for the level of biotin incorporation (Chapter 23, Section 7). 

The following protocol describes both of these HABA-based tests for determining the level of biotinylation. 

The post-translational biotinylation of biotin-containing enzymes is absolutely required for the activation of these enzymes. Indeed, biotinylation of biotin-containing enzymes is a potential locus for the regulation of their activities. We have previously shown that biotinylation of MCCase is an important regulatory mechanism by which the activity of this enzyme is determined in leaves and roots of tomato. Specifically, we found that even though the biotinylated subunit of MCCase accumulates to equal levels in roots and leaves, in the latter a significant proportion of the protein accumulated in its apo-form, which explained the lower MCCase activity that was found in extracts of leaves in comparison to roots. 

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