Competitive Labeling

Competitive labeling is a method of chemical modification for determining the pKa and the reactivity of functional groups in proteins. The extent of modification of a particular protein group at various pH values is compared to that of the same group in a standard compound. This gives the reactivity of the protein group relative to its intrinsic reactivity, and the pH-dependence gives the pK_a value. These data reflect the accessibility of the group in the folded protein, which provides information about the chemical reactivity of the group and its environment in the folded protein. This indirectly gives conformational information about the protein. Conformational transitions associated with function, association, and ligand binding can be monitored with this method.

 Competitive labeling was originally employed in a study of the amino groups in elastase (1). A mixture of elastase and phenylalanine (the standard compound) was acetylated with a small amount of 3H-labeled acetic anhydride. It is important that the amount of the labeling agent is less than that of the groups being modified. Under these conditions, the relative amounts of label in the standard and in the specific protein group will depend upon their relative ionization and reactivities. The fraction of the reactive, nonionized form of the amino group (a_i) and the ratio of reactivity (r = k_i/k_standard) of the ith group are derived from the known pK_a of the standard (a_standard( and the measured ratio of labeling: 

The label on the ith group could be determined by the radioactivity of the appropriate peptide separated by peptide mapping. The values on the right side of the equation are obtained

experimentally at each pH and are plotted against pH. The values of the pK_a and of r of the protein group are obtained by theoretical curve fitting.

Besides using acetic anhydride to react with amino groups, 1-fluoro-2,4-dinitrobenzene has been used to react with amino groups and with histidine and tyrosine residues, and iodoacetate with thiol groups. A small agent is most suitable for this purpose of modification. Double labeling with 3H-label, followed by ¹³C-labeling after denaturation gives a much more sensitive analysis. In this case, the same group in the denatured protein is the standard.

References

1. H. Kaplan, K. J. Stevenson, and B. S. Hartley (1971) Biochem. J. 124, 289–299. 

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DNA Looping and Repression of araBAD

Binding Sites of the ara Regulatory Proteins

Endoplasmic Reticulum-Associated Degradation and the Unfolded Protein Response

Protein Folding and Modifications in the Endoplasmic Reticulum

Protein Modifications

Regulating AraC Synthesis

Repression by AraC

Detection and Isolation of AraC Protein

Genetics of the Arabinose System

The Eukaryotic Genome

Nucleic Acids and Their Organization in Eukaryotic, Prokaryotic, and Viral Genomes