Modification of lactose repressor protein with 2-chloromercuri-4-nitrophenol
Yang, Diana Sou Tung
Matthews, Kathleen S.
Master of Arts
The reaction of the lactose repressor protein of Escherichia coli with 2-chloromercuri-4-nitrophenol (MNP) has been investigated. MNP reacts specifically with thiol groups in proteins. When a thiol reacts with a mercurinitrophenol, the ionization state of nitrophenol may be altered due to environmental changes, and this in turn affects its spectral characteristics. It is possible from the spectral changes observed to detect altered ionization and/or absorption characteristics of the chromophore and thereby to detect conformational changes in the protein. Titrations of lactose repressor with MNP were carried out under a variety of conditions. Similar results were obtained in the presence of inducer, anti-inducer, or neutral ligand. The equivalence point of the titration curve corresponded to 2 moles MNP/mole repressor monomer. This implies that there are 2 sulfhydryl groups which react with MNP under all conditions. The third sulfhydryl group in native repressor monomer did not react and is apparently completely buried under all conditions. A slight difference of environment between the two sulfhydryl groups which bind to MNP has been observed. The binding activities of the modified protein were measured under various conditions to determine any effects of modification. No significant loss of IPTG binding activity or operator binding activity was observed. Therefore, the two cysteine residues which MNP can modify do not interfere with either the inducer binding site or the operator binding site. Difference spectra of MNP-repressor in the presence of inducer have been measured. The spectral changes apparently result from a conformation change which alters the environment of nitrophenol group. However, when anti-inducer is bound to the repressor no spectral changes are observed. Since inducers and anti-inducers have same binding site, this implies inducer binding and anti-inducer binding have different effects on the protein structure. The pK of free MNP was determined to be 6.75. The combined pK of the two mercurinitrophenol groups bound to cysteine residues was found to be ~8.; and the combined pK of MNP-repressor in the presence of IPTG was found to be ~7.9. Reaction rates of MNP with repressor were measured. No significant differences were observed in the reaction rates in the presence of inducer or anti-inducer. When the concentration of MNP was increased, the reaction also increased. It can be determined from these measurements that MNP reacts with the 2 sulfhydryl groups with different rate constants.