Inorganic photochemical protein scissors

Authors

Jyotsna Thota, University of Connecticut

Date of Completion

January 2005

Keywords

Chemistry, General

Degree

Ph.D.

Abstract

Reagents that can cleave protein backbone at predictable sites can be useful in protein structural studies. For example, ligand binding sites and metal binding sites can be located using such reagents. This dissertation focuses on developing such inorganic photochemical reagents that cleave proteins with high selectivity and efficiency. Site-selective photocleavage of chicken egg white lysozyme by hexaamminecobalt(III) chloride ([Co(NH₃) ₆]⁺³), pentaamminechlorocobalt(III) chloride ([Co(NH ₃)₅Cl]⁺²), pentaamminebromocobalt(III) bromide ([Co(NH₃)₅Br]⁺²) and tetraamminecarbonatocobalt(III) nitrate ([Co(NH₃)₄CO₃]⁺¹) is demonstrated here for the first time. Fluoresce, ¹H NMR and ITC studies indicate that the Co(III) complexes interact with the protein. ^ On irradiating at 310 nm, the above-mentioned complexes cleave lysozyme into fragments of molecular masses approximately 11.8 kDa and 2.5 kDa. The presence of these two sharp product bands is monitored by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). 50 minute irradiations of lysozyme (75 μM) with [Co(NH₃)₆]⁺³ (5 mM), [Co(NH₃)₅Cl]⁺² (2.5 mM), ([Co(NH₃)₅Br]⁺² (0.6 mM), [Co(NH ₃)₄CO₃]⁺¹ (2.5 mM) gave rise to 15%, 30%, 29% and 26% respectively. ^ Product yields increased with irradiation time and complex concentration. Irradiation at wavelengths greater than 310 nm up to 390 nm gave rise to products in the case of [Co(NH₃)₅Cl]⁺², [Co(NH ₃)₅Br]⁺² and [Co(NH₃)₄CO ₃]⁺¹ but not [Co(NH₃)₆] ⁺³ indicating that population of charge transfer states of the complexes produces radical intermediates that mediate the protein cleavage. ^ N-terminal sequencing of the larger fragment and smaller fragment from photocleavage of consists of the N-terminus of lysozyme and the sequence VAXRN indicating that the cleavage occurs between Trp 108 and Val 109. Flash photolysis data indicate that NH₃⁺•, CO₃ ^-•, and Br• are produced and presence of protein quenches the radicals during the irradiation by excimer laser (308 nm). 310 nm irradiation of lysozyme and [Co(NH₃)₆]⁺³ in the presence of Mg⁺² (0.2 M) decreased the intensity of the product bands indicating that [Co(NH₃)₆]⁺³, and Mg ⁺² compete for a common binding site on the protein. The cleavage site indicated above is in close proximity to a possible Mg⁺² binding site on lysozyme and thus protein cleavage by Co(III) complexes could be used as a methodology to locate magnesium binding sites on proteins. ^

Recommended Citation

Thota, Jyotsna, "Inorganic photochemical protein scissors" (2005). Doctoral Dissertations. AAI3193747.
https://digitalcommons.lib.uconn.edu/dissertations/AAI3193747