Document Type

Dissertation

Date of Award

1977

Keywords

Copper oxide, Ribonucleases

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Gilbert E. Janauer

Second Advisor

Stanley K. Madan

Third Advisor

Bruce McDuffie

Abstract

The binding of Cu(II) ion to ribonuclease A at various sodium perchlorate concentrations was studied as a function of Cu(II) ion concentration and pH using a Cu(II) ion electrode. Spectrophotometric studies of the same systems were also carried out. The copper binding studies were carried out at pH 6.0, 8.4, and 9.0 at 25°C in solutions of 0 to 6 M sodium perchlorate. The sodium perchlorate served not only to provide a constant ionic medium but also to unfold the protein at the higher concentrations. The mid-point sodium perchlorate concentration for unfolding was 2.5 M at pH 8.4 and 0.9 M at pH 9.0.

The number of Cu(II) ions required to saturate the binding sites of ribonuclease A at each pH and NaCl04 concentration was estimated by potentiometric observation of the Cu(OH)2 precipitation. The potentiometric experiments also made possible the determination of protons dissociated from the protein as a consequence of Cu(II) ion binding.

The static accessibility of the backbone peptide groups of ribonuclease A to the solvent in the native and unfolded state was estimated from the number of Cu(II) ions bound and the number of protons dissociated at pH 8.4. At sodium perchlorate molalities below 2.5 M where ribonuclease A is in its native state, 6% of the backbone peptide units are accessible to the solvent while the accessibility of the peptide backbone units in the unfolded state is 19%. These values are in approximate agreement with data obtained previously by different methods. At pH 9.0, 72% of the 163 binding sites which are available are complexed to Cu(II) ion at high NaClO4 concentrations.

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