The Inactivation of Enzymes by Ultraviolet Light. V. The Disruption of Specific Cystines in Ribonuclease

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Abstract
Titrations with p-chloromercuribenzoate (pCMB) or C14-iodoacetic acid (C14-IAA) show a direct correlation between the increase in titrable SH groups and destruction of ribonuclease (RNase) activity by 2537-A light: there is an increase of about 2.0 SH groups per molecule inactivated. A similar behavior was reported for trypsin; however, unlike trypsin, the residual enzymatic activity measured in irradiated RNase is not affected significantly by pCMB or urea added prior to assay. Autoradiographs of fragmented RNase reacted with C14-IAA show that one or at most 2 specific cystines are disrupted by ultraviolet doses sufficient to destroy 50% of the enzymatic activity. Determinations of the C14 associated with different fragments indicate that the various cystines are disrupted at quite different rates. These results seem consistent with a previous hypothesis that inactivation is initiated by the disruption of a specific cluster of secondary and tertiary bonds and inconsistent with the notion that ultraviolet inactivation depends on random destruction of amino acid residues.