The Inactivation of Enzymes by Ultraviolet Light,‐iv. The Nature and Involvement of Cystine Disruption*

Abstract

Two divergent theories of the mechanisms involved in U.V. inactivation of enzymes have been developed over a period of years. The one proposes that the random destruction of any amino acid residue causes inactivation. The second emphasizes the importance of the disruption of a cluster of specific cystines and hydrogen bonds responsible for the spatial integrity of the active center. Consistent with the latter postulates, previous studies in this series have shown that the number of cystines disrupted in trypsin is correlated with loss in enzymic activity and in ribonuclease the disruption of cystines is very non‐random. In the present experiments only 1–1.1 titrable (with pCMB) SH groups are formed for each cystine disrupted. The available evidence suggests that about 0.1 of the disrupting events produce two SH groups: 0.2 lead to the formation of one ‐SH and one ‐SOnH: and about 0.7 involve the cleavage of a C‐S bond with the appearance of one ‐SH plus an oxide (perhaps ‐C = O) of the C moiety. The disruption of cystine is such that neither serine, alanine, glycine nor H2S is formed in large amounts. The kinetics of SH production emphasize that the various cystines in RNase and trypsin have unequal radiation sensitivities; some must differ by at least a factor of 10. Tentatively it is concluded that u.v. absorbed directly by cystines (ca 20 per cent) causes random, symmetric breakage of S‐S, whereas quanta absorbed in aromatic residues (ca 80 per cent) lead to efficient energy transfer and localization in chromophores adjacent to critical cystines followed by addition of hydrogen to specific C‐S bonds resulting in their cleavage.

High probability of disrupting a disulphide bridge mediated by an endogenous excited tryptophan residue

“…The photosensitivity of protein-bound cystine residues has been shown to depend on their microenvironment (Augenstein and Ghiron 1961;Dose and Rajewsky 1962;Augenstein and Riley 1964;Dose 1964Dose , 1967Fiore and Dose 1965;Grist et al 1965;Risi et al 1967;Koudelka and Augenstein 1968). This correlates with the observation by Petersen et al (1999) that cystine residues (cysteines involved in disulphide bridges) have a clear preference for aromatic residues as spatial neighbors.…”

ACTION SPECTRUM AND QUANTUM YIELD FOR THE PHOTOINACTIVATION OF MNEMIOPSIN, A BIOLUMINESCENT PHOTOPROTEIN FROM THE CTENOPHORE MNEMIOPSIS SP.*

“…Certain well-characterized proteins such as lysozyme, trypsin. and chymotrypsin have servcd as models for protein photoinactivation mechanisms, In these proteins absorption of light by tryptophan residues has been implicated in the destruction of essential cystine groups (Augenstein and Riley, 1564;Risi et al, 1967: Jagger. 1567Dose, 1967;Vladimirov et al, 1570;Sellers and Ghiron, 1973).…”

Ultraviolet Irradiation Produces Loss of Saxitoxin Binding to Sodium Channels in Rat Synaptosomes

“…A small conformational change in this narrow part of the channel which is involved in ion selection and dehydration could easily lead to an irreversible block in ion movement. The photochemical mechanism might involve primary photon absorbtion by tryptophan with direct energy transfer to an adjacent reactive disulfide bond (Augenstein and Riley, 1964). Interestingly, the active binding conformation of the solubilized garfish olfactory nerve TTX receptor has been chemically shown to be dependent upon the integrity of a disulfide bond (Benzer and Raftery, 1973).…”