The Inactivation of Trypsin by Ultraviolet Light, I. The Correlation of Inactivation With the Disruption of Constituent Cystine

Augenstine, L. G.; Ghiron, Camillo A.

doi:10.1073/pnas.47.10.1530

Cited by 50 publications

(6 citation statements)

References 23 publications

(6 reference statements)

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“…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.…”

mentioning

confidence: 63%

“…It was also found that the effective destruction of cystine, tryptophan, tyrosine, and phenylalanine occurs on UV irradiation of proteins (Kazutomo 1955; Augenstine and Ghiron 1961; McLaren and Luse 1961; Hideko and Kazutomo 1963; Dose 1964; Vladimirov and Roshchupkin 1964; Cooper and Davidson 1965; Fujimoro 1966; Piras and Vallee 1966; Vladimirov et al 1970). 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 1964, 1967; Fiore 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.…”

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confidence: 99%

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

et al. 2002

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It is well known that ultraviolet (UV) radiation may reduce or even abolish the biological activity of proteins and enzymes. UV light, as a component of sunlight, is illuminating all light-exposed parts of living organisms, partly composed of proteins and enzymes. Although a considerable amount of empirical evidence for UV damage has been compiled, no deeper understanding of this important phenomenon has yet emerged. The present paper presents a detailed analysis of a classical example of UV-induced changes in three-dimensional structure and activity of a model enzyme, cutinase from Fusarium solani pisi. The effect of illumination duration and power has been investigated. A photon-induced mechanism responsible for structural and functional changes is proposed. Tryptophan excitation energy disrupts a neighboring disulphide bridge, which in turn leads to altered biological activity and stability. The loss of the disulphide bridge has a pronounced effect on the fluorescence quantum yield, which has been monitored as a function of illumination power. A general theoretical model for slow two-state chemical exchange is formulated, which allows for calculation of both the mean number of photons involved in the process and the ratio between the quantum yields of the two states. It is clear from the present data that the likelihood for UV damage of proteins is directly proportional to the intensity of the UV radiation. Consistent with the loss of the disulphide bridge, a complex pH-dependent change in the fluorescence lifetimes is observed. Earlier studies in this laboratory indicate that proteins are prone to such UV-induced radiation damage because tryptophan residues typically are located as next spatial neighbors to disulphide bridges. We believe that these observations may have far-reaching implications for protein stability and for assessing the true risks involved in increasing UV radiation loads on living organisms.Keywords: tryptophan fluorescence lifetime; fluorescence quenching; disulphide (disulfide) bridges; photochemical reaction; protein structure damaged by UV light; SS bond disruption; indole; theoretical model Two divergent theories of the mechanisms involved in ultraviolet (UV) inactivation of enzymes have been developed over a period of years. One theory proposes that the random destruction of any amino acid residue causes inactivation (Augenstein and Riley 1964). The second emphasizes the importance of the disruption of a cluster of specific cystine residues (cysteines involved in disulphide bridges; Augenstein and Riley 1964). It was also found that the effective destruction of cystine, tryptophan, tyrosine, and phenylalanine occurs on UV irradiation of proteins (Kazutomo

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

et al. 2002

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“…Photoinduced oxidative damage can occur directly upon absorption of UV radiation by aromatic residues or indirectly through the generation of reactive oxygen species (ROS) upon absorption of visible light by photosensitizers (Sgarbossa et al, 2003). Less well documented is the sensitivity of disulfide bonds to UV damage (Augenstine, 1962;Augenstine and Ghiron, 1961;Grist et al, 1965). The exact mechanism by which disulfide bonds are destroyed remains unclear; however, recent evidence suggests that aromatic residues close to disulfide bonds are important (Neves-Petersen et al, 2002).…”

Section: Iðhklþmentioning

confidence: 99%

Phasing Macromolecular Structures with UV-Induced Structural Changes

Nanao

Ravelli

2006

Structure

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Experimental phasing of macromolecular crystal structures relies on the accurate measurement of two or more sets of reflections from isomorphous crystals, where the scattering power of a few atoms is different for each set. Recently, it was demonstrated that X-ray-induced intensity differences can also contain phasing information, exploiting specific structural changes characteristic of X-ray damage. This method (radiation damage-induced phasing; RIP) has the advantage that it can be performed on a single crystal of the native macromolecule. However, a drawback is that X-rays introduce many small changes to both solvent and macromolecule. In this study, ultraviolet (UV) radiation has been used to induce specific changes in the macromolecule alone, leading to a larger contrast between radiation-susceptible and nonsusceptible sites. Unlike X-ray RIP, UV RIP does not require the use of a synchrotron. The method has been demonstrated for a series of macromolecules.

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“…Auganstine et al· (114,129), have studied the ultraviolet and ionising It has been found that a general relationship exists between molecular weight and quantum yields for ultraviolet inactivation (123,124).…”

Section: Possible Methods Of Enhancemen T Of Photodynam Ic Actionmentioning

confidence: 99%

Quantum Yield Studies of the Photodynamic Inactivation of Trypsin

Glad¹,

Spikes²

1964

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The Inactivation of Trypsin by Ultraviolet Light, I. The Correlation of Inactivation With the Disruption of Constituent Cystine

Cited by 50 publications

References 23 publications

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

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

Phasing Macromolecular Structures with UV-Induced Structural Changes

Quantum Yield Studies of the Photodynamic Inactivation of Trypsin

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