A Small-Angle X-Ray Scattering Study on Pre-Irradiated Malate Synthase. The Influence of Formate, Superoxide Dismutase, and Catalase on the X-Ray Induced Aggregation of the Enzyme

Abstract

The sulfhydryl enzyme malate synthase from baker’s yeast was X-irradiated with 6 kGy in air-saturated aqueous solution (enzyme concentration: ≃ 10 mg/ml; volume: 120 μl), in the absence or presence of the specific scavengers formate, superoxide dismutase, and catalase. After X-irradiation, a small aliquot of the irradiated solutions was tested for enzymic activity while the main portion was investigated by means of small-angle X-ray scattering. Additionally, an unir­radiated sample without additives was investigated as a reference. Experiments yielded the fol­lowing results: 1. X-irradiation in the absence of the mentioned scavengers caused considerable aggregation, fragmentation, and inactivation of the enzyme. The dose Dt37 for total (= repairable + non­-repayable) inactivation resulted as 4.4 kGy. The mean radius of gyration was found to be about 13 nm. The mean degree of aggregation was obtained as 5.7, without correction for fragmenta­tion. An estimation based on the thickness factor revealed that about 19% of material might be strongly fragmented. When this amount of fragments was accordingly taken into account, a value of 7.1 was obtained as an upper limit for the mean degree of aggregation. The observed retention of the thickness factor and the finding of two different cross-section factors are in full accord with the two-dimensional aggregation model established previously (Zipper and Durchschlag, Radiat. Environ. Biophys. 18, 99 - 121 (1980)). 2. The presence of catalytic amounts of superoxide dismutase and/or catalase, in the absence of formate, during X-irradiation reduced both aggregation and inactivation significantly. 3. The presence of formate (10 or 100 mᴍ) during X-irradiation led to a strong decrease of aggregation and inactivation. This effect was more pronounced with the higher formate concen­tration or when superoxide dismutase and/or catalase were simultaneously present during X-irradiation. The presence of formate also reduced the amount of fragments significantly. 4. The results clearly show that the aggregation and inactivation of malate synthase upon X-irradiation in aqueous solution are mainly caused by OH·; to a minor extent O·̄2 and H2O2 are additionally involved in the damaging processes.

Quantifying radiation damage in biomolecular small-angle X-ray scattering

“…Cryocooling samples to 100 K has been shown to reduce radiation damage rates in SAXS (Meisburger et al, 2013;Hopkins et al, 2015), but substantial methodological development is required before cryocooling can be accepted for routine use. Despite the importance of radiation damage as a limiting factor in SAXS, early efforts using laboratory X-ray sources (Zipper & Durchschlag, 1980a,b,c, 1981Zipper et al, 1980Zipper et al, , 1985Zipper & Kriechbaum, 1986) have been followed by only two systematic, quantitative studies at synchrotron sources (Kuwamoto et al, 2004;Jeffries et al, 2015). With recent and planned upgrades to already bright third-generation sources and construction of high-brightness fourth-generation sources, understanding, quantifying and ultimately minimizing radiation damage in biological SAXS will be essential to efficient use and full exploitation of these sources.…”