In the past the technique of small‐angle scattering has been a powerful tool for studying conformational changes of proteins which occur, for example, upon binding with ligands. Results obtained by different authors from X‐ray and neutron experiments on a variety of proteins and under various conditions have been compiled. This offers the possibility of comparing the extent of changes in the molecular parameters investigated (e.g. change of the radius of gyration). Problems encountered with the detection of small changes are discussed. As an example, conformational changes of the enzyme citrate synthase upon substrate binding (oxaloacetate) are presented. X‐ray crystallography had already found distinct changes between open and closed forms of the enzyme. Small‐angle X‐ray scattering studies registered slight changes of some parameters in solution. These changes could be paralleled with the results of other solution techniques (UV absorption, fluorescence and circular dichroism spectroscopy, analytical ultracentrifugation). The results found for citrate synthase are also compared with previous findings for malate synthase, an enzyme of similar enzymatic function. Above all, this study shows that care has to be taken when studying small conformational changes. It is absolutely necessary to use different methods and conditions and to study the problem from different points of view to avoid pitfalls.

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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.