Small-angle X-ray scattering studies on the X-ray induced aggregation of malate synthase

Abstract

Summary.Malate synthase was investigated by the small-angle X-ray scattering technique in aqueous solution. Measurements extending for several hours revealed a continuous increase of the intensity in the innermost portion of the scattering curve. There is clear evidence that this increase was caused by an X-ray induced aggregation of enzyme particles during the performance of the small-angle X-ray scattering experiment. The monitoring of the aggregation process in situ by means of small-angle X-ray scattering led to a model of the way how the aggregation might proceed. The analysis of the scattering curves of malate synthase taken at various stages of aggregation established the retention of the thickness factor of the native enzyme and the occurrence of one and later on of two cross-section factors. The process of aggregation was also reflected by the increase of extension of the distance distribution function. According to these results, th~ first step of aggregation might be a linear side-by-side association of the oblate enzyme particles, a process which is followed by a twodimensional aggregation. An aggregation in the third dimension was not observed during the time covered by our experiment. The predominance of aggregation in only one or two dimensions was corroborated by comparison of appropriate theoretical scattering curves with the experimental curves. The theoretical scattering curves for this comparison were obtained by averaging over the properly weighted scattering curves calculated for various species of hypothetical aggregates. The time dependence of the apparent mean radius of gyration was used to compare the aggregation of enzyme samples that were irradiated under different experimental conditions. It turned out that by addition of dithiothreitol to the enzyme solutions as well as in the presence of the substrates (acetyl-CoA, glyoxylate) or of a substrate analogue (pyruvate) or of ethanol the rate of aggregation is reduced. Enzymic activity was found to decrease about exponentially with increasing X-ray dose. The presence of dithiothreitol or of the substrate glyoxylate or of the substrate analogue pyruvate protects the enzyme against X-ray induced inactivation. The substrate acetyl-CoA does not exhibit a comparable protective effect against 0301-634X/80/0018/0099/$ 04.60 100 P. Zipper and H. Durchschlag inactivation. Measurements of enzymic activity and small-angle X-ray scattering on samples, which had been X-irradiated with a defined dose prior to the measurements, established two different series of efficiency for the protection of the enzyme against aggregation (pyruvate > glyoxylate > acetyl-CoA) and inactivation (glyoxylate > pyruvate ,> acetyl-CoA). The results showed that there is no direct relation between the extent of aggregation and the loss of enzymic activity.

The Structure of 11‐S Globulins from Sunflower and Rape Seed

“…No changes of the protein scattering curves were observed during the irradiation, so that radiation-induced aggregations can be ruled out [19]. Fig.…”

Detection of small conformational changes of proteins by small-angle scattering

“…(vii) A further serious problem, especially in the case of SAXS, is the occurrence of radiation damage due to the attack of the radiolysis products of water (OH and 02-radicals especially will be formed in aerated solution, together with H202). This, above all, may lead to aggregation of the proteins, but also to fragmentation and partial unfolding (Zipper & Durchschlag, 1980;Zipper, Wilting, Kriechbaum & Durchschlag, 1985). Such damage may be avoided by a series of precautions (Zipper & Durchschlag, 1981;Zipper, Kriechbaum, Wilting & Durchschlag, 1986): e.g.…”

Combined SAXS/UV–vis/Raman as a Diagnostic and Structure Resolving Tool in Materials and Life Sciences Applications

“…Earlier work on X-ray induced radiation damage − suggests that the indirect effects of radiation, i.e., the reaction of radical and nonradical products of water radiolysis with the protein, are mainly responsible for radiation damage. In our experiment, we cannot observe the effects of radicals, but the likely small (catalytic) changes in the side chain residues (UV spectra) may be indicative of such radical activities.…”