STUDIES OF INSULIN CRYSTALS AT LOW TEMPERATURES: EFFECTS ON MOSAIC CHARACTER AND RADIATION SENSITIVITY

Abstract

The low-temperature X-ray diffraction studies of orthorhombic insulin citrate crystals reported here were initially undertaken because of the rapid deterioration at room temperature of certain insulin crystals containing heavy-metal cations. The effects of cooling on mosaic character and radiation sensitivity were studied. Two temperature ranges were employed: (a) below 150?C and (b) 00C to 13?C. In normal laboratory practice orthorhombic insulin citrate crystals are grown and stored at 1 i 1?0C. Immersion studies in heavy-atom reagents are also made at this temperature. All X-ray diffraction work is, however, carried out at room temperature ('-21 ?C). We have found that orthorhombic insulin citrate crystals may be cooled rapidly to below -1500 C without impairing the X-ray diffraction pattern. Rapid cooling does, however, enhance the mosaic character of these crystals. X-ray diffraction studies at 00C and 130C have provided evidence for marked reduction in radiation sensitivity in certain heavy-atom-containing crystals. General Background.-Although metal-free insulin crystals are somewhat more sensitive to radiation damage (nickel-filtered CuKa) at room temperature than at 00C, the radiation sensitivity at room temperature is not marked enough to prevent the collection of intensity data by counter techniques.' Furthermore, these crystals do not deteriorate on standing at room temperature for several weeks without irradiation. However, after immersion (1 i 1?C) in buffered solutions containing certain salts with heavy-metal cations (including uranyl) insulin crystals deteriorate rapidly at room temperature (sometimes within 1 day or less of exposure) after only the minimal radiation exposure (minutes) necessary to monitor the state of the crystal. The deterioration has been observed both in the presence and in the absence of significant changes in X-ray diffraction intensity distribution after immersion. It is not markedly accelerated by continuous X-ray irradiation. This deterioration was originally attributed to greatly enhanced radiation damage, as laboratory practice avoids keeping the crystals at room temperature prior to X-ray photography. Some apparent enhancement in radiation damage is also observed with a second group of metal-containing crystals which do not show the striking and rapid room-temperature deterioration reported above. Before the deterioration in the first class of crystals was recognized as largely of thermal origin, studies were made of both classes of metal-containing crystals into which radiation protectors had been introduced. These provided marginal evidence of protection. There was no evidence of protection when the crystals were studied under oxygen-free conditions. In order to collect X-ray data from the crystals which deteriorate rapidly at room temperature, it would have been necessary to build an apparatus to maintain