Crystalline preparations of rhombohedral porcine insulin as studied by electron diffraction

Porcine rhombohedral insulin crystals, mainly of the varieties containing 2.5 and 0.8% Zn, were studied by electron diffraction in the fixed, embedded, and thin-sectioned state. Stable and reproducible diffraction patterns (resolution limit 0.8 nm) were obtained only with 2.5% Zn-insulin crystals. Suitable fixatives were combinations of OsO 4 and glutaraldehyde; suitable embedding media were an epoxy resin and a glutaraldehyde-urea resin. The crystals, in the embedded state, were trigonal (rhombohedral cell) with a H = 7.4 nm, c = 3.1 nm as measured in the diffraction patterns. In discussing these values, a number of possible errors are considered, i.e., reciprocal lattice spikes, section compression, and mass loss from sections due to irradiation. For comparison wet unfixed 2.5% Zn-insulin crystals were also studied in X-ray powder patterns (Guinier, low angle), yielding a H = 8.2 nm, c = 3.3 nm (rhombohedral cell). These values indicate a close resemblance to porcine 2 Zn-insulin crystals (0.4% Zn), apparently shown here for the first time. Confidence intervals for a H and c were computed by use of their asymptotic distribution. The results are discussed in view of electron microscope studies of intracellular protein crystals, where, in conclusion, a reduction in length of approximately 8% is to be expected in reference to the hydrated state.