CRYSTALLIZATION OF AN FV FRAGMENT FROM A HUMAN-IGM CRYOGLOBULIN BY A MICROSEEDING TECHNIQUE

In contrast to many fragments of human IgM immunoglobulins, V(L)-V(H) heterodimer (Fv) from a patient (Pot) with macroglobulinemia produces large single crystals in relatively short periods of time (days). It was therefore convenient to segregate the crystal production into the following three processes for study under a variety of conditions: (1) the preparation of seeds, (2) the enlargement of steeds into microcrystals, and (3) the growth of microcrystals into large crystals suitable for X-ray diffraction. The protein could be crystallized in solutions of polyethylene glycol (PEG) ranging in molecular weight from 600 to 8000. PEG 600 was chosen for the present work because of its lower viscosity. Seeds stable for several hours could be prepared by washing previously grown crystals with 32% (v/v) PEG 600. If stored for more than a day, the seeds formed aggregates which eventually appeared as a cluster of crystals growing in different orientations. When freshly prepared see is were added to solutions of 40 mg of protein per ml in 25% PEG 600, however, microcrystals with distinctive polyhedral habits could be observed at x 250 magnification in 20-30 min. After 1 2 h. an optional number of these microcrystals could be transferred to ''sitting drops'' of protein in PEG 600 for the final growth period of 2-10 days. The shapes of the crystals could be controlled by adjusting the concentration of PEG 600. In 22% PEG the crystals assumed their ''equilibrium shape'' of a bipyramid; at 25% the crystals were truncated bipyramids; and at higher concentrations the crystals were small cubes or even unstable rounded forms associated with ''normal growth'' in all directions. If one end was in contact with a glass or plastic surface, the crystal developed into a pyramid instead of a bipyramid. The system could be further manipulated to study growth rates, as well as the processes leading to the formation of depressions, dislocations and skeletal crystals.