THE CRYSTALLIZATION OF BIOLOGICAL MACROMOLECULES FROM PRECIPITATES - EVIDENCE FOR OSTWALD RIPENING

Crystals were obtained by different methods under conditions where nuc leation and growth occur from precipitated macromolecular material. Th e phenomenon was observed with compounds of different size and nature, such as thaumatin, concanavalin A, an alpha-amylase, a thermostable a spartyl-tRNA synthetase, the nucleo-protein complex between a tRNA(Asp ) transcript and its cognate yeast aspartyl-tRNA synthetase, and tomat o bushy stunt virus, In each system, after a rather rapid precipitatio n step at high supersaturation lasting one to several days, a few micr ocrystals appear after prolonged equilibration at constant temperature . With alpha-amylase, the virus and the thermostable synthetase, cryst allization is accompanied by appearance of depletion zones around the growing crystals and growth of the largest crystals at the expense of the smaller ones. These features are evidences for crystal growth by O stwald ripening. In the case of thaumatin, concanavalin A and the nucl eo-protein complex, crystallization occurs by a phase transition mecha nism since it is never accompanied by the disappearance of the smalles t crystals. A careful analysis with thermostable aspartyl-tRNA synthet ase indicates that its crystallization at 4 degrees C under high super saturation starts by a phase transition mechanism with the formation o f small crystals within an amorphous protein precipitate. Ostwald ripe ning follows over a period of up to three/four months with a growth ra te of about 0.8 Angstrom/s that is 13 times slower than that of crysta ls growing at 20 degrees C in the absence of precipitate without ripen ing. At the end of the ripening process at 4 degrees C, only one uniqu e synthetase crystal remains per microassay with dimensions as large a s 1 mm.