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The nucleation of protein crystals is reconsidered taking into account the specificity of the protein molecules. In contrast to the homogeneous surface properties of small molecules, the protein molecule surface is highly inhomogeneous. Over their surfaces proteins exhibit high anisotropic distribution of patches, which are able to form crystalline bonds, the crystallization patch representing only a small fraction of the total surface of the protein molecule. Therefore, an appropriate spatial orientation of the colliding protein molecules is required in order to create a crystalline cluster. This scenario decreases considerably the success ratio of the attempt frequency for crystal nucleation. On the other hand a heterogeneous nucleation of (protein) crystals may be accelerated due to the arrival on some support of under-critical clusters that are formed in bulk solution; when arriving there they may acquire the property of critical nuclei. Thus, a plausible explanation of important peculiarities of protein crystal nucleation, as inferred from the experimental data, is suggested.

Number density of insulin crystals versus nucleation time dependences were measured simultaneously, during the same experiment, at four typical places: in solution bulk, at the glass support, at the air/solution interface, and at the solution/glass/air boundary. Stationary nucleation rates were determined from the linear parts of the corresponding plots, and energy barriers for nucleus formation and nucleus sizes were estimated. A key finding of the present investigation was that, surprisingly, the lowest energy barrier (3.8 Â 10 -13 erg), and correspondingly the smallest nucleus size (six insulin molecules), were calculated not for some kind of heterogeneous substrate, but for insulin crystals nucleated in the solution bulk; in both cases, the critical nuclei were formed from preliminary built Zn-insulin hexamers. The interpretation of these results is that no true homogeneous but rather heterogeneous (surface) insulin crystal nucleation is taking place also in the bulk solution. The nuclei form on some foreign particles of biological origin that are present in every protein solution.

Although they require surprisingly high supersaturations, both nucleation and growth of protein crystals proceed substantially more slowly as compared to small molecule crystallization. The slow nucleation of the protein crystals is explained by the steric restriction for association of the protein molecules that is due to their highly inhomogeneous surfaces. Over their surfaces, the proteins exhibit a limited number of discrete patches that are the only attractive portions on the molecule. A simple model for protein crystal nucleation has been devised on this basis, which enables calculations of nucleus form, energy barrier, and probability for nucleus formation. It turns out that the unusually high supersaturations used experimentally are necessary for the formation of relatively small nuclei, because the probability for bigger fluctuations is negligibly small. On the other hand, the big size of the protein molecules contributes additionally for the slow growth of the protein crystals. It causes huge Burgers vectors of the dislocations. In turn, this leads to a locally increased chemical potential and an effective decrease of the local supersaturation at the step source.

By using a supersaturation gradient along a protein solution contained in a glass capillary tube, we modified the classical double pulse technique, thus substantially accelerating the procedure of measurement of nucleation parameters. Data for the number of crystal nuclei, n vs nucleation time, t, were obtained for hen-eggwhite lysozyme, chosen as a model because of the availability of reliable solubility data in the literature. The stationary nucleation rate and the nucleation time lag have been measured. Quantitative data for the work required for nucleus formation (A k = 4.3x10 -13 erg) and the size of the critical cluster (three molecules) were also obtained. Besides, it was observed that Ostwald ripening seems to play an important role for nucleation times longer than 150 min. Using the same technique, semi-quantitative investigations were performed with porcine pancreatic trypsin.

The heterogeneous nucleation of hen-egg-white lysozyme (HEWL) crystals has been repeatedly investigated using a double-(thermal)-pulse technique, thus detaching nucleation from growth stage. n(t) dependencies of the nucleus number n, on templates of poly-L-lysine, vs time, t were plotted and the steady-state nucleation rates I were determined. They were compared with the results obtained earlier for surfaces rendered hydrophobic (by means of hexamethyl-disilazane) as well as for bare glass surfaces. In the present paper we determine the number of HEWL molecules in the (heterogeneously formed) critical nucleus. It turned out that it is build of 3 (to 4) HEWL molecules on glass substrate and 8 molecules for both hexamethyl-disilazane and poly-L-lysine templates. The energy A k required for heterogeneous formation of a critical nucleus on poly-L-lysine has been calculated, on the basis of the steady-state nucleation rates I. Intermolecular binding energy in the HEWL crystal lattice has been estimated again (approximately 10 -9 erg/molecule). This time the basis was the adhesion of HEWL crystals to poly-L-lysine substrate (compare D. TZEKOVA, S. DIMITROVA, C.N. NANEV, J. Cryst. Growth, 196 (2), 226 (1999)).