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.

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Insulin (INS) was encapsulated into complexes with poly(ethylene glycol)-block-poly(L-lysine) (PEG-b-PLys), which is a polypeptide-based block copolymer (a neutral-cationic block polyelectrolyte). The particular cationic-neutral block copolymer can complex INS molecules in aqueous media via electrostatic interactions. Light-scattering techniques are used to study the complexation process and structure of the hybrid nanoparticles in a series of buffers, as a function of protein concentration. The physicochemical and structural characteristics of the complexes depend on the ionic strength of the aqueous medium, while the concentration of PEG-b-PLys was constant through the series of solutions. As INS concentration increased the size distribution of the complexes decreased, especially at the highest ionic strength. The size/structure of complexes diluted in biological medium indicated that the copolymer imparts stealth properties and colloidal and biological stability to the complexes, features that could in turn affect the clearance properties in vivo. Therefore, these studies could be a rational roadmap for designing the optimum complexes/effective nanocarriers for proteins and peptides.

Modifying the classical double pulse technique, by using a supersaturation gradient along an insulin solution contained in a glass capillary tube, we found conditions appropriate for the direct measurement of nucleation parameters. The nucleation time lag has been measured. Data for the number of crystal nuclei versus the nucleation time were obtained for this hormone. Insulin was chosen as a model protein because of the availability of solubility data in the literature. A comparison with the results for hen-egg-white lysozyme, HEWL was performed.

Nucleation of lysozyme crystals in quiescent solutions at a regime of progressive nucleation is investigated under an optical microscope at conditions of constant supersaturation. A method based on the stochastic nature of crystal nucleation and using discrete time sampling of small solution volumes for the presence or absence of detectable crystals is developed. It allows probabilities for crystal detection to be experimentally estimated. One hundred single samplings were used for each probability determination for 18 time intervals and six lysozyme concentrations. Fitting of a particular probability function to experimentally obtained data made possible the direct evaluation of stationary rates for lysozyme crystal nucleation, the time for growth of supernuclei to a detectable size and probability distribution of nucleation times. Obtained stationary nucleation rates were then used for the calculation of other nucleation parameters, such as the kinetic nucleation factor, nucleus size, work for nucleus formation and effective specific surface energy of the nucleus. The experimental method itself is simple and adaptable and can be used for crystal nucleation studies of arbitrary soluble substances with known solubility at particular solution conditions.

The degree of adhesion of protein crystals, heterogeneously nucleated and grown on different supports (e.g. glass plates and plates coated with poly-L-lysine, hexamethyl-disilazane and silicon) is measured directly with a purposely-developed technique. The sticking force crystal/support is determined by means of a flexible glass fibre, which bending is calibrated by means of series of weights. In this way an elastic constant, specific for each glass fiber is determined individually. Appropriate glass fibres with relative bending less than 10% (Hook's law) are used. The force which is necessary to be exerted, by means of a micro-manipulator, in order to detach the crystal from the support is taken as a quantitative measure for the adhesion strength. Forces between 10 N cm -2 and 1 N cm -2 for differently oriented tetragonal hen-egg-white lysozyme and cubic ferritin crystals, and 0.1 N cm -2 for rhombohedral (porcine) insulin and orthorhombic trypsin crystals are measured. The tetragonal HEWL and rhombohedral insulin crystals show anisotropy of the adhesion strength. In contrast, the cubic ferritin crystals are isotropic also in this respect. For comparison purposes adhesion measurements are performed with NaCl and sugar crystals. An attempt is made to evaluate also the adhesion energy of the protein crystals.

Crystals from apoferritin which is an iron-free form of protein ferritin were obtained from protein mixtures lysozyme/apoferritin using sedimentation under high gravity. Solution containing apoferritin at concentration as high as 5mg/ml in the presence of 25mg/ml lysozyme and overlaid on 5%(w/v) CdSO 4 in 0,2M/L NaAC, pH=5 still favors apoferritin crystal formation under normal gravity conditions, but at apoferritin concentrations <0,5mg/ml (~1,14µM/L) in 25mg/ml (~1,71mM/L) lysozyme only the sedimentation in a centrifuge appears to be useful for separating the apoferritin molecules from the mixture followed by apoferritin crystallization in the same system. The very high molecule number ratio (~1:103 ) of two proteins is used to stress on the observed effect.

We report on the use of alternating crystallization for deposition of layers of different (though closely related) proteins in a single crystal. Investigations were carried out with the unique protein couple consisting of two forms of ferritin, apoferritin and holoferritin from horse spleen, which, despite being of quite different molecular masses, still possess identical organic shells. Crystals of both proteins were used as substrates for subsequent contiguous growth of the partner protein in perfect alignment. We observed continuous growth of combined (onion-like) single crystals; artificial structures of biological macromolecules can be designed in this way. The homoepitaxial layered growth shows in an unambiguous way that protein crystallization depends only on the surface protein conformation and amino-acid composition, but not on the internal molecule structure. The limitations of protein crystal growth for designing layered structures of biological macromolecules were revealed by growing of heterogeneous protein crystals onto pre-existing protein crystalline substrates. Tetragonal crystals of hen egg-white lysozyme were grown onto cubic apoferritin crystals used as substrates. It was observed that the lysozyme crystals were not lattice-matched to the 'host' apoferritin crystals; this led to mere aggregates of different crystals.