Protein crystallization solubility determination

Insulin suspensions. When the hormone is injected as a suspension of insulin-containing particles, its dissolution and release in subcutaneous tissue are retarded (rapid, intermediate, and slow insulins). Suitable particles can be obtained by precipitation of apolar, poorly water-soluble complexes consisting of anionic insulin and cationic partners, e.g the polycationic protein protamine or the compound aminoqui-nuride (Surfen). In the presence of zinc and acetate ions, insulin crystallizes crystal size determines the rate of dissolution. Intermediate insulin preparations (NPH or isophane, lente or zinc insulin) act for 18 to 26 h, slow preparations (protamine zinc insulin, ultralente or extended zinc insulin) for up to 36 h. 

An exception to the approach for determining protein solubility, that is based on concentrating the protein, occurs when the protein can be crystallized, in which case, the solubility of the protein is established by the protein concentration in the solution phase that is in equilibrium with the crystals. Though the crystals could also be dried and dissolved in other solvent systems to determine solubility in those systems, protein crystals carry salt and other ions, and may render different solubilities depending on the method of preparation. 

The selectivity of ion channels is often determined by only a handful of amino acids. Thus, the amino acids Glu-Glu-Glu-Glu (EEEE) determine the selectivity of the calcium channel of the heart [9] the amino acids Asp-Glu-Lys-Ala (DEKA) determine the selectivity of the sodium channel of nerve and muscle cells [10]. The structure of ion channels is notoriously difficult to determine because channel proteins do not easily crystal-hze. They are normally found in hpid membranes, and so methods suitable for crystallizing soluble proteins are not too helpful. Several structures have been determined, most notably, the KcsA potassium channel [4]. 

There are two types of electron transport those involving flavoproteins and iron-sulfur proteins, and those requiring only flavoproteins. The X-ray crystal structure of the soluble cytochrome P450 from Pseudomonas putida grown on camphor (P-450-CAM) has been determined (Poulos et ah, 1985), as have several others. The haem group is deeply embedded in the hydrophobic interior of the protein, and the identity of the proximal haem iron ligand, based on earlier spectroscopic studies (Mason et ah, 1965) is confirmed as a specific cysteine residue. 

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