Chemical apparatus and process disinfecting – deodorizing – preser – Physical type apparatus – Crystallizer
Reexamination Certificate
2000-04-26
2001-07-10
Utech, Benjamin L. (Department: 1765)
Chemical apparatus and process disinfecting, deodorizing, preser
Physical type apparatus
Crystallizer
C422S253000, C117S206000
Reexamination Certificate
active
06258331
ABSTRACT:
TECHNICAL FIELD
The present invention relates to an apparatus for crystallization of macromolecules. Particularly, the present invention relates to the technique of providing an apparatus for crystallization of various biological macromolecules such as proteins and nucleic acids by the combination of a material such a semiconductor substrate whose valence electrons are controlled with a substrate of another material such as glass.
BACKGROUND ART
For the understanding of specific properties and functions in various types of biological macromolecules such as protein and complexes thereof, detailed steric structures thereof are indispensable information. From the basic biochemical standpoint, for example, information on the three-dimensional structure of protein or the like becomes the basis to understand the mechanism of functional expression in the biochemistry system with an enzyme or hormone. Particularly in the fields of pharmacy, genetic engineering, and chemical engineering among the industrial circles, the three-dimensional structure provides information indispensable for rational molecular design to facilitate drug designing, protein engineering, biochemical synthesis and the like.
As to the method of obtaining a three-dimensional steric structure of biological macromolecules at the atomic level, X-ray crystal structural analysis is the most cogent and precise means at present. The speed for analysis is significantly improving by virtue of the drastic increasing in the processing speed of computers in addition to reduction in the time for measurement and improvement in the measuring accuracy resulting from the recent hardware improvement of X-ray light sources and analyzers. It is expected that the three-dimensional structure will be clarified mainly depending upon this method.
In order to determine the three-dimensional structure of biological macromolecules by X-ray crystal structural analysis, it is essential to crystallize the target substance after extraction and purification. At present, there is neither technique nor apparatus that can be applied to every substance to achieve crystallization. In a conventional crystallization process, trial and error has been repeated relying on intuition and experience. In order to obtain a crystal of a biological macromolecule, the process for crystal growth has been required great numbers of experimental conditions, and it has been a serious bottleneck in the field of X-ray crystallographic analysis.
In a conventional process for crystallization of a biological macromolecule such as protein, a treatment for eliminating a solvent from an aqueous or non-aqueous solution containing the macromolecule is basically carried out, so that the resulting supersaturated state can reduce the solubility, leading to crystal growth. That is similar to the crystal growth process for low molecular weight compounds such as inorganic salts. As typical methods thereof, a batch method, dialysis, and diffusion are known. These methods are used depending upon the type, quantity, property, and the like of the sample.
In the batch method, a precipitant for eliminating the water of hydration is directly added to a solution containing a biological macromolecule to reduce its solubility, leading to its conversion into a solid phase. In this method, solid ammonium sulfate, for example, is often used. This method is disadvantageous in that a large amount of sample solution is required, fine adjustment of the salt concentration and pH is difficult, skill is required in operation, and reproducibility is low. In the dialysis method that is a improved one to eliminate some faults of the batch method, a solution including a biological macromolecule is filled in a sealed dialytic tube, and the pH or the like of the dialytic tube surrounding liquid such as a buffer solution is altered to induce crystallization. This method allows adjustment of the salt concentration and difference in pH of the inner and outer solutions at an arbitrary speed to facilitate the research for the crystallization conditions. One of the diffusion methods such as a gas-liquid phase diffusion method is shown in
FIG. 39. A
droplet
397
of a sample solution is placed on sample holders
393
a
and
393
b
. The droplet
397
and precipitant solutions
394
a
and
394
b
are retained in containers
391
a
and
391
b
, respectively, sealed by a cap
392
, whereby the volatile components of the droplet and the precipitant solutions are vaporized into equilibrium. More preferable conditions can be obtained using different precipitants in a plurality of containers as shown in the drawing. In a liquid-liquid phase diffusion method, a droplet
407
of the mother liquor including the target substance and a droplet
404
of a precipitant are placed approximately 5 mm apart on a substrate
401
, as shown in FIGS.
40
(
a
) and
40
(
b
). A thin liquid channel
406
is formed between the droplets by the tip of a needle or the like. Mutual diffusion through the channel
406
promotes crystallization. These diffusion methods are advantageous over the batch method in that the required amount of solution is extremely small.
However, there are still various problems as described above in the crystallization process of biological macromolecules such as proteins.
Many biological macromolecules do not have good crystallinity, so that a single crystal of them cannot be easily formed in a large size. This is probably because of the fact that the biological macromolecules generally having a great molecular weight are highly susceptible to gravity, causing convection in the solution (cf, F. Rosenberger, J. Cryst. Growth, 76, 618 (1986)). The small crystal nucleus of the biological macromolecules precipitates by its own weight, whereby convection occurs around the molecules or the crystal nucleus in the solution. The reduction in the concentration of the molecules also causes local convection at the surface of the grown crystal in the solution. The generated convection moves the grown crystal in the solution. Particularly around the crystal, the molecular supply layer is significantly reduced by the convection in the solution. Accordingly, the crystal growth rate is reduced, and anisotropic growth occurs at the crystal plane, so that crystallization is inhibited.
The crystal of biological macromolecules may contain a larger amount (≧50% by volume) of solvent (mainly water from mother liquor) as compared with the crystal of other substances. The solvent is disorderly and readily movable in the intermolecular clearances of the crystal. Though the size of the molecules is relatively large, there is little packing contact between the molecules in a wide range of the crystal, and the weak bond by the van der Waals force between the molecules or the hydrogen bond via the water molecule simply contributes to the contact. These are also related to the inhibited crystallization.
The biological macromolecules may be very sensitive to the conditions for crystallization. Although the biological macromoleculars may be stabilized in the solvent by the interaction between the molecular surfaces, the charge distribution on the molecular surface and particularly the conformation of amino acids around the molecular surface may significantly vary with the environmental factor such as pH, ion strength, and temperature of the solution, the type and dielectric constant of the buffer solution, and the like. Therefore, the crystallization is a multi-parameter process with a complicated combination of various conditions. Thus, a universal crystallization technique that can be effective for any substance is under development. Especially, crystallization of hydrophobic proteins that may have more biochemical interest as compared with water-soluble proteins is very difficult. Only a few cases have been successful in crystallization of hydrophobic proteins and the analysis thereof with high resolution.
The resulting amount of biological macromolecules may often be quite small. For example, when a protein such as an enzyme is extracted from
Anderson Matthew
Barnes & Thornburg
Sumitomo Metal Industries Ltd.
Utech Benjamin L.
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