Method and apparatus for two-dimensional measurement of concentration of biological macromolecule solution

Abstract: A method of making a two-dimensional measurement of the concentration of a biological macromolecule aqueous solution and an apparatus for carrying out this method are disclosed. By charging the biological macromolecule aqueous solution and an aqueous crystallizing agent solution into a crystallizing cell, irradiating the crystallizing cell with an ultraviolet light from an ultraviolet light source, two-dimensionally scanning the ultraviolet light transmitted through the crystallizing cell by a linear sensor to detect the quantity of the transmitted light, and carrying out a conversion of the detected quantity of the transmitted light to the protein concentration, the process of a crystallization of the biological macromolecule from biological macromolecule aqueous solution can be easily and accurately two-dimensionally measured.

Description: The present invention relates to a method and an apparatus for making a two-dimensional measurement of the concentration of a biological macromolecule solution. Such a method and apparatus enable the crystallization of a biological macromolecule to be observed and two-dimensionally scanned. Biological macromolecules such as proteins, polypeptides and nucleic acids exert important biological functions such as energy metabolism, structure construction and information transmission. It is known that these functions of biological macromolecules result from the structures thereof and, therefore, the need for investigation of the three-dimensional structure of biological macromolecules has increased. In general, NMR (nuclear magnetic resonance), X-ray diffraction, and the like are used for analyzing the three-dimensional structure of biological macromolecules, although X-ray diffraction is now attracting particular attention because it enables the three-dimensional structure of a biological macromolecule to be directly analyzed. To accomplish X-ray diffractometry, it is important to obtain a biological macromolecule crystal having a structure of reduced disorder and a high quality. However, it is difficult routinely to obtain a high-quality crystal of a biological macromolecule because the crystallization conditions differ between respective biological macromolecules, and thus the optimum conditions must be determined by trial and error. Furthermore, since knowledge about the growth process conditions of crystal nuclei of biological macromolecule crystals is limited, it is difficult to obtain a biological macromolecule crystal having a high quality. Recently, trials have been initiated to examine in detail the growth process of a crystal of a biological macromolecule, especially a protein (see, for example, Azuma et al., J. Crystal Growth, 98 , 371-376, 1989). In this thesis, the process of crystallization of a protein is traced by measuring changes of the refractive index in the vicinity of the crystal, by utilizing a Mach-Zehender interference optical system. According to this method, it becomes possible to carry out a two-dimensional measurement of the concentration gradient in the diffusion boundary layer around a growing crystal as a function of concentration of lysozyme concentration. However, the method of measuring the protein concentration by the differential interference optical system has several problems. First, since the protein concentration is measured based on the refractive index, there is a risk of mistaking a change in the solute concentration in a crystal mother liquor for a change in the protein concentration. Second, since it is necessary to measure the space between interference fringes in the vicinity of the crystal and the mother liquor having a concentration gradient in the diffusion boundary layer around a growing crystal interface to convert the refractive index to the protein concentration, a complicated operation such as an image processing is necessary by transforming the refractive index gradient using Lorentz-Lorentz formula. A two-dimensional measuring apparatus utilizing ultraviolet rays recently has been developed as an analysis and measurement apparatus. An example of the two-dimensional measurement apparatus utilizing ultraviolet rays is an ultraviolet microscope. An apparatus is known which is constructed by combining this ultraviolet microscope with a microscope photometer (MPM) system, so that intercellular calcium concentration and DNA quantity can be measured. For example, an MPM System is supplied by Carl Travis Co. This apparatus, however, is not designed for the analysis of a biological macromolecule crystal, and it comprises a heavy, large, and delicate specific optical system. Thus, although a point light measurement of microscope system can be performed, it is not possible to perform promptly a light measurement over a wide range. Accordingly, the development of a method of two-dimensionally tracing the crystallization process of a biological macromolecule, easily and promptly, and of measuring the concentration of a biological macromolecule concentration, and a simple and small apparatus for carrying out this method, is desired. Schlichta. J. Crystal Growth, 76 , No. 3, 656-662, 1986 reviews, inconclusively, the feasibility of using optical techniques for mapping the convection, temperature, and solute concentration in the solution around a growing protein crystal. The present invention has been completed as the result of research made with a view to solving the foregoing problems, and an object of the present invention is to provide a method of two-dimensionally scanning the concentration of a biological macromolecule in a biological macromolecule aqueous solution, more specifically the crystallization process of a biological macromolecule from biological macromolecule, such as myoglobin, aqueous solution, and a simple apparatus for carrying out this method. In accordance with one aspect of the present invention, there is provided a method of measuring in two dimensions the concentration of a solution of a biological macromolecule, which comprises: supplying said solution of a biological macromolecule and a solution of a crystallizing agent into a crystallizing cell; irradiating the crystallizing cell with ultraviolet light from an ultraviolet light source; detecting the quantity of ultraviolet light transmitted through the crystallizing cell in two dimensions by scanning the crystallizing cell using (a) a linear image sensor and a moving unit for moving the linear image sensor or (b) a two-dimensional image sensor; and converting the detected quantity of transmitted light to the concentration of said solution of a biological macromolecule. In accordance with another aspect of the present invention, there is provided apparatus for the measurement in two dimensions of the concentration of a biological macromolecule solution, which comprises: an ultraviolet light source unit, a crystallizing cell, an absorbance-detecting unit comprising (a) a linear image sensor and a moving unit for moving the linear image sensor or (b) a two-dimensional image sensor, and a computing and controlling unit for a subtraction or differentiation of the quantity of the transmitted light from the absorbance-detecting unit, the ultraviolet light source unit, the crystallizing cell and the absorbance detecting unit being arranged in the recited order. For a better understanding of the invention, and to show how the same may be carried into effect, reference will now be made, by way of example, to the accompanying drawings, in which: Figure 1 illustrates the structure of apparatus in accordance with the present invention; Fig. 2(A) illustrates a protein-crystallizing cell used in the free interface method, and Fig. 2(B) illustrates a protein-crystallizing cell used in the dialysis diffusion method; Fig. 3 is a perspective view of a main part of one embodiment of the present invention; and, Fig. 4 illustrates the point of measuring the width of a quartz cell. In the present invention, it has been noted that many biological macromolecules show peculiar absorbances to light of the ultraviolet ray region, and thus the present method and apparatus are constructed so that two-dimensional scanning of the ultraviolet light absorbance by detecting the light transmitted through the cell can be accomplished. Therefore, the measurement of the process of growth of a protein crystal, more specifically the protein concentration, can be performed two-dimensionally. The subject of the measurement in the present invention includes proteins and nucleic acids. An inorganic salt such as a neutral salt or an organic compound such as an alcohol is preferably used as the crystallizing agent. For example, inorganic salts such as ammonium sulphate, NaCℓ, KCℓ, sodium sulphate, a phosphate and caesium chloride are preferably used, and organic compounds such as polyethylene glycol, water-miscible solvents (for example, methanol and ethanol), dioxane and methylpentane-diol are preferably used. The present invention will now be described in detail with reference to the following examples, that by no means limit the scope of the invention. Example 1 Figure 1 illustrates a block diagram of the structure of apparatus in accordance with one embodiment of the invention. As seen from this structural diagram, the apparatus comprises a light source unit capable of uniformly irradiating a measurement range with a specific ultraviolet light of, for example, 280nm or 254nm, a cell for carrying out crystallization, a detecting unit (absorbance-detecting unit) for measuring a change in the biological macromolecule concentration, caused by nucleus formation and/or crystal growth, and a computing and controlling unit for converting the output of the detecting unit to the concentration of the biological macromolecule. A linear image sensor-moving or driving unit is connected to the absorbance-detecting unit, as described below, to co-operate with the computing and controlling unit. The ultraviolet light source unit comprises an ultraviolet light source and a spectral element. For example, a deuterium discharge tube and a low-pressure mercury lamp are used as the light source. This low-pressure mercury lamp has a life time of about 10,000 hours. The spectral element comprises, for example, a monochromator or an interference filter. A stabilizing power source unit is connected to the ultraviolet light source unit, to maintain the voltage at a constant level. The stabilizing power source device can be fabricated by utilizing a known fluorescent lamp-lighting circuit. An ultraviolet light having a specific wavelength necessary for the measurement of the concentration is obtained in the following manner. Namely, a light having a wavelength of about 280 nm (wavelength specific to a protein) is obtained by using phosphor and an interference filter, and an emission line having a wavelength of 254 nm, generated by a low-pressure mercury lamp, is used as the light having a wavelength of 260 nm (wavelength specific to a nucleic acid). An ultraviolet light-transmitting face (4 mm x 10 mm) of a crystallizing cell described below is uniformly irradiated with the above-mentioned light source device. The protein-crystallizing cell is designed to be used for the free interface method and the dialysis diffusion method, having a different crystal growth system, as described below with reference to Fig. 2. Figure 2(A) illustrates a protein-crystallizing cell used for the free interface method, and Fig. 2(B) illustrates a protein-crystallizing cell used for the dialysis diffusion method. According to the free interface method, a liquid-liquid interface is formed between the aqueous solution of the crystallizing agent and the aqueous solution of the protein, to form a concentration gradient. In this method, branches 11, 12 are arranged in the crystallizing cell 1, as shown in Fig. 2(A), independently to introduce the aqueous crystallizing agent solution and the aqueous protein solution into the crystallizing cell 1. A partition plate 2, 2' is attached to each branch 11, 12. One end of the cell 1 is opened to discharge the overflowing solution. According to this method, the partition plate 2 is opened to allow the aqueous crystallizing agent solution to fill the cell 1 through a conduit 11 arranged at one end of the crystallizing cell 1. The partition plate 2 is then closed and the partition plate 2' is opened to allow the aqueous protein solution to flow into the cell 1 through the branch 12. When the liquid-liquid interface of the crystallizing agent solution and the protein solution is located almost at the center of the cell 1, the partition plate 2' is then closed. The crystallizing agent solution overflowing during this operation is discharged from a drain tube 13. According to the dialysis diffusion method, a concentration gradient is formed between both sides of a membrane. In this method, as shown in Fig. 2(B), a permeable membrane 3 is attached at almost the center of the crystallizing cell 1'. The pore size of the permeable membrane 3 is such that passage of the crystallizing agent therethrough is allowed but passage of the protein is not allowed. The interior of the cell is divided into two chambers 1P', 1A' by this permeable membrane 3. One end 12 of the cell is opened to introduce the protein solution into chamber 1P' and an opening 15 for discharging the overflowing solution is provided. The other end 11 of the cell is opened to introduce the crystallizing agent solution into the chamber 1A' and an opening 14 is provided to discharge the overflowing solution [see Fig. 2(B) and Fig. 3]. According to this method, for example, the experiment can be carried out while simultaneously introducing the crystallizing agent solution and the protein solution into the cell 1'. In each of the foregoing methods, a gradient of the protein concentration is formed from the vicinity of the liquid-liquid free interface region or the permeable membrane and, by procedures described below, the point of the concentration gradient where a crystal of the protein is first formed is determined. Obviously, a method other than the above-mentioned methods, such as a stationary batchwise method, can be similarly adopted. In the present example, a quartz glass is used as the material of the cell for measuring the ultraviolet light absorbance. In view of the used protein concentration (ordinarily 1 to 5 mg/mℓ), the cell thickness (optical path length) is adjusted to 2 mm. In view of the size of a primary scanning sensor, the length and width of the crystallizing cell are adjusted to 10 mm and 4 mm respectively. Because of poor processability of the quartz glass, a rectangular parallelepiped shape is adopted for the cell. The amounts of the protein solution and the crystallizing agent solution used are preferably 0.3 mℓ and 1.5 mℓ, respectively, in the dialysis diffusion method, and each of the amounts of both solutions is preferably 0.3 mℓ in the dialysis diffusion method. A high ultraviolet ray spectral sensitivity and a good space resolving power are required for the absorbance-detecting unit. Accordingly, a linear image sensor (for example, S3923-1024Q supplied by Hamamatsu Photonics) is used. This sensor can scan a range of 25.6 mm at a resolving power of 25 µm. A drive circuit C4704 for the MOS Linear Image Sensor and a Pulse Generator C4091, each supplied by Hamamatsu Photonics, are used as the drive circuit. The output is taken into a storage oscilloscope. To scan a two-dimensional region of 4 mm x 10 mm by the linear image sensor, it is necessary to move the linear image sensor at a pitch of 25 µm, and thus a mechanical stage for a microscope (not shown) is used. By using this apparatus, the region of 4 mm x 10 mm can be scanned at a pitch of 25 µm (see Fig. 3). The computing and controlling unit controls the movement of the linear image sensor by controlling and driving the mechanical stage and the computation of the output from the ultraviolet ray-detecting portion. The computation in the ultraviolet ray-detecting portion is a differentiation or subtraction between the output signal of the empty crystallizing cell and the output signal of the cell at the time of measurement. By this computation, the irradiation intensity unevenness can be eliminated. Furthermore, in this computing unit, the conversion of the absorbance to the concentration can be performed. Example 2 In this example, a method of measuring the protein concentration of an aqueous solution of myoglobin is illustrated. The experiment was carried out according to the free interface method wherein an aqueous ammonium sulphate solution (97% saturation) (used as the crystallizing agent) and a 1.0% aqueous myoglobin solution (dissolved in 55% saturation ammonium sulphate aqueous solution) were independently charged in syringes, were connected through a three-way cock, and were put into a quartz glass cell [see Fig. 2(A)] in sequence. In this way, a liquid-liquid interface is formed between the aqueous ammonium sulphate solution and the aqueous myoglobin solution. The system was kept stationary and the experiment was carried out at room temperature. The quantity of transmitted light of the empty quartz cell was measured just before injection of the aqueous solutions into the quartz cell, and the linear image sensor was moved intermittently at a pitch of 25 µm by the mechanical stage. Measurement was conducted over the entire region of 4 mm x 10 mm of the quartz cell, to obtain data of 64,000 points. The measurement points in the quartz cell are shown in Fig. 4. The obtained data was subjected as the quantity of the transmitted light in the empty cell to 16-bit analog-digital (AD) conversion and was stored in a microcomputer. After the start of the experiment, data was obtained at intervals of 2 hours, to trace the crystallization process. Data obtained at the respective measurement points was accumulated as common logarithms (absorbances A) of the ratio of the data in the empty cell (Io) to the transmitted light quantity (I) at each time. The absorbance can be converted to the molar concentration of the protein according to the following Beer-Lambert equation: log (Io/I) = A = k x C x ℓ wherein k is the molar absorption constant, c represents the molar concentration, and ℓ is the optical path length. More simply, the conversion can be performed by using the formula of protein concentration (mg/mℓ) = 1.45 x A. The value of 1.45 in the above-mentioned formula is the constant for converting the absorbance to the concentration expressed in the unit of mg/mℓ. The values of absorbance obtained after an elapse of 24 hours and 48 hours from the point of the start of the experiment are shown in Tables 1 and 2. The value (4250) given at the corner of each of Tables 1 and 2 indicates the left-upper measurement point, and a symbol " " indicates a scale-out. From the results shown in Table 1, the following can be seen. The crystallization of myoglobin starts after the elapse of about 24 hours from the initiation of the experiment. The crystallization begins in the form of a band at a position 2 to 3 mm apart from the liquid-liquid interface (see absorbance values at measurement points of 4253 to 4255). The formation of a crystal nucleus in the protein solution is judged from a sharp rise of the absorbance and a scale-out of the absorbance (because the measurement becomes impossible) (see absorbance values at measurement points of 4653, 4654, 4655, 5051, 5053 and 5054 in Table 2). Also, the increase of the protein concentration in the protein solution is observed in the peripheral portion of the crystal nucleus. The manner of the growth of the crystal nucleus can be confirmed by tracing the number of measurement points occupied by the crystal. In Table 2, it is seen that the crystal nucleus grows at the scale-out point. According to the present invention, due to the above-mentioned structure, various effects as mentioned below can be attained. First, the process of crystallization of a biological macromolecule can be two-dimensionally traced. Note, by using apparatus of certain embodiments in accordance with the present invention, the process of crystallization of a biological macromolecule such as a protein can be measured at a space resolving power of 25 µm and a concentration difference resolving power of 1/100 of the initial concentration within a dynamic range of 5 to 50 mg/ml. The optical system of the apparatus of the present invention is much simpler than that of the conventional apparatus such as MPM system for measuring the protein concentration, and the size thereof can be reduced and the weight lowered. Therefore, a measurement error caused by a change of the salt concentration, which is a defect of the conventional apparatus, can be completely eliminated. Furthermore, conversion to the concentration can be performed more easily than in the conventional apparatus. Still further, the apparatus of the present invention can be applied to various crystallization processes.

Claims: A method of measuring in two dimensions the concentration of a solution of a biological macromolecule, characterized by the steps of: supplying said solution of a biological macromolecule and a solution of a crystallizing agent into a crystallizing cell; irradiating the crystallizing cell with ultraviolet light from an ultraviolet light source; detecting the quantity of ultraviolet light transmitted through the crystallizing cell in two dimensions by scanning the crystallizing cell using (a) a linear image sensor and a moving unit for moving the linear image sensor or (b) a two-dimensional image sensor; and converting the detected quantity of transmitted light to the concentration of said solution of a biological macromolecule. A method as claimed in claim 1, wherein the solution of a biological macromolecule is a protein solution. A method as claimed in claim 1 or claim 2, wherein the quantity of transmitted light is detected by a one-dimensionally arranged photodiode. A method as claimed in claim 1, 2 or 3, wherein the detected quantity of transmitted light is converted to the concentration of said solution of a biological macromolecule by calculating the light absorbance in the crystallizing cell from the ratio of the detected quantity of transmitted light from the crystallizing cell to the detected quantity of transmitted light from an empty crystallizing cell, and converting the calculated absorbance into the concentration of the biological macromolecule solution. A method as claimed in claim 4, wherein said sensor is arranged to calculate said absorbance. A method as claimed in claim 1, wherein the quantity of transmitted light is detected with respect to an empty measurement cell, and with respect to each picture element scanned, a differential computation being performed while holding measurement data thereby obtained. A method according to any preceding claim, wherein said solution of said biological macromolecule is an aqueous solution and said solution of crystallizing agent is an aqueous solution. Apparatus for the measurement in two dimensions of the concentration of a biological macromolecule solution, comprising an ultraviolet light source unit, a crystallizing cell, an absorbance-detecting unit comprising (a) a linear image sensor and a moving unit for moving the linear image sensor or (b) a two-dimensional image sensor, and a computing and controlling unit for a subtraction or differentiation of the quantity of the transmitted light from the absorbance-detecting unit, the ultraviolet light source unit, the crystallizing cell and the absorbance detecting unit being arranged in the recited order. Apparatus as claimed in claim 8, wherein the ultraviolet light source unit comprises an ultraviolet ray source and a spectral unit. Apparatus as claimed in claim 8 or claim 9, wherein the ultraviolet light source unit is a deuterium discharge tube or a low-pressure mercury lamp. Apparatus as claimed in claim 9 or claim 10, wherein the spectral unit is a monochromator or an interference filter.

Inventors: ASANO KOJI, FUJITA SHOZO, OKA KOTARO, ASANO, KOJI, FUJITA, SHOZO, OKA, KOTARO, ASANO, KOJI, C/O FUJITSU LIMITED, Fujita, Shozo, c/o Fujitsu Limited, OKA, KOTARO, C/O FUJITSU LIMITED
Applicants: FUJITSU LTD, FUJITSU LIMITED
Application year: 1991
Main classification: C30B7
IPC codes: G01N25, G01N21, B01D9, G01N33