Chemistry: analytical and immunological testing – Rate of reaction determination
Reexamination Certificate
2000-05-22
2002-08-06
Snay, Jeffrey (Department: 1743)
Chemistry: analytical and immunological testing
Rate of reaction determination
C436S086000, C436S094000, C436S171000
Reexamination Certificate
active
06429015
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to a method for identifying active substances and a device for executing the method.
In “Fourier-Transform Infrared Spectroscopic Studies on Avidin Secondary Structure and Complexation with Biotin and Biotin-Lipid Assemblies,” Biophysical Journal Vol. 71 (1996), pp. 840-847, M. J. Swamy, T. Heimburg and D. Marsh describe efforts to explain the structure of complexes of the protein avidin with biotin and biotin-lipid through Fourier-transform infrared spectroscopy (FTIR). In this work, FTIR spectra of the avidin are first recorded in heavy water (D
2
O). Then, avidin with biotin or biotin-lipid is mixed with buffered D
2
O as a solvent and stored for several hours at room temperature, which apparently should produce the highest yield of the resulting avidin complex. The FTIR spectra of the complex are recorded again. Differential spectra are formed from the spectra of the avidin and the spectra of the avidin complex. Because this work only focuses on the structure of the avidin complexes, no time-dependent spectra are recorded. In all instances, the described FTIR spectra reflect states of equilibrium. The vibrational spectra of bonded deuterium is recorded—shifted due to the higher mass—as opposed to the vibrational spectra of bonded, normal hydrogen.
Consequently, a comparatively thick cuvette (50 &mgr;m) can be used. The question of whether a chemical compound, such as a protein, may form at a coordination point with a specific ligand is of no consequence in this work, because the fact that a complex of avidin with biotin forms was already known.
FTIR studies for determining structures of protein complexes are also described by M. Gonzales, et al. in “Interaction of Biotin with Streptavidin,” The Journal of Biological Chemistry, Vol. 272, No. 17 (1997), 11288-11294. The spectra were recorded with an H
2
O buffer as well as a D
2
O buffer; in the case of H
2
O, the layer thickness was 6 &mgr;m, and 50 &mgr;m in the case of D
2
O. The goal of the study was to ascertain the thermal stability of biotin and the biotin-streptavidin complex. The thermal denaturing was represented in chronologically consecutive spectra. In contrast, the formation of the complexes was not investigated with spectrometry.
In “Redox-linked conformational changes in proteins detected by a combination of infrared spectroscopy and protein electrochemistry—Evaluation of the technique with cytochrome c,” Eur. J. Biochem. 187, 565-572 (1990), D. Moss, E. Nabedryk, J. Breton and W. Mantele report on an electrochemical reduction and a subsequent re-oxidation of the protein cytochrome c. Cytochrome c is provided in a layer thickness of 10 to 15 &mgr;m to preclude the IR absorption of water in the medium infrared range. The reduction and subsequent re-oxidation were proven with the aid of FTIR spectroscopy. Differential spectra of the reduced and re-oxidized state are shown. Because the cuvette only contained cytochrome c, no definitive statements could be made about the formation of protein complexes.
The publication by A. J. White, K. Drabble and C. W. Wharton: “A stopped-flow apparatus for infrared spectroscopy of aqueous solutions,” Biochem. J. (1995) 306, 843-849 describes an apparatus for executing the so-called “stopped-flow” method, in which the reagents are sprayed into a cuvette with sprayers, and mixed. According to the authors, HPLC valves have proven unsuitable due to the necessary high pressure and the high viscosity of peptides. This apparatus was used to record FTIR spectra in the temporal range of 6.25 seconds to 966 seconds after the mixing of 12C═O- and 13C═O-cinnamoyl chymotrypsin with a deacylating agent in a D
2
O buffer; the optical layer thickness was 50 &mgr;m. Differential spectra were formed from the spectra of 12C═O- and 13C═O-cinnamoyl chymotrypsin. The “Conclusions” include the statement that it is not possible to create a “stopped-flow” IR transmission cuvette that permits the use of (non-deuterated) water, because the heavy absorption of water at 1640 cm
−
requires a layer thickness of 5 &mgr;m (the writings incorrectly state ‘5 mm’).
Q. H. Gibson and L. Milnes provide a detailed description of the “stopped-flow” method in “Apparatus for Rapid and Sensitive Spectrophotometry,” Biochem. J. (1964) 91, 161-171.
The large dead volume of the apparatuses due to the use of sprayers is a general drawback of the “stopped-flow” methods. Mass-screening methods, therefore, cannot be implemented with such apparatuses, notably because the microtitration plate provided with 96 depressions of 400 &mgr;l each is the standard model for automated methods; refer to J. R. Broach and J. Thorner: “High-throughput screening for drug discovery,” Nature Vol. 384 Supp. Nov. 7, 1996, which offers an overview of mass-screening methods. With regard to ascertaining the bondability of a ligand to the receptor of a peptide, Broach and Thorner cite a method in which Eu
2+
at the ligand and allophycocyanin at the receptor are covalently bonded. Through the formation of a receptor-ligand complex, Eu
2+
closely approaches allophycocyanin, resulting in an energy transfer that can be detected as a fluorescence signal.
SUMMARY OF THE INVENTION
It is an object of the invention to develop a method for identifying active substances that permits the low-cost detection of the formation of complexes between reactants in the smallest-possible volume, flexibly and quickly and with reproducible results. The method is also intended to have the capability of being automated. It is a further object to provide a device for executing the method.
The above and further objects are accomplished according to the invention by the provision of a method comprising: mixing at least two reactants that form a reactant complex; recording an IR spectrum of individual reactants that have not yet been converted in the mixture at a first time; recording at least one further IR spectrum at a second time for detecting the reactant complex; forming a differential spectrum for the two IR spectra recorded at different times; and selecting the reactants whose differential spectrum has a band structure as active substances.
According to the invention, the active substances are identified through the investigation of the bondability of a reactant, such as a ligand, to at least one further reactant, such as a protein. The reactants produce a mixture from which an IR or FTIR spectrum is recorded at least at two different times. The ligand measurement can be effected in an aqueous solution, in which case the conventional use of deuterated solutions, such as deuterated water or a deuterated buffer, is not absolutely necessary. Depending on the viscosity and the physical-chemical properties of the reactants, the use of a different or further solvent may be indicated; a deuterated buffer or deuterated solvent can be omitted.
The mixture can be produced in accordance with the cited state of the technology. The use of high-pressure pumps (up to about 400 bar) and the loop valves known from HPLC technology is preferred, however.
The mixture should preferably be applied in a layer thickness of 1 to 25 &mgr;m, especially 8 to 15 &mgr;m. The mixture is advantageously produced on the way to and/or in an IR cuvette with a corresponding optical thickness.
Usually, one endeavors to produce a complete mixture from the organic compound and the reagent. Because most of the reactions of organic compounds take place slowly, the time required to produce an optimum mixture and record the first IR or FTIR spectrum is typically sufficiently short. If, however, the speed of reactions between the reactants is high, it may be advisable to record the first IR or FTIR spectrum with an incomplete mixture to prevent a substantial reaction conversion at this time.
In the recording of the first IR or FTIR spectrum, the reactants must still be at least partially unconverted, so the formation of the complex can be detected in the second or further IR or FTIR spectrum. Ideally, the reaction of the reactan
Füchsle Kathrin
Masuch Ralf
Moss David
Platsch Herbert
Forschungszentrum Karlsruhe GmbH
Kinberg Robert
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