Data processing: measuring – calibrating – or testing – Measurement system in a specific environment – Chemical analysis
Reexamination Certificate
1998-05-01
2001-08-07
Hoff, Marc S. (Department: 2857)
Data processing: measuring, calibrating, or testing
Measurement system in a specific environment
Chemical analysis
C702S028000
Reexamination Certificate
active
06272436
ABSTRACT:
BACKGROUND OF INVENTION
1. Field of Invention
This invention relates to a new method of spectroscopic analysis to determine the fraction of conversion of a physical configuration of a system of interest from a first configuration to a second configuration, and in particular, a method to determine respective fractions of two different forms of a molecule in solution. It has broad applicability to the fields of medicine, agriculture, and biotechnology.
2. Description of the Related Art
The field of spectroscopic analysis seeks, among other characteristics, the configuration of molecules of interest. A molecule often studied is the protein and a configuration of interest is its three dimensional structure. As those in the art recognize, a protein exhibits a specific three-dimensional structure which is critical to activity and function. That three-dimensional structure (also known as the native or folded state) is sensitive to a variety of factors, such as pH, temperature, pressure, or the presence of a denaturant such as urea.
Spectroscopic analysis of proteins is made possible by a protein's ability to absorb light over a wide spectrum and to re-emit it in a characteristic fashion as well as the change in absorbance and emission due to a perturbation of the structure. The three most popular modes of spectroscopic analysis today are based on the absorbance/emission of visible light (the absorbance spectrum), differential absorbance of polarized light (circular dichroism), and fluorescent light. All three modes are sensitive to the three-dimensional structure of a protein. For example, fluorescence emission intensity can be used to gauge the change of molecular configuration as function of some parameter expected to effect the stability of the test molecule. As noted above, this parameter might be pH, some denaturant such as urea, or an extensive property of the system such as temperature or pressure. Practitioners have also used the ellipticity signal at 222 nanometers from circular dichroism (&thgr;
222
, CD) to estimate the extent of unfolding in proteins having a significant &agr;-helical content.
Nonetheless, each of these methods suffers in adequately testing the proportions of folded state. Fluorescence intensity analysis is highly temperature sensitive. Fluorescence analysis depends on the assumption that the magnitude of a spectroscopic signal at one wavelength will remain a constant function of whether the molecule is in its folded or unfolded form. Thus, the analysis requires that two constant signals exist, one for each form, and that each constant signal be independent of the variation of the perturbing parameter. If this does not occur, as is the case when temperature is the perturbing factor, a correction must be applied. One correction commonly applied is based on Taylor series expansions of unknown functions multiplied by an exponentially declining temperature term, E. A. Permyakov,
The Luminescent Spectroscopy of Proteins
(CRC Press 1993) at pp. 99-107. Although some in the art assert that protein structure as a result of heat denaturation can be studied on a quantitative basis using data so corrected, such data frequently yields coefficients which predict infinite emission intensities in the temperature range from 0° C. to 100° C. Thus the method is often not as reliable as desired over the important temperature range which includes both cold and heat denaturation.
Another important method for determining the denaturation of the protein structure, or extent of exposure of fluorophores to the environment external to the protein, is fluorescence quenching, Joseph Lakowicz,
Principles of Fluorescence Spectroscopy
(Plenum Press 1983) at pp. 279-284. In this method the protein is exposed to varying levels of a quencher such as iodide which cannot interact with fluorophores buried in the protein interior. The percentage of exposed fluorophores can then be calculated using the Stern-Volmer equation:
F
0
Δ
F
=
(
1
f
a
*
1
K
sv
*
1
[
Q
]
)
=
1
f
a
,
where F
0
is initial fluorescence intensity at the test wavelength at zero concentration of quencher Q, &Dgr;F is the initial fluorescence intensity minus the fluorescence intensity at a given concentration of quencher, f
a
is the fraction of initial fluorescence accessible to the quencher, K
SV
is the Stern-Volmer constant, and [Q] is the concentration of quencher Q. However, this cannot easily be related to the percent of protein unfolded because it provides no information about the spectral characteristics of the unquenched fluorophores. Thus, if a protein has two domains, which can be referred to as A and B, with the same number of buried fluorophores, then A may heat denature at a lower temperature than B, but B may cold denature at a higher temperature than A. In this case the Stern-Volmer analysis will show that the protein appears to be increasing the percentage of exposed fluorophores at low temperature and the percent increased exposure could be scaled, using interpolation or normalization techniques, between the initial value and the heat denatured value to estimate a percent of unfolded protein. But, in fact, a different part of the protein would be unfolding.
In the case of &thgr;
222
measurements of proteins, one must assume that the shift at that wavelength or the change in signal accurately reflects the helical content of the protein, that the change in helical content reflects accurately the fraction of unfolded protein, and that the protein domain which is unfolding is where the helices are.
It has been generally accepted for quite some time that the helical signal depends strongly on the length of the helices even when total helical content is constant, Y. H. Chen, et al.,
Biochemistry
13:3350 (1974). Also, helices are often quite stable at low temperature as shown by J. M. Scholtz et al.,
Proc. Natl. Acad. Sci.,
USA 88:2854 (1991). Thus low temperature tertiary unfolding may leave helical secondary structures intact and the helices may appear longer, i.e., have larger magnitude CD signals, such that the secondary structure may appear even more unlike the denatured state than that from the protein at physiological temperatures. Under these circumstances, &thgr;
222
measurements will appear to indicate increasing stability of the protein, an erroneous conclusion.
Thus, there is a need in the art for a spectroscopic analysis that minimizes dependence on the independently varied parameter, such as temperature such that it need not rely on corrections and assumptions that cannot be substantiated. Moreover, there is a need for an analysis that can be used simultaneously with several of the common modes of spectroscopy presently in use.
SUMMARY OF THE INVENTION
To fulfill the above needs, it is an object of the present invention to provide a method for determining the respective fractions of a folded form and an unfolded form of a molecule in solution, in order to estimate a fraction of an unfolded form of a molecule in solution and to determine apparent free energy of a conversion reaction between a folded form and an unfolded form of a molecule in solution.
Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.
Accordingly, to achieve the above object, the present invention, as embodied and broadly defined herein, provides a method of determining the respective fractions of a folded form and an unfolded form of a molecule in solution using differential spectral topographic analysis (DISTA), comprising summing absolute differences in topography between each of two reference spectra and a sample spectrum taken at different magnitudes of at least one perturbation parameter, plotting curves of the sums versus the at least one perturbation parameter,
Hirsh Allen G
Litvinovitch Serguei
Mehl Patrick M.
Tsonev Latchezar I.
Barbee Manuel L.
Finnegan Henderson Farabow Garrett & Dunner
Henry M. Jackson Foundation for the Military Medicine
Hoff Marc S.
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