Arrayable thermal assays

Details Arrayable thermal assays Arrayable thermal assays

1999-11-02

2002-06-11

Gutierrez, Diego (Department: 2859)

Thermal measuring and testing

Differential thermal analysis

Detail of sample holder or support therefor

C374S011000, C374S033000, C374S179000, C422S051000, C136S204000, C136S224000, C436S147000, C435S288400

Reexamination Certificate

active

06402369

ABSTRACT:

This invention relates to arrayable thermal or calorimetric measurement systems allowing precise, reproducible calorimetric assays for physical characterization of biochemical products, as well as for use in pharmaceutical and biotechnology product development. Specifically, this invention uses a novel calorimetric method and apparatus employing base thermopiles in thermal communication with a plurality of samples, to provide thermal signatures, such as changes in specific heat capacity or capacitance, to differentiate or rank biological or chemical samples. This allows systems having arrays of cells, and using facile automated handling, such as by use of disposable tubes, microtiter plates, robotic transports, and the like to perform thermophysical assays, such as for sample screening or to characterize chemical or biological activity.
Biological calorimetry is a well known technique used as a marker to identify systems, or to detect phase transformations or reactions such as binding of ligands to proteins (see A. E. Beezer,
Biological Calorimetry, ©
1980, Academic Press, New York). Useful measurements for calorimetric assays include the derivative with respect to temperature of a unique state function, the enthalpy H, yielding the heat capacity
C
p
=d H
(
T
)
p
/dT
  (1)
at constant pressure p. Direct calorimetric measurements allow determination of the heat capacity C
p
of a sample, measured in kJ/K or similar units. By determining heat capacity in a selected temperature region, one can determine the enthalpy H, and in turn other state functions such as the sample entropy S, and the Gibbs Function G. See P. W. Atkins,
Physical Chemistry,
6
th
Ed., W. H. Freeman and Company, New York © 1997, ISBN 0716728710. It is useful to note that heat capacity is an intensive property, independent of the quantity or shape of the substance under consideration. The corresponding extensive property is known as heat capacitance, in analogy with electrical capacitance, and is a function of the quantity of substance involved.
Many processes involving biopolymers and proteins take place with detectable changes in apparent heat capacities of the reacting species. A molecule or biochemical system that has or obtains many translational, rotational, and vibrational degrees of freedom will have a high heat capacity Cp while a simpler (e.g., folded protein) system will have a lower heat capacity. Determining the heat capacity C
p
therefore yields an important thermophysical property, and can be used for assays and structure determinations for solutions, proteins, and biological samples. Such assays and structure determinations can be useful for sample screening and biochemical product synthesis.
Six possible sources of large heat capacity (and entropy) changes have been identified for processes involving proteins (Julian M. Sturtevant,
Proc. Natl. Acad. Sci. USA
, Vol 74, No. 6, pp.2236-2240, June 1977). Protein structure changes such as unfolding can produce large changes in heat capacity, such as unfolding of &agr;-Chymotrypsin, which yields a change in heat capacity C
p
of +3080 cal/K/mol at neutral pH. Processes include hydrophobic effects, where nonpolar groups raise the heat capacities of solutes in aqueous solutions; electrostatic effects, where creation of positive and negative charges in aqueous solutions leads to a negative change in heat capacity; breaking of hydrogen bonds with increasing temperature, where heat capacity increases; intramolecular vibrations, affected by chemical changes such as unfolding or ligand binding, where an increase in the number of easily excitable internal vibrational modes results in heat capacity increases; or changes in equilibria, where an actual shift with temperature of an equilibrium between two or more states will appear experimentally as a contribution to the heat capacity.
Current views about protein-ligand interactions state that electrostatic forces drive the binding of charged species and that burial of hydrophobic and polar surfaces influences or controls the heat capacity changes associated with the reaction. However, concerning interactions of a protein with a monovalent cation where electrostatic forces are expected to be significant due to the ionic nature of the ligand, heat capacity changes are expected to be small due to the small surface area involved in the protein-ligand recognition event. It has been found, however, that with the physiologically important interaction of Na+ with thrombin, binding is characterized by a modest dependence on ionic strength, but a large negative heat capacity change of −1.1 ±0.1 kcal/mol/K (see Guinto, Cera,
Biochemistry, Vol
35, No 27, pp. 880-8804). It is proposed that this change is linked to electrostatic effects can reveal a binding or folding event where water molecules are buried, resulting in significant heat capacity changes independent of changes in exposed hydrophobic surface or coupled conformational transitions (rotations about a single chemical bond). Generally, monovalent cation binding to proteins is a widespread phenomenon and can play an important role in enhancing catalytic activity of enzymes. Potassium ion binding to proteins is typically accomplished mainly through two mechanisms. In one mechanism, K+ forms a ternary complex with the enzyme and substrate (e.g., ATPases). In another mechanism, as seen in pyruvate kinase, K+ binds to a distinct site and influences the activity of the enzyme in an allosteric fashion, thereby causing a change in the function of the enzyme. Sodium binding can also be important, for example, Na+ activated enzymes are involved in blood coagulation and complement cascades.
Solvation of charged and polar groups is typically accompanied by a negative heat capacity change which is small and only known for simple molecules. Heat capacity of water molecules sequestered in the interior of a protein is significantly lower than in bulk water, because of reduced mobility and more ordered structure. Burial of water molecules linked to ligand binding or protein folding can result in large negative heat capacity changes, which can be detected in an assay using the disclosed invention.
As another example, the binding of L-aribinose and D-galactose to the L-aribinose-binding protein of Escherichia coli has been studied by isothermal and scanning calorimetry (see Fukada, Sturtevant,
Journal of Biological Chemistry
, Vol. 258, No. 21 pp. 13193-13198, 1983). It is found that the binding reaction with arabinose is characterized by an enthalpy change of −15.3 kcal/mol, with a large decrease in apparent heat capacity of −0.44 kcal/mol/K. However, determination methods used are painstaking, typically done with two samples at a time (such as an experimental sample and a control or reference sample), and involve elaborate experimental and chemical procedures.
Thermophysical assays have the advantage of not requiring the use of any external or added agents, such as fluorescent or radioactive tags, which can cause damaging or unknown perturbations on a biochemical system. The heat capacity C
p
is also an equilibrium property that can be used to great advantage. Thus, while a small molecule ligand that does not bind to a protein should have a negligible effect on the heat capacity of a system, a small molecule that does bind usually causes a permanent change in molecular degrees of freedom, and hence the heat capacity C
p
of the system. Many measurements can thus be made at leisure, after the binding event.
Thermophysical assays can also elucidate cellular processes. One especially important cellular protein, calmodulin (CAM), appears to be at the junction of many signal transduction processes. CAM appears to be able to modulate many distinct processes because it can exist in a large ensemble of different structural states, presenting a corresponding large ensemble of chemical interaction sites. As with most molecular structures, each distinct state is likely to have a characteristic heat capacity C
p
Elu

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