Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving hydrolase
Reexamination Certificate
2000-04-26
2001-09-04
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving hydrolase
C435S004000, C436S066000
Reexamination Certificate
active
06284481
ABSTRACT:
The present invention relates to an assay for detecting hemoglobin degradation.
Several parasites are known to degrade host cell hemoglobin by means of their proteases, for example, the human malarial parasite
Plasmodium falciparum
. It has a limited capacity to de novo synthesize amino acids or to take them up from its immediate environment. Thus, this parasite uses the host erythrocyte hemoglobin as a major nutrient source. It consumes 25%-75% of the host cell hemoglobin during a short segment of its intra-erythrocytic life cycle. This massive catabolism is probably an ordered process which occurs in a unique acidic organelle called the digestive vacuole, at a pH optimum of ~5. At least three vacuolar proteases (two aspartic and one cysteine) are known to be involved in the breakdown of human hemoglobin to its constituent amino acids. One of the aspartic proteases, Plasmepsin I has been indicated in the initiation of hemoglobin degradation by making the first cleavage and unwinding the molecule, so that further proteolysis can proceed efficiently. A second aspartic protease, Plasmepsin II presumably cleaves hemoglobin with an overlapping specificity. The cysteine protease, Falcipain is also involved in an early step of hemoglobin degradation. All three proteases Plasmepsin I, II & Falcipain cleave denatured hemoglobin in vitro.
Several different assays exist for monitoring hemoglobin degradation by parasitic proteases, e.g. as described by Daniel E. Goldberg et al. in “Hemoglobin degradation in the malaria parasite
Plasmodium falciparun
: An ordered process in a unique organelle”, Biochemistry (1990), 87:2931-2935.
For example, there is the Centricon Assay for Proteolysis in which varying concentrations of enzyme (protease) extract are added to an incubation mixture containing radioactively labelled [
3
H] hemoglobin and sodium citrate buffer (pH 5). After incubation at 37° C. for one hour, the reaction is stopped by addition of iced guanidine hydrochloride. After 15 minutes on ice, the mixture is loaded onto a Centricon 10 kDa filter and centrifuged at 5000 g for one hour. The filtrate is then assayed for radioactivity. The assay has been shown to be linear with time and protein concentration.
In the Trichloroacetic Acid (TCA) Assay for Proteolysis hemoglobin degradation is assessed by measuring production of TCA-soluble fragments. The Centricon assay incubation mix is used with citrate/phosphate buffer at varying pH. TCA precipitation is performed by addition of iced unlabelled hemoglobin and iced 20% TCA. After 30 minutes on ice, the mixture is centrifuged at 16,000 g for 15 minutes and the supernatant is assayed for radioactivity.
Hemoglobin degradation may also be assessed using SDS-PAGE Analysis. Human hemoglobin is used as a substrate in the Centricon assay incubation mix. The reaction is stopped by adding SDS sample buffer and boiling for 3 minutes. The samples are then run on a 20% SDS-PAGE which is developed using Silver Staining.
All of the above known assay systems have the drawback that they serve only as end-point assays. Furthermore, because of their nature, such assays are often time consuming.
Assays in which the proteolytic activities of the parasitic proteases can be monitored continuously and directly are also known in the art. These assays employ synthetic substrates, mostly oligo-peptides or peptidomimics, that are designed to simulate the natural cleavage sites of a given protease, e.g., Plasmepsin II as described by Jeffrey Hill et al. in “High level expression and characterisation of Plasmepsin II, an aspartic proteinase from
Plasmodium falciparum
”, Federation of European Biochemical Societies (FEBS) Letters (1994), 352:155-158.
Whilst such assays using synthetic substrates have the benefit that they are generally quicker to carry out than those mentioned above in which hemoglobin is used as the substrate, the synthetic substrates are expensive and are required in high concentration. Furthermore, in the context of ligand binding studies, it has been found that these assays only work well with pure ligands and not ligand mixtures.
Therefore, it would be desirable to develop an assay for detecting hemoglobin degradation that is quick and efficient, that uses hemoglobin as the substrate and that can be reliably used with a variety of ligand sources.
In accordance with the present invention, there is provided a spectrophotometric assay for detecting hemoglobin degradation which comprises:
a) preparing a mixture comprising hemoglobin and a proenzyme of a hemoglobin-degrading enzyme, wherein the mixture has a pH in the range from 7.5 to 7.6,
b) measuring the absorbance of the mixture at a wavelength (&lgr;) in the range from 404 to 407 nm,
c) acidifying the mixture to activate the hemoglobin-degrading enzyme,
d) incubating the mixture of step c),
e) measuring the absorbance of the mixture of step d) at a wavelength (&lgr;) in the range from 404 to 407 nm,
f) adding a base to the mixture of step e) to effect renaturation of undegraded hemoglobin,
g) incubating the mixture of step f), and
h) measuring the absorbance of the mixture of step g) at a wavelength (&lgr;) in the range from 404 to 407 nm.
The present assay is based on and makes use of the spectral properties of hemoglobin. The tetra-pyrrole nucleus of hemoglobin is responsible for a characteristic absorption band between 400 to 410 nm exhibited by hemoproteins, and this is referred to as the Soret band. Human hemoglobin, in its native form, absorbs at a wavelength (&lgr;
max
)=405 to 406 nm. In step a) of the assay, the hemoglobin is present in its native state. On addition of acid in step c), the proenzyme is converted to active hemoglobin-degrading enzyme and the hemoglobin is partially denatured. Upon activation, the hemoglobin-degrading enzyme begins to cleave the partially denatured hemoglobin resulting in removal of the heme moiety and an observed drop in absorbance at 405 to 406 nm. Base is added in step f) to effect renaturation of undegraded hemoglobin (the term “undegraded hemoglobin” meaning hemoglobin that has not been cleaved by the hemoglobin-degrading enzyme), resulting in an observed increase in absorbance at 405 to 406 nm. By monitoring the changes in absorption in the range from 404 to 407 nm, the present assay provides a direct method for assessing hemoglobin degradation.
REFERENCES:
patent: WO-200028391A (2000-08-01), None
Goldberg et al., J. Exp. Med., 1991, 173(4), 961-969.
Brindley, P.J., Mol. Biochem. Parasitol., 1997, 89(1), 1-9.
Ridley, R., J. Pharm. Pharmacol., 1997, 49(2), 43-48.
Hill et al., FEBS Lett., 1994, 352(2), 155-158.
Datta Santanu
Rane Rajendra
Astrazeneca AB
Leary Louise N.
White & Case LLP
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