RU2384842C2 - METHOD FOR QUANTITATIVE DETERMINATION OF PEPTIDES AND DENATURED PROTEINS, NON-COVALENTLY BONDED TO 70 kDa HEAT-SHOCK PROTEIN - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, biopharmacology, laboratory diagnostics and can be used in medicine, immunology and oncology for vaccine standardisation and for research purposes. To realise the method, a solution of adenosine triphosphate is added to the analysed sample until attaining final concentration of 1-5 mM. Incubation is then carried out at temperature 25-40°C for 15-60 minutes, with subsequent colorimetric determination of the amount of the formed inorganic phosphate and calculation of the fraction of complexes in the preparation using the formula:

The method can be used to standardise vaccines based on HSP70 independent of the method of obtaining the vaccines. The given method is also suitable for use in scientific research, for analysing purity of recombinant protein HSP70, its properties, as well as investigating its reaction with proteins and peptides.

EFFECT: use of the invention simplifies quantitative determination and widens the range of determined peptides and denatured proteins which are non-covalently bonded to HSP70 independent of their structure.

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Description

The invention relates to the field of biochemistry, biopharmacology, laboratory diagnostics and can be used in medicine, immunology and oncology to standardize vaccines and for research purposes.

Heat shock proteins of 70 kDa (HSP70) are used to create drugs with the ability to elicit a specific immune response against various peptide antigenic determinants non-covalently associated with HSP70. Based on complexes of peptide fragments with HSP70, the creation of antiviral and antitumor vaccines is possible {Schmitt E, Gehrmann M, Brunet M, Multhoff G, Garrido C. Intracellular and extracellular functions of heat shock proteins: repercussions in cancer therapy. J Leukoc Biol. 2007 Jan; 81 (1); 15-27. Epub 2006 Aug 24}. Moreover, the effectiveness of the vaccine depends on the amount of bound peptide, as well as on the antigenic properties of these peptides. An increase in the amount of HSP70 bound peptide has been shown to increase vaccine efficacy {Flechtner JB, Cohane KP, Mehta S, Slusarewicz P, Leonard AK, Barber BH, Levey DL, Andjelic S. High-affinity interactions between peptides and heat shock protein 70 augment CD8 + T lymphocyte immune responses. J Immunol. 2006 Jul 15; 177 (2): 1017-27., MacAry PA, Javid B, Floto RA, Smith KG, Oehlmann W, Singh M, Lehner PJ. HSP70 peptide binding mutants separate antigen delivery from dendritic cell stimulation. Immunity. 2004 Jan; 20 (1): 95-106}.

A known method of measuring the amount of HSP70 bound peptide using a radioactive label {Moroi Y., Mayhew M., Trcka J., Hoe M.H., Takechi Y., Hartl F.U., Rothman J.E., Houghton A.N. Induction of cellular immunity by immunization with novel hybrid peptides complexed to heat shock protein 70 PNAS, 2000; 97: 3485-90}. The disadvantages of this method are, firstly, the need to work with radioactive isotopes, and secondly, the ability to measure with it only pre-labeled peptides. In addition, the radioactivity of labeled peptides does not make it possible to use them as part of immunotherapeutic preparations, which makes the described method unsuitable for use in standardizing vaccines based on HSP70 complexes with peptides.

A known method for the quantification of complexes of peptides with HSP70 by direct spectrophotometric measurement of the amount of peptide after separation using HPLC {Grossmann ME, Madden BJ, Gao F, Pang YP, Carpenter JE, McCormick D, Young CY. Proteomics shows Hsp70 does not bind peptide sequences indiscriminately in vivo. Exp Cell Res. 2004 Jul 1; 297 (1): 108-17}. This method is well suited for the quantification of complexes of HSP70 with known denatured proteins {Palleros DR, Welch WJ, Fink AL. Interaction of hsp70 with unfolded proteins: effects of temperature and nucleotides on the kinetics of binding. Proc Nail Acad Sci USA. 1991 Jul 1; 88 (13): 5719-23}, since these complexes can differ significantly in molecular weight from free HSP70. The absence of specific absorption spectra of the peptides makes it possible to erroneously determine other substances, for example, nucleotides, as peptides. The specific optical absorption of the peptides depends on the composition and relative position of amino acids. This makes it possible to determine the number of peptides of only a known structure or peptides for which the standard setting is possible. In addition, the formulation of this analysis requires special technical equipment and additional training of specialists conducting it.

Closest to the proposed invention is a method for estimating the amount of HSP70 bound peptide using a fluorescent label {Becker T, Hartl FU, Wieland F. CD40, an extracellular receptor for binding and uptake of Hsp70-peptide complexes. J Cell Biol. 2002 Sep 30; 158 (7): 1277-85}. This method involves the selection of an aliquot of the drug containing a known amount of protein, and the direct determination of the amount of labeled peptide by optical absorption of the label. The proportion of complexes in the protein preparation is defined as the quotient of these values (example 3). This method allows you to measure the number of complexes with HSP70 only one peptide containing a pre-entered label. The reaction of introducing a fluorescent label does not always have a sufficient yield, which limits the use of this method in conditions of peptide deficiency.

The objective of the invention is to simplify the method of quantitative determination and expansion of the spectrum of detectable non-covalently associated with HSP70 peptides and denatured proteins, regardless of their structure.

The problem is solved by the method consisting in the fact that a solution of adenosine triphosphate (ATP) is added to the test sample to a final concentration of 1-5 mM, incubated at a temperature of 25-40 ° C for 15-60 minutes, followed by colorimetric determination of the amount of inorganic phosphate formed and calculating the proportion of complexes in the HSP70 preparation according to the formula:

where η (%) is the fraction of HSP70 complexes with peptides in the preparation;

- количество образовавшегося неорганического фосфата, нмоль;

- the amount of inorganic phosphate formed, nmol;

t is the incubation time of the mixture, min;

m is the mass of HSP70 taken for analysis, mg,

k is the coefficient, which is calculated according to the calibration graph.

The calibration graph is built according to measurements of complexes of HSP70 with known labeled peptides obtained by the described method and obtained using a fluorescent label (example 1, 3).

The method is based on increasing the ATPase activity of proteins of the HSP70 family with specific non-covalent binding to a peptide or denatured protein. The increase in ATPase activity in this case is the result of only conformational changes in the protein and does not depend on the nature of the ligand (example 2).

The advantage of the claimed technical solution is, firstly, the ability to measure the number associated with HSP70 as labeled and unlabeled peptides. Secondly, the ability to determine the number of complexes in the HSP70 preparation, regardless of the qualitative composition of the peptides and the method of preparation. Thirdly, the claimed technical solution does not require preliminary modification of the analyzed drug and can be used to standardize drugs based on HSP70 complexes. Fourth, the proposed technical solution is based on well-known analytical methods and does not require additional training of specialists performing this analysis, or working with radioactive compounds.

The list of examples illustrating the invention

Example 1. Figure 1 shows a calibration graph for the recombinant protein HSP70A1B, reflecting the dependence of the ATPase activity of the preparation HSP70A1B (nmol / mg · min) on the proportion of complexes with peptides in the protein preparation (in%) obtained by the method described in example 3 Dots indicate the values obtained by measuring the ATPase activity of HSP70A1B preparations with a known fraction of labeled peptides. The correlation coefficient of the smoothing line r is 0.99 with a confidence probability of p <0.01. Statistical processing of measurement results was performed using the Statistica software package

Example 2. Figure 2 shows the absence of significant differences with a confidence probability of 0.95 between the dependence of ATPase activity on the proportion of complexes in the HSP70 preparation with peptides of various structures, measured by fluorescence label. The abscissa axis shows the proportions of the complexes (in%) in the studied samples of HSP70A1B protein preparations with the known labeled peptides of the compositions YMLDLQPETT (hollow squares) and ALFDIESKV (hollow circles). On the ordinate axis, the corresponding ATPase activity values (nmol / mg · min), measured in samples, are noted. Solid lines indicate smoothing lines separately for preparations of complexes of the HSP70A1B protein with a peptide of the composition YMLDLQPETT and a peptide of the composition ALFDIESKV. Dotted lines indicate confidence intervals (p = 0.95) for each line. Statistical processing of the measurement results was performed using the Statistica software package. Each line is within the confidence interval of the other line, which graphically reflects the absence of differences between the smoothing lines.

Example 3. The construction of a calibration graph using fluorescently labeled peptides.

.HSP70A1B protein complexes with the well-known peptides YMLDLQPETT and ALFDIESKV labeled with fluorescein isothiocyanate (FITC) are prepared by incubating a 5-fold excess of labeled peptide with HSP70 in a buffer solution containing 0.05 M Tris-HCl pH 8.0 Dose phosphate, 2 m phosphate for 30, 60, 90 and 120 minutes The free peptide is separated by ultrafiltration through a membrane passing molecules with a molecular weight of up to 10 kDa. The amount of labeled peptide in an aliquot of the purified preparation of the complexes is immediately determined by direct spectrophotometric method according to the absorption of the label at a wavelength of λ = 495 nm. The proportion of complexes in the protein preparation is determined by the following formula:

.

, где m - масса белка в аликвоте.Another aliquot containing about 10 μg of protein was mixed with a 3-fold volume of a buffer solution containing 50 mM Tris-HCl pH 8.0, 2 mM ATP, and incubated for 30 min at 37 ° C. Then add a 4-fold volume of a coloring reagent containing 2 parts of an aqueous solution of malachite green (0.8 mg / ml), 2 parts of an aqueous solution of polyvinyl alcohol (23.2 mg / ml of water), 1 part of a solution of (NH ₄ ) ₆ Mo ₇ O ₂₄ · 4H ₂ O (57 mg / ml) in 6N HCl and 2 parts of water, stirred and immediately measured optical density at a wavelength of λ = 630 nm. The ATPase activity of the preparation of HSP70 complexes with the peptide is determined by the formula:

where m is the mass of protein in an aliquot.

Based on the data obtained, a calibration graph is constructed that represents the dependence of the ATPase activity of the preparation, in nmol · mg ^-1 · min ^-1 , of HSP70 complexes on their content in the preparation, in% (Fig. 1). The dependence on the calibration graph is linear, the correlation coefficient is r = 0.99 at a significance level of p <0.01. The proposed method for the quantitative determination of HSP70-related peptides, thus, has accuracy at the level of the prototype.

Example 4. Quantification of Recombinant HSP70 Associated Individual Peptide.

Preparations of HSP70 complexes with the ITDQVPFSV peptide are obtained [by incubating a 5-fold excess of the peptide with 40 μg of recombinant HSP70A1B in buffer solutions containing 0.05 M Tris-HCl pH 6.0 and 0.05 M Tris-HCl pH 8.0, respectively. The complexes are separated from the free peptide by Ni-CAM sepharose. Aliquots containing about 10 μg of protein are selected and the ATPase activity is measured as described in Example 3. The measured absorbance values correspond to the ATPase activity of 5.4 nmol · mg ^-1 · min ^-1 and 4.1 nmol · mg ^-1 · Min ^-1, respectively. These values are interpreted using the calibration graph obtained in Example 1. The preparations studied in this way contain, respectively, 7.8% and 6.0% of complexes of recombinant HSP70A1B with the known peptide ITDQVPFSV.

Example 5. Determination of the degree of contamination of recombinant HSP70 bacterial peptides.

Recombinant HSP70A1B dialyzed from 0.05 M Tris-HCl pH 8.0 buffer solution isolated from biomass of the E. coli producer strain using chelate chromatography, purified from impurities, and then an aliquot corresponding to about 10 μg of protein was selected for determination of ATPase activity. ATP was added to the remaining solution to a concentration of 2 mM and the pH was adjusted to 8.0, after which it was filtered through a membrane passing molecules with a molecular weight of 10 kDa. An aliquot corresponding to about 10 μg of protein is taken, and the ATPase activity of the samples taken is determined as described in Example 3. The measured optical density values correspond to the ATPase activity of 2.6 nmol · mg ^-1 · min ^-1 and 0 nmol · mg ^{- 1} · min ^-1 in the first and second cases, respectively. These values are interpreted using the calibration graph obtained in Example 3. The study shows that the recombinant HSP70A1B preparation contained 3.7% complexes with bacterial peptides before incubation with ATP and was practically free of them after that.

Example 6. Joint determination of HSP70-linked labeled and unlabeled peptides.

The preparation of HSP70A1B protein complexes with ALFDIESKV and YMLDLQPETT peptides labeled with FITC was obtained by incubating a 5-fold excess of a mixture of labeled and unlabeled peptides (1: 1) with HSP70 in 0.05 M Tris-HCl buffer solution pH 8.0, 1 mM ADP for 60 min . Free peptides are separated g by ultrafiltration through a membrane passing molecules with a molecular weight of up to 10 kDa. The amount of labeled peptide in an aliquot of the purified preparation of the complexes is immediately determined by direct spectrophotometric method by absorbing the label at a wavelength of λ = 495 nm, as described in example 3.

A portion containing about 10 μg of protein is selected from the same aliquot and the ATPase activity of the preparation is determined as described in Example 1. The measured optical density corresponds to the ATPase activity of 6.7 nmol · mg ^-1 · min ^-1 , which corresponds to 9.8 % complexes of HSP70 with peptides. The proportion of HSP70 complexes with the FITC-labeled peptide YMLDLQPETT is 4.5%. The proportion of complexes with unlabeled ALFDIESKV peptide is thus 5.3%.

Example 7. Quantification of HSP70 bound denatured protein.

The preparation of complexes of HSP70A1B with denatured lactalbumin (RCMLA) is obtained by incubation of 8 μg of RCMLA with 40 μg of recombinant HSP70A1B in 0.05 M Tris-HCl pH 8.0 buffer solution. The complexes are separated from free RCMLA on Ni-CAM Sepharose. An aliquot containing about 10 μg of HSP70A1B is taken and the ATPase activity is measured as described in Example 3. The measured optical density corresponds to the ATPase activity of 12.3 nmol · mg ^-1 · min ^-1 . This value is interpreted using the calibration graph obtained in Example 3. Thus, the studied preparation contains 17.8% of the complexes of recombinant HSP70A1B with the denatured RCMLA protein.

Claims (1)

A method for the quantitative determination of peptides and denatured proteins non-covalently associated with a heat shock protein of 70 kDa, characterized in that an adenosine triphosphate solution is added to the test sample to a final concentration of 1-5 mM, incubated at a temperature of 25-40 ° C for 15-60 minutes with subsequent colorimetric determination of the amount of inorganic phosphate formed and the calculation of the proportion of complexes in the preparation according to the formula

,
where η (%) is the fraction of HSP70 complexes with peptides in the preparation;

- the amount of inorganic phosphate formed, nmol;
t is the incubation time of the mixture, min;
m is the mass of HSP70 taken for analysis, mg;
k is the coefficient, which is calculated according to the calibration graph.

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