HU204081B - Process for producing myeloperoxidase enzyme - Google Patents

Abstract

The invention relates to a process for the preparation of the enzyme myeloperoxidase by purification and activation of human leucocytes, disruption and extraction of the resulting material and purification of the enzyme, using as starting material a leucocyte-rich blood fraction (buffy coat) which derives from human blood and has already been used to prepare, inter alia, interferon or interleukin, employing as activator Sendai viruses, concanavalin A, phytohaemagglutinin, Lens culinaris lectin or Ca<++> ionophore, and carrying out the chromatography step for purifying the enzyme on a column packed with carboxymethylated controlled pore glass.

Description

The present invention relates to a process for the production of the enzyme myeloperoxidase by purifying, activating, and purifying human white blood cells by using buffy coats already used as starting materials for the production of interferon or interleukin, and by purifying the fin carried out.

The myeloperoxidase enzyme can be used as a diagnostic agent in cases of infarction, as the extent of infarction is proportional to the level of tissue myeloperoxidase. This major utilization of the enzyme is reported in the Journal of Cardiovascular Pharmacology 7, 1154-1160 (1985), RavenPress, New York. The association between inflammatory disease and myeloperoxidase levels is discussed in Blood Vol 60, No. 3 (September) [1982]. Biochemistry 3 [9], 1234-38 [1964], on the ethnicity of the enzyme myeloperoxidase, whereby white blood cells obtained by purifying buffy coat from healthy donors are digested by trypsin digestion, are precipitated with 50% cold ethanol. From the precipitate, the enzyme is extracted with phosphate buffer and purified by chromatography on an XE-64 type Amberlite column.

Archives of Biochemistry and Biophysics 245 [1], 167-173 (1986) also extract buffy blood cells from buffycoat. The enzyme from the extract is purified first on DEAE-Sepharose and then on a ConA-Sepharose affinity column.

In the literature cited above, methods for isolating the enzyme, on the one hand, are non-farm-scale methods and, on the other hand, buffy coat white blood cells are not treated to increase the amount of intracellular enzyme.

The process of the present invention differs from the literature in two important respects

1.The intracellular myeloperoxidase content of white blood cells is increased by the use of activating agents.

Activating agents may include Sendai virus, Concanavalin-A Phytohemagglutinin, Lens cullinaris lectin, Ca ²⁺ ionophores or forhormyristate acetate. A particularly preferred form of activation is the use of white blood cells previously used for the production of interferons or interleukins as an enzyme source; in fact, it has been observed that the induction of these leukotins by one of the above activators also significantly increases the cellular enzyme content. The "waste" white blood cells produced during aleukokine production are thus more preferred sources of the myeloperoxidase enzyme than untreated fresh cells.

2. Purification of the enzyme is carried out with a controlled pore size glass bead containing carboxymethyl functional groups ("CM-CPG - henceforth used in the English patent to denote columnar kits") for other types of ion exchangers or unstituent CPGs. it is capable of absorbing the enzyme with multiple capacities.

Accordingly, the process of the present invention is for the preparation of the enzyme myeloperoxidase by purifying white blood cells obtained from human buffycoat by hemolysis with ammonium chloride, digesting the resulting cells and purifying the enzyme. The starting material used is white blood cells used for the production of interferon or interleukin, the sending agent used is Sendai virus 10, Concanavalin At, Phytohemagglutinin, Lens cullinaris lectin or Ca ²⁺ ionophore, and the column is purified by chromatography on CMCP. The invention provides an average protein specific activity of 70-100E / mg. In contrast, the commercial myeloperoxidase preparation (Calbiochem) has an activity of only 20 U / mg protein.

Protein Blood Cell Purification, Activation, Protein Blood Cells Reversal, and Enzyme Purification are illustrated by the following examples, without limiting our need to

Example 1

Production of myeloperoxidase enzyme from white blood cells produced as a by-product of interferon production

(a) Purification of white blood cells

The starting material is the so-called. A buffy coat is used, which is a fraction of the white blood cell rich in the separation of whole blood. Contains 1 buffy coat 1, 4x10 ⁹ white blood cells; volume about 40 mL A concentrated version of this is commercially available with a volume of 10 mL. In addition to white blood cells, buffy-coat also contains blood cells and blood plasma. These materials should be disposed of during cleaning.

To 100 buffy coats (4000ml and 1000ml respectively), add three times the volume of 0.83% ammonium chloride solution cooled to +4 'C and allow to stand on ice for 10 minutes until the red blood cells have dissolved. Thereafter, white blood cells from the suspension are pelleted by centrifugation (1400 rpm; 20 minutes), the supernatant is discarded, and the adherent cells are resuspended in PBS buffer (approximately 400 mL).

In PBS). The cell suspension volume of approximately 500 ml, the cell concentration 3-4xl0 ⁸ cells / ml.

The buffer composition of PBS is as follows:

component concentration (mg / ml) NaCI 8800 potassium chloride 220 disodium hydrogen phosphate. .I ₂ H ₂ O 3500 sodium dihydrogen phosphate. Ή9Ο pH 7.2-7.4 224

The ammonium chloride treatment was repeated except that 9 volumes of 0.83% ammonium chloride solution were added to 1 volume of cell suspension.

gQ The cells thus obtained were again in 400 ml PBS buffer

Resuspend; the volume of the cell suspension is about 500 ml.

b) Activity

During activation, white blood cells are induced to produce myeloperoxidase enzyme.

500 ml of a white blood cell suspension containing approximately 1.4 x ^{10 7} cells are added to 10,000 ml of medium having the following composition:

(RCG / l)

CaCl ₂ 200 Fe (NO ₃ ) ₃ .9H ₂ O 0.1 KC1. 400 MgSO ₄ .7H ₂ O 200 NaCl 6400 NaHCO ₃ 2000 NaH ₂ PO ₄ .H ₂ O 120 glucose 4500 phenol red 10 human serum free from gamma globulin (Agamma serum ") 1000

Activation is performed with 15 ml of 100 HE / ml Sendai virus. Sendai virus, a known activator of intracellular α-DFN production, is a commercially available virus preparation, which, however, has been produced by self-propagation in SPF eggs (sterile, pathogen-free, sterile).

After incubation, cells are removed from the culture medium by centrifugation (2000 g, 30 min).

The supernatant is crude α-interferon. The separated cells were resuspended in PBS buffer to give a volume of 500 ml.

(c) Detection and separation of pituitary blood cells

The purpose of the digestion is to recover the enzyme myeloperoxidase from the cells. The cell suspension is frozen and thawed seven times in succession. Freezing is performed at -70 ° C and thawed at room temperature. This causes the cells to disintegrate and the enzyme into solution. Cell debris was separated from the enzyme-containing solution by centrifugation (20,000 g, 60 min).

d) Purification of myeloperoxidase enzyme

Low molecular weight impurities are separated by ultrafiltration in continuous operation.

The enzyme-containing solution has a value of 100,000 cut-offs. Diafiltrated using a "hollow fiber" filter insert (AMICON DC-4). This filter cartridge holds molecules larger than 100 kD, permeable to smaller molecules.

Enzyme activity is found in the fraction of molecular weight greater than 100,000 D. The volume of the resulting enzyme-containing fraction was adjusted to about 200 mL.

Deposition of impurities by changing the pH

To the above 200 mL fraction was added 200 mL of 0.1 M potassium citrate (pH 4); 400 ml of a solution at pH 4.5 are obtained. As a result of the changed pH, some of the contaminating proteins are precipitated. The precipitate was removed by centrifugation at 7000 g for 30 minutes. Approximately 350 ml of the supernatant obtained contains the enzyme.

Chromatography on CM-CPG (Serva)

Absorption 1: 350 ml of pH 4.5 enzyme-containing fraction was pressed onto a C16 / 20 chromatography column containing 100 ml / h of a flow rate of 100 ml / h and recycled at the bottom of the column to enhance the adsorption efficiency. The adsorption is thus carried out overnight at 0 - (+ 4) '.

Wash 2: Wash the cartridge with 60 mL of 0.1 M potassium citrate (pH 4); the column material does not show enzymatic activity, discard.

Wash 3 Π: Wash the cartridge with 60 mL of PBS buffer and apply the wash.

4. Elution: The myeloperoxidase was eluted from the column with 1.5 M NaQ + 0.5 M Tris pH 8.2; the activity appears in a single sharp fraction with a volume of about 20 ml having an activity of 10-150 U / ml.

(e) Determination of the amount of myeloperoxidase enzyme

The amount of enzyme is usually expressed in units of enzyme based on its activity.

Definition: 1 unit is the amount of enzyme which liberates 1 pMH ₂ O ₂ in a minute at 25 ° C, pH 6.00.

The rate of H 2 decomposition can be monitored spectrophotometrically. The exact method is described in the following literature:

G. Allan, P. Bhattacherjee, CJD, Brook, N. G. Read,

A. S. Parké, J. Cardiovasc. Pharm. 7, 1154-1160 [1985].

(f) Further purification by gel chromatography

The 20 ml enzyme-containing fraction obtained from the CM-CPG osriol was loaded onto a K50 / 100 column containing 1800 ml of an ULTROGEL AcA 54 gel filtration acrylamide-based packing, manufactured by LKB, marketed by Pharmacia AB, and eluted with 2 MNaCl / PBS. In the fraction of molecular weight 6040-8040, myeloperoxidase is clearly obtained. The column is pre-calibrated using molecular weight standards to select the appropriate fractions.

The average activity of the obtained products was 2000E; specific purity of the product is between 70-100E / mg protein. The concentration of the resulting solution was 100E / ml.

Example 2

The procedure described in Example 1 was followed, except that Concanavalin A was used as activating agent instead of Sendai virus at a concentration of 15 pg / ml.

Example 3

Example 1 except that Phytohemagglutinin was used as activating agent at a concentration of 10 pg / ml instead of Sendai virus.

HU 204081 Β

Example 4

Example 1 except that Lens culinaris Lectin was used as the activating agent in a concentration of 30 µg / ml instead of Sendai virus.

Example 5

Exemplary of Example 1 is the use of Ca ²⁺ ionophore as the activating agent, Ca ²⁺ chelate complexing agent A23137.

Claims (1)

A process for producing myeloperoxidase enzyme from human buffy coat by purification of white blood cells obtained by ammonium chloride hemolysis, digestion of the resulting cells and purification of the enzyme, characterized in that At, Phytohemagglutinin, Lens cullinaris lectin or Ca ²⁺ ionophore is used and the chromatographic step is carried out on a controlled pore size glass bead column packed with carboxymethyl functional groups. Published by: National Office of Inventions, Budapest Responsible publisher: dr. Gabriella Swoboda