RU2271219C1 - Method for production of thrombin-like enzyme from snake venom - Google Patents

Abstract

FIELD: medicine, pharmaceutical industry, in particular production of pharmaceutical and diagnosis preparations from snake venom.

SUBSTANCE: venom dissolved in buffer solution is subjected to step-by-step chromatography on Sephadex G-100; cation-exchange resin Toyopearl 650 M with linear gradient of sodium chloride concentration and total gradient volume providing the most complete detection and separate elution of thrombin-like enzymes. Then said enzymes are separately subjected to recromatography, wherein linear gradients of sodium chloride concentration are defined based on elution conditions of respective thrombin-like enzymes on the previous step, and simultaneously total volumes of said gradients are increased up to level that provides additional separation of adjacent thrombin-like enzymes. Target product in homogenous state is produced after separate purification of thrombin-like enzymes on ABA-TSK sorbent.

EFFECT: method of increased yield; target product of improved activity, useful in evaluation of blood coagulation time or treatment and prophylaxis of thrombosis and related conditions.

1 ex, 5 tbl

Description

The invention relates to medicine and the pharmaceutical industry, in particular to the production of medicinal and diagnostic preparations from snake venom, and can be used for diagnostic and therapeutic purposes, in particular, for estimating blood coagulation time or for treating and preventing thrombosis and similar pathologies.

A known method of obtaining thrombin-like enzymes (enzymes with coagulating activity) from raw venom of the snakes of the genus Agkistrodon (Soloviev D.A., Ugarova T.P. Isolation and characterization of α-specific thrombin-like enzymes from the venoms of common moth (Agkistrodon halys halys) and orientalis (Central Asian subspecies Agkistrodon bromhoffii) // Biochemistry. - 1993 - vol. 58 - N8. 1221-1233.), in which the venom of snakes A. halys halys and A. bromhoffii are used as a raw material source. The method includes dissolving the poison in a buffer solution and subsequent purification using a sorbent - cybacron agarose. The method allows to separate and purify only 2 thrombin-like enzymes from the same source. The yield of active fraction in protein is 1.1-5.1%. The level of coagulating activity of the resulting enzymatic preparations does not exceed 450 NIH / mg protein.

A known method of producing thrombin-like enzymes from snake venom Trimeresurus okinavensis by fractionation of the poisoned buffer with Sephadex G-100, chromatography on CM-Toyopearl 650M in two stages and final purification on Mono Q gel (Nose S., Shimohigashi Y. et al. Purification and characterization of a coagulant enzyme, okinaxobin II, from Trimeresurus okinavensis (Himehabu snake) venom which releases fibrinopeptides A and B // Toxicon. - 1994. - V.32, N12. - P. 1509-1520). The method allows to separate and purify only 2 thrombin-like enzymes from the same source. The yield according to the level of the total coagulating active fraction for the protein is 5.15%. The level of specific coagulating activity of the resulting enzymatic preparations does not exceed 41.8 NIH / mg protein.

A known method for the isolation of thrombin-like enzymes from Agkistrodon saxatilis poison, in which crude poison dissolved in a buffer, is subjected to gradual fractionation on Q-Sepharose, Sephadex G-75 and Mono Q gel (Koh Y., Chung K., Kim D. Biochemical characterization of a thrombin-like enzyme and a fibrinolytic serine protease from snake (Agkistrodon saxatilis) venom // Toxicon. - 2001. - V.39, N4. - P.555-560). The method allows you to select only one thrombin-like enzyme from the same source. The yield according to the level of the total coagulating active fraction for the protein is 8.15%. The level of coagulating activity of the drug is 41.8 NIH / mg protein.

A known method for the isolation of thrombin-like enzymes from Agkistrodon acutus poison, in which the poison dissolved in the buffer is subjected to chromatography on DEAE-Sepharose, Sephacryl S-200 and CM-Sepharose (Huang Q., Teng M., Niu L. Purification and characterization of two fibrinogen- clotting enzymes from five-pace snake (Agkistrodon acutus) venom // Toxicon. - 1999.- V.37, N7. - P.999-1013). The method allows you to select only 2 thrombin-like enzymes with a level of coagulating activity not higher than 370 NIH / mg protein.

A known method for the isolation of thrombin-like enzymes from Agkistrodon acutus poison, in which the poison dissolved in the buffer is subjected to separation on a DEAE-Sephadex A-50 anion exchanger, and then chromatographed twice on Sephadex G-200 (Quyang C., Hong J. et al. Purification and properties of the thrombin-like principle of Agkistrodon acutus venom and its comparison with bovine thrombin. // Thromb.Diath.haemorrh. - 1971. - V.26 - P.224-234). The method allows you to select only one thrombin-like enzyme with a level of coagulating activity of 345 NIH / mg protein and a degree of purification of 13 times.

A known method of obtaining thrombin-like enzymes from crude venom of the snake Agkistrodon acutus, in which the poison dissolved in a buffer (pH 7.2) is subjected to sequential chromatography on Sephadex G-75, an anion exchanger DEAE-Sephadex A-50 with a gradient; sodium chloride 0.01-0.6 M, sorbent Heparin-Sepharose CL-6B (gradient of sodium chloride NaCl 0.1-0.3; 0.5 M) and, in a separate case, additionally on Sephadex G-100 (Komori Y. , Nikai T. et al. A comparative study of clotting factors from the venom of Agkistrodon acutus collected in China and Taiwan // Comp. Biochem. Physiol. - 1987. - V.88B, N2. - P.643-649). The method allows you to select only 2 thrombin-like enzymes from the poison A. acutus. At the final stage, 2-20.7 mg of the protein active drug is obtained from 2 g of the original poison. The level of specific coagulating activity of the resulting enzymatic preparations is 3-24.42 NIH / mg protein.

The disadvantage of the prototype is both a low level of specific coagulating activity of the target product, and a low quantitative yield of enzymes. The prototype does not allow you to select more than two enzymes from one source.

The objective of the invention is to provide a method for producing thrombin-like enzymes, which allows to increase the quantitative yield of enzymes and the final coagulating activity of the target product by increasing the number of secreted enzymes and their subsequent separate purification to obtain the target product in a homogeneous state.

The problem is solved in that in the known method for producing thrombin-like enzymes from snake venom by chromatography, according to the invention, the poison dissolved in the buffer solution is subjected to stepwise chromatography on Sephadex G-100, on a SM-Toyopearl 650M cation exchanger with a linear sodium chloride concentration gradient and a full gradient volume that provide the most complete identification and separate elution of thrombin-like enzymes, then separate rechromatography of these enzymes is carried out, setting linear gradients of concentration sodium chloride radio conditions of respective elute thrombin-like enzyme in the previous step while increasing the total volume of these gradients to levels which provides additional separation of adjacent thrombin enzymes, to give the desired product in a homogeneous condition after the separate cleaning enzyme thrombin on the sorbent ABA-TSK.

Studies using Agkistrodon acutus snake venom showed that chromatography on Sephadex G-100 allows for more complete removal of high molecular weight and low molecular weight inhibitors of thrombin-like enzymes and other ballast components from the crude venom that prevent the detection of complete coagulating activity at the initial stage of purification. As a result, already at this stage of purification, the level of detectable coagulating activity rises by 2-13 times in comparison with the prototype. The soft ion-exchange chromatography conditions on CM-Toyopearl 650M allow, by varying the linear gradient of the concentration of sodium chloride (NaCl gradient) and its total volume, to separate up to seven thrombin-like enzymes, eluting in a certain order along the increase in the concentration of NaCl. The dependence of the amount of thrombin-like enzymes obtained on the NaCl gradient and its total volume is presented in Table 1.

Table 1
Quantitative yield of enzymes from the venom of the snake Agkistrodon acutus Gradient NaCl (M) 0-0.2 0-0.3 0-0.4 0-0.6 0.1-0.4 0.15-0.4 The total volume of the gradient of NaCl (ml) 500 750 1000 1500 750 625 Number of obtained enzymes (peaks) 6 7 7 6 5 1-2

Studies have shown that with a linear NaCl gradient from 0 M to 0.3-0.4 M and a full gradient volume of 750 ml - 1000 ml, respectively, up to 7 enzymes are released. This is the most complete number of enzymes obtained from the venom of the snake Agkistrodon acutus of the proposed method. With a NaCl gradient of 0.1-0.4 and a full gradient of 750 ml, only 5 enzymes are released, and with a gradient of 0.15-0.4 M and a full volume of 625 ml, only 1-2 peaks of coagulating activity are obtained, i.e. the separation of enzymes is possible and, accordingly, the coagulating activity of the target product does not increase. Separate elution allows the separation of thrombin-like enzymes with high activity from those with low activity. Moreover, the more thrombin-like enzymes are isolated, the more accurately highly active are released. Repeated cation exchange chromatography (rechromatography) was carried out on a CM-Toyopearl 650M separately for each thrombin-like enzyme, with a slower increase in the concentration of NaCl due to an increase in the total volume of the NaCl gradient by 1.4-1.6 times. The boundaries of the linear NaCl gradient are determined based on the elution conditions of the corresponding thrombin-like enzyme in the previous step. This allows you to remove from the intermediate fractions of the enzyme as neighboring eluting enzymes, and the remaining inhibitory and ballast proteins. Such additional purification of each of the enzymes on CM-Toyopearl 650M under conditions corresponding only to their individual physicochemical characteristics leads to an increase in the level of activity (for the most active ones, by 11-15 times, with a yield of 10-19%). At the final stage of purification, the preparation of each enzyme is subjected to chromatography on an ABA-TSK sorbent. This sorbent contains a serine protease inhibitor as a ligand. From available sources, the authors are not aware of the use of ABA-TSK sorbent for the purification of thrombin-like enzymes. Studies have shown that the sorbent ABA-TSK is highly specific for thrombin-like enzymes. Each enzyme has its own ballast proteins. In this regard, at the last stage of purification, when it is necessary to remove from the individual intermediate preparation of the enzyme the ballast and inhibitory proteins accompanying only this enzyme (and not their mixtures), individual chromatography conditions are selected for each enzyme. As a result, the level of activity of the target product during purification of the most active enzymes rises to 1100 NIH / mg and higher. Chromatography of individual enzymes, rather than mixtures thereof, greatly simplifies the control of chromatography and allows you to increase the coagulating activity of the target product by separately purifying each of the highly active thrombin-like enzymes to 8-14%. In addition, separate purification makes it possible to isolate in a homogeneous state new thrombin-like enzymes that differ in their physicochemical properties.

The method can be carried out as follows.

The crude venom of Agkistrodon acutus snake is dissolved in 0.05 M ammonium acetate buffer (pH 6.8). Insoluble components are removed by centrifugation at 10,000-15,000 g for 20-30 minutes. The supernatant was chromatographed on a Sephadex G-100 column (90-100 cm high), filled with the same buffer. The eluting fractions having coagulating activity are pooled and introduced into a CM-Toyopearl 650M column (110-120 cm high) filled with 0.05 M ammonium acetate buffer (pH 6.5). Elution is carried out with a linear NaCl gradient from 0 M to 0.3-0.6 M. The solutions making up the linear NaCl gradient are prepared in a ratio of 1: 1 with a total gradient volume of 750 ml - 1500 ml, respectively. The fractions obtained as a result of elution having coagulating activity are combined according to their corresponding elution peak. Peaks in the profile of coagulating activity reflect the site of elution of enzymes from the column CM-Toyopearl 650M. From this moment, the combined fractions are numbered according to the order of elution from the column (for example, as E _n , where n = 1, 2 ... 7, ... and so on) and the enzyme E _n (where n = 1, 2, is conditionally called). .. 7 ... and so on). The pooled fractions of each enzyme were equilibrated with 0.05 M ammonium acetate buffer (pH 6.5) and NaCl ions were removed using an ultrafiltration membrane. At this stage, it is possible to carry out complete removal of salt ions and lyophilization of the drug for the purpose of delayed subsequent cleaning. The resulting balanced solution of the partially purified enzyme was subjected to repeated chromatography (rechromatography) on a CM-Toyopearl 650M column under the same conditions as in the previous step, setting only new boundaries for the NaCl gradient. The boundaries of the NaCl gradient at this stage are set based on the elution conditions of the corresponding thrombin-like enzyme at the previous purification step (namely, during the first chromatography of CM-Toyopearl 650M). Each of the solutions that make up the NaCl gradient is prepared 1.4-1.6 times more than in the previous purification step, and in the ratio 1: 1 to each other. After elution, the fractions corresponding to the base of the main peak of the coagulating activity profile are combined. This procedure leads to the final separation of the enzyme E _n from the neighboring elution enzymes E _n-1 and E _{n + 1} . The pooled fractions of the enzyme E _{n are} equilibrated with the buffer of the next step, removing NaCl ions using an ultrafiltration membrane. At this stage, it is possible to carry out complete removal of salt ions and lyophilization of the drug for the purpose of delayed subsequent cleaning. After the preparation described above, the enzyme preparation E _n is introduced into the ABA-TSK column of the sorbent and the elution is performed with a smooth NaCl gradient from 0 M to 0.5 M. The eluting fractions having coagulating activity are combined. The resulting preparation is an individual thrombin-like enzyme E _n purified from a homogeneous state from Agkistrodon acutus snake venom. This drug can be purified from salt ions and then lyophilized for storage.

The method is illustrated by the following example.

Example. 500 mg of raw venom of A. acutus snake is dissolved in 3 ml of 0.05 M ammonium acetate buffer (pH 6.8) and centrifuged at 12000 g for 20 minutes. The supernatant was applied to a Sephadex G-100 column (2.8 x 97 cm) buffered with the same solution. Elution is carried out with the same solution at a rate of 17 ml / hour. The size of the collected fractions 5 ml. Fractions containing coagulating activity are identified by determining the clotting time of fibrinogen (to 0.27 ml of 5% bull fibrinogen, 0.03 ml of eluate is added). 13 fractions were collected. The total volume of 65 ml. It was introduced into a CM-Toyopearl 650M column (1.7 × 115.5 cm), equilibrated with 0.05 M ammonium acetate buffer (pH 6.5), and then eluted with a linear NaCl gradient from 0 M to 0.4 M. The gradient components are prepared in a volume of 500 ml each. Elution is carried out at a rate of 30 ml / hour. The volume of collected fractions of 2.5 ml. In the range of fractions No. 120-212 of the eluate, 7 peaks of coagulating activity (conditionally designated 1, 2 ... 7) are detected. The fractions corresponding to each peak are combined and separate purification is carried out by rechromatography and chromatography on a sorbent. The results of chromatography are presented on the example of obtaining enzymes designated E ₃ , E ₅ , E ₆ and E ₇ .

table 2
Obtaining the enzyme E ₆ . Stage name Total protein (mg) Total activity (NIH) Specific Activity (NIH / mg) Degree of purification Exit (%) Raw poison 1454 - - - - Buffer dissolution 1000 0 0 - - Sephadex G-100 174 8874 51 one one hundred CM-Toyopearl 650M 7.17 3850 537 10.5 43 CM-Toyopearl 650M 1.55 1211 781 15.3 fourteen ABA-TSK 0.77 1247 1102 21.6 10

To obtain the E ₆ enzyme, 8 fractions (No. 179-186) corresponding to the E ₆ enzyme are combined, NaCl is removed and equilibrated with ammonium acetate buffer (pH 6.5) using an ultrafiltration membrane. The resulting solution of the E ₆ enzyme was again introduced into a CM-Toyopearl 650M column (1.7 × 115.5 cm), equilibrated with 0.05 M ammonium acetate buffer (pH 6.5). Elution is carried out by a linear NaCl gradient from 0.04 M to 0.14 M in accordance with the elution conditions of the enzyme E ₆ in the previous step. The gradient components are prepared in a volume of 750 ml each. The remaining conditions of rechromatography are the same. Fractions of the eluate No. 335-375 have coagulating activity and correspond to the enzyme E ₆ . These fractions are combined and, using an ultrafiltration membrane, balance with 0.03 M ammonium acetate buffer (pH 7.0). The resulting protein solution is introduced into the column of the sorbent ABA-TSK (size 1 × 10 cm). Elution is performed with a NaCl gradient from 0 M to 0.5 M. The elution rate is 1 ml / min. The volume of fractions is 1 ml. Fractions having a coagulating activity ranging in the range of 61-75 min, represent a homogeneous thrombin-like enzyme E ₇ from the snake venom Agkistrodon acutus. This product shows one lane for polyacrylamide gel electrophoresis with sodium dodecyl sulfate. The results of the determination of coagulating activity during the purification process are presented in Table 2 (data recalculated for 1454 mg of crude poison).

Table 3
Obtaining the enzyme E ₇ Stage name Total protein (mg) Total activity (NIH) Specific Activity (NIH / mg) Degree of purification Exit (%) Raw poison 1454 - - - Buffer dissolution 1000 0 0 - - Sephadex G-100 174 8874 51 one one hundred SM-Toyopearl 650M 17.6 3520 200 3.9 40 SM-Toyopearl 650M 2.46 1700 691 13.5 19 ABA-TSK 0.97 1247 1286 25,2 fourteen

To obtain the E ₇ enzyme, the 23 fractions (No. 187-209) corresponding to the E ₇ enzyme are combined, NaCl is removed and equilibrated with ammonium acetate buffer (pH 6.5) using an ultrafiltration membrane. The resulting enzyme solution E _{7 was} again introduced into a CM-Toyopearl 650M column (1.7 × 115.5 cm), equilibrated with 0.05 M ammonium acetate buffer (pH 6.5).

Elution is carried out with a linear NaCl gradient from 0.05 M to 0.15 M in accordance with the elution conditions in the previous step. The gradient components are prepared in a volume of 750 ml each. The remaining conditions of rechromatography are the same. Fractions of the eluate No. 235-270 have coagulating activity and correspond to the enzyme E ₇ . These fractions are combined and, using an ultrafiltration membrane, equilibrated with 0.03 M ammonium acetate buffer (pH 7.0). The resulting protein solution is introduced into the column (1 × 10 cm in size) of the ABA-TSK sorbent. Elution is performed with a NaCl gradient from 0 M to 0.5 M. The elution rate is 1 ml / min. The volume of fractions is 1 ml. Fractions having a coagulating activity ranging in the range of 25-45 min represent the thrombin-like enzyme E ₇ from the venom of the snake Agkistrodon acutus. This product shows one lane in polyacrylamide gel electrophoresis with sodium dodecyl sulfate. The results of the determination of coagulating activity during the purification process are presented in Table 3 (data recalculated for 1454 mg of crude poison).

Table 4
Obtaining the enzyme E ₅ Stage name Total protein (mg) Total activity (NIH) Specific Activity (NIH / mg) Degree of purification Exit (%) Raw poison 1454 - - - - Buffer dissolution 1000 0 0 - - Sephadex G-100 174 8874 51 1,0 one hundred CM-Toyopearl 650M 6.87 1065 155 3.0 12 CM-Toyopearl 650M 1,61 963 598 11.7 eleven ABA-TSK 0.48 672 1399 27.4 8

To obtain the E ₅ enzyme, 9 fractions (No. 170-178) corresponding to the E ₅ enzyme are combined, NaCl is removed and equilibrated with ammonium acetate buffer (pH 6.5) using an ultrafiltration membrane. The resulting solution of enzyme E _{5 was} again introduced into a CM-Toyopearl 650 M column (1.7 × 115.5 cm), equilibrated with 0.05 M ammonium acetate buffer (pH 6.5). Elution is carried out by a linear NaCl gradient from 0.03 M to 0.13 M in accordance with the elution conditions of the enzyme E ₅ in the previous step. The gradient components are prepared in a volume of 750 ml each. The remaining conditions of rechromatography are the same. Fractions of eluate No. 247-303 have coagulating activity and correspond to the enzyme E ₅ . These fractions are combined and, using an ultrafiltration membrane, equilibrated with 0.03 M ammonium acetate buffer (pH 7.0). The resulting protein solution was introduced into an ABA-TSK sorbent column (1 × 10 cm in size). Elution is performed with a NaCl gradient from 0 M to 0.5 M. The rate is 1 ml / min. The volume of fractions is 1 ml. Fractions having a coagulating activity ranging from 19-38 min represent the homogeneous thrombin-like enzyme E ₅ from the venom of the snake Agkistrodon acutus. This product shows one lane in polyacrylamide gel electrophoresis with sodium dodecyl sulfate. The results of the determination of coagulating activity during the purification process are presented in Table 4 (data recalculated for 1454 mg of crude poison).

Table 5
Obtaining the enzyme E ₃ Stage name Total protein (mg) Total activity (NIH) Specific Activity (NIH / mg) Degree of purification Exit (%) Raw poison 1454 - - - - Buffer dissolution 1000 0 0 - - Sephadex G-100 168 8874 51 one one hundred CM-Toyopearl 650M 25.8 671 26 0.5 8 CM-Toyopearl 650M 2.96 243 82 1,6 3 ABA-TSK 0.41 118 288 5,6 1.4

To obtain the E ₃ enzyme, 7 fractions (No. 155-161), corresponding to the E ₃ enzyme, are combined, NaCl is removed and balanced with ammonium acetate buffer (pH 6.5) using an ultrafiltration membrane. The resulting solution of enzyme E _{3 was} again introduced into a CM-Toyopearl 650M column (1.7 × 115.5 cm), equilibrated with 0.05 M ammonium acetate buffer (pH 6.5). Elution is carried out with a linear NaCl gradient from 0.01 M to 0.11 M. The components of the gradient are prepared in a volume of 750 ml each. The remaining conditions of rechromatography are the same. Fractions of eluate No. 268-282 have coagulating activity and correspond to the enzyme E ₃ . These fractions are combined and, using an ultrafiltration membrane, equilibrated with 0.03 M ammonium acetate buffer (pH 7.0). The resulting protein solution is introduced into the column of the sorbent ABA-TSK (size 1 × 10 cm). Elution is performed with a NaCl gradient from 0 M to 0.5 M. The elution rate is 1 ml / min. The volume of fractions is 1 ml. Fractions having coagulating activity represent the homogeneous thrombin-like enzyme E ₃ from the venom of the snake Agkistrodon acutus. This product shows one lane for polyacrylamide gel electrophoresis with sodium dodecyl sulfate. The results of the determination of coagulating activity during the purification process are presented in Table 5 (data recalculated for 1454 mg of crude poison).

From table.2-5 it is seen that already at the stage of chromatography on Sephadex G-100, the level of coagulating activity increases by 2-13 times compared with the coagulating activity of the target product in the prototype. As noted above, this may be due to the removal of high molecular weight and low molecular weight inhibitors of thrombin-like enzymes. The cleaning effect is further enhanced by the chromatography and rechromatography steps on the CM-Toyopearl 650M. The latter is associated with the separation of enzymes with high activity from those with low activity. Moreover, the method identifies and allows you to select more than 2 enzymes from the venom of the snake Agkistrodon acutus, the possibility of which is completely absent in the prototype. Work with an individual enzyme probably greatly facilitates the conduct and additional purification at the last stage (ABA-TSK sorbent), when the activity level increases by 100%.

Thus, due to the separate purification of the most active enzymes, the method allows to increase the activity level of the final preparation of the thrombin-like enzyme from the venom of Acustrodon acutus snake by more than 45-55 times (up to 1100-1400 NIH / mg) compared to the prototype. The yield of the final enzyme preparation is at least 8-14%, which is 8-14 times higher than that of the prototype. Isolation of the full spectrum of enzymes provides the targeted creation of polyvalent serum to neutralize the effects of snake bites, and can also serve as an experimental model for studying the mechanism of action of thrombin.

The method can be used to obtain thrombin-like enzymes from the poisons of other snakes. The optimal conditions for chromatography are selected in preliminary experiments for each specific poison.

Claims (1)

A method of producing thrombin-like enzymes from snake venom of the genus Agkistrodon acutus, comprising dissolving the poison in a buffer solution, separating soluble components from insoluble components and subsequent stepwise chromatography on Sephadex, on an ion exchanger with a linear gradient of sodium chloride concentration and on a sorbent, characterized in that it is dissolved in a buffer the solution is separated by centrifugation from insoluble components at 10000-15000 g, then the supernatant is chromatographed on a Sephadex G-100, fractions containing coagulir The active activity is identified, collected, chromatography is carried out on a CM-Toyopearl 650 M cation exchanger with a linear gradient of sodium chloride concentration from 0 to 0.3-0.4 M with a total gradient volume of 750 ml to 1000 ml, respectively, and the fractions corresponding to each of the separated enzymes, repeated chromatography is carried out on a CM-Toyopearl 650 M cation exchanger separately for each thrombin-like enzyme with an increase in the total volume of sodium chloride gradients by 1.4-1.6 times, and the gradient boundaries are set based on the elution conditions of the corresponding thrombin-like enzyme in the previous step, and the target product is obtained in a homogeneous state after separate purification of the thrombin-like enzymes on the ABA-TSK sorbent with a smooth gradient of sodium chloride from 0 to 0.5 M.