Dynamic continuous flow enzyme reactor

• US 4,865,984 A
• Filed: 02/08/1988
• Issued: 09/12/1989
• Est. Priority Date: 02/08/1988
• Status: Expired due to Fees

• Current Assignee: Mount Sinai School of Medicine of New York University
• Original Assignee: Mount Sinai School of Medicine of New York University
• Inventors: Nemerson, Yale, Turitto, Vincent
• Primary Examiner(s): Jones, Larry
• Application Number:
  US07/154,083
• Time in Patent Office: 582 Days
• Field of Search: 435/288, 435/310, 435/174, 435/284, 128/DIG. 22
• US Class Current: 435/287.1
• CPC Class Codes: C12M 21/18 Apparatus specially designed for the use of free, immobilized or carrier-bound enzymes
• CPC Class Codes: C12M 23/06 Tubular C12M23/08, C12M23/16 take precedence
• CPC Class Codes: C12Q 1/56 involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
• CPC Class Codes: G01N 2333/96444 Factor X (3.4.21.6)
• CPC Class Codes: G01N 2333/96447 Factor VII (3.4.21.21)
• CPC Class Codes: G01N 2333/96463 Blood coagulation factors not provided for in a preceding group or according to more than one of the proceeding groups
• CPC Class Codes: G01N 2333/974 Thrombin
• CPC Class Codes: G01N 2405/04 Phospholipids, i.e. phosphoglycerides

Abstract

A dynamic continuous flow enzyme reactor and method for carrying out and analyzing phospholipid-dependent enzyme reactions are provided. The enzyme reactor is a tubular member preferably a capillary tube, coated on its inner surface with a planar phospholipid bilayer membrane, optionally and preferably containing an enzyme or enzyme cofactor. The reactor is connectable at one end to a means for delivering fluid flow reagents to the reactor and is connectable at a second end to a means for analyzing an effluent exiting from the reactor. Preferably the enzyme cofactor in the phospholipid membrane is purified tissue factor and the fluid reagents are inactive blood clotting factors which become activated upon interaction with the phospholipid membrane through a tissue factor-mediated reaction.

Claims

1. A dynamic continuous flow enzyme reactor for carrying out and analyzing phospholipid-dependent enzymatic reactions which comprises a tubular housing coated on its inner surface with a planar phospholipid bilayer membrane, the housing being connectable at one opening to a means for delivering fluid flow reagents thereto and connectable at a second opening to a means for collecting and analyzing an effluent fluid therefrom.

2. The enzyme reactor of claim 1 wherein the housing is a capillary tube.

3. The enzyme reactor of claim 1 wherein the housing is a capillary tube having an inner diameter of about 0.1 to 1.1 mm and a length of about 1.0 to 15 cm.

4. The enzyme reactor of claim 1 wherein the phospholipid membrane comprises at least one additional component selected from the group consisting of enzymes and enzyme cofactors.

5. The enzyme reactor of claim 4 wherein the cofactor is tissue factor.

6. The enzyme reactor of claim 5 wherein the phospholipid membrane contains tissue factor in a ratio of approximately 1-10 moles of tissue factor per 100,000 moles of phospholipid.

7. The enzyme reactor of claim 1 wherein the phospholipid membrane comprises neutral and acidic phospholipids.

8. The enzyme reactor of claim 1 wherein the phospholipid membrane comprises phosphatidylcholine and phosphatidylserine.

9. The enzyme reactor of claim 1 wherein the phospholipid membrane comprises a mixture of 60-100% phosphatidylcholine and 0-40% phosphatidylserine.

10. The enzyme reactor of claim 1 wherein the phospholipid membrane comprises a mixture of 70% phosphatidylcholine and 30% phosphatidylserine.

11. A method for continuously carrying out and measuring dynamic enzyme reactions comprising:

(a) delivering reagents for performing an enzymatic reaction at a defined flow rate to an inlet in a dynamic continuous enzyme flow reactor comprising a tubular housing which is coated on its inner surface with a planar phospholipid bilayer membrane, wherein the reagents and membrane components are separately inactive for performing the enzymatic reaction, but together yield an enzymatically active system;

(b) pumping the reagents through the enzyme reactor at a constant and defined flow rate so that an enzyme reaction mediated by contact of the reagents with the phospholipid membrane takes place in the enzyme reactor;

(c) collecting a product of the enzyme reaction in an effluent from an exit in the enzyme reactor; and

(d) measuring the amount of product formed during the enzyme reaction by means of a suitable assay.

12. Method according to claim 11 wherein the phospholipid membrane further comprises at least one additional component selected from the group consisting of enzymes and enzyme cofactors.

13. Method according to claim 12 wherein the cofactor is tissue factor.

14. Method according to claim 13 wherein the reagents comprise blood clotting factors and calcium ions.

15. Method according to claim 14 wherein the blood clotting factors comprise a blood clotting factor selected from the group consisting of factor VII and factor VII_a, together with at least one factor selected from the group consisting of factor IX, factor X, factor V, factor VIII, prothrombin and whole plasma.

16. Method according to claim 14 wherein the blood clotting factors are factor VII and factor IX.

17. Method according to claim 14 wherein the blood clotting factors are factor VII_a and factor IX.

18. Method according to claim 14 wherein the blood clotting factors are factor VII and factor X.

19. Method according to claim 14 wherein the blood clotting factors are factor VII_a and factor X.

20. Method according to claim 14 wherein the blood clotting factors are factor VII, factor X, factor V and prothrombin.

21. Method according to claim 14 wherein the blood clotting factors are factor VII_a, factor X, factor V and prothrombin.

22. Method according to claim 11 wherein the assay for measuring the amount of product is selected from the group consisting of radioassays, chromogenic assays and fluorogenic assays.

23. Method according to claim 11 further comprising adding a chromogenic or fluorogenic substrate specific for the product of the reaction to the effluent before measuring the amount of product formed.

24. Method according to claim 11 further comprising splitting the product effluent from the enzyme reactor such that the amount of more than one product formed in the enzyme reactor can be measured.