IP Library › Granted Patent US 6,977,142
Granted Patent B2
US 6,977,142 · App. 10/669,382 · Granted Dec 20, 2005

High-throughput turbidometric assay for screening inhibitors of protein disulfide isomerase activity

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Quick Facts
Patent No.
US 6,977,142
App. No.
10/669,382
Granted
Dec 20, 2005
Kind
B2
Abstract

Methods for performing an end-point assay of protein disulfide isomerase activity. The method may be based on the enzyme-catalyzed reduction of insulin in the presence of dithiothreitol; measuring the aggregation of reduced insulin chains at 650 nm; and using hydrogen peroxide as a stop reagent.

Claims (17)

1. A method of performing an end point assay for protein disulfide isomerase (PDI) activity, the method comprising:

combining in a reaction mixture: a source of PDI activity; a substrate for PDI activity; and a reducing agent, wherein a plurality of said assays are performed in parallel;

incubating said reaction mixture for a period of time sufficient to reduce disulfide bonds present in said substrate;

stopping said reaction with hydrogen peroxide; and

taking the optical density measurement of said reaction mixture, wherein an increase in said optical density as compared to said optical density of a negative control is indicative of the presence of PDI activity.

2. The method according to claim 1 , wherein said substrate is selected from the group consisting of insulin and HIV gp120.

3. The method according to claim 2 , wherein said substrate is present at a concentration of from 0.075 to 0.3 mM.

4. The method according to claim 1 , wherein said reducing agent is dithiothreitol (DTT).

5. The method according to claim 4 , wherein said DTT is present at a concentration of from 0.1 to 2 mM.

6. The method according to claim 1 , wherein said hydrogen peroxide is added to a final concentration of from 10 to 500 mM.

7. The method according to claim 1 , wherein said reaction mixture is incubated for a period of time from 15 to 60 minutes.

8. The method according to claim 1 , wherein said optical density is read at 650 nm.

9. The method according to claim 1 , wherein said reaction mixture comprises less than 2% dimethyl sulfoxide.

10. The method according to claim 1 , wherein said assays are performed in a microtiter plate.

11. The method according to claim 1 , wherein said source of PDI activity is a biological sample.

12. The method according to claim 1 , wherein a recombinantly produced source of PDI activity is combined with said reaction mixture, and said reaction mixture further comprises a sample comprising a candidate PDI modulating agent.

13. The method according to claim 12 , wherein said PDI is provided at a concentration of 0.5 μg/ml to 100 μg/ml.