METHODS OF ISOLATING AND DETECTING METAL IONS FROM PROTEINS

• US 20180067094A1
• Filed: 09/02/2016
• Published: 03/08/2018
• Est. Priority Date: 09/02/2016
• Status: Active Grant

• Current Assignee: Zepto Life Technology Incorporated
• Original Assignee: Zepto Life Technology, LLC
• Inventors: Sinha, Joy, Aguirre, Gerson
• Granted Patent: US 10,451,599 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 38/482 Serine endopeptidases (3.4.21)
• CPC Class Codes: C12N 9/50 Proteinases , e.g. Endopeptidases (3.4.21-3.4.25)
• CPC Class Codes: C12N 9/6408 Serine endopeptidases (3.4.21)
• CPC Class Codes: C12N 9/6424 Serine endopeptidases (3.4.21)
• CPC Class Codes: C12Y 304/21 Serine endopeptidases (3.4.21)
• CPC Class Codes: G01N 33/04 Dairy products
• CPC Class Codes: G01N 33/20 Metals
• CPC Class Codes: G01N 33/84 involving inorganic compounds or pH

Abstract

A method of detecting a metal ion in a protein-containing sample includes adding a protein degrading enzyme to the protein-containing sample to form an enzyme degradation product, adding an acid to the enzyme degradation product to provide a mixture, filtering the mixture to provide a supernatant, extracting the supernatant with an organic solvent to remove organic solvent soluble byproducts to provide a washed aqueous layer, and detecting the metal ion in the washed aqueous layer. The method is amenable to the detection of heavy metal ions in complex products such as milk. A kit includes reagents for performing the method.

Claims

1. A method of detecting a metal ion in a protein-containing sample comprising:

adding a protein degrading enzyme to the protein-containing sample to form an enzyme degradation product;

adding an acid to the enzyme degradation product to provide a mixture;

filtering the mixture to provide a supernatant;

extracting the supernatant with an organic solvent to remove organic solvent soluble byproducts to provide a washed aqueous layer; and

detecting the metal ion in the washed aqueous layer.

2. The method of claim 1, wherein the protein-containing sample is liquid milk.

3. The method of claim 1, wherein the protein-containing sample is powdered milk.

4. The method of claim 1, wherein the protein-containing sample is a solution of a protein powder.

5. The method of claim 1, wherein the protein degrading enzyme is a proteinase K enzyme.

6. The method of claim 5, further comprising a denaturing agent.

7. The method of claim 6, wherein the denaturing agent is an anionic surfactant, a urea, a chelating agents, a sulfhydryl reagent, a serine protease, or combinations thereof.

8. The method of claim 1, wherein the acid is concentrated nitric acid.

9. The method of claim 1, wherein the filtering step is performed with a 3 micron to 5 micron pore size filter.

10. The method of claim 1, wherein the detecting step is performed by fluorescence detection.

11. The method of claim 1, wherein the detecting step is performed by giant magnetoresistance measurements.

12. A method of detecting a heavy metal ion in a milk sample comprising:

adding a proteinase K enzyme to the milk sample to form an enzyme degradation product;

adding nitric acid to the enzyme degradation product to provide a de-emulsified mixture;

filtering the de-emulsified mixture to provide a supernatant;

extracting the supernatant with chloroform to remove chloroform soluble byproducts to provide a washed aqueous layer; and

detecting the heavy metal ion in the washed aqueous layer.

13. The method of claim 12, wherein the heavy metal ion is lead.

14. The method of claim 12, wherein the heavy metal ion is uranium.

15. A kit comprising:

a container for holding a protein-containing sample;

a proteinase K reagent;

a nitric acid reagent;

a chloroform reagent; and

instructions for performing the isolation of metal ions from the protein-containing sample.

16. The kit of claim 15, wherein the kit is a small scale kit for performing the isolation of metal ions in the field.

17. The kit of claim 15, further comprising a hand held detection device for detecting metal ions.

18. The kit of claim 17, where the hand held detection device employs fluorescence detection or detection based on giant magnetoresistance (GMR).

19. The kit of claim 15, further comprising other protein denaturing agents.

20. The kit of claim 19, wherein the further protein denaturing agents comprise at least one selected from the group consisting of sodium dodecyl sulfate (SDS), urea, ethylenediamine tetraacetic acid, trypsin, and chymotrypsin.