Arginine hydrochloride enhances chaperone-like activity of alpha crystallin

• US 20070009956A1
• Filed: 12/30/2003
• Published: 01/11/2007
• Est. Priority Date: 12/30/2003
• Status: Abandoned Application

• Current Assignee: Council of Scientific & Industrial Research
• Original Assignee: Council of Scientific & Industrial Research
• Inventors: Rao, Chintalagiri, Raman, Bakthisaran, Rao, Kunchala, Ramakrishna, Tangirala, Srinivas, Volety
• Application Number:
  US10/528,443
• Publication Number:
  US 20070009956A1
• Time in Patent Office: Days
• US Class Current: 435/7.100
• CPC Class Codes: A61K 2300/00 Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
• CPC Class Codes: A61K 31/197 the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid carnitine A61K31/205
• CPC Class Codes: A61K 38/28 Insulins
• CPC Class Codes: C07K 14/47 from mammals

Abstract

The present invention provides that arginine, a biologically compatible molecule that is known to bind to the peptide backbone and negatively charged side-chains, increases the chaperone-like activity of a-crystallin significantly. Arginine, interestingly, restores the activity of mutant protein to a considerable extent. The invention shows the effect of arginine on the structural changes of a-crystallin by circular dichroism, fluorescence and glycerol gradient sedimentation. The invention shows that arginine brings about subtle changes in the tertiary structure and significant changes in the quaternary structure of a-crystallin and enhances its chaperone activity significantly. This invention thus has role in designing strategies to improve chaperone function for therapeutic applications.

Claims

1. A method for enhancing molecular chaperone activity of α

-crystalline (comprising of forms α

-crystalline and α

B-crystalline) with a biological compatible amino acid molecule of Arginine Hydrochloride, said method comprising the steps of;

(a) isolating and purifying α

-crystalline from calf eye lenses by convention methods (as described in reference

     24), and

(b) reacting α

-crystalline in the presence of phosphate buffer of pH 7.4 with Arg.HCl and insulin or ξ

-crystalline in presence or absence of DTT, and

(c) observing the enhancement in chaperone activity of α

-crystalline in presence of Arg.HCl by fluorescence spectrophotometer.

2. A method as claimed in claim 1, wherein Arginine hydrochloride (Arg.HCl) binds to the peptide backbone and negatively charged side chains of α

-crystalline to enhance chaperone activity.

3. A method as claimed in claim 1, wherein Arg.HCl is in the range of about 50 to 350 mM.

4. A method as claimed in claim 3, wherein Arg.HCl is in the range of about 100 to 300 mM.

5. A method as claimed in claim 1, wherein Arg.HCl enhances the chaperone activity of α

-crystalline by about 95%.

6. A method as claimed in claim 5, wherein Arg.HCl enhancs the chaperone activity of α

-crystalline by about 90%.

7. A method as claimed in claim 1, wherein Arg.HCl enhance the chaperone activity of α

-crystalline by about 90% in presence of various aggregation systems.

8. A method as claimed in claim 7, wherein Arg.HCl enhance the chaperone activity of α

-crystalline by about 81% in presence of various aggregation systems.

9. A method as claimed in claim 1 and 7, wherein aggregation systems maybe selected from group comprising of insulin, ξ

-crystallin and related compounds.

10. A method as claimed in claim 1, wherein Arg.HCl at a temperature of about 30°

C. protects the α

-crystalline by about 35%.

11. A method as claimed in claim 1, wherein Arg.HCl at a temperature of about 30°

C. protects the α

-crystalline by about 28%.

12. A method as claimed in claim 1, wherein Arg.HCl brings about subtle changes in the tertialry structure and significant changes in the quaternary structure of both homo-multimers or hetero-multimers of α

A-crystalline and α

B-crystalline to enhance the chaperone activity.

13. A method as claimed in claims 1 and 12, wherein presence of Arg.HCl the molecular mass of α

-crystalline is reduced ˜

360 kDa thereby bringing about subtle changes in the tertialry structure and significant changes in the quaternary structure of both homo-multimers or hetero-multimers of α

A-crystalline and α

B-crystalline to enhance the chaperone activity.

14. A method as claimed in claim 1, wherein wild type and mutant α

A-crystalline are less sensitive to Arg.HCl than α

B-crystalline, thereby enhancing the chaperone activity.

15. A method as claimed in claims 1 and 14, wherein protection of mutant α

B-crystalline (R120α

B-crystallin) is about 80% in presence of Arg.HCl.

16. A method as claimed in claim 15, wherein protection of mutant α

B-crystalline (R120α

B-crystallin) is about 75% in presence of Arg.HCl.

17. A method as claimed in claim 1, wherein Arg.HCl enhances the α

-crystalline chaperone activity by about 45% by exposing the hydrophobic surfaces of α

-crystalline.

18. A method as claimed in claim 14, wherein Arg.HCl enhances the α

-crystalline chaperone activity by about 38% by exposing the hydrophobic surfaces of α

-crystalline.