FORM -2
THE PATENTS ACT, 1970 (39 of 1970)
COMPLETE SPECIFICATION
(See Section 10) RULE 13
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*tMPROVED PROCESS Of INIIIDfflQN 1
HINDUSTAN LEVER LIMITED, a company incorporated under the Indian Companies Act, 1913 and having its registered office at Hindustan Lever House, 165/166, Backbay Reclamation, Mumbai -400 020, Maharashtra, India
The following specification particularly describes the nature of the invention and the manner in which it is to be performed.



FIELD OF THE INVENTION
The present invention relates to a method of treating intra or extra cellular proteinase in laboratory studies and in particular to a method of inhibiting/inactivating intra or extra cellular proteinase in isolated tissues/cell suspensions or extracts with melanin monomers and derivatives.
BACKGROUND ART
Proteinases are basically enzymes that are active in catalyzing the break down of peptide bonds which process is conventionally known as proteolysis. Proteases are usually compartmentalised in organelles like lysosomes and endosomes so that they do not hydrolyse proteins nonspecifically. During protein purification and isolation from tissues and biological samples, proteases get liberated in the homogenate, resulting in a low yield of the desired protein. Addition of protease inhibitors like leupeptin, phenyl methyl sulphonyl fluoride to restrict proteolysis is generally an accepted protocol.
OBJECT OF THE INVENTION
It has thus been the basic objective of the present invention to provide a method of inhibiting/inactivating intra or extra cellular proteinase in laboratory studies.
It is another objective of the present invention to provide a method of inhibiting/inactivating intra or extra cellular proteinase which would favour identifying the characteristics and mechanism of complex formation of isolated
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proteinase with monomers or melanin monomers/polymers for varied end uses/applications.
Yet another object of the present invention is directed to simulate conditions for in-vitro treating of intra or extra cellular proteins which would provide for proteins complex formation in combination with monomers or melanin monomers / polymers to thereby facilitate the study of the nature of such proteins complex formation including the crystallographic data of such complexes which could further facilitate clear appreciation of the nature of bonding of the binding sites in proteins complexes for varied end applications/uses.
SUMMARY OF THE INVENTION
Thus according to the basic aspect of the present invention there is provided a method for inactivating/inhibiting intra or extra cellular proteins in laboratory studies comprising subjecting the said intra or extra cellular proteins to treatment with melanin monomers or their derivatives.
The method for inactivating/inhibiting intra or extra cellular proteins particularly refer to intra or extra cellular proteins in isolated tissues/cell suspensions or extracts and any other similar form encountered in laboratory studies.
Thus according to another aspect of the present invention there is provided a method for providing isolated proteins complexes consisting of:


i. isolating the selected proteins
ii. crystallization of the thus isolated proteins;
iii. subjecting the thus crystallized isolated proteins to treatment with
melanin monomers or their derivatives to thereby provide the isolated
proteins complex.
In accordance with a preferred aspect of the present invention the method of
providing the isolated proteins complexes consisting of:
i. isolating Proteins K;
ii. crystallization of the thus isolated Proteins K.
iii. subjecting the thus crystallized isolated Proteins K to said treatment with melanin monomers or their derivatives by soaking in Dopa solution for 3-4 hours to thereby provide the isolated proteins K complex.
The melanin monomers can be generated in situ by combining a precursor of melanin formation with an enzyme or an oxidanror af mixture thereof.
Precursors of melanin formation are selected from Tyrosine, Dihydroxy phenyl alanine (DOPA), catechol, other phenolic derivatives (unsubstituted hydroxyl groups in the 3rd & 4th position); and is employed at levels in the range 0.005mM to 3M.
Enzymes are selected from tyrosinase, polyphenol oxidase, is employed at levels in the range 0.1 unit/ml to 7500 units/ml.
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. Oxidants are selected from Hydrogen peroxide, and other peroxide derivatives and is employed at levels in the range 0.1 mM to 3M.
It is particularly preferred that the precursor of melanin is in the range 0.01 mM to 1M and more preferably 0.01 mM to 100mM. The concentration of the enzyme 0.2 units/ml to 2500 units/ml or 0.2mM to 1M of the oxidant or a free radical generating system or a mixture thereof and more preferably 0.2 units/ml to 250 units/ml.of an enzyme or 0.2MM to 100mM oxidant or a free radical generating system or a mixture thereof.
In the above process of the invention the melanin monomers are preferably prepared by inducing auto oxidation of Dopa solution at pH of 5 to 10 preferably 7.0 - 8.0 incubated for 1 to 6 hours preferably 5 hours.
It is found by way of the above process of the invention that it is possible to form proteinase complexes from isolated proteinases especially between isolated proteinase K and the melanin monomers/oligomers. Importantly, the above process of the invention providing for isolated proteinase complexes confirm the binding sites and that proteinase can be adapted to bind to melanin monomers.
It is thus possible following the above process of the invention to achieve obtaining isolated proteinase complexes following an in-vitro process which would enable modulating the nature of binding between isolated proteinases and mefanin monomers/oligomers.
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The details of the invention its objects and advantages are explained hereunder in greater detail in relation to non-limiting exemplary illustrations by way of the following examples:
EXAMPLES
Binding of Proteinase K bv melanin monomers:
Example 1:
Preparation of isolated Proteinase K. complex
Under this example, proteinase K complex with monomers or the melanin
monomers/oligomers was obtained as detailed hereunder:
Isolation and Purification of the Proteinase K.:
Proteinase K was isolated from Bacillus spp. using G75 gel filtration column chromatography. N-terminal sequence of the above protein was determined up to 14 amino acids residues to confirm its identity and is as follows:
E, A, A/H, K, S, E, I, A/H, A, R, Y, N, D, L
Crystallization of isolated proteinase K.
The crystallization of the proteinase K was next effected using micro dialysis method.
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Proteinase K solution of 10mg/ml in 50mM Tris-CI and 1mM CaCl2 at pH of 6.5 was dialyzed against 1M of NaN03 to thereby obtain the proteinase K crystals.
Preparation of Dopachrome solution
Dopa solution at pH of 7.0 was prepared by auto oxidation of the solution at said pH incubated for 5 hours.
Method of treating the crystallized proteinase K to obtain the complex :-
The crystals of proteinase K were soaked in Dopa solution (prepared as above) for 3-4 hours. Crystals were next washed in 3 M NaN03 to remove the external black precipitate to finally obtain the desired Proteinase K complex based on the isolated Proteinase K.
The thus obtained complexes based on Proteinase K under Examples 1 above were studied using Denzo and refined using X-plot and CCP4 packages.
The crystallographic data on the crystals of the complexes under Examples 1 above are noted hereunder in Table 1.
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TABLE 1

Crystallographic data Example 1
Proteinase K + DOPA complex
System Tetragonal
Space group P43 212
A 68.319
B 68.319
C 108.292
V 5.1 X105
VmA3/Da 2.18A3/da
Solvent content 44%
Total no.of reflections collected 32082
Total no. of unique reflections 17087
Current R factor 0.23
Following the above method of the invention, the isolated proteinase crystals as obtained above, were further studied to identify the nature of the binding sites in the isolated proteinase source i.e. the Proteinase K of the Example 1. Accompanying Figures 1 and 2 further illustrate the nature ot the binding sites achieved between the proteinase and the melanin monomer in the crystal form under Examples 1.
As represented in Figure 1 and 2 there is a single Indolequinone binding site in case of isolated Proteinase K
The above demonstrates that it is possible that isolated proteinases such as proteinase K, can bind to melanin monomers and there are specific binding


sites for the formation of isolated proteinase complexes following the above method of the invention. This binding site is at the active site of the proteinase.
The method can be adapted to carry out generation of isolated proteinase complexes which would enable effective use and application of the number and the nature of the binding sites in possible variants of isolated proteinase complexes with melanin monomers/intermediates. The method is also directed to serve in modulating the generation of proteinase complexes from isolated proteinases.
Example 2:
Inhibition of Proteinase K bv melanin monomers
Dihydroxy phenyl alanine (DOPA) and tyrosinase in an acetate buffer (pH 5.0) was allowed to react for 2 hours at about 25°C and only melanin intermediates less than 3000 daltons were used. Bovine Serum Albumin (BSA) was used as the model substrate and proteinase K (PK) as the proteolytic enzyme for its hydrolysis. To demonstrate the effect of melanin intermediates on the inhibition of proteinase, enzyme proteinase K was preincubated with and without melanin intermediates (Ml) and BSA was used as a substrate and the reaction was allowed to proceed. After the reaction was complete the proteins were precipitated with 10% trichloro acetic acid and washed with diethyl ether. The unreacted protein was analysed on 10% Sodium Dodecyl Sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The results obtained as regards the extent of proteolytic activity achieved is represented in the figure
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3. The intensity of the band marked (A) in the figure represents the level of BSA unreacted.
Figure 3. shows the following lanes.

Lane 1 Lane 2 Lane 3 Lape 3
PK + MI BSA PK + BSA (PK +MI) +BSA „

i '
* t

As clearly apparent from said Figure 3 the digestion of BSA when the enzyme proteinase K was incubated with melanin oligomers lead to restricted proteolysis. The band corresponding to undigested BSA was clearly more intense when proteolysis was assayed in the presence of melanin oligomers.
The above results clearly go to show that the melanin intermediates inhibit proteolysis by inactivating enzyme proteinase.

■\th
Dated 30m December 2002

Hindustan Lever Limited
(S Venkatramani) Patents Manager


WE CLAIM:
1. A method for inactivating/inhibiting intra or extra cellular proteinase in
laboratory purposes comprising subjecting the said intror extra cellular
proteinase to treatment with melanin monomers or their derivatives.
2. A method as claimed in claim 1 wherein said laboratory activities comprise the said step of inactivating/inhibiting intrcvor extra cellular proteinase in experiments to test and/or evaluate and/or produce a product or any process.
3. A method as claimed in claims 1 or 2 wherein the intra or extra cellular proteinase used comprise intra or extra cellular proteinase in isolated tissues/cell suspensions and/or extracts and/or any other similar form encountered in laboratory purposes.
4. A method for providing isolated proteinase complexes comprising of :
i. isolating the selected proteinase;
ii. crystallization of the thus isolated proteinase;
iii. subjecting the thus crystallized isolated proteinase to treatment as claimed in claim 1 with melanin monomers or their derivatives to thereby provide the isolated proteinase complex.
5. A method or providing the isolated proteinase complexed comprising of:
i. isolating Proteinase K;
ii. crystallization of the thus isolated Proteinase K;
iii. subjecting the thus crystallized isolated Proteinase K to said treatment as claimed in claim 1 with melanin monomers or their derivatives by soaking in DOPA solution for 3-4 hours to thereby provide the isolated proteinase K complex.
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A method as claimed in anyone of claims 1 to 5 wherein said melanin monomers used are generated in situ by combining a precursor of melanin formation with an enzyme or an oxidant or a mixture thereof.
A method as claimed in claim 6 wherein said precursors of melanin formation are selected from Tyrosine, Dihydroxy phenyl alanine (DOPA), catechol, other phenolic derivatives (unsubstituted hydroxyl groups in the 3rd and 4th position).
A method as claimed in claim 7 wherein precursors of melanin formation are employed at levels in the range 0.005mM to 3M.
A method as claimed in anyone of claims 6 to 8 wherein said enzymes are preferably selected from tyrosinase and polyphenol oxidase.
A method as claimed in claim 9 wherein said enzymes are used at levels in the range 0.1 unit/ml to 7500 units/ml.
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11. A method as claimed in anyone of claims 6 to 10 wherein said oxidant is preferably selected from Hydrogen peroxide and other peroxide derivatives.
12. A method as claimed in claim 11 wherein said oxidant is used at levels in the range 0.1 mM to 3M.
13. A method as claimed in anyone of claims 1 to 12 comprising precursor of melanin used in the range 0.01 mM to 1M and more preferably 0.01 mM to 100mM, the concentration of the enzyme 0.2 units/ml to 2500 units/ml or 0.2mM to 1M of the oxidant or a free radical generating system or a mixture thereof and more preferably 0.2 units/ml to 250 units/ml of an enzyme or or 0.2mM to 100mM oxidant or a free radical generating system or a mixture thereof.
14. A method as claimed in anyone of claims 1 to 13 wherein the melanin monomers are preferably prepared by inducing auto oxidation of DOPA solution at pH of 5 to 10 preferably 7.0 - 8.0 incubated for 1 to 6 hours preferably 5 hours.
15. A method of inactivating/inhibiting intra or extra cellular proteinase for laboratory purposes substantially as hereindescribed and illustrated with reference to the accompanying examples.
Dated this 29th day of December 2003
Dr. Sanchita Ganguli Of S.MAJUMDAR & CO. Applicant's Agent