CS258607B1

CS258607B1 - Method of isolation of SH-amino acids, peptides and proteins

Info

Publication number: CS258607B1
Application number: CS851639A
Authority: CS
Inventors: Dusan Podhradsky; Gejza Suchar; Pavol Kristian; Stanislav Krcmar; Sona Huncikova
Original assignee: Dusan Podhradsky; Gejza Suchar; Pavol Kristian; Stanislav Krcmar; Sona Huncikova
Priority date: 1985-03-08
Filing date: 1985-03-08
Publication date: 1988-09-16
Also published as: CS163985A1

Abstract

Riešenie sa týká spůsobu oddelenia sulfhydrilových od nesulfhydrilových zložiek zó zmesi proteínov kovalentnóu chromatografiou. Podstata riešenia spočívá v tom, že vzorka sa naneste na kolónu obsahujúcu ako sorbent 2-nitrofenylhydrazónpropándinitrilové deriváty polymérov obecného vzorca I, kde X znamená Y—O—CO alebo Y—NH—CO a Y je anhydroglukózový polymér, polyhydroxymetylmetakrylát, gél na báze agarózy alebo celulóza zá přítomnosti tlmivého roztoku o pH až 5 a po přetečení celého objemu Vzorky sa kolóna premyje tlmivým roztokom o pH 7 až 8.The solution relates to a method of separating sulfhydryl from non-sulfhydryl components from a protein mixture by covalent chromatography. The essence of the solution lies in the fact that the sample is applied to a column containing as a sorbent 2-nitrophenylhydrazonepropanedinitrile derivatives of polymers of the general formula I, where X means Y—O—CO or Y—NH—CO and Y is an anhydroglucose polymer, polyhydroxymethyl methacrylate, agarose-based gel or cellulose in the presence of a buffer solution with a pH of up to 5 and after the entire volume of the sample has overflowed, the column is washed with a buffer solution with a pH of 7 to 8.

Description

2586 3

The invention relates to a process for the isolation of SH-amino acids, peptides and proteins from a mixture comprising sulfhydril and non-sulfhydril components of the proteins by covalent chromatography. SH-groups have a materially essential meaning in the structure (PJ Jocelyn: Biochemistry of SH-group, Academic Press, London-NY 1972). They are part of the enzyme aprotein structures, oxidation-reduction, catalytic, detoxification processes, etc. are involved in peptides. In general, peptide and protein isolation is a complex problem. One of the possibilities of its solution is the immobilization of these substances on various sorbents through SH-groups of the incorporated cysteine (K. Brocklehurst: Int. J. Biochem. 10, 259 (1979), D. Podhradský, P. Crisantium: Chem., 35, 567 [1981]) However, these isolation methods run into the problem of adsorption from a carrier, which is usually accomplished by displacement with another thiol, thereby impairing the isolated product in some way. however, the reversible covalent chromatography for the isolation of SH is very promising.

The issue of greater use of covalent chromatography is mainly related to a suitable ligand that specifically and reliably reacts with SH-groups of amino acids, peptides and proteins. A phenylhydrazone-propanedinitrile type ligand has been described which, upon binding to cellulose, allows the capture of low molecular weight thiols (V. Marko, P. Gemeiner, M. Antalik, E. Sturdik, L. Drobnice, P. Kristian: Reactive Polymers1, 91 (1983). however, such a carrier 074 occurs only at high levels of H (about 12), when most SH groups are oxidized to the disulfide, and in proteins, such immobilization leads to irreversible structural changes, and activation is lost in enzymes.

The above disadvantages are substantially hindered by the method of isolating the SH-amino acids, peptides and proteins of the present invention by providing a sananesium sample on a column containing 2-nitrophenyl hydrazone propanedinitrile derivatives of the formula IXN * C ( Wherein X is Y-O-CO or Y-NH-COa Y is an allylhydroglucose polymer, polyhydroxymethyl methacrylate, agarose or cellulose gel in the presence of a buffer of pH 4 to 5, and after the entire sample volume has overflowed, the column is washed with a pH 7 to 8 buffer solution. The advantage of this is that the pKa value of their amino groups is in the range of 5.3- 5,6, principally means that the absorbing substance absorbs at pH 4-5 and releases at pH 7-8. The binding and release of SH-substances to polymers can be represented by the equation λ ”NH ~ N ~ C (Cn) z + R ~ N0z

wherein X is the aforesaid.

The described method of isolating SH-amino acids, peptides and proteins of the invention has the advantage that: the binding and release of the substance is carried out simply by changing the pH in the 4-8 mode, thereby retaining the SH-groups of the immobilized substance, the displacement does not use any other substance that would contaminate the sample, further the regeneration of the carrier is omitted, the equilibration of the column to pH binding is sufficient and, finally, due to the high reactivity of the ligand, the binding ability of the carrier is very high. Přikladl

Isolation of SH-amino acids, peptides and proteins

A sample containing glutathione, cysteine, or other SH-protein at a concentration of at most 2 mg / ml (e.g., an aqueous extract of fermentation, a deproteinated homogenate of animal or plant tissues, etc.) is applied to a 10 cm column of carrier with a 2-nitro-phenylhydrazone-propanedinitrile ligand, which was equilibrated to a pH of about 4. After washing with a buffer of the same pH,

Claims

258607 5 ry contains 0.01% EDTA, all SH-group-free components are removed from the column. After changing the pH to 8, the H 2 -amino acids, peptides or proteins are eluted from the column. is 96-97%

2 Separation of SH-Proteins 2 mg of proteins, which are a mixture of sulfhydril anesulfhydril components, are dissolved in pH 4-5 buffer. After loading the sample on a column with 2-nitrophenylhydrazone-propanedinitrile ligand, proteins that do not contain proteins are separated at pH 4-5. SH groups of incorporated cysteine. After gradual change of buffer to pH 8, SH-proteins are displaced. Detection from the column of outgoing components is done by UV254 or UVzoe

3 Concentrating the enzyme 2 mg of the enzyme containing the SH-groups [e.g. urease, oxidoreductase and the like) are dissolved in 5 dm 3 of pH 4-5 buffer containing 0.01% EDTA. Said enzyme solution is pumped through the column via a peristaltic pump, at a flow rate of 2 ml / min. After pumping the entire volume on the column, the captured enzyme releases the buffer at pH 7-8. By measuring the enzyme activity, it is possible to detect a 100-fold concentration of the enzyme. PEEDMET A method of isolating SH-acids, peptides and proteins from a mixture comprising sulfhydride and non-sulfhydril components of proteins by cationic chromatography using a phenylhydrazone propanedinitrile ligand carrier, wherein the sample is coated with a colloid containing 2-nitrophenyl hydrazone propanedinitrile derivatives of the polymer as the sorbent Formula I wherein X is Y-O-CO or Y-NH-COa Y is an anhydroglucose polymer, polyhydroxymethyl methacrylate, agarose or cellulose gel in the presence of a buffer of pH 4-5, wash the column with pH 7 to 8 buffer. NH — N = C (CN) 2 NO 2 (0