DD225718A1 - CLEANING PROCESS OF SULFATASE BY AFFINITY CHROMATOGRAPHY - Google Patents

Abstract

The method can find application in medicine and biotechnology. The aim of the invention is the development of a simple, specific purification process from sulfatase-containing solutions. According to the invention, this is achieved by contacting sulfatase substrates bound to a high molecular weight carrier with a sulfatase-containing solution in the batch or column process and eluting the bound sulfatase from the carrier.

Description

Field of application of the invention

The invention relates to purification processes for sulfatases which can be used in medicine and biotechnology.

Characteristic of the known technical solutions

For the isolation and purification of proteins and enzymes in many cases chromatographic methods are used. Affinity chromatography has become particularly important. Furthermore, it is characterized by its high specificity. The affinity chromatography is characterized in that a ligand, which forms a complex with the protein or enzyme to be isolated or purified, is bound to an insoluble matrix (high molecular carrier). The direct covalent binding of a small ligand to the high molecular weight carrier leads to steric hindrance between the high molecular weight carrier and the enzyme that is supposed to bind to the ligand. Therefore, a spacer is chemically inserted between the high molecular weight carrier and ligands.

The complex of ligand and protein or enzyme formed must then be cleaved under specific conditions to be isolated or purified for the protein or enzyme, respectively, in order to recover these biological macromolecules.

An affinity chromatographic method for the isolation of human liver sulfatase is described in the literature (Agogbua, SIO & Wynn, CH, Biochem Soc Trans 3, 1975, 405-408, Agogbua, SIO & Wynn, CH, Biochem J. 153 (1976) 415-421). Here, after diazotization of the 4-aminobenzamidoethyl derivative of Sepharose 4β, 4-hydroxy-2-nitrophenyl sulfate is coupled at pH 10.0. Although our own investigations also revealed a reddish gel, which does not contain sulphate groups. The structure of the affinity gel indicated by the authors thus seems quite doubtful, especially since the electrophilic substitution of 4-hydroxy-2-nitrophenyl sulfate in the 4-position to the nitro group is to take place. Furthermore, the acting in the diazotization agent H ₂ NO ₂ or ON-OSOf also attack other present in the gel N-electrophile. In later publications, the affinity gel synthesized by the above authors is no longer used to isolate sulfatases.

The isolation and purification processes of sulfatases, which take place in several and time-consuming steps, have hitherto been carried out, inter alia, via anion or cation exchange chromatography on diethylaminoethylcellulose or carboxymethylcellulose (Farooqui, AA, Clin Chim Acta 100 [1980] 285-299, Fluharty, A. L & Edmond, J., Meth. Enzymol. 50 [1978] 537-547; Waheed, A. & Van Etten, RL, Arch. Biochem. Biophys. 194 [1979] 215-225) or Cibacron-β-leu-Sepharose ( Ahmad, A., Surolia, A. & Bachhawat, BK, Biochim, Biophys, Acta 481 [1977] 542-549). Both the lectin concanavalin-A and the dye Cibacron Blue F3G-A have a broad binding spectrum, in particular with respect to glycoproteins (enzymes). The previously used chromatographic separation methods for the isolation of sulfatases are thus not specific.

Object of the invention

The aim of the invention is to develop a simplified, specific purification process for sulfatases from sulfatase-containing solutions.

Explanation of the essence of the invention

For the isolation or purification of sulfatase according to the invention sulfatase substrates are bound to a high molecular weight carrier, which are immobilized not degradable by sulfatase and brought into contact with a sulfatase-containing solution. The interaction of the enzyme solution with the immobilized substrates takes place at temperatures of 4 ° C. to room temperature and at pH values of 5-7.

The cleaning method can be carried out in both batch and column processes. In the column method, the bound sulfatase is eluted by increasing the ionic strength of the eluent using a ünearen gradient of 0-1 mol / l NaCl. In the batch process, the bound sulfatase is eluted with the aid of the starting buffer containing 1 mol / l NaCl. From the protein fraction with sulfatase activity, the purified sulfatase is then obtained by dialysis after lyophilization.

Suitable sulfatase substrates for the purposes of the invention are those substances which contain functional groups, such as NH ₂ -orDerr -COOH groups, which allow covalent bonding via a spacer to the support. Preferably, the substrates such as 5-amino-2-hydroxyphenylsulfate, 5-carboxy-2-hydroxyphenylsulfate and (1 ', 4'-butanedicarboxylic acid-1'-amido) -2-hydroxyphenylsulfate. Methods for producing such substances are known. Suitable spacer molecules for the purposes of the invention are those substances which contain functional groups which allow covalent bonding both to the sulfatase substrates and to the support. Preferably, the spacer molecules, such as 6-aminohexanoic acid, adipic dihydrazide or their multiples. Suitable carrier materials in the context of the invention are those high-polymer substances which are insoluble, macroporous and easily activated by conventional and known methods

are. The activation of agarose with BrCN and the reaction with 6-aminohexanoic acid is analogous; Cuatrecasas, J. Biol. Chem. 245 (1970) 3059-3065. Up to a concentration of 5 mmol / l 5-amino-2-hydroxyphenylsuifat, the affinity gel has a high binding capacity of 2.2 mg protein / μΓηοΙ covalently bound 5-amino-2-hydroxyphenylsulfat or 6.6 mg protein / ml gel over the sulfatase.

The suifatase products prepared by these methods have 11.5 times (column method) and 6.5 times (batch method) higher activity than the commercially available sulfatase. In contrast to the purification methods used in the literature (ion exchange chromatography and affinity chromatography on Con-A-Sepharose or Cibacron-Blue-Sepharose), the method described here is simpler and more specific. _i \

embodiments Example 1:

33 ml of agarose, which are activated with BrCN and reacted with 6-aminohexanoic acid, in an 80 ml suspension of 0.2 mol / i KCl with 0.13 g (0.5 mmol) of 5-amino-2-hydroxyphenylsulfat added and adjusted to pH4.5 with 1 mol / l HCl. Subsequently, with vigorous stirring, 0.19 g (1.0 mmol) of N-ethyl-N'-O-dimethylaminopropyD-carbodiimide hydrochloride are added in portions within 10 minutes and the pH between 4.5-4.7 by addition of 1 mol / l NaOH or 1 mol / l HCI kept. After 2-3 hours, the pH is relatively constant, and the suspension is shaken for a further 24 hours at room temperature. After aspiration, the affinity gel is washed successively with 600 ml of 0.2 mol / l KCl and 1 200 ml of water.

For affinity chromatography, 9.6 ml of gel are washed several times with a pyridine / acetic acid buffer, 0.05 mol / l, pH 6.5, and filled into a small column (15.0 cm height χ 0.9 cm diameter). 8.6 mg sulfatase (Helix pomatia) with a protein content of 2.4 mg are dissolved in 1.5 ml pyridine / acetic acid buffer, 0.05 mol / l pH 6.5 and applied to the column. The flow rate is 3.0 ml per hour, and each 3.0 ml fractions are collected. After equilibrating the column with 45.0 ml of starting buffer, the ionic strength of the eluent is increased from 0-1 mol / l NaCl with a linear gradient over 32 hours, and the elution is then terminated. In the collected fractions, the sulfatase activity is determined using the substrate 2-hydroxy-5-nitrophenyl sulfate and the protein content using the Coomassie Brilliant Blue method. The sulfatase eluted in fraction 22 (protein content: 0.3 mg) has an 11.5-fold higher specific activity after dialysis than the starting sulfatase.

Example 2:

0.5 ml of the affinity gel synthesized according to Example 1 is washed several times with a pyridine / acetic acid buffer, 0.05 mol / l, pH 6.0, and suspended in 1.0 ml of the same buffer. After the addition of 1.0 m | a sulfatase-containing solution (2.9 mg protein) is no longer detected after stirring for 2 hours at room temperature in the supernatant sulfatase activity. The gel is washed with the starting buffer and the bound sulphate is released by elution with the aid of the starting buffer containing 1.0 mol / l NaCl (protein content 0.35 mg). After dialysis certain specific sulfatase activity is 6.5 times higher than in the starting solution.

Schedule:

Purification method of sulfatase according to claim

Column method Binding of sulphate to sub state s high molecular weight, insoluble carrier Addition of sulfatase containing solution activation Batch process]:. "I I Bindong the spacer affinity

Xquilibri eren

Sluieren

DialysLeren

lyophilization

j purified sulfatase

Claims (6)

Invention claims: 1. purification process of sulfatase by affinity chromatography, characterized in that one binds sulfatase substrates to a high molecular weight carrier, with a sulfatase-containing solution in a batch or column method in contact and the bound sulfatase eluted from the carrier in a conventional manner. 2. A purification process according to claim 1, characterized in that 5-amino-2-hydroxyphenylsulfat, 5-carboxy-2-hydroxyphenylsulfat or derivatives of these compounds are used as sulfatase substrates. 3. A purification process according to claim 1 and 2, characterized in that the sulfatase substrates are bound to the carrier via a spacer, preferably via 6-aminohexanoic acid or adipic dihydrazide or their multiples. 4. Cleaning method according to claim 1-3, characterized in that are used as the carrier agarose or polyacrylamide. 5. A purification process according to claim 1-4, characterized in that 5-amino-2-hydroxyphenylsülfat to Agaiose, after BrCN activation and reaction with 6-aminohexanoic acid is condensed to a degree of binding of 5mmol / l or 100mmol / kg. See 1 page table