CS252790B1 - A method of separating amino acids from peptides and proteins - Google Patents

Abstract

The solution relates to the separation of amino acids from peptides and proteins from the end of the peptide chain containing free carboxyl groups of amino acids. It is intended for determining the sequence of peptides and proteins. An aqueous solution, buffered to pH 5.0 to 8.0, containing a peptide or protein, is subjected to the action of the carboxypeptidase enzyme, immobilized on a polyhydroxyethyl methacrylate gel, in an ultrafiltration cell equipped with a membrane permeable only to low-molecular substances, such as amino acids released by carboxypeptidase from peptides or proteins, at an enzyme-substrate ratio of 1 to 200 to 1 to 2,000.

Description

The invention relates to a method for separating amino acids from peptides and proteins from the end of a peptide chain containing free carboxyl groups of amino acids for determining the sequence of peptides and proteins.

In biochemistry, there is a great need to determine the primary structures of proteins, that is, the sequence of amino acids that are bound together by peptide bonding to form peptides or proteins. Known methods are used for this procedure, all of which are a compromise between the accuracy of the assay and the amount of material to be used for the assay. One known method utilizes specific proteolytic enzymes, so-called carboxypeptidases. These enzymes cleave individual amino acids from that end of the peptide or protein having a free carboxyl group.

The so-called conventional terminal amino acid methods used so far are to determine the content of individual amino acids in a solution containing the polypeptide and enzyme of interest, i.e., the carboxypeptidase. The medium in which the assay is performed is buffered to a pH close to the optimum effect of the respective carboxypeptidase.

In the original reaction mixture, which initially contains only the polypeptide and the carboxypeptidase, the increasing concentration of amino acids released by the present carboxypeptidase is monitored from the peptides into solution.

In the classical procedure, the step affecting the accuracy of the method is precisely the processing of the aliquots to be analyzed for amino acid analysis. In said classical procedure, the non-227,790 advantage is the necessity of precipitation with trichloroacetic acid, subsequent concentration of the precipitate by centrifugation, extraction with ether and evaporation. Only after these operations is the sample ready for amino acid analysis.

According to the present invention, there is provided a method of separating amino acids from peptides and proteins from the end of a peptide chain to determine the sequence of peptides and proteins, using a carboxypeptidase enzyme, characterized in that an aqueous solution buffered to pH 5.0 to 8.0 containing the peptide or protein is treated with a carboxypeptidase enzyme immobilized on a polyhydroxyethyl methacrylate gel in an ultrafiltration cell equipped with a membrane permitting only low molecular weight substances, such as amino acids, released by the carboxypeptidase from peptides or proteins at an enzyme-substrate ratio of 1 to 200 to 1 in 2,000.

The basic advantage of the process according to the invention is that the process is substantially simplified, since all treatment of the sample prior to amino acid analysis consists only in its evaporation. A further advantage is that the carboxypeptidase is immobilized on a solid support and therefore does not autolysis it, furthermore it can be easily separated from the mixture. Due to the strength of the material used, there is no danger of crushing and the carboxypeptidase can be easily reused. This procedure is considerably shorter compared to conventional sequencing, by about half and the whole process can be easily automated.

The process according to the invention is described in more detail below with reference to specific examples.

Example 1

To use carboxypeptidase Y for sequential scrubbing, the carboxypeptidase was immobilized on a poly-hydroxyethyl methacrylate gel with immobilized Konkavalin A. The immobilized carboxypeptidase was added to 3 ml of protein solution (ribonuclease A). The protein concentration was 10 mg in 3 ml of 0.1 M sodium acetate pH 6.0 with 0.5% sodium lauryl sulfate. Solution

The 252,790 protein was placed in a small ultrafiltration cell (10 ml volume) with a membrane permeable membrane with a molecular weight below 2000. At appropriate time intervals, an aliquot (0.2 ml) was taken at nitrogen pressure and used directly for the amino acid after evaporation. analysis.

This method yields results of the same quality as the classical procedure.

Example 2

The same procedure using ultrafiltration cell was used for sequential work with carboxypeptidase A and carboxypeptidesis

B.

Example 5

Under the conditions of Example 1, protein digestion with immobilized carboxypeptidase was performed. 1 ml protein solution (0.1 M sodium acetate, pH 6.0 with 0.5% sodium lauryl sulphate containing 2 _of µg ribonuclease A) was subjected to carboxypeptidase Y digestion immobilized on polyhydroxyethyl methacrylate gel with immobilized concanavalin A. 200 µg of carrier was used. containing 0.1 .mu.g of carboxypeptidase Y. A 2 ml ultrafiltration cell was used, fitted with a membrane permeable material with a molecular weight below 2000, and 0.05 ml samples were taken.

Claims (1)

SUBJECT OF THE INVENTION A method for separating amino acids from peptides and proteins from the end of a peptide chain for determining peptide and protein sequences using a carboxypeptidase enzyme, characterized in that an aqueous solution buffered to pH 5.0 to 8.0 containing the peptide or protein is treated with an enzyme a carboxypeptidase immobilized on a polyhydroxyethyl methacrylate gel in an ultrafiltration cell equipped with a membrane permitting only low molecular weight substances, such as amino acids released by the carboxypeptidase from the peptide or protein, at an enzyme-substrate ratio of 1 to 200 to 1 to 2,000¾.