Analysis of N-terminal amino acids of proteins by dansulfonylation method
Analysis of N-terminal amino acids of proteins by dansulfonylation method
The reaction of α-amino group of protein with dansulfonyl chloride (DNS-Cl is a fluorescent substance) produces DNS-protein, which can be hydrolyzed to produce DNS-amino acid. The purpose of this experiment is to understand the basic principle of analyzing the N-terminal amino acids of proteins by dansulfonyl chloride, and to master the hydrolysis of proteins and the operation method of polyamide film chromatography.
Operation method
Dan sulfonylation
Principle
The α-amino group of protein reacts with dansulfonyl chloride (DNS-Cl is a fluorescent substance) to produce DNS-protein, which is hydrolyzed to produce DNS-amino acids. DNS-amino acids can be analyzed by polyamide film chromatography to determine the N-terminal amino acids of proteins. This method is highly sensitive and can also be used to determine the amino acid composition of proteins. The adsorption effect of polyamide on polar substances is that it can form hydrogen bonds with the adsorbed substances, and the strength of such hydrogen bonds determines the size of the adsorption capacity between the separated substance and the polyamide film. During chromatography, the layer spreader competes with the separated substance to form hydrogen bonds on the surface of the polyamide film. Therefore, by selecting the appropriate spreading agent, a continuous process of adsorption, desorption, re-adsorption and re-desorption of the separated material on the surface of the polyamide can lead to the separation of the separated material.
Materials and Instruments
Protein Move 1. Danesulfonylation of standard amino acids For more product details, please visit Aladdin Scientific website.
Amino acids Acetone Dan sulfonyl chloride Hydrochloric acid Sodium bicarbonate Formic acid Water Benzene Glacial acetic acid Ethyl acetate Methanol Glacial acetic acid Trisodium phosphate Ethanol Triethylamine
Vacuum dryer Hydrolysis tube Oven Ultraviolet analyzing lamp Glass test tube Polyamide film
Weigh 2.3 μmol of the chromatographically pure amino acid. 3μmol 层析纯的氨基酸, 溶于0 . 5 ml of 0.5 mol/ L sodium bicarbonate solution. 2 mol/ L sodium bicarbonate solution. Dissolve 0.5 ml in 0.2 mol/L sodium bicarbonate solution. 1 ml in a stoppered glass test tube, add 0.1 ml DNS-Cl acetone solution, check the pH, if necessary, with triethylamine pH 9 . 0～9 . 9.0～9.5 , and At room temperature (about 25 ℃) for 2 ~ 4 h. Then dilute 10 times with nonionized water, and store in a dark place. Store in dark place. The standard DNS-amino acid profile was obtained by chromatography. 2.
2. DNS of protein N-terminal amino acids
Take 0.5 mg of protein sample. Take 0.5 mg of protein sample and put it in a glass tube with stopper. Dissolve the sample in a small amount of water. Add 0.5 ml of 0.5 ml of 0.5 ml of 0.5 mg of protein. 5 ml of 0.2 mol/L sodium bicarbonate solution. 2 mol/ L sodium bicarbonate solution. Add 0.5 ml of DNS-Cl Acetone solution was added and adjusted to pH 9.0 to 9.0 with triethylamine. 0~9 . 5, plugged, and placed in an oven at 40 ℃ for 2 h, or at room temperature. The reaction was carried out in an oven at 40 ℃ for 2 h, or at room temperature (about 25 ℃) for 2-4 h, to produce DNS-protein.
3. Hydrolysis of DNS-protein
At the end of the DNS reaction, acetone was evaporated, and 0.5 ml of 6 mol/ L salt was added. 5 ml 6 mol/ L hydrochloric acid to dissolve DNS-protein. acid to dissolve DNS-protein. All of them were transferred into the hydrolysis tube, and the tube was sealed under vacuum and placed in the oven at 111 ℃ for 18~24 h. The hydrolysis was carried out in the oven. After opening the tube, evaporate the hydrochloric acid, add a small amount of water, and then evaporate to dryness. Repeat Repeat 2～3 times to remove hydrochloric acid. 4.
4. Extraction of DNS-amino acid
Add 0.5 ml of water to the above hydrolyzed product and extract with 1.5 ml of water. Add 0.5 ml of water and adjust to pH 2-3 with 1 mol/L hydrochloric acid. Add 0.5 ml of ethyl acetate for extraction. Add 0.5 ml of ethyl acetate for extraction, and the separation can be carried out in a long burette. Repeat Repeat the extraction 2~3 times, combine the upper layers of the extraction solution in a small test tube, withdraw the ethyl acetate, and set aside in a desiccator. Place in a desiccator.
5. Chromatography and detection of DNS-amino acids
Generated DNS-amino acids and standard DNS-amino acids were subjected to polyamide film chromatography. chromatography.
(1) Preparation of polyamide film
Cut the polyamide film into 7 cm×7 cm squares, and draw an interconnecting line at 0.5 cm from the edge. The polyamide film was cut into 7 cm×7 cm squares, and two base lines were drawn 0.5 cm from the edge. Draw two perpendicular baselines at 0.5 cm from the edge, with the intersection point as the origin. For single-phase chromatography, only one baseline is drawn. If only single-phase chromatography, only one baseline is drawn, and one sample point is drawn every 1 cm on the baseline.
(2) Sampling
Sample with capillary tube, point on the point sample position, point sample diameter should be less than 2 mm. If the sample is taken more than once, it should be taken once and blown dry once.
(3) Unfolding
Roll the polyamide film into a cylinder, the sample is in the cylinder, hooped with a line The sample is inside the cylinder and fixed with a wire loop. Put it in a small dialysis tank (can be replaced by a petri dish in a small dryer), and put it in the tank (petri dish). Inside the tank (Petri dish), put 5~10 ml of unfolding solvent Ⅰ, and unfold it in such a way that the front edge of the solvent reaches 0.5 ml from the top. The solvent is spread until the front edge of the solvent reaches 0.5 cm from the top (about 20 cm). The membrane is unfolded until the front edge of the solvent reaches about 0.5 cm from the tip (about 20 min). Remove the membrane and blow dry. For bi-directional chromatography, blow dry the membrane completely after the first direction (sometimes it needs to be dried overnight to blow dry sufficiently). For two-way chromatography, after the first direction of chromatography is completed and completely blown dry (sometimes it is necessary to air-dry overnight to blow dry sufficiently), turn the polyamide film sheet by 90° and spread it with unfolding solvent II. Spreading solvent II. In order to distinguish DNS-Threonine or DNS-Aspartic Acid from DNS-Glutamic Acid, the film can be spread in solvent II. To distinguish DNS-threonine or DNS-aspartic acid from DNS-glutamic acid, the film can be unfolded with Solvent II, blown dry, and then unfolded with Solvent III in the same direction. After unfolding in Solvent II, blow dry and then unfold in the same direction with Solvent III, only half of the height of the unfolding is sufficient. In order to distinguish between ε-DNS-lysine, α-DNS-histidine and DNS-glutamic acid, it was necessary to spread them in the same direction with solvent III. DNS-lysine, α-DNS-histidine and DNS-arginine, they should be unfolded in solvent II, blown dry, and then unfolded in solvent IV in the same direction. After unfolding in solvent Ⅱ, blow dry and then unfold in solvent Ⅳ in the same direction.
(4) Detection of DNS-amino acids
After unfolding, remove the film, blow it dry with a hair dryer, and examine it under a UV lamp at 360 nm or 280 nm. DNS-amino acids show yellow fluorescence. In addition, there are other colors of heterogeneous dots, such as DNS-amino acids. DNS-amino acid showed yellow fluorescence, and there were also other colors of heterogeneous dots, such as DNS-OH, which showed green fluorescence. The chromatographic spectrum of the sample was compared with the standard DNS-amino acid chromatographic spectrum. The chromatographic spectrum of the sample can be compared with that of the standard DNS-amino acid to identify the type of DNS-amino acid in the sample.