CN102241753A

CN102241753A - Fluorescence labeling method for microcystins

Info

Publication number: CN102241753A
Application number: CN2011101170326A
Authority: CN
Inventors: 黎双飞; 刘芳; 周连宁; 胡章立; 吴锦霞; 谢艳梅
Original assignee: Shenzhen University
Current assignee: Shenzhen University
Priority date: 2011-05-06
Filing date: 2011-05-06
Publication date: 2011-11-16

Abstract

The invention provides a fluorescence labeling method for microcystins, comprising the following steps of: A, under the conditions of nitrogen gas shielding for pH and shaking at room temperature, reacting the microcystins with cysteine based on mol ratio in a 5% K2CO3 solution to obtain the cysteine derivatives of microcystins; B, purifying by a chromatograph under set conditions to remove the excessive cysteine from the reactant liquid to obtain purified MC-Cys; C, reacting the purified MC-Cys with fluorescein isothiocyanate (FITC) based on mol ratio in a borate buffer solution, performing oscillating reaction at room temperature to obtain microcystins coupled with the FITC; and D, purifying by a chromatograph under set conditions to remove the excessive FITC from the reactant liquid to obtain purified MC-Cys-FITC. According to the method which is easy and simple to operate, the cysteine is used as an intermediate connector for connecting the microcystins with the fluorescein isothiocyanate to perform fluorescence labeling on the microcystins, the purity of the obtained MC-Cys-FITC is 95%, and a direct experimental evidence is provided for studying the microcystins on toxicology.

Description

The fluorescently-labeled method of a kind of Microcystin

Technical field

The present invention relates to the fluorescence labeling method of algae toxin, be specifically related to the preparation method of a kind of Microcystin-halfcystine-lsothiocyanates (MC-Cys-FITC).Utilize fluorescently-labeled algae toxin and laser scanning co-focusing microtechnique combined utilization, can observe directly the algae toxin approach of accumulative position, transhipment in vivo, for research algae toxin transporting pathway is in vivo established certain basis.

Background technology

Along with water pollution increases the weight of gradually, eutrophication appears and the blue-green alga bloom that causes is general day by day, and (microcystins MCs) pollutes and has become the environmental problem that everybody shows great attention to the Microcystin in the water body environment.MCs is a kind of secondary metabolism thing that is produced by blue-green algae, has strong carcinogenesis, its molecular structure complexity, stable in properties, traditional treatment process is difficult to effectively dispel, this has constituted great threat to human health, therefore become pressing for of concern human health to deeply carrying out in the research aspect the Microcystin toxicology, mainly concentrate at present the liver acceptor of Microcystin, the transhipment principle of toxin, the research of the aspects such as molecular mechanism of the especially short tumour of the molecule mechanism of detoxifying function, but all be indirect Microcystin toxicology is studied, lack enough experimental evidences.Therefore, urgent need is a kind of can directly show the Microcystin novel method of accumulative position, transporting pathway and molecule mechanism in vivo.

Summary of the invention

The objective of the invention is to be to provide a kind of Microcystin (MC-LR) fluorescently-labeled method, easy to implement the method, easy and simple to handle, be to use halfcystine (Cys) Microcystin and fluorescein isothiocyanate (FITC) to be linked together specifically, Microcystin is carried out fluorescent mark as intermediate connector.Adopt marking method of the present invention effectively MC-LR and FITC to be linked together, and to obtain purity be 95% MC-Cys-FITC, for the research of Microcystin toxicology aspect provides the direct experiment evidence.

In order to realize above-mentioned purpose, the present invention adopts following technical measures:

The fluorescently-labeled method of a kind of Microcystin (MC-LR) the steps include:

A, under the condition of protection of the inflated with nitrogen of pH9-13 and room temperature (identical below 20-30 ℃) concussion, with Microcystin (MC-LR) and halfcystine with 1: 20 mol ratio at 5% K ₂CO ₃React 9-11h in the solution, obtain the cysteine derivative (MC-Cys) of Microcystin;

B, by high performance liquid chromatograph (HPLC) and certain preparative chromatography condition is set carries out purifying, remove halfcystine excessive in the reaction solution, obtain the MC-Cys of purifying;

C, MC-Cys behind the purifying and fluorescein isothiocyanate (FITC) are reacted in borate buffer solution with 1: 2 mol ratio, pH 8.5-9.5, oscillatory reaction 9-11h at room temperature obtains the algae toxin MC-Cys-FITC of coupling FITC;

D, by high performance liquid chromatograph (HPLC) and certain preparative chromatography condition is set carries out purifying, remove FITC excessive in the reaction solution, obtain the conjugates (MC-Cys-FITC) of the algae toxin-halfcystine-fluorescein isothiocyanate of purifying.

Described B step preparative chromatography condition is:

Moving phase: A:H ₂O+0.1%TFA; B:ACN+0.1%TFA, flow velocity: 15mL/min detects wavelength: 238nm, sample size 30mL, detector: visible ultraviolet spectrometry detector, preparative column: XTerra Prep MS C ₁₈Column (19 * 50mm), HPLC chromatographic separation gradient condition such as table 1:

Table 1 chromatographic separation gradient condition

Described D step preparative chromatography condition is:

Moving phase: A:25nM ammonium acetate; B:CAN, flow velocity: 15mL/min detects wavelength: 238nm, sample size 30mL, detector: visible ultraviolet spectrometry detector, preparative column: XTerra Prep MS C ₁₈Column (19 * 50mm), HPLC chromatographic separation gradient condition such as table 2:

Table 2 chromatographic separation gradient condition

The present invention compared with prior art has the following advantages and effect:

First halfcystine is a kind of essential amino acid, there is not toxicity, and molecular weight is less, there are these three kinds of functional groups of carboxyl, amino and sulfydryl to react, halfcystine is linked together Microcystin and fluorescein isothiocyanate as intermediate connector, overcome directly bonded problem of fluorescein and Microcystin.

Its two importantly carry out purifying by preparative chromatography condition of the present invention after, can obtain purity up to 95% MC-Cys-FITC, thereby remove the interference that the free fluorescein isothiocyanate is brought.

Description of drawings

Fig. 1 is the structure iron of a kind of Microcystin (MC-LR).

Fig. 2 is the reaction formula that a kind of MC is connected with Cys.

Fig. 3 is the reaction formula that a kind of MC-Cys is connected with FITC.

Fig. 4 is a kind of color atlas of MC-Cys reaction solution.

Show among the figure that MC-Cys goes out the peak at 12.3min, calculate that the purity of the MC-Cys behind the purifying is 94% according to area normalization method.

Fig. 5 is that the mass spectrum of the MC-Cys behind a kind of purifying is identified figure.

Molecular ion peak is m/z1116.4 among the figure, and molecular weight is 1116, and is consistent with the theoretical molecular of MC-Cys, so think that the reactant that purifying comes out is MC-Cys.

Fig. 6 is a kind of color atlas of MC-Cys-FITC reaction solution.

Show among the figure that MC-Cys-FITC goes out the peak at 14.1min, calculate that the purity of the MC-Cys behind the purifying is 95% according to area normalization method.

Fig. 7 is that the mass spectrum of the MC-Cys-FITC behind a kind of purifying is identified figure.

Molecular ion peak is m/z 1505.2 among the figure, and molecular weight is 1505, and is consistent with the theoretical molecular of MC-Cy-FITC, so think that the reactant that purifying comes out is MC-Cys-FITC.

Embodiment

The first step: the front adds being connected of Chinese (MC) and halfcystine (Cys), with MC-LR and halfcystine with 1: 20 mol ratio at 5% K ₂CO ₃In reaction,

pH

9 or 10 or 11 or 12 or 12.10 or 12.4 or 12.7 or 13, inflated with nitrogen protection, reaction 9 or 10 or 11h under the room temperature concussion obtains the cysteine derivative MC-Cys of MC.

Second step: the purifying of MC-Cys, contain excessive Cys in the reaction solution of MC-LR and Cys, if be not removed, excessive Cys will react with FITC, thereby produce a lot of side reactions, follow-up evaluation, separation and purifying are brought very big trouble, so earlier by high performance liquid chromatograph (HPLC) and certain preparative chromatography condition is set carries out purifying, remove halfcystine excessive in the reaction solution, obtain the MC-Cys of purifying.

Described preparative chromatography condition is:

Moving phase: A:H ₂O+0.1%TFA, B:ACN+0.1%TFA, flow velocity: 15mL/min detects wavelength: 238nm, sample size 30mL, detector: visible ultraviolet spectrometry detector, preparative column: XTerra Prep MS C ₁₈Column (19 * 50mm), HPLC chromatographic separation gradient condition such as table 1:

Table 1 chromatographic separation gradient condition

Time/min	Mobile phase A %	Mobile phase B %
			0	90	10
5	72	28
			8	70	30
8.5	70	30
			9	10	90
10	10	90
			10.5	90	10

The 3rd step: the MC-Cys behind the second step purifying is carried out mass spectrum through mass spectrograph identify that molecular ion peak shown in Figure 5 is m/z1116.4, molecular weight is 1116, and is consistent with the theoretical molecular of MC-Cys, so think that the reactant that purifying comes out is MC-Cys.

The 4th step: MC-Cys is connected with FITC's, MC-Cys behind the purifying and FITC are reacted in borate buffer solution with 1: 2 mol ratio, pH 8.5 or 8.7 or 9 or 9.2 or 9.5, oscillatory reaction at room temperature 9 or 10 or 11h obtains the algae toxin MC-Cys-FITC of coupling FITC.

The 5th step: the purifying of MC-Cys-FITC, in the reaction solution of previous step, fully react in order to guarantee MC-Cys, FITC is excessive, in order to remove FITC excessive in the reaction solution, needs to carry out purifying with HPLC.

Described preparative chromatography condition is:

Moving phase: the A:25nM ammonium acetate, B:CAN, flow velocity: 15mL/min detects wavelength: 238nm, sample size 30mL, detector: visible ultraviolet spectrometry detector, preparative column: XTerra Prep MS C ₁₈Column (19 * 50mm), HPLC chromatographic separation gradient condition such as table 2:

Table 2 chromatographic separation gradient condition

Time/min	Mobile phase A %	Mobile phase B %
			0	90	10
5	74	26
			12	70	30
12.5	70	30
			13	10	90
14	10	90
			14.5	90	10

The 6th step: the MC-Cys-FITC behind the 5th step purifying is carried out mass spectrum through mass spectrograph identify, molecular ion peak shown in Figure 7 is m/z 1505.2, molecular weight is 1505, and is consistent with the theoretical molecular of MC-Cy-FITC, so think that the reactant that purifying comes out is MC-Cys-FITC.

Claims

1. the fluorescently-labeled method of Microcystin the steps include:

A, under the condition of protection of the inflated with nitrogen of pH9-13 and room temperature concussion, with Microcystin and halfcystine with the mol ratio of 1:20 at 5% K ₂CO ₃React 9-11 h in the solution, obtain the cysteine derivative of Microcystin;

B, by high performance liquid chromatograph and certain preparative chromatography condition is set carries out purifying, remove Cys excessive in the reaction solution, obtain the MC-Cys of purifying;

C, MC-Cys behind the purifying and fluorescein isothiocyanate are reacted in borate buffer solution with the mol ratio of 1:2, pH 8.5-9.5, oscillatory reaction 9-11 h at room temperature obtains the algae toxin MC-Cys-FITC of coupling FITC;

D, by high performance liquid chromatograph and certain preparative chromatography condition is set carries out purifying, remove FITC excessive in the reaction solution, obtain the conjugates of the algae toxin-halfcystine-fluorescein isothiocyanate of purifying;

Described (B) step preparative chromatography condition is:

Moving phase: A:H ₂O+0.1% TFA; B:ACN+0.1% TFA, flow velocity: 15 mL/min, detect wavelength: 238 nm, sample size 30 mL, detector: visible ultraviolet spectrometry detector, preparative column: XTerra Prep MS C ₁₈Column, HPLC chromatographic separation gradient condition is:

Time/min mobile phase A % Mobile phase B %

O 90 10

5 72 28

8 70 30

8.5 70 30

9 10 90

10 10 90

10.5 90 10

Described (D) step preparative chromatography condition is:

Moving phase: A:25 nM ammonium acetate; B:CAN, flow velocity: 15 mL/min, detect wavelength: 238 nm, sample size 30 mL, detector: visible ultraviolet spectrometry detector, preparative column: XTerra Prep MS C ₁₈Column, HPLC chromatographic separation gradient condition is:

Time/min mobile phase A % Mobile phase B %

O 90 10

5 74 26

12 70 30

12.5 70 30

13 10 90

14 10 90

14.5 90 10。