CN108226263A - A kind of capillary isoelectric focusing detection method of urate oxidase - Google Patents
A kind of capillary isoelectric focusing detection method of urate oxidase Download PDFInfo
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- CN108226263A CN108226263A CN201611151762.7A CN201611151762A CN108226263A CN 108226263 A CN108226263 A CN 108226263A CN 201611151762 A CN201611151762 A CN 201611151762A CN 108226263 A CN108226263 A CN 108226263A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44756—Apparatus specially adapted therefor
- G01N27/44795—Isoelectric focusing
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
Abstract
The present invention relates to a kind of capillary Isoelectric Focusing Methods for detecting urate oxidase, it specifically includes and mixes pharmalyte, capillary isoelectric focusing gel, urea, anodic stabilization agent, cathode stabilizers, isoelectric point marker and urate oxidase sample according to a certain concentration ratio, it is splined on coatings capillary pipe, under suitable temperature, voltage and focal time, it is detected by capillary electrophoresis, can realize each component in protein sample to be analyzed and efficiently separate.This method is reproducible, available for the quality control of Urate oxidase drug in process of production.
Description
Technical field
The present invention relates to a kind of capillary isoelectric focusing detection methods, should for measuring urate oxidase charge heterogeneity
Detection method can carry out good Analysis of quality control to Urate oxidase drug.
Background technology
Uric acid is birds, in reptiles and primate body including people purine metabolism final product because
Lack the urine that allantoin, carbon dioxide and hydrogen peroxide are further broken into using molecular oxygen as receptor catalysis uric acid in these animal bodies
Acid oxidase.Uric acid as human body purine metabolism final product through kidney excretion, when uric acid generation more than renal metabolism ability or
Person can will cause Plasma Uric Acid significantly to increase when kidney is in pathological state, form hyperuricemia.Due to urea and
Its esters low solubility in blood and easy deposition allow for hyperuricemia and can cause or aggravate a variety of diseases, such as：
Acute inflammation and pain caused by uric acid crystal is deposited on periphery joint, synovial membrane when continuing hyperuricemia are the main diseases of gout
Cause；Uric acid can be not only proliferated with stimulated vascular smooth muscle cell, will also lead to endothelial dysfunction；Blood plasma high lithemia is also
The important risk factor of the angiocardiopathies such as atherosclerosis；Uric acid is deposited on renal tissue, is that cause acute renal failure, kidney small
The main reason for pipe damage, IgA ephritis, therefore hyperuricemia can bring own health all multi-risk Systems.
The classical scheme of anti-trioxypurine be with the xanthine oxidase inhibitors class such as allopurinol drug reduce uric acid generation or
Promote uric acid excretion with drugs such as probenecid, Benzbromarones.The water solubility of xanthine is less than uric acid, and accumulation has potential danger
Danger, xanthine oxidase inhibitor curative effect when initial uric acid concentration is too high are very poor, moreover it is possible to cause super quick syndrome, show as sending out
Heat, the necrolysis of toxicity epithelial cell, hepatitis and acidophil increase, and the death rate is up to 20%, in addition this kind of medicine and probenecid, benzene
The uricosurics such as bromine Malong excretion class medicine has apparent liver renal toxicity.Therefore, classical anti-trioxypurine therapeutic strategy not enough safety and
Curative effect is also not ideal enough.The excellent water-soluble and kidney of allantoin becomes urate oxidase the efficient discharge capacity of allantoin
Treat the ideal medicament of hyperuricemia and its secondary disease：Clinical research shows that urate oxidase can rapidly and efficiently reduce serum urine
Acid, and almost without toxic side effect；When treating tumor lysis syndrome, urate oxidase is more safer and more effective than allopurinol；
For treating gout, the uric acid of energy fast decoupled joint deposition is so as to eliminate the inflammation of initiation and skin injury.
Urate oxidase is as a kind of novel anti-trioxypurine drug, one of which recombined Aspergillus flavus uricoxidase (commodity
Name：Rasburicase) ratified to treat tumor lysis syndrome for clinical prevention, therefore urate oxidase is with good by America and Europe
Druggability.Certainly as albumen kind biological product, one of an important factor for good method of quality control is its patent medicine.The present invention
A kind of analysis method to urate oxidase with the control of good and stabilised quality will be provided.
The urate oxidase that the present invention uses is that genomic DNA is extracted from candida utili, by PCR amplification, if
Specific primer is counted, urate oxidase gene is detached from candida utili genome, and inserts it into Escherichia coli table
Up in plasmid, recombination candida utili urate oxidase transformant is obtained, then candida utili urate oxidase will be recombinated
Transformant is transformed into Escherichia coli, obtain can high efficient expression urate oxidase colibacillus engineering strain.Using fermentation
Culture, the thalline of harvest expression recombination recombination candida utili urate oxidase, purifies by cellular lysate, column chromatography
To the recombination candida utili urate oxidase (referring to patent of invention ZL02819387.3) of purifying.Different types of uric acid oxygen
Change enzyme has the identical mode of action and similar physicochemical property, therefore the detection method of the present invention is similary for the effect of uric acid
Suitable for other urate oxidases.
Invention content
The purpose of the present invention is to provide a kind of new urate oxidase charge heterogeneities based on capillary electrophoresis technique
Analysis method, suitable for the Analysis of quality control of Urate oxidase drug.
Detection method includes the following steps for capillary isoelectric focusing of the present invention：
(1) by pharmalyte, capillary isoelectric focusing gel, urea, anodic stabilization agent, cathode stabilizers, isoelectric point mark
Note object and urate oxidase sample to be analyzed are mixed, and form mixed liquor；
(2) mixed liquor in step (1) is splined on coatings capillary pipe, under suitable temperature, voltage and focal time, led to
Capillary electrophoresis detection is crossed, obtains isoelectric focusing spectrogram；
(3) standard song is worth to according to the transit time of isoelectric point marker and pI in the isoelectric focusing collection of illustrative plates of step (2)
Line determines the pI values of protein sample each component to be analyzed；
(4) according to the isoelectric focusing profile information of step (2), the content of protein sample each component to be analyzed is determined.
In the step (1), pharmalyte includes：Pharmalyte3-10、Pharmalyte5-8、
Pharmalyte6.7-7.7、Pharmalyte8-10.5、Servalyt4-9、Servalyt5-7、Servalyt6-9、
One or more in Servalyt3-10, the volume range in pharmalyte in mixed liquor is 1%-5%；
A concentration of 0mol/L-4.86mol/L of the urea in mixed liquor；
Anodic stabilization agent be iminodiacetic acid (salt) acid solution, a concentration of 2.97mmol/L-5.11mmol/L in mixed liquor；
Cathode stabilizers be L-arginine solution, a concentration of 74.35mmol/L-127.80mmol/ in mixed liquor
L；
Isoelectric point marker, the small molecule isoelectric point marker provided including Beckman companies；Beckman companies provide
Small molecule isoelectric point marker include：pI4.1、pI5.5、pI9.5、pI10.0；
The concentration range of protein sample to be analyzed in mixed liquor is 0.1mg/mL-1mg/mL.
In the step (2), institute is included using coatings capillary pipe：The FC coatings capillary pipes of Agilent companies, Beckman
The neutral coatings capillary pipe or N-CHO coatings capillary pipes of company or other similar non-covalent bondings or covalent bonding coating
Capillary；
In the step (2), used capillary column temperature is 10-40 DEG C, preferably 10 DEG C, 25 DEG C, 30 DEG C；
In the step (2), used capillary voltage is 15-50kv, preferably 15kv, 25kv, 30kv, 40kv；
In the step (2), used focal time is 10-30 minutes.
The present invention can realize the following contents：
1st, it by selecting suitable experiment condition, realizes each component in protein sample to be analyzed and efficiently separates, and
Determine the corresponding isoelectric point of each component in collection of illustrative plates.
2nd, it can determine that the relative amount of each component in collection of illustrative plates.
3rd, method is reproducible.
Description of the drawings
The abscissa of attached drawing used is chronomere Minute, ordinate UV, absorption intensity unit AU.
Fig. 1 shows urate oxidase CE-SDS electrophoresis purity collection of illustrative plates.
Fig. 2 shows the urate oxidase capillary isoelectric focusing collection of illustrative plates under the conditions of embodiment 2.
Fig. 3 shows under the conditions of embodiment 3 (wherein 2 μ l of 200mmol/L iminodiacetic acid (salt)s acid solution) urate oxidase
Capillary isoelectric focusing collection of illustrative plates.
Fig. 4 shows under the conditions of embodiment 3 (wherein 10 μ l of 200mmol/L iminodiacetic acid (salt)s acid solution) urate oxidase
Capillary isoelectric focusing collection of illustrative plates.
Fig. 5 shows under the conditions of embodiment 3 (wherein 20 μ l of 200mmol/L iminodiacetic acid (salt)s acid solution) urate oxidase
Capillary isoelectric focusing collection of illustrative plates.
Fig. 6 shows under the conditions of embodiment 3 (wherein 50 μ l of 200mmol/L iminodiacetic acid (salt)s acid solution) urate oxidase
Capillary isoelectric focusing collection of illustrative plates.
Fig. 7 shows that under the conditions of embodiment 3 (wherein 100 μ l of 200mmol/L iminodiacetic acid (salt)s acid solution) uric acid aoxidizes
Enzyme capillary isoelectric focusing collection of illustrative plates.
Fig. 8 shows under the conditions of embodiment 4 (wherein 10 μ l of 500mmol/L L-arginines solution) urate oxidase hair
Capillary isoelectrofocusing collection of illustrative plates.
Fig. 9 shows under the conditions of embodiment 4 (wherein 20 μ l of 500mmol/L L-arginines solution) urate oxidase hair
Capillary isoelectrofocusing collection of illustrative plates.
Figure 10 shows under the conditions of embodiment 4 (wherein 30 μ l of 500mmol/L L-arginines solution) urate oxidase
Capillary isoelectric focusing collection of illustrative plates.
Figure 11 shows that under the conditions of embodiment 5 (wherein 20 μ l, 200mmol/L of 500mmol/L L-arginines solution is sub-
2 μ l of aminoacetaldehyde diethyl acid solution) urate oxidase capillary isoelectric focusing collection of illustrative plates.
Figure 12 shows that under the conditions of embodiment 5 (wherein 40 μ l, 200mmol/L of 500mmol/L L-arginines solution is sub-
4 μ l of aminoacetaldehyde diethyl acid solution) urate oxidase capillary isoelectric focusing collection of illustrative plates.
Figure 13 shows that under the conditions of embodiment 5 (wherein 60 μ l, 200mmol/L of 500mmol/L L-arginines solution is sub-
6 μ l of aminoacetaldehyde diethyl acid solution) urate oxidase capillary isoelectric focusing collection of illustrative plates.
Figure 14 shows that under the conditions of embodiment 5 (wherein 80 μ l, 200mmol/L of 500mmol/L L-arginines solution is sub-
8 μ l of aminoacetaldehyde diethyl acid solution) urate oxidase capillary isoelectric focusing collection of illustrative plates.
Figure 15 shows under the conditions of embodiment 5 (wherein 100 μ l, 200mmol/L of 500mmol/L L-arginines solution
10 μ l of iminodiacetic acid (salt) acid solution) urate oxidase capillary isoelectric focusing collection of illustrative plates.
Figure 16 shows under the conditions of embodiment 6 (wherein 30kV is focused on 15 minutes) urate oxidase capillary isoelectric focusing
Collection of illustrative plates.
Figure 17 shows under the conditions of embodiment 6 voltolisation such as (wherein 30kV is focused on 17.5 minutes) urate oxidase capillary
Burnt collection of illustrative plates.
Figure 18 shows under the conditions of embodiment 6 (wherein 30kV is focused on 20 minutes) urate oxidase capillary isoelectric focusing
Collection of illustrative plates.
Figure 19 shows under the conditions of embodiment 6 voltolisation such as (wherein 30kV is focused on 22.5 minutes) urate oxidase capillary
Burnt collection of illustrative plates.
Figure 20 shows under the conditions of embodiment 6 (wherein 30kV is focused on 25 minutes) urate oxidase capillary isoelectric focusing
Collection of illustrative plates.
Figure 21 shows under the conditions of embodiment 7 (wherein 200 μ l of 0mol/L urea-cIEF gel solutions) urate oxidase
Capillary isoelectric focusing collection of illustrative plates.
Figure 22 shows under the conditions of embodiment 7 (wherein 200 μ l of 1mol/L urea-cIEF gel solutions) urate oxidase
Capillary isoelectric focusing collection of illustrative plates.
Figure 23 shows under the conditions of embodiment 7 (wherein 200 μ l of 2mol/L urea-cIEF gel solutions) urate oxidase
Capillary isoelectric focusing collection of illustrative plates.
Figure 24 shows under the conditions of embodiment 7 (wherein 200 μ l of 3mol/L urea-cIEF gel solutions) urate oxidase
Capillary isoelectric focusing collection of illustrative plates.
Figure 25 shows under the conditions of embodiment 7 (wherein 200 μ l of 4mol/L urea-cIEF gel solutions) urate oxidase
Capillary isoelectric focusing collection of illustrative plates.
Figure 26 shows under the conditions of embodiment 7 (wherein 200 μ l of 6mol/L urea-cIEF gel solutions) urate oxidase
Capillary isoelectric focusing collection of illustrative plates.
Figure 27 shows under the conditions of embodiment 8 (wherein 200 μ l of 3mol/L urea-cIEF gel solutions) urate oxidase
Capillary isoelectric focusing collection of illustrative plates.
Figure 28 shows under the conditions of embodiment 8 (wherein 200 μ l of 6mol/L urea-cIEF gel solutions) urate oxidase
Capillary isoelectric focusing collection of illustrative plates.
Figure 29 shows that under the conditions of embodiment 9 (wherein 20 μ l, 200mmol/L of 500mmol/L L-arginines solution is sub-
2 μ l of aminoacetaldehyde diethyl acid solution) urate oxidase capillary isoelectric focusing collection of illustrative plates.
Figure 30 shows that under the conditions of embodiment 9 (wherein 40 μ l, 200mmol/L of 500mmol/L L-arginines solution is sub-
4 μ l of aminoacetaldehyde diethyl acid solution) urate oxidase capillary isoelectric focusing collection of illustrative plates.
Figure 31 shows that under the conditions of embodiment 9 (wherein 60 μ l, 200mmol/L of 500mmol/L L-arginines solution is sub-
6 μ l of aminoacetaldehyde diethyl acid solution) urate oxidase capillary isoelectric focusing collection of illustrative plates.
Figure 32 shows that under the conditions of embodiment 9 (wherein 80 μ l, 200mmol/L of 500mmol/L L-arginines solution is sub-
8 μ l of aminoacetaldehyde diethyl acid solution) urate oxidase capillary isoelectric focusing collection of illustrative plates.
Figure 33 shows the urate oxidase capillary isoelectric focusing collection of illustrative plates under the conditions of embodiment 10.
Figure 34 shows continuous three batches of urate oxidase capillary isoelectric focusings superposition collection of illustrative plates.
Specific embodiment
Following embodiment instrument and reagent are routine business purchase unless otherwise indicated.
Embodiment 1
First, the preparation of urate oxidase stoste
Urate oxidase engineered strain is prepared according to patent of invention ZL02819387.3.
Recovery urate oxidase engineering bacteria, 37 DEG C of inclined-plane are cultivated 17~24 hours.Then strain is inoculated into shaking flask, 37
DEG C, 200~300rpm shaking cultures.As the OD of bacterium solution600When being 2.0~5.0, it is seeded to fermentation tank culture.450~700rpm,
PH7.0 fermentation tank cultures keep dissolved oxygen >=50%, OD600IPTG induced expressions are added in for 7.5~9.5 2 hours.Bacterium is collected by centrifugation
Body is resuspended thalline using lysate, then handles suspension using homogenizer, suspension and collects supernatant after centrifugal treating.Pass through
Hydrophobic chromatography and ion exchange obtain pure urate oxidase stoste.
2nd, the purity detecting of stoste
Urate oxidase is subjected to CE-SDS analyses, detects sample purity, testing result refers to Fig. 1.
Embodiment 2
First, experiment condition
Capillary isoelectric focusing loading mixed liquor is prepared according to table 1, is placed in Beckman companies PA800plus capillaries electricity
It swims in the autosampler of instrument, setting column temperature is 30 DEG C, is analyzed using N-CHO coatings capillary pipes (Beckman companies),
15kV is focused on 15 minutes, and 25kV is analyzed 30 minutes, is detected under the UV detectors of 280nm wavelength.
1 capillary isoelectric focusing sample solution of table matches
| Reagent name | Reagent volume (μ l) | Final concentration |
| 3mol/L urea-cIEF gel solutions | 200 | 2.43mol/L |
| 3-10 carrier ampholytes (Pharmalyte3-10) | 12 | |
| 500mmol/L L-arginine cathode stabilization solution | 20 | 40.49mmol/L |
| 200mmol/L iminodiacetic acid anodic stabilization solution | 2 | 1.62mmol/L |
| 10.0 protein labelings of pI | 1 | - |
| 5.5 protein labelings of pI | 1 | - |
| 4.1 protein labelings of pI | 1 | - |
| 10mg/mL protein samples | 10 | 0.40mg/mL |
2nd, data analysis
Capillary isoelectric focusing collection of illustrative plates finds more heterogeneous peak as shown in Fig. 2, analyze urate oxidase stoste,
Effective Analysis of quality control can not be carried out to it.
Embodiment 3
First, experiment condition
Capillary isoelectric focusing loading mixed liquor is prepared according to table 2, the anodic stabilization of different volumes is separately added into according to table 3
Agent, mixing are placed in the autosampler of Beckman companies PA800plus capillary electrophoresis, and setting column temperature is 30 DEG C, is adopted
It is analyzed with N-CHO coatings capillary pipes (Beckman companies), 15kV is focused on 15 minutes, and 25kV is analyzed 30 minutes, in 280nm
It is detected under the UV detectors of wavelength.
2 capillary isoelectric focusing sample solution of table matches
| Reagent name | Reagent volume (μ l) | Final concentration |
| 3mol/L urea-cIEF gel solutions | 200 | 1.74-2.43mol/L |
| 3-10 carrier ampholytes (Pharmalyte3-10) | 12 | - |
| 500mmol/L L-arginine cathode stabilization solution | 20 | 28.99-40.49mmol/L |
| 10.0 protein labelings of pI | 1 | - |
| 5.5 protein labelings of pI | 1 | - |
| 4.1 protein labelings of pI | 1 | - |
| 10mg/mL protein samples | 10 | 0.29-0.40mg/mL |
3 anodic stabilization agent of table matches
2nd, data analysis
Capillary isoelectric focusing collection of illustrative plates is shown in Fig. 3 to Fig. 7, and anodic stabilization agent volume successively increases.As shown in the figure, anodic stabilization
Agent volume can't detect sample peak when increasing to 10 μ l.Concentration gradient experiment is carried out using the anodic stabilization agent of 200mmol/L
Grope, do not generate good effect.
Embodiment 4
First, experiment condition
Capillary isoelectric focusing loading mixed liquor is prepared according to table 4, the cathode stabilization of different volumes is separately added into according to table 5
Agent, mixing are placed in the autosampler of Beckman companies PA800plus capillary electrophoresis, and setting column temperature is 30 DEG C, is adopted
It is analyzed with N-CHO coatings capillary pipes (Beckman companies), 30kV is focused on 15 minutes, and 30kV is analyzed 30 minutes, in 280nm
It is detected under the UV detectors of wavelength.
4 capillary isoelectric focusing sample solution of table matches
| Reagent name | Reagent volume (μ l) | Final concentration |
| 3mol/L urea-cIEF gel solutions | 200 | 2.33-2.53mol/L |
| 3-10 carrier ampholytes (Pharmalyte3-10) | 12 | - |
| 200mmol/L iminodiacetic acid anodic stabilization solution | 2 | 1.56-1.69mmol/L |
| 10.0 protein labelings of pI | 1 | - |
| 5.5 protein labelings of pI | 1 | - |
| 4.1 protein labelings of pI | 1 | - |
| 10mg/mL protein samples | 10 | 0.39-0.42mg/mL |
5 anodic stabilization agent of table matches
2nd, data analysis
Capillary isoelectric focusing collection of illustrative plates is shown in Fig. 8 to Figure 10, only touches the cathode stabilizers of 500mmol/L progress concentration gradient
Rope does not generate good effect.
Embodiment 5
First, experiment condition
Capillary isoelectric focusing loading mixed liquor is prepared according to table 6, different volumes anodic stabilization agent is separately added into according to table 7
And cathode stabilizers, mixing are placed in the autosampler of Beckman companies PA800plus capillary electrophoresis, set column
Temperature is 10 DEG C, is analyzed using N-CHO coatings capillary pipes (Beckman companies), and 30kV is focused on 15 minutes, and 30kV analyzes 30 points
Clock is detected under 280nm.
6 capillary isoelectric focusing sample solution of table matches
| Reagent name | Reagent volume (μ l) | Final concentration |
| 3mol/L urea-cIEF gel solutions | 200 | 1.79-2.43mol/L |
| 3-10 carrier ampholytes (Pharmalyte3-10) | 12 | - |
| 10.0 protein labelings of pI | 1 | - |
| 5.5 protein labelings of pI | 1 | - |
| 4.1 protein labelings of pI | 1 | - |
| 10mg/mL protein samples | 10 | 0.30-0.40mg/mL |
7 cathode/anode stabilizer of table matches
2nd, data analysis
Capillary isoelectric focusing collection of illustrative plates is shown in Figure 11 to Figure 15, and anode and cathode stabilizer volume is multiplied simultaneously.As schemed
Show, reduced as stabilizer concentration increases the heterogeneous peak of sample, stabilizer volume can't detect sample peak when being 5N.Stabilizer volume
For that can reach preferable when 2~4N (i.e. anolyte is 2.97~5.11mmol/L, and catholyte is 74.35~127.80mmol/L)
Experiment effect, final concentration ranging from 1.92~2.23mol/L of the corresponding 3mol/L urea of the concentration range in system.1N
Stabilizer concentration with 5N is without the detection for being suitble to urate oxidase sample.
We will be also with it is experimentally confirmed that the stabilizer concentration less than 1N will detect more signal peaks so that uric acid aoxidizes
Enzyme is difficult to carry out quality control.Stabilizer concentration higher than 5N makes sample not detect signal.
We are still produced and the present embodiment class using the anode and cathode stabilizer of the identical final concentration of various concentration configuration
As experiment effect.The selection for illustrating cathode/anode stabilizer proportioning final concentration is the key that realize the present invention.
Embodiment 6
First, experiment condition
Capillary isoelectric focusing loading mixed liquor is prepared according to table 8, is placed in Beckman companies PA800plus capillaries electricity
It swims in the autosampler of instrument, setting column temperature is 25 DEG C, is analyzed using N-CHO coatings capillary pipes (Beckman companies),
30kV is focused on 15 minutes, 17.5 minutes, 20 minutes, 22.5 minutes and 25 minutes respectively, and 30kV is analyzed 30 minutes, under 280nm
It is detected.
8 capillary isoelectric focusing sample solution of table matches
2nd, data analysis
Capillary isoelectric focusing collection of illustrative plates is shown in Figure 16 to Figure 20, extends focal time successively, and urate oxidase peak shape is not bright
Aobvious variation, the change of focal time do not influence the Analysis of quality control to uric acid enzyme sample.But our subsequent experimentals confirm that 30kV gathers
The burnt time will influence experiment effect less than 10 minutes.Therefore, it is suggested that used focal time is 10-30 minutes.
We have carried out related experiment to the selection of capillary voltage again, are sequentially adjusted in gathering using 15~50kV different voltages
The burnt time can also reach the result similar to the present embodiment.It will lead to long focal time less than 15kV voltages, and then
Influence working efficiency.It will be easily damaged higher than 50kV voltages capillary, and increase experimental cost.
Embodiment 7
First, experiment condition
Capillary isoelectric focusing loading mixed liquor is prepared according to table 9, various concentration urea liquid is separately added into according to table 10,
Mixing is placed in the autosampler of Beckman companies PA800plus capillary electrophoresis, and setting column temperature is 30 DEG C, is used
N-CHO coatings capillary pipes (Beckman companies) are analyzed, and 30kV is focused on 10 minutes, and 30kV is analyzed 30 minutes, under 280nm
It is detected.
9 capillary isoelectric focusing sample solution of table matches
10 urea-cIEF gel solutions of table
| Reagent name | Reagent volume (μ l) | Final concentration |
| 0mol/L urea-cIEF gel solutions | 200 | 0mol/L |
| 1mol/L urea-cIEF gel solutions | 200 | 0.81mol/L |
| 2mol/L urea-cIEF gel solutions | 200 | 1.62mol/L |
| 3mol/L urea-cIEF gel solutions | 200 | 2.43mol/L |
| 4mol/L urea-cIEF gel solutions | 200 | 3.24mol/L |
| 6mol/L urea-cIEF gel solutions | 200 | 4.86mol/L |
2nd, data analysis
Capillary isoelectric focusing collection of illustrative plates is shown in Figure 21 to Figure 26, is successively increased as depicted with urea concentration, and uricase is different
Mass peak is reduced.Previous embodiment is it has proven convenient that the concentration of appropriate increase anode and cathode stabilizer can effectively reduce uricase
Heterogeneity, but stabilizer is expensive.The concentration that the present embodiment confirms to increase urea can also effectively reduce urease
It is heterogeneous.Being added without urea can increase the absorption peak of sample, use urea more than 6mol/L concentration so that increase with liquid difficulty
Add.
Embodiment 8
First, experiment condition
Capillary isoelectric focusing loading mixed liquor is prepared according to table 11, it is molten to be separately added into various concentration urea according to table 12
Liquid, mixing are placed in the autosampler of Beckman companies PA800plus capillary electrophoresis, and setting column temperature is 25 DEG C, is adopted
It is analyzed with neutral coatings capillary pipe (Beckman companies), 40kV is focused on 10 minutes, and 40kV is analyzed 30 minutes, under 280nm
It is detected.
11 capillary isoelectric focusing sample solution of table matches
12 urea-cIEF gel solutions of table
| Reagent name | Reagent volume (μ l) | Final concentration |
| 3mol/L urea-cIEF gel solutions | 200 | 2.43mol/L |
| 6mol/L urea-cIEF gel solutions | 200 | 4.86mol/L |
2nd, data analysis
Capillary isoelectric focusing collection of illustrative plates is as shown in Figure 27 to Figure 28, and as urea concentration increases, the heterogeneous peak of uricase is shown
It writes and reduces.
Embodiment 9
First, experiment condition
Capillary isoelectric focusing loading mixed liquor is prepared according to table 13, different volumes anodic stabilization is separately added into according to table 14
Agent and cathode stabilizers, mixing are placed in the autosampler of Beckman companies PA800plus capillary electrophoresis, setting
Column temperature is 25 DEG C, is analyzed using neutral coatings capillary pipe (Beckman companies), and 25kV is focused on 10 minutes, 25kV analyses 30
Minute, it is detected under 280nm.
13 capillary isoelectric focusing sample solution of table matches
| Reagent name | Reagent volume (μ l) | Final concentration |
| 6mol/L urea-cIEF gel solutions | 200 | 3.83-4.86mol/L |
| 3-10 carrier ampholytes (Pharmalyte3-10) | 12 | - |
| 10.0 protein labelings of pI | 1 | - |
| 5.5 protein labelings of pI | 1 | - |
| 4.1 protein labelings of pI | 1 | - |
| 10mg/mL protein samples | 10 | 0.32-0.40mg/mL |
14 cathode/anode stabilizer of table matches
2nd, data analysis
Capillary isoelectric focusing collection of illustrative plates is as shown in Figure 29 to Figure 32, using experimental program similar to Example 5.By urea
Concentration rise to 6mol/L, for stabilizer in the range of low concentration, that is, 2N~4N, the end of corresponding anodic stabilization agent is dense
2.97~5.11mmol/L is spent, 74.3~127.80mmol/L of final concentration of cathode stabilizers can still obtain preferable reality
It tests as a result, final concentration ranging from 3.83~4.46mol/L of the corresponding 6mol/L urea of the concentration range in system.
Isoelectric point marker has selected pI9.5 again, can equally achieve the effect that similar to pI10.Different manufacturers wait electricity
Point marker can achieve the effect that identical.
Embodiment 10
First, experiment condition
Capillary isoelectric focusing loading mixed liquor is prepared according to table 15, is placed in Beckman companies PA800plus capillaries electricity
It swims in the autosampler of instrument, setting column temperature is 30 DEG C, is analyzed using N-CHO coatings capillary pipes (Beckman companies),
15kV is focused on 15 minutes, and 20kV is analyzed 30 minutes, is detected under 280nm.
15 capillary isoelectric focusing sample solution of table matches
2nd, data analysis
Capillary isoelectric focusing collection of illustrative plates is as shown in figure 33, and urate oxidase realizes complete baseline separation, and common property gives birth to 4
Component peaks.The isoelectric point of urate oxidase each component and percentage composition (being shown in Table 16) are can detect that using the method after the optimization,
And then effective Analysis of quality control is carried out to urate oxidase drug.
The isoelectric point and percentage composition of 16 urate oxidase each component of table
| Target peak | Transit time | Peak area | Percentage | Isoelectric point |
| 1 | 39.017 | 435618 | 33.67 | 7.26 |
| 2 | 39.208 | 470038 | 36.33 | 7.14 |
| 3 | 39.425 | 251615 | 19.45 | 7.00 |
| 4 | 39.733 | 136366 | 10.54 | 6.80 |
The manufacturer of capillary is investigated again under the same conditions, the coating of Agilent and Beckman productions
Capillary can generate similar experimental result, can use.As long as the coating that electroosmotic flow theoretically can effectively be inhibited to generate
Capillary is suitable for this experiment, and electroosmotic flow can generate experiment larger interference.
We have investigated producer and the range of pharmalyte again, the pharmalyte of GE and SERVA companies same range can be with
Similar experimental result is generated, when the pharmalyte of 3-10 ranges to be mixed with the carrier of other ranges to the reservation that can make peak
Between or peak capacity change, related experiment result can also effectively to urate oxidase drug carry out Analysis of quality control.
Urate oxidase is acceptable in 0.1~1mg/ml after mixing, less than the analog value of 0.1mg/ml detectors
Relatively low, the cycles of concentration higher than 1mg/ml urate oxidases increases, and sample is more sticky so that accurately pipettes the more difficult of change.
Embodiment 11
First, experiment condition
Capillary isoelectric focusing loading mixed liquor is prepared according to table 15, is placed in Beckman companies PA800plus capillaries electricity
It swims in the autosampler of instrument, setting column temperature is 30 DEG C, is analyzed using N-CHO coatings capillary pipes (Beckman companies),
15kV is focused on 15 minutes, and 20kV is analyzed 30 minutes, is detected under 280nm.
2nd, data analysis
Capillary isoelectric focusing collection of illustrative plates is as shown in figure 34, continuously analyzes three batches of urate oxidases, realizes complete base
Line detaches, and common property gives birth to 4 component peaks.Illustrate that the method after the optimization is more stable, can detect that uric acid aoxidizes using this method
The isoelectric point and percentage composition of enzyme each component, so as to the quality testing and control being used in production.
The isoelectric point and percentage composition of 17 3 batches of urate oxidase each components of table
Claims (10)
1. a kind of capillary isoelectric focusing detection method of urate oxidase, the method includes the stepss：
(1) by pharmalyte, capillary isoelectric focusing gel, urea, anodic stabilization agent, cathode stabilizers, isoelectric point marker
It is mixed with urate oxidase sample to be analyzed, forms mixed liquor,
(2) mixed liquor in step (1) is splined on coatings capillary pipe, under suitable temperature, voltage and focal time, passes through hair
Capillary electrophoresis instrument detects, and obtains isoelectric focusing spectrogram,
(3) standard curve is worth to according to the transit time of isoelectric point marker and pI in the isoelectric focusing collection of illustrative plates of step (2), really
Determine the pI values of urate oxidase sample each component,
(4) according to the isoelectric focusing profile information of step (2), the content of protein sample each component to be analyzed is determined.
2. the method as described in claim 1, which is characterized in that a concentration of 0mol/L- of the urea of step (1) in mixed liquor
4.86mol/L。
3. the method as described in claim 1, which is characterized in that the anodic stabilization agent of step (1) is iminodiacetic acid (salt) acid solution,
A concentration of 2.97mmol/L-5.11mmol/L in mixed liquor, cathode stabilizers are L-arginine solution, in mixed liquor
A concentration of 74.35mmol/L-127.80mmol/L.
4. the method as described in claim 1-3, which is characterized in that the ranging from 3-10 of the pharmalyte of step (1), both sexes carry
Volume range of the body in mixed liquor be 1%-5%, pharmalyte be selected from Pharmalyte3-10, Pharmalyte5-8,
Pharmalyte6.7-7.7、Pharmalyte8-10.5、Servalyt4-9、Servalyt5-7、Servalyt6-9、
It is one or more in Servalyt3-10.
5. the method as described in claim 1-3, which is characterized in that the isoelectric point marker ranging from pI4.0 of step (1) is extremely
It is one or more in pI10.0, preferably pI4.1, pI5.5, pI9.5, pI10.0.
6. the method as described in claim 1-3, which is characterized in that the urate oxidase sample of step (1) is in mixed liquor
Final concentration ranging from 0.1mg/mL-1mg/mL.
7. the method as described in claim 1-3, which is characterized in that the capillary column temperature of step (2) is 10-40 DEG C, is used
Capillary voltage for 15-50kv, focal time is 10-30 minutes.
8. the method as described in claim 1-7, which is characterized in that the urea of step (1) is a concentration of in mixed liquor
1.92mol/L-2.23mol/L, anodic stabilization agent be iminodiacetic acid (salt) acid solution, a concentration of 2.97mmol/ in mixed liquor
L-5.11mmol/L, cathode stabilizers be L-arginine solution, a concentration of 74.35mmol/L- in mixed liquor
127.80mmol/L。
9. the method as described in claim 1-7, which is characterized in that the urea of step (1) is a concentration of in mixed liquor
3.83mol/L-4.46mol/L, anodic stabilization agent be iminodiacetic acid (salt) acid solution, a concentration of 2.97mmol/ in mixed liquor
L-5.11mmol/L, cathode stabilizers be L-arginine solution, a concentration of 74.35mmol/L- in mixed liquor
127.80mmol/L。
10. a kind of capillary isoelectric focusing detection method of urate oxidase, the method includes the stepss：
(1) by carrier ampholyte, 4.12mol/L urea-cIEF gel solutions, 103.09mmol/L L-arginine cathodes
Stablizing solution, 4.12mmol/L iminodiacetic acid anodic stabilization solution, pI 4.1, pI 5.5 and 10.0 protein labelings of pI,
0.34mg/mL urate oxidases sample mixes, and forms mixed liquor,
(2) mixed liquor in step (1) is splined on coatings capillary pipe, setting column temperature is 30 DEG C, and 15kV is focused on 15 minutes, 20kV
Analysis 30 minutes, is detected under 280nm, obtains isoelectric focusing spectrogram,
(3) standard curve is worth to according to the transit time of isoelectric point marker and pI in the isoelectric focusing collection of illustrative plates of step (2), really
Determine the pI values of urate oxidase sample each component,
(4) according to the isoelectric focusing profile information of step (2), the content of protein sample each component to be analyzed is determined.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103756983A (en) * | 2014-01-10 | 2014-04-30 | 临沂大学 | Preparation method of uricase of natural mammals |
| CN104411383A (en) * | 2012-05-03 | 2015-03-11 | 米迪缪尼有限公司 | Method for analyzing sample components |
| CN105412942A (en) * | 2015-12-23 | 2016-03-23 | 沈阳三生制药有限责任公司 | Pegylated recombined candida utilis urate oxidase freeze-drying injection |
-
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104411383A (en) * | 2012-05-03 | 2015-03-11 | 米迪缪尼有限公司 | Method for analyzing sample components |
| CN103756983A (en) * | 2014-01-10 | 2014-04-30 | 临沂大学 | Preparation method of uricase of natural mammals |
| CN105412942A (en) * | 2015-12-23 | 2016-03-23 | 沈阳三生制药有限责任公司 | Pegylated recombined candida utilis urate oxidase freeze-drying injection |
Non-Patent Citations (3)
| Title |
|---|
| KHADIJEH ALISHAH 等: "Utilizing intein-mediated protein cleaving for purification of uricase, a multimeric enzyme", 《ENZYME AND MICROBIAL TECHNOLOGY》 * |
| KNUD LARSEN: "Purification of Nodule-Specific Uricase From Soybean by Arginine-Sepharose Affinity Chromatography", 《PREPARATIVE BIOCHEMISTRY》 * |
| 孟尧 等: "朊圆酵母尿酸酶的基本特性研究", 《四川大学学报( 自然科学版)》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115541686A (en) * | 2022-11-25 | 2022-12-30 | 黑龙江飞鹤乳业有限公司 | Dairy product identification method |
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