CN106319027A - Visualized enzyme activity determination method and determinator thereof - Google Patents
Visualized enzyme activity determination method and determinator thereof Download PDFInfo
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- CN106319027A CN106319027A CN201610568796.XA CN201610568796A CN106319027A CN 106319027 A CN106319027 A CN 106319027A CN 201610568796 A CN201610568796 A CN 201610568796A CN 106319027 A CN106319027 A CN 106319027A
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- test tube
- syringe
- solution
- catalase
- reaction
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
- C12Q1/30—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving catalase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/902—Oxidoreductases (1.)
- G01N2333/908—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
Abstract
The invention discloses a visualized enzyme activity determination method which comprises the following steps: sequentially putting 7 test tubes with equal catalase solutions, of which the pH values are respectively regulated to 4, 5, 6, 7, 8, 9 and 10 with a HCl solution and a NaOH solution, on a test tube rack, respectively adding 10ml of a 30% H2O2 solution, and sealing the orifices with rubber plugs; respectively absorbing 10ml of a prepared pH catalase solution by 7 injectors, and putting on the test tube rack; putting a charging bracket on the test tube rack; and pressing the charging bracket down to inject the reagents in the injectors into the test tubes, and lifting the hand so that the charging bracket is sprung up by the spring. When the reaction starts, the injectors are slowly lifted under the extrusion effects of gases produced by the reactions, and the lifting heights are different due to different reactions, thereby forming a visualized reaction mathematic curve. The determination method is simple to operate, and can directly and vividly display the pH-dependent enzyme activity curve.
Description
Technical field
The present invention relates to a kind of bioconversion medium test method, be applicable to carry out PH affect the reality of enzymatic activity particularly to a kind of
Test visualization activity determination method and the analyzer thereof of demonstration.
Technical background
" PH impact on enzymatic activity " is that people teaches an edition teaching material " biological required 1 molecule and cell " the 5th chapter Section 1 " enzyme
Feature " one joint in inquiry experiment, learn this experiment well, three big characteristics of enzyme can not only be better understood from, after being also study
The important contents such as ATP, photosynthesis, Cellular respiration have established solid basis.But, people teaches edition teaching material for this experiment only
Enzymatic activity is affected schematic diagram by PH, does not has concrete experimental provision and operating procedure, although the teaching material of some version gives experiment
Device and operating procedure, but the most complicated, and the most bad operation, success rate is the most extremely low.
Summary of the invention
For above-mentioned problems of the prior art and defect, it is an object of the invention to provide visualization enzymatic activity and survey
Determining method, this assay method is the most simple to operate, and can directly, vivo embody the curve that enzymatic activity is affected by PH.
Realize foregoing invention purpose and be the technical scheme is that a kind of visualization enzyme assay side, specifically include down
Row step:
1) take 7 clean small beakers, be separately added into the Catalase solution of equivalent, by Hcl solution and NaOH solution
Respectively its PH is adjusted to 4,5,6,7,8,9,10；
2) take 7 clean test tubes, respectively numbering 4,5,6,7,8,9,10, be placed on the lower floor support of test tube rack in order
On, it is separately added into the H of 10ml30% to it2O2Solution, with rubber closure ferrule；
3) taking 7 clean syringes, the catalase that PH is 5,6,7,8,9 that absorption 10ml configures respectively is molten
Liquid, is placed in the upper layer bracket of test tube rack；
4) respectively in the rubber closure of the syringe needle side of the being inserted under test tube of syringe, then material-feeding rack is caught；
5) firmly material-feeding rack is pressed downwards, in whole for the reagent in syringe injecting tubes, unclamp hands, charging
Frame is bounced by spring；
6) reaction starts, and syringe starts slowly to lift due to the squeezing action of the gas that reaction is caused, due to reaction
Difference, the height lifted is the most different, thus forms one and react mathematic curve intuitively.
It is a still further object of the present invention to provide a kind of enzyme assay instrument, including test tube support, syringe, test tube, institute
State test tube to be inserted on the lower floor support of test tube support；Described syringe is inserted in the upper layer bracket of test tube support, and its syringe needle inserts
On plug piston at the test tube mouth of pipe, the upper end of the both sides side arm of described test tube support offers cylindrical hole, material-feeding rack two
The side arm on limit stretches in the hole being provided with spring just.
Described syringe and a test tube row symmetrical above and below are provided with several.
The operation principle of the visualization activity determination method of the present invention: hydrogen peroxide meeting under catalatic catalysis
Quickly producing a large amount of oxygen, and most of enzyme is protein, therefore the activity of enzyme is affected the most notable by environmental PH.Logical
Normal various enzyme only just shows its activity in the range of certain PH.Show its catalysis activity the highest time pH value be referred to as this enzyme
Optimal pH, during below or above optimal pH, the activity of enzyme is gradually lowered.Thus can be according to the oxygen produced in the identical time
The size of how many judgement catalase activities.Following device oxygen produces the most, and air pressure is the biggest, syringe ascensional range
The biggest, directly can reflect such as " enzymatic activity is affected schematic diagram by PH " curve.
The present invention visualizes activity determination method compared with prior art, has the advantage that
(1) save the traditional experiment time greatly, and reduce experimental implementation difficulty, simplify laboratory operating procedures；
(2) solve the enzyme by different PH process to be simultaneously injected in reaction.
(3) can the flat-footed parabola observed in a device in mathematical coordinates.By abstract mathematical thinking shape
Elephant be rendered as it is observed that concrete model；
(4) multi-functional: to install, dismantle simply, when not doing this experiment, be segmented into two this enzymatic activitys of common test tube frame
Analyzer；Simple in construction, feature richness, it is simple to promote.
Accompanying drawing illustrates:
Fig. 1 is the structural representation of the present invention a kind of enzyme assay instrument；
Fig. 2 is the work sheet of the present invention a kind of enzyme assay instrument；
Fig. 3 is the result figure of the present invention a kind of enzyme assay instrument.
Detailed description of the invention:
Below in conjunction with the accompanying drawings the present invention is embodied as example to be described in further detail.
Embodiment 1 one kinds visualization enzyme assay side, specifically includes the following step:
1) take 7 clean small beakers, be separately added into the Catalase solution of equivalent, by Hcl solution and NaOH solution
Respectively its PH is adjusted to 4,5,6,7,8,9,10；
2) take 7 clean test tubes, respectively numbering 4,5,6,7,8,9,10, be placed on the lower floor support of test tube rack in order
On, it is separately added into the H of 10ml30% to it2O2Solution, with rubber closure ferrule；
3) taking 7 clean syringes, the catalase that PH is 5,6,7,8,9 that absorption 10ml configures respectively is molten
Liquid, is placed in the upper layer bracket of test tube rack；
4) respectively in the rubber closure of the syringe needle side of the being inserted under test tube of syringe, then material-feeding rack is caught；
5) firmly material-feeding rack is pressed downwards, in whole for the reagent in syringe injecting tubes, unclamp hands, charging
Frame is bounced by spring；
6) reaction starts, and syringe starts slowly to lift due to the squeezing action of the gas that reaction is caused, due to reaction
Difference, the height lifted is the most different, thus forms one and react mathematic curve intuitively.
2 one kinds of enzyme assay instrument of embodiment
The structural representation of Fig. 1 enzyme assay of the present invention instrument, including test tube support 1, syringe 7, test tube 8, described examination
Pipe 8 is inserted on the lower floor support 2 of test tube support 1；Described syringe 7 is inserted in the upper layer bracket 5 of test tube support 1, and its syringe needle is inserted
Entering on plug piston at test tube 8 mouth of pipe, the upper end of the both sides side arm 3 of described test tube support 1 offers cylindrical hole, charging
The side arm on frame 6 both sides stretches in the hole being provided with spring 4 just, and described syringe 7 and test tube 8 row symmetrical above and below are provided with some
Individual.
First the test tube 8 having added reaction substrate 30% hydrogenperoxide steam generator is placed on reactant fixed support 1, then will
The syringe 7 adding different PH process Catalase solution inserts the test tube 8 (mouth of pipe is plugged with rubber closure) of correspondence, with test tube even
Connect and be integrally formed, conveniently enzymatic solution is added test tube.
Install material-feeding rack 6, extruding material-feeding rack (such as Fig. 2) so that the enzymatic solution in syringe 7 is simultaneously introduced test tube
8, react, produce oxygen, oxygen gas increases, and in test tube 8, pressure increases, and gas push syringe 7 piston moves, due to
The enzymatic activity that different PH process is different, and gas production rate is different, so in the unit interval, piston moving speed different.Make
Syringe piston presents a parabola (such as Fig. 3).
Test example 1 present invention visualizes experimental principle and the device of activity determination method
1.1 experimental principle
Hydrogen peroxide can quickly produce a large amount of oxygen under catalatic catalysis, and most of enzyme is albumen
Matter, therefore the activity of enzyme is affected the most notable by environmental PH.Usual various enzyme only just shows it in the range of certain PH
Activity.Show its catalysis activity the highest time pH value be referred to as the optimal pH of this enzyme, during below or above optimal pH, the activity of enzyme
It is gradually lowered.Thus can be according to the size of how many judgement catalase activities of the oxygen produced in the identical time.As follows
Device oxygen produces the most, and air pressure is the biggest, and syringe ascensional range is the biggest, directly can reflect as " enzymatic activity is affected by PH
Schematic diagram " curve.
Fig. 1 is shown in by 1.2 experimental provisions.
2 experimental articles and preparation
2.1 experimental article
2.1.1 experiment material and apparatus:
Fresh Rhizoma Solani tuber osi (containing catalase), test tube, test tube plug, test tube rack, small beaker, graduated cylinder, dropper, PH reagent paper, water
Really cutter, juice extractor, syringe
2.1.2 experiment reagent:
The H2O2 solution of 30%, Hcl solution, NaOH solution
2.2 Preparatory work of experiment
2.2.1 Catalase solution is prepared
It is cut into small pieces after one fresh Rhizoma Solani tuber osi (about 200g) is removed the peel, puts into juice extractor, add the clear of about 800ml
Water, squeezes the juice.
Catalase solution under the most differently configured ph value
Take 7 clean small beakers, be separately added into the Catalase solution of equivalent, divide with Hcl solution and NaOH solution
Its PH is not adjusted to 4,5,6,7 (pH value of former Catalase solution substantially close to 7), 8,9,10.
3 experimental procedures
3.1 take 7 clean test tubes, respectively numbering 4,5,6,7,8,9,10, are placed in order on test tube rack, to it respectively
Add the H2O2 solution of 10ml30%.
Take 7 clean syringes, draw the Catalase solution that PH is 5,6,7,8,9 that 10ml configures respectively,
By syringe needle test tube plug beyond the Great Wall, inserting corresponding sizer jack, test tube plug stoppered test tube, such syringe is with test tube even
Connect and be integrally formed (such as Fig. 2), conveniently enzymatic solution is joined in corresponding test tube.
Install material-feeding rack, extrude material-feeding rack, on earth after unclamp immediately so that the enzymatic solution in syringe adds simultaneously
Enter test tube, observe gas in syringe and rise variation tendency.
4 experimental results and analysis
From experimental result it can be seen that the fierceness of catalase and hydroperoxidation after different PH processes
Degree is significantly different, and the response strength of pH value 4-7 is gradually increased, and the response strength of pH value 7-10 is gradually lowered,
Show that catalase activity can gradually reduce, and there is an optimum PH centre, i.e. along with acidity strengthens or alkalescence enhancing
Catalase activity is maximum.The amplitude that the different syringes produced according to gas rise is different, naturally reflects enzyme
The curve (shown in Fig. 3 upper curve) that activity is affected by PH.
5 device advantages:
5.1 convenient experimental operation are simple
Senior high school period now, major part school does not the most carry out this experiment, if it is possible to from simple description of test problem, energy
The enough less time is to the understanding of a kind of perception of student, then such experiment, for teaching of senior high school, is a kind of good
Resource.As long as consumption is suitable, just it is observed that experimental phenomena clearly within one minute, everyone can succeed.
5.2 consumptions that can well control enzyme and the time of addition enzyme
The consumption of enzyme is the key factor affecting this experiment, and syringe can well control consumption, thus successful
Get rid of the interference that this is tested by the consumption of enzyme.And enzyme can also be controlled be simultaneously introduced test tube.
5.3 read data easily, and curve is the most directly perceived
This device can also simply measure the volume producing oxygen, graduated syringe, just sees its climb
It is.
5.4 experimental costs are low
Experimental article has in common lab, and even without syringe, each big pharmacy is the most all
Can buy, the most cheaply, and can reuse.If it can, can also collect discarded, twice laid, but
Must sterilize.
Claims (3)
1. a visualization activity determination method, it is characterised in that specifically include the following step:
1) take 7 clean small beakers, be separately added into the Catalase solution of equivalent, by Hcl solution and NaOH solution difference
Its PH is adjusted to 4,5,6,7,8,9,10；
2) take 7 clean test tubes, respectively numbering 4,5,6,7,8,9,10, be placed in order on the lower floor support of test tube rack, to
It is separately added into the H of 10ml30%2O2Solution rubber closure seals；
3) take 7 clean syringes, draw the Catalase solution that PH is 5,6,7,8,9 that 10ml configures respectively, put
In the upper layer bracket of test tube rack；
4) respectively in the rubber closure of the syringe needle side of the being inserted under test tube of syringe, then material-feeding rack is caught；
5) firmly material-feeding rack is pressed downwards, in whole for the reagent in syringe injecting tubes, unclamp hands, material-feeding rack quilt
Spring bounces；
6) reaction starts, and syringe starts slowly to lift due to the squeezing action of the gas that reaction is caused, due to reaction difference,
The height lifted is the most different, thus forms one and react mathematic curve intuitively.
2. an enzyme assay instrument, including test tube support (1), syringe (7), test tube (8), it is characterised in that described test tube
(8) it is inserted on the lower floor support (2) of test tube support (1)；Described syringe (7) is inserted in the upper layer bracket (5) of test tube support (1)
On, its syringe needle inserts on plug piston at test tube (8) mouth of pipe, and the upper end of the both sides side arm (3) of described test tube support (1) is offered
Cylindrical hole, the side arm on material-feeding rack (6) both sides is had just to stretch in the hole being provided with spring (4).
Enzyme assay instrument the most according to claim 2, it is characterised in that described syringe (7) and test tube (8) are the most right
A row is claimed to be provided with several.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610568796.XA CN106319027A (en) | 2016-07-13 | 2016-07-13 | Visualized enzyme activity determination method and determinator thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610568796.XA CN106319027A (en) | 2016-07-13 | 2016-07-13 | Visualized enzyme activity determination method and determinator thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106086157A (en) * | 2016-06-21 | 2016-11-09 | 俞率成 | Catalase activity analyzer and using method thereof |
| CN114984896A (en) * | 2022-03-30 | 2022-09-02 | 北京擎科生物科技有限公司 | Oligonucleotide synthesis device and oligonucleotide synthesis method |
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| CN202394415U (en) * | 2011-12-23 | 2012-08-22 | 赵双双 | Demonstrating device for exploring influence on activity of enzyme by different factors |
| CN203276656U (en) * | 2013-04-19 | 2013-11-06 | 景宁山凤皇工艺制品有限公司 | Reactor for experiment for studying influence of pH on catalase |
| CN205803496U (en) * | 2016-07-13 | 2016-12-14 | 高静 | A kind of enzyme assay instrument |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN202394415U (en) * | 2011-12-23 | 2012-08-22 | 赵双双 | Demonstrating device for exploring influence on activity of enzyme by different factors |
| CN203276656U (en) * | 2013-04-19 | 2013-11-06 | 景宁山凤皇工艺制品有限公司 | Reactor for experiment for studying influence of pH on catalase |
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| 冯莉琴: "探究"pH 对过氧化氢酶的影响"活动研究", 《中学教学参考》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106086157A (en) * | 2016-06-21 | 2016-11-09 | 俞率成 | Catalase activity analyzer and using method thereof |
| CN114984896A (en) * | 2022-03-30 | 2022-09-02 | 北京擎科生物科技有限公司 | Oligonucleotide synthesis device and oligonucleotide synthesis method |
| CN114984896B (en) * | 2022-03-30 | 2023-08-18 | 北京擎科生物科技股份有限公司 | Oligonucleotide synthesis device and oligonucleotide synthesis method |
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Application publication date: 20170111 |