CN101776589B - Method for measuring purine with ultraviolet spectrophotometer - Google Patents

Method for measuring purine with ultraviolet spectrophotometer Download PDF

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CN101776589B
CN101776589B CN2010101025135A CN201010102513A CN101776589B CN 101776589 B CN101776589 B CN 101776589B CN 2010101025135 A CN2010101025135 A CN 2010101025135A CN 201010102513 A CN201010102513 A CN 201010102513A CN 101776589 B CN101776589 B CN 101776589B
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purine
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单安山
乔国华
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Northeast Agricultural University
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Abstract

The invention provides a method for measuring purine with an ultraviolet spectrophotometer, which comprises the following steps: accurately weighing 0.5g of fully dried chyme sample (dried with a freeze dryer), and placing the chymus sample in a 25ml test tube with scales; adding 2.5ml of perchloric acid, and placing the mixture in a constant-temperature water bath of 90-95 DEG C, and culturing for 1 hour; then adding 17.5ml of acetic acid buffer, placing the mixture in a constant-temperature water bath of 90-95 DEG C again, culturing for 10-15 minutes, and filtering; taking 0.5ml of filtrate, transferring the filtrate into a centrifuge tube with the volume being 15ml, and centrifuging at a speed of 4000rpm for 15 minutes; culturing in the constant-temperature water bath of 90-95 DEG C for 30 minutes; and carrying out colorimetry with an ultraviolet spectrophotometer in the place 260nm away, thereby obtaining the purine concentration expressed by a RNA equivalent. The invention has the advantages of simple operation, high repeatability and accurate numerical values. The content of microprotein in the duodenal chyme is determined through measuring the purine content in the chyme with the ultraviolet spectrophotometer, thereby providing a methodological basis for determination of the protein content in the metabolism of dairy cows.

Description

The method of measuring purine with ultraviolet spectrophotometer
(1) technical field
The present invention relates to animal science, is exactly a kind of method of measuring purine with ultraviolet spectrophotometer specifically.
(2) background technology
Dairy cow diet duodenum metabolizable protein (Metablizable protein, MP) comprise rumen bypass protein (the Rumen undegradable protein of daily ration, RUP), rumen microorganism albumen (Microbial crude protein, MCP) and intrinsic protein (Endogenous crudeprotein, ECP) three parts.Rumen microorganism albumen accounts for 60～80% of metabolizable protein, and therefore quantitatively rumen microorganism albumen synthetic quantity is most important.The alloxuric nitrogen method that Zinn and Owen (1986) are proposed is because of it is easy, quick, experimentation cost is low and easy to operately adopted doubt (Perez etal, 1997 that also are subjected to some scholars because of the purine recovery is lower simultaneously by some scholars; Makker et al, 1999; Gonzalaz et al, 2003,2004).This method discharges purine based on perchloric acid to the hydrolysis of rumen microorganism, precipitates purine with silver nitrate again, measures absorbance with spectrophotometer 260nm at last.With the object of the pure product of yeast rna as research, mix with other material respectively in the method, the recovery of measuring purine at last is more than 90%.But, with intracorporal method (In vivo) measure sample that rumen microorganism albumen duodenum quantity delivered obtains be rumen microorganism freeze-drying sample (Lyophilized rumen microbes, LRM).Microorganism in the rumen microorganism freeze-drying sample is that the form with thalline exists, and has comprised other material simultaneously again, and composition is comparatively complicated.
(3) summary of the invention
The object of the present invention is to provide a kind of method of measuring purine with ultraviolet spectrophotometer.
The object of the present invention is achieved like this: the method for described measuring purine with ultraviolet spectrophotometer, and step is as follows:
Step 1: the chyme sample that accurately takes by weighing 0.5 gram fully dry (using the freeze dryer drying), or separating obtained chyme microorganism part sample, place in the 25ml tool plug band scale test tube, note: sample must be dry, otherwise can cause nucleic acid hydrolysis incomplete;
Step 2: add 2.5ml perchloric acid (HClO 4, 1M), tightly cover bottleneck, place it in 90～95 ℃ interior the cultivation 1 hour of water bath with thermostatic control then, this moment, sample was charing, formed black induration, it is broken to harden, in order to reacting completely;
Step 3: add 17.5ml acetate buffer (NH again 4H 2PO 4, 0.0285M), fully shake, tightly cover bottleneck, place 90～95 ℃ interior the cultivation 10～15 minutes of water bath with thermostatic control again, then by No. 4 filter paper filterings of Whatman, this moment, pH value of filtrate was about 2;
Step 4: get 0.5ml filtrate and transfer in the centrifuge tube that volume is 15ml, add 0.5ml AgNO respectively 3(0.4M) with 9ml first kind pH damping fluid (NH 4H 2PO 4, 0.2M), placed the dark place then at least 30 minutes, preferably its placement is spent the night (5 ℃), can improve the accuracy of analysis;
Step 5: in 4000rpm centrifugal 15 minutes, abandoning supernatant carefully noted stirring the bottle sediment at the end then;
Step 6: with pH be 2 distillation washing centrifuging gained sediment once, use with the same method of step 5 centrifugal, abandoning supernatant;
Step 7: add the hydrochloric acid solution of 10ml 0.5N, fully shake, make till itself and the sediment mixing;
Step 8: apparatus plug sealing centrifuge tube is placed on 90～95 ℃ interior the cultivation 30 minutes of water bath with thermostatic control;
Step 9: in 260nm place colorimetric, the purine concentration that records is represented with the RNA equivalent with ultraviolet spectrophotometer;
Step 10: the formulation of yeast rna typical curve: accurately take by weighing 0.05,0.10,0.15,0.20,0.25,0.30,0.40 0.50 gram yeast rna places respectively in the tool plug band scale test tube of 8 25ml, method of operating by purine content in the above-mentioned analyzing samples is handled, at last in 260nm place colorimetric;
Step 11: data statistic analysis adopts SAS 2003 ANOVA to carry out multiple ratio.
The present invention has convenient easily, easy and simple to handle, repeatability is high, the numerical value characteristic of accurate, measure purine content in the chyme by ultraviolet spectrophotometer, thereby determine the content of microprotein in the duodenum chyme, can be for determining that accurately milk cow metabolizable protein content provides methodology basis accurately.
(4) embodiment
Below the invention will be further described.
Embodiment 1: the method for a kind of measuring purine with ultraviolet spectrophotometer of the present invention, and step is as follows:
Step 1: the chyme sample that accurately takes by weighing 0.5 gram fully dry (using the freeze dryer drying), or separating obtained chyme microorganism part sample, place in the 25ml tool plug band scale test tube, note: sample must be dry, otherwise can cause nucleic acid hydrolysis incomplete;
Step 2: add 2.5ml perchloric acid (HClO 4, 1M), tightly cover bottleneck, place it in 90～95 ℃ interior the cultivation 1 hour of water bath with thermostatic control then, this moment, sample was charing, formed black induration, it is broken to harden, in order to reacting completely;
Step 3: add 17.5ml acetate buffer (NH again 4H 2PO 4, 0.0285M), fully shake, tightly cover bottleneck, place 90～95 ℃ interior the cultivation 10～15 minutes of water bath with thermostatic control again, then by No. 4 filter paper filterings of Whatman, this moment, pH value of filtrate was about 2;
Step 4: get 0.5ml filtrate and transfer in the centrifuge tube that volume is 15ml, add 0.5ml AgNO respectively 3(0.4M) with 9ml first kind pH damping fluid (NH 4H 2PO 4, 0.2M), placed the dark place then at least 30 minutes, preferably its placement is spent the night (5 ℃), can improve the accuracy of analysis;
Step 5: in 4000rpm centrifugal 15 minutes, abandoning supernatant carefully noted stirring the bottle sediment at the end then;
Step 6: with pH be 2 distillation washing centrifuging gained sediment once, use with the same method of step 5 centrifugal, abandoning supernatant;
Step 7: add the hydrochloric acid solution of 10ml 0.5N, fully shake, make till itself and the sediment mixing;
Step 8: apparatus plug sealing centrifuge tube is placed on 90～95 ℃ interior the cultivation 30 minutes of water bath with thermostatic control;
Step 9: in 260nm place colorimetric, the purine concentration that records is represented with the RNA equivalent with ultraviolet spectrophotometer;
Step 10: the formulation of yeast rna typical curve: accurately take by weighing 0.05,0.10,0.15,0.20,0.25,0.30,0.40 0.50 gram yeast rna places respectively in the tool plug band scale test tube of 8 25ml, method of operating by purine content in the above-mentioned analyzing samples is handled, at last in 260nm place colorimetric;
Step 11: data statistic analysis adopts SAS 2003ANOVA to carry out multiple ratio.
Embodiment 2: the method for measuring purine with ultraviolet spectrophotometer of the present invention is a purine recovery ultraviolet spectrophotometer method, and purine recovery ultraviolet spectrophotometer method and liquid phase chromatography comparative study are as follows:
The preparation of 1 sample
1.1 the preparation of rumen microorganism freeze-drying sample
Raise morning and got rumen content 1L in back 2 hours.Two-layer cotton filtration is kept at 4 ℃ and is filled with CO 2The wide-necked bottle of 1L band plug in 30 minutes.Draw center section with pipette and do not contain the low weight even matter liquid of particle of heavier particle of lower floor and upper strata.4 ℃, the centrifugal 20min of 20000 * g.With distillation washing bacterium ball, centrifugal again, last freeze-drying.
1.2 the preparation of neutral detergent fiber
Adopt the method for Van Soest et al (1991) to prepare medium-sized washing the fibre.Distilled water with heat thoroughly cleans neutral detergent.In baking oven, dry sheep's hay.
1.3 do not digest the preparation of sheep's hay and microorganism freeze-drying sample
Sheep's hay 500mg is (Menke et al., 1979) fermentation 24h under external (In vitro) self-control fermentor condition.Fermentation is afterwards with fermentation liquor centrifugal 30min under 20000 * g, freeze-drying after the supernatant discarded.Contain indigested sheep's hay and rumen microorganism in this freeze-drying sample (Undigested residue).
1) the Menke liquid making method is as follows:
The preparation of nutrient solution is carried out according to the method for (1979) such as Menke.Before test Niu Chen raises, from every bovine rumen, extract the 150ml rumen fluid respectively, mix, with 4 layers of filtered through gauze, the rumen fluid injection after filtering is continued to feed CO 21L glass bottle in, vial remains in 39 ℃ of water-baths.
Add the 600ml mixed-culture medium then.Mixed-culture medium is by 400ml water, 0.1ml nutrient solution A (13.2g CaCl 22H 2O, 10.0g MnCl 24H 2O, 1.0CoCl 26H 2O, 8.0gFeCl 36H 2O is dissolved in the 100ml water).
200ml nutrient solution B (39g NaHCO 3With 2g NH 4HCO 3Be dissolved in the 1L water).
200ml nutrient solution C (5.7g Na 2HPO 4, 6.2g KH 2PO 4, 0.6g Mg 2SO 47H 2O is dissolved in the 1L water), 1ml resin reddish black (0.1%) and 40ml reducing solution (95ml water, 4mlNaOH and 0.625g Na 2S9H 2O).
Mixed-culture medium feeds CO before use 2, place 39 ℃ of water-baths.Suck 30ml rumen fluid and nutrient solution mixed liquor in each syringe, the bubble that sucks is discharged, then injector head is used the sealing of self-control plug.Then syringe is placed 39 ℃ of water-baths, start shaking table, begin fermentation.
2 analytical approachs
2.1 adopt the method in Zinn (1986) year
1) accurately take by weighing the fully chyme sample of drying (using the freeze dryer drying) of 0.5 gram, or separating obtained chyme microorganism part sample, place in the 25ml tool plug band scale test tube.Attention: sample must be dry, otherwise can cause nucleic acid hydrolysis incomplete.
2) add 2.5ml perchloric acid (HClO 4, 12M, 70%), tightly cover bottleneck, place it in 90～95 ℃ interior the cultivation 1 hour of water bath with thermostatic control then.This moment, sample was charing, formed black induration.It is broken to harden, in order to reacting completely.
3) add 17.5ml acetate buffer (NH again 4H 2PO 4, 0.0285M), fully shake, tightly cover bottleneck, place 90～95 ℃ interior the cultivation 10～15 minutes of water bath with thermostatic control again, then by No. 4 filter paper filterings of Whatman, this moment, pH value of filtrate was about 2.
4) getting 0.5ml filtrate transfers in the centrifuge tube that volume is 15ml.Add 0.5mlAgNO respectively 3(0.4M) with 9ml first kind pH damping fluid (NH 4H 2PO 4, 0.2M).Placed the dark place then at least 30 minutes.Preferably its placement is spent the night (5 ℃), can improve the accuracy of analysis.
5) in 4000rpm centrifugal 15 minutes, abandoning supernatant carefully then.Note not stirring the bottle sediment at the end.
6) with pH be 2 distillation washing centrifuging gained sediment once.With centrifugal with same method of the 5th step, abandoning supernatant.
7) add the hydrochloric acid solution of 10ml 0.5N, fully shake, make till itself and the sediment mixing.
8) apparatus plug sealing centrifuge tube is placed on 90～95 ℃ interior the cultivation 30 minutes of water bath with thermostatic control.
9) with ultraviolet spectrophotometer in 260nm place colorimetric, the purine concentration that records is represented with the RNA equivalent.
Yeast rna (Shanghai Inst. of Biochemistry, Chinese Academy of Sciences)
The formulation of typical curve: accurately take by weighing 0.05,0.10,0.15,0.20,0.25,0.30,0.40,0.50 gram yeast rna, place respectively in the tool plug band scale test tube of 8 25ml, handle, at last in 260nm place colorimetric by the method for operating of purine content in the above-mentioned analyzing samples.
2.2HPLC measure the adenine and the guanine recovery under the condition
Adenine and guanine (available from Sigma company) be formulated as contain the solution that adenine and guanine are 1000 μ M, get 2.5ml and add 47.5ml distilled water, get 15 μ l sample introduction analyses.
Get rumen microorganism freeze-drying sample 100mg, perhaps mix with other material of 200mg, it is 5 groups that sample is divided into, and establishes 3 each repetition for every group.Place 25ml to have the test tube of screw cap.
Processing is respectively:
1) adds 2.5ml 12M HClO 4, 90 ℃ of water-bath 1h, cool to room temperature;
2) add 2.5ml 1M HClO 4, 90 ℃ of water-bath 1h, cool to room temperature;
3) add 2.5ml 2M HClO 4, 90 ℃ of water-bath 1h, cool to room temperature;
4) add 2.5ml 12M HClO 4, 121 ℃ of water-bath 2h, cool to room temperature.
Adjusting the pH value of solution value with 8M NaOH is 6.5, adds the HPLC solution A to 10ml.The centrifugal 10min of 3000g gets 15 μ l sample introduction analyses behind 0.45 membrane filtration.
Liquid phase chromatogram condition
Solution (A) 10mM NH 2HPO 4, use 2.86M NH 4It is 6 that OH adjusts the pH value.Solution (B) second cyanogen 150ml joins 600ml 12.5mM NH 2HPO 4In the solution, use 2.86MNH 4It is 6 that OH adjusts the pH value.All solution is all through 0.45 μ m membrane filtration, and in the ultrasonic oscillation device degasification 15min.
Liquid spectrum post solution gradient condition is: in the 30min solution B is risen to 100% from 0%, solution A rises to 100% behind the 40min behind 5min, and flow velocity is 0.8ml/min under the room temperature condition, and sample size is 15 μ l.
3 data statistic analysis
Adopt SAS 2003ANOVA to carry out multiple ratio.
4 purine recovery ultraviolet spectrophotometer methods and liquid phase chromatography comparative study 4.1 different disposal temperature and times are to the influence of the rumen microorganism freeze-drying sample purine recovery
Table 4-1 shows, adopts the method for Zinn (1986) to measure rumen microorganism freeze-drying sample and cellulose, and neutral detergent fiber and starch mix the back purine recovery to be had only about 50%.Rumen microorganism freeze-drying sample with do not digest the recovery that food-residue and microorganism mix purine and have only about 50% too.Cause this structural reason probably to be because prepared sample at 12M HClO4,90 ℃, does not have hydrolysis complete under the 1h condition.Therefore, the method for 121 ℃ of hydrolysis 2h has been adopted in this test simultaneously.The result shows, at ultraviolet spectrophotometer 260nm place, absorbance after 121 ℃ of pyroprocessing is than 90 ℃, and the absorbance under the 1h condition after the hydrolysis significantly improves, and the amount of 260nm place absorbance and rumen microorganism freeze-drying sample has presented good linear relationship (table 4-1).
Yeast rna and neutral detergent fiber and starch mix the recovery of back purine respectively than higher, have reached 92.2% and 98.3% respectively.This each result of study is very close with Zinn (1986) results reported.
Table 4-1 different disposal temperature and time is to the influence of the rumen microorganism freeze-drying sample purine recovery
Figure GSA00000018215800071
Figure GSA00000018215800081
Figure GSA00000018215800091
1LRM lyophilized rumen microbes rumen microorganism freeze-drying sample.
2AUR is through the residue of artificial rumen fermentor 24 hours fermentation after centrifugal.
3y＝-0.01633+0.00926×mg?LRM(R2＝0.99，n＝3).
4y＝-0.01433+0.01526×mg?LRM(R2＝0.99，n＝3).
4.2 different perchloric acid concentration are to the influence of the rumen microorganism freeze-drying sample purine recovery
Under 90 ℃ of conditions of same hydrolysis temperature, use 1M respectively, 2M and 12M HClO4 are hydrolyzed.The result shows that the absorbance at 260nm place is higher under 12M HClO4 condition
(table 3-2).Under 90 ℃ of conditions of same hydrolysis temperature, use 1M respectively, the absorbance after 2M HClO4 is hydrolyzed is very approaching.
The different perchloric acid concentration of table 4-2 are to the influence of the rumen microorganism freeze-drying sample purine recovery
Figure GSA00000018215800092
1LRM lyophilized rumen microbes, the recovery under the rumen microorganism freeze-drying sample .4.3 liquid chromatography for measuring rumen microorganism freeze-drying sample different disposal condition
Table 4-3 shows that 12M HClO4 adopts 90 ℃ respectively, and 1h and 121 ℃ adopt 1M respectively under 2h hydrolysis and 90 ℃ of conditions, 2M hydrolysis 1h.Measure rumen microorganism freeze-drying sample and cellulose with liquid chromatography (HPLC), neutral detergent fiber and starch mix the back purine recovery and have reached about 100%.Yet under 121 ℃ of conditions, the absolute yield of adenine is on the low side.Show that under the hot conditions, adenine has been subjected to destruction.Liquid chromatography result shows, lower HClO4 concentration (1M, 2M) and under 90 ℃ of conditions, the hydrolysis of nucleic acid is completely, the purine recovery can reach more than 95%.

Claims (1)

1. the method for a measuring purine with ultraviolet spectrophotometer, it is characterized in that: step is as follows:
Step 1: accurately take by weighing 0.5 gram with the fully dry chyme sample of freeze dryer, place in the 25ml tool plug band scale test tube, described sample must be dry, otherwise can cause nucleic acid hydrolysis incomplete;
Step 2: add 2.5ml 1M HClO 4, tightly cover bottleneck, place it in 90～95 ℃ interior the cultivation 1 hour of water bath with thermostatic control then, this moment, sample was charing, formed black induration, it is broken to harden, in order to reacting completely;
Step 3: add 17.5ml 0.0285M NH again 4H 2PO 4, fully shake, tightly cover bottleneck, place 90～95 ℃ interior the cultivation 10～15 minutes of water bath with thermostatic control again, then by the Whatman4 filter paper filtering, this moment, pH value of filtrate was about 2;
Step 4: get 0.5ml filtrate and transfer in the centrifuge tube that volume is 15ml, add 0.5ml 0.4M AgNO respectively 3With 9ml 0.2M NH 4H 2PO 4First kind pH damping fluid placed the dark place at least 30 minutes then, under 5 ℃ its placement was spent the night, and improved the accuracy of analyzing;
Step 5: in 4000rpm centrifugal 15 minutes, abandoning supernatant was carefully stirred the bottle sediment at the end then;
Step 6: with pH be 2 distillation washing centrifuging gained sediment once, use with the same method of step 5 centrifugal, abandoning supernatant;
Step 7: add the hydrochloric acid solution of 10ml 0.5N, fully shake, make till itself and the sediment mixing;
Step 8: apparatus plug sealing centrifuge tube is placed on 90～95 ℃ interior the cultivation 30 minutes of water bath with thermostatic control;
Step 9: in 260nm place colorimetric, the purine concentration that records is represented with the RNA equivalent with ultraviolet spectrophotometer;
Step 10: the formulation of yeast rna typical curve: accurately take by weighing 0.05,0.10,0.15,0.20,0.25,0.30,0.40 0.50 gram yeast rna places respectively in the tool plug band scale test tube of 8 25ml, method of operating by purine content in the above-mentioned analyzing samples is handled, at last in 260nm place colorimetric;
Step 11: data statistic analysis adopts SAS 2003ANOVA to carry out multiple ratio.
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CN101320030A (en) * 2008-07-21 2008-12-10 中国农业科学院蜜蜂研究所 Method for measuring royal jelly

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CN101320030A (en) * 2008-07-21 2008-12-10 中国农业科学院蜜蜂研究所 Method for measuring royal jelly

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