WO2019140677A1 - 一种prpp的定量检测方法 - Google Patents

一种prpp的定量检测方法 Download PDF

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WO2019140677A1
WO2019140677A1 PCT/CN2018/073577 CN2018073577W WO2019140677A1 WO 2019140677 A1 WO2019140677 A1 WO 2019140677A1 CN 2018073577 W CN2018073577 W CN 2018073577W WO 2019140677 A1 WO2019140677 A1 WO 2019140677A1
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prpp
phase
solution
quantitatively detecting
column
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PCT/CN2018/073577
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戴柱
宋锦华
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邦泰合盛生物科技（深圳）有限公司
邦泰生物工程(深圳)有限公司
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Priority to CN201880038047.3A priority Critical patent/CN110770580B/zh
Priority to PCT/CN2018/073577 priority patent/WO2019140677A1/zh
Publication of WO2019140677A1 publication Critical patent/WO2019140677A1/zh

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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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

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  • the invention relates to the technical field of a method for detecting a specific substance, and in particular to a method for quantitatively detecting PRPP.
  • PRPP is the abbreviation of Phosphoribosyl pyrophosphate, which is called 5-phosphate ribose-1-pyrophosphate in Chinese, and has the chemical formula C 5 H 13 O 14 P 3 . It is an activated form of ribose C1, which is produced by ribose-5-phosphate and ATP catalyzed by ribose phosphate pyrophosphate kinase. It is an extremely important precursor for nucleotide synthesis and is involved in the de novo of purine nucleotides and pyrimidine nucleotides. Synthetic and remedial synthesis, also involved in the synthesis of certain nucleotide coenzymes such as coenzyme I and coenzyme II, as well as certain amino acids such as histidine and tryptophan.
  • the quantitative detection of PRPP is mostly carried out by enzymatic reaction.
  • the principle is to convert PRPP into IMP by HGPRT enzyme, then convert IMP to NADH by IMPDH enzyme, and then use NADH to have maximum UV absorption at 340 nm.
  • the current PRPP detection kit commonly used on the market is to use this principle, the reaction process is shown below.
  • the method for detecting PRPP by enzymatic method has the following disadvantages: 1.
  • the operation is complicated, and requires two steps of enzymatic reaction and one-step ultraviolet detection; 2.
  • the cost is high, for example, a PRPP detection kit is sold at a price of 5000-10000 yuan; 3.
  • the present invention aims to provide a new quantitative detection method for PRPP, which aims to solve the technical problems of the existing PRPP enzymatic detection, which is complicated in operation, expensive, and unstable in detection results.
  • the present invention provides a quantitative detection method of PRPP, which is characterized by HPLC, and the chromatographic conditions are as follows: the column is triazole-bonded silica gel as a filler, and the mobile phase is composed of phase A and phase B. Composition, the phase A is a buffer of pH 6.0-6.8, the phase B is a solvent having a polarity of 4-7, which is miscible with water and capable of dissolving PRPP, and the volume of the phase A in the mobile phase The percentage is 35-50% and is detected by a differential detector.
  • the buffer is a tribasic acid or a tribasic acid salt.
  • the buffer is phosphate or citric acid.
  • the phosphate buffer is at a concentration of 50-150 mM.
  • the phase B is methanol, ethanol, acetonitrile or isopropanol.
  • the column temperature of the column is 20-50 ° C
  • the column temperature of the column is 30 °C.
  • the mobile phase has a flow rate of from 0.5 to 1.5 ml/min.
  • the method has a detection range of from 0.05 to 2 mg/ml.
  • the invention adopts HPLC to quantitatively detect PRPP, and adopts PRPP as a direct detection object, without multi-step transformation, simple operation, no enzyme is needed, and the cost is low, and is not tested.
  • the effect of sample and enzyme activity was good, the recovery was more than 98%, the linear relationship was good, the correlation coefficient R was greater than 0.99, the injection was repeated 6 times, and the peak area RSD was less than 5%.
  • Figure 1 is a chromatogram of the blank solution in Example 1;
  • Figure 2 is a chromatogram of the standard solution in Example 1;
  • Figure 3 is a chromatogram of the sample solution in Example 1;
  • Figure 4 is a chromatogram of the blank solution in Example 2.
  • Figure 5 is a chromatogram of the standard solution in Example 2.
  • Figure 6 is a chromatogram of the sample solution in Example 2.
  • Figure 7 is a chromatogram of the blank solution in Example 3.
  • Figure 8 is a chromatogram of the standard solution in Example 3.
  • Figure 9 is a chromatogram of the sample solution in Example 3.
  • the content of PRPP was determined by HPLC as follows:
  • Injection volume 20 ⁇ L
  • Detector Differential detector.
  • Blank solution the mobile phase is used as a blank solution
  • Standard solution weigh the appropriate amount of PRPP standard, dissolve in the mobile phase, and calibrate the PRPP standard solution with a concentration of 0.022mol/L;
  • Sample solution is the reaction solution of PRPP of Bangtai Bioengineering (Shenzhen) Co., Ltd., containing PRPP, D-ribose, 5-phosphate ribose, AMP, ADP, and the like.
  • the chromatogram of the standard solution is shown in Figure 2.
  • the figure shows that the main solution has a retention time of 14.331, the main peak area is 7903.6, the theoretical plate number is 4232, the symmetry factor is 0.74, and the separation from the previous adjacent peak is 7.54. , the separation from the next adjacent peak is 7.66;
  • the chromatogram of the sample solution is shown in Figure 3.
  • the figure shows that the main solution retention time of the sample solution is 14.524, the theoretical plate number is 3954, the symmetry factor is 1.26, and the separation from the previous adjacent peak is 5.05, and the latter phase.
  • the resolution of the adjacent peak was 7.31.
  • the content of PRPP was determined by HPLC as follows:
  • Injection volume 20 ⁇ L
  • Detector Differential detector.
  • Blank solution the mobile phase is used as a blank solution
  • Standard solution weigh the appropriate amount of PRPP standard, dissolve in the mobile phase, and calibrate the PRPP standard solution with a concentration of 0.022mol/L;
  • Sample solution is the reaction solution of PRPP of Bangtai Bioengineering (Shenzhen) Co., Ltd., containing PRPP, D-ribose, 5-phosphate ribose, AMP, ADP, and the like.
  • the chromatogram of the standard solution is shown in Figure 5.
  • the figure shows that the main solution has a retention time of 9.080, a peak area of 10512.0, a theoretical plate number of 2912, a symmetry factor of 0.54, and a resolution of 7.54 from the previous adjacent peak. ;
  • the chromatogram of the sample solution is shown in Figure 6.
  • the figure shows that the retention time of the main peak of the sample solution is 9.087, the peak area is 89627.2, the number of theoretical plates is 3263, the symmetry factor is 0.57, and the resolution from the previous adjacent peak is 1.53.
  • the separation from the next adjacent peak is 12.91.
  • the peak area RSD was 1.21%.
  • the content of PRPP was determined by HPLC as follows:
  • Injection volume 20 ⁇ L
  • Detector Differential detector.
  • Blank solution the mobile phase is used as a blank solution
  • Standard solution weigh the appropriate amount of PRPP standard, dissolve in the mobile phase, and calibrate the PRPP standard solution with a concentration of 0.022mol/L;
  • Sample solution is the reaction solution of PRPP of Bangtai Bioengineering (Shenzhen) Co., Ltd., containing PRPP, D-ribose, 5-phosphate ribose, AMP, ADP, and the like.
  • the chromatogram of the standard solution is shown in Figure 8.
  • the figure shows that the main solution has a retention time of 7.667, a peak area of 96382.2, a theoretical plate number of 2970, a symmetry factor of 1.13, and a resolution of 1.21 from the previous adjacent peak. , the degree of separation from the next adjacent peak is 2.67;
  • the chromatogram of the sample solution is shown in Figure 9.
  • the sample shows that the retention time of the main peak of the sample solution is 7.644, the peak area is 77066.7, the number of theoretical plates is 3155, the symmetry factor is 1.05, and the resolution from the previous adjacent peak is 1.77. .
  • the peak area RSD was 2.08%.

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  • Life Sciences & Earth Sciences (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

一种PRPP的定量检测方法，涉及特定物质的检测方法的技术领域。该方法采用HPLC进行检测，色谱条件如下：色谱柱以三唑基键合硅胶为填料，流动相由A相和B相组成，A相为pH6.0-6.8的缓冲液，B相为极性4-7、能与水互溶且能溶解PRPP的溶剂，A相在流动相中的体积百分比为35-50％，采用示差检测器进行检测。

Description

一种PRPP的定量检测方法 技术领域
本发明涉及特定物质的检测方法的技术领域，具体涉及对PRPP的定量检测方法。
 背景技术
PRPP是Phosphoribosyl pyrophosphate的英文缩写，中文全称为5-磷酸核糖-1-焦磷酸，化学式为C 5H 13O 14P 3。它是核糖C1的活化形式，由核糖-5-磷酸与ATP在核糖磷酸焦磷酸激酶催化下生成，是核苷酸合成的极其重要的前体，参与嘌呤核苷酸与嘧啶核苷酸的从头合成和补救合成，也参与某些核苷酸类辅酶如辅酶I和辅酶II、以及某些氨基酸如组氨酸和色氨酸的合成。
在科研和工业生产中，以PRPP作为原料、中间体或产品的反应多是通过对PRPP的检测来监测反应进行的程度或者特定物质的转化率，因此，对PRPP的定量检测对于科研和工业生产而言，有着十分重要的意义。
目前对PRPP的定量检测多是采用酶促反应来进行的，其原理是利用HGPRT酶使PRPP转化为IMP，然后利用IMPDH酶使IMP转化为NADH，然后再利用NADH在340nm下有最大紫外吸收的性质来间接定量分析，目前市场上常见的PRPP检测试剂盒就是利用这个原理，其反应过程如下式所示。
 
Figure 64750dest_path_image001
技术问题
酶法检测PRPP的方法具有如下缺点：1、操作复杂，需要经过两步酶促反应和一步紫外检测；2、成本昂贵，譬如一个PRPP检测试剂盒售价高达5000-10000元；3、检测结果不稳定，受其他因素影响较大，重复性差，譬如，若酶促反应不完全，则检测结果会偏低，若待测样品中本就含有IMP或者NADH，则检测结果会偏高。
 技术解决方案
鉴于上述现有技术的不足，本发明的目的在于提供一种新的PRPP的定量检测方法，旨在解决现有的PRPP酶法检测存在的操作复杂、成本昂贵、检测结果不稳定的技术问题。
为实现上述目的，本发明提供了一种PRPP的定量检测方法，其特征在于，采用HPLC进行检测，色谱条件如下：色谱柱以三唑基键合硅胶为填料，流动相由A相和B相组成，所述A相为pH6.0-6.8的缓冲液，所述B相为极性4-7、能与水互溶且能溶解PRPP的溶剂，所述A相在所述流动相中的体积百分比为35-50%，采用示差检测器进行检测。
优选地，所述缓冲液为三元酸或三元酸盐。
更优选地，所述缓冲液为磷酸盐或柠檬酸。
更优选地，所述磷酸盐缓冲液的浓度为50-150mM。
优选地，所述B相为甲醇、乙醇、乙腈或者异丙醇。
优选地，所述色谱柱的柱温为20-50℃
更优选地，所述色谱柱的柱温为30℃。
优选地，所述流动相的流速为0.5-1.5ml/min。
优选地，所述方法的检测范围为0.05-2mg/ml。
 有益效果
与现有的PRPP定量检测方法相比，本发明采用HPLC来定量检测PRPP，是以PRPP为直接检测对象，无需经过多步转化，操作简单，不需要用到酶，成本低，不受待测样品和酶活性的影响，重复性好，加标回收率大于98%，线性关系良好，相关系数R大于0.99，重复进样6次，峰面积RSD小于5%。
 附图说明
图1是实施例1中空白溶液的色谱图；
图2是实施例1中标准溶液的色谱图；
图3是实施例1中样品溶液的色谱图；
图4是实施例2中空白溶液的色谱图；
图5是实施例2中标准溶液的色谱图；
图6是实施例2中样品溶液的色谱图；
图7是实施例3中空白溶液的色谱图；
图8是实施例3中标准溶液的色谱图；
图9是实施例3中样品溶液的色谱图。
 本发明的实施方式
下面结合具体实施例和附图对本发明做进一步的详细说明，以下实施例是对本发明的解释，本发明并不局限于以下实施例。
以下实施例中所使用的原料及试剂，除特别指明的以外，均可从市场中购入。
实施例1
采用HPLC检测PRPP的含量，方法如下：
1、色谱条件
仪器：Agilent 1260高效液相色谱仪；
色谱柱：三唑基键合硅胶柱；
流动相：磷酸盐缓冲液（50mM，pH6.0）:甲醇=50:50（v:v）；
流速：1ml/min；
柱温：30℃；
进样量：20μL；
检测器：示差检测器。
2、溶液配制
空白溶液：以流动相作为空白溶液；
标准溶液：称取PRPP标准品适量，溶解于流动相中，标定浓度为0.022mol/L的PRPP标准溶液；
样品溶液：样品溶液为邦泰生物工程（深圳）有限公司的PRPP的反应液，含PRPP、D-核糖、5-磷酸核糖、AMP、ADP等。
3、检测结果
空白溶液的色谱图如附图1所示，图中显示：空白溶液对主峰无干扰；
标准溶液的色谱图如附图2所示，图中显示：标准溶液主峰保留时间为14.331，主峰面积为7903.6，理论塔板数4232，对称因子0.74，与前一个相邻峰的分离度为7.54，与后一个相邻峰的分离度为7.66；
样品溶液的色谱图如附图3所示，图中显示：样品溶液主峰保留时间为14.524，理论塔板数3954，对称因子1.26，与前一个相邻峰的分离度为5.05，与后一个相邻峰的分离度为7.31。
此检测方法的加标回收率98.6%，线性关系良好，相关系数R=99.8%，重复进样6次，峰面积RSD为3.3%。
实施例2
采用HPLC检测PRPP的含量，方法如下：
1、色谱条件
仪器：Agilent 1260高效液相色谱仪；
色谱柱：三唑基键合硅胶柱；
流动相：磷酸盐缓冲液（100mM，pH6.6）:甲醇=45:55（v:v）；
流速：1ml/min；
柱温：30℃；
进样量：20μL；
检测器：示差检测器。
2、溶液配制
空白溶液：以流动相作为空白溶液；
标准溶液：称取PRPP标准品适量，溶解于流动相中，标定浓度为0.022mol/L的PRPP标准溶液；
样品溶液：样品溶液为邦泰生物工程（深圳）有限公司的PRPP的反应液，含PRPP、D-核糖、5-磷酸核糖、AMP、ADP等。
3、检测结果
空白溶液的色谱图如附图4所示，图中显示：空白溶液对主峰无干扰；
标准溶液的色谱图如附图5所示，图中显示：标准溶液主峰保留时间为9.080，峰面积为10512.0，理论塔板数2912，对称因子0.54，与前一个相邻峰的分离度为7.54；
样品溶液的色谱图如附图6所示，图中显示：样品溶液主峰保留时间为9.087，峰面积为89627.2，理论塔板数3263，对称因子0.57，与前一个相邻峰的分离度为1.53，与后一个相邻峰的分离度为12.91。
此检测方法的加标回收率101.03%，线性关系良好，相关系数R=0.998，重复进样6次，峰面积RSD为1.21%。
实施例3
采用HPLC检测PRPP的含量，方法如下：
1、色谱条件
仪器：Agilent 1260高效液相色谱仪；
色谱柱：三唑基键合硅胶柱；
流动相：磷酸盐缓冲液（150mM，pH6.8）:甲醇=35:65（v:v）；
流速：1ml/min；
柱温：30℃；
进样量：20μL；
检测器：示差检测器。
2、溶液配制
空白溶液：以流动相作为空白溶液；
标准溶液：称取PRPP标准品适量，溶解于流动相中，标定浓度为0.022mol/L的PRPP标准溶液；
样品溶液：样品溶液为邦泰生物工程（深圳）有限公司的PRPP的反应液，含PRPP、D-核糖、5-磷酸核糖、AMP、ADP等。
3、检测结果
空白溶液的色谱图如附图7所示，图中显示：空白溶液对主峰无干扰；
标准溶液的色谱图如附图8所示，图中显示：标准溶液主峰保留时间为7.667，峰面积为96382.2，理论塔板数2970，对称因子1.13，与前一个相邻峰的分离度为1.21，与后一个相邻峰的分离度为2.67；
样品溶液的色谱图如附图9所示，图中显示：样品溶液主峰保留时间为7.644，峰面积为77066.7，理论塔板数3155，对称因子1.05，与前一个相邻峰的分离度为1.77。
此检测方法的加标回收率98.10%，线性关系良好，相关系数R=0.994，重复进样6次，峰面积RSD为2.08%。

Claims (9)

  1. 一种PRPP的定量检测方法，其特征在于，采用HPLC进行检测，色谱条件如下：色谱柱以三唑基键合硅胶为填料，流动相由A相和B相组成，所述A相为pH6.0-6.8的缓冲液，所述B相为极性4-7、能与水互溶且能溶解PRPP的溶剂，所述A相在所述流动相中的体积百分比为35-50%，采用示差检测器进行检测。
  2. 根据权利要求1所述的PRPP的定量检测方法，其特征在于： 所述缓冲液为三元酸或三元酸盐。
  3. 根据权利要求2所述的PRPP的定量检测方法，其特征在于： 所述缓冲液为磷酸盐或柠檬酸。
  4. 根据权利要求3所述的PRPP的定量检测方法，其特征在于：所述磷酸盐缓冲液的浓度为50-150mM。
  5. 根据权利要求1所述的PRPP的定量检测方法，其特征在于： 所述B相为甲醇、乙醇、乙腈或者异丙醇。
  6. 根据权利要求1所述的PRPP的定量检测方法，其特征在于：所述色谱柱的柱温为20-50℃。
  7. 根据权利要求6所述的PRPP的定量检测方法，其特征在于：所述色谱柱的柱温为30℃。
  8. 根据权利要求1所述的PRPP的定量检测方法，其特征在于：所述流动相的流速为0.5-1.5ml/min。
  9. 根据权利要求1所述的PRPP的定量检测方法，其特征在于：所述方法的检测范围为0.05-2mg/ml。
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CN112831482A (zh) * 2019-12-27 2021-05-25 中国科学院天津工业生物技术研究所 一种快速检测prpp的方法及其使用的生物传感器

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016136620A1 (ja) * 2015-02-24 2016-09-01 国立研究開発法人科学技術振興機構 補酵素の製造方法及び補酵素製造用形質転換体セット

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PENG, LAN ET AL.: "Estimation of Phosphoribosylpyrophosphate (PRPP) Synthetase Activity in Human Red Blood Cells by Ion-pair HPLC", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 13, no. 1, 28 February 1997 (1997-02-28) *
SAKUMA, R. ET AL.: "Phosphoribosylpyrophosphate synthetase in human erythrocytes: Assay and kinetic studies using high", CLINICA CHIMICA ACTA, vol. 203, no. 2, 3, 16 December 1991 (1991-12-16), pages 143 - 152, XP023395880 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112831482A (zh) * 2019-12-27 2021-05-25 中国科学院天津工业生物技术研究所 一种快速检测prpp的方法及其使用的生物传感器
CN112831482B (zh) * 2019-12-27 2022-04-15 中国科学院天津工业生物技术研究所 一种快速检测prpp的方法及其使用的生物传感器

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