{"title":"使用次黄嘌呤-鸟嘌呤磷酸核糖基转移酶的酶循环法：焦磷酸盐的高灵敏度测定。","authors":"Shigeru Ueda, Yoshiaki Yasutake, Tatsuya Hirata, Takehiko Sahara and Shin-ichi Sakasegawa","doi":"10.1039/D4AY01692K","DOIUrl":null,"url":null,"abstract":"<p >We developed a novel enzyme cycling method using hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) from <em>Hungateiclostridium thermocellum</em>. This method exploits the reversible nature of the HGPRT reaction, catalyzing both forward and reverse reactions in the presence of excess inosine 5′-monophosphate (IMP) and guanine (Gua), similar to the established purine nucleoside phosphorylase (PNP)-based method. Real-time detection was achieved by coupling the reaction with commercially available xanthine dehydrogenase (XDH) (EC 1.17.1.4) in the presence of nicotinamide adenine dinucleotide (NAD<small><sup>+</sup></small>). The reaction exhibited high efficiency, with a cycling rate constant of approximately 60 min<small><sup>−1</sup></small> or higher at an HGPRT concentration of 1 U mL<small><sup>−1</sup></small>. Additionally, we demonstrated a preliminary application of this XDH-coupled enzyme cycling reaction for the determination of pyrophosphate (PPi).</p>","PeriodicalId":64,"journal":{"name":"Analytical Methods","volume":" 12","pages":" 2600-2606"},"PeriodicalIF":2.7000,"publicationDate":"2025-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Enzyme cycling method using hypoxanthine-guanine phosphoribosyltransferase: a highly sensitive assay for pyrophosphate†\",\"authors\":\"Shigeru Ueda, Yoshiaki Yasutake, Tatsuya Hirata, Takehiko Sahara and Shin-ichi Sakasegawa\",\"doi\":\"10.1039/D4AY01692K\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p >We developed a novel enzyme cycling method using hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) from <em>Hungateiclostridium thermocellum</em>. This method exploits the reversible nature of the HGPRT reaction, catalyzing both forward and reverse reactions in the presence of excess inosine 5′-monophosphate (IMP) and guanine (Gua), similar to the established purine nucleoside phosphorylase (PNP)-based method. Real-time detection was achieved by coupling the reaction with commercially available xanthine dehydrogenase (XDH) (EC 1.17.1.4) in the presence of nicotinamide adenine dinucleotide (NAD<small><sup>+</sup></small>). The reaction exhibited high efficiency, with a cycling rate constant of approximately 60 min<small><sup>−1</sup></small> or higher at an HGPRT concentration of 1 U mL<small><sup>−1</sup></small>. Additionally, we demonstrated a preliminary application of this XDH-coupled enzyme cycling reaction for the determination of pyrophosphate (PPi).</p>\",\"PeriodicalId\":64,\"journal\":{\"name\":\"Analytical Methods\",\"volume\":\" 12\",\"pages\":\" 2600-2606\"},\"PeriodicalIF\":2.7000,\"publicationDate\":\"2025-03-11\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Analytical Methods\",\"FirstCategoryId\":\"92\",\"ListUrlMain\":\"https://pubs.rsc.org/en/content/articlelanding/2025/ay/d4ay01692k\",\"RegionNum\":3,\"RegionCategory\":\"化学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"CHEMISTRY, ANALYTICAL\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Analytical Methods","FirstCategoryId":"92","ListUrlMain":"https://pubs.rsc.org/en/content/articlelanding/2025/ay/d4ay01692k","RegionNum":3,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"CHEMISTRY, ANALYTICAL","Score":null,"Total":0}
引用次数: 0
摘要
本研究利用黄芽胞杆菌的次黄嘌呤-鸟嘌呤磷酸核糖基转移酶（HGPRT） （EC 2.4.2.8）建立了一种新的酶循环方法。该方法利用了HGPRT反应的可逆性质，在过量的肌苷5'-单磷酸（IMP）和鸟嘌呤（Gua）存在下催化正反反应，类似于基于嘌呤核苷磷酸化酶（PNP）的方法。在烟酰胺腺嘌呤二核苷酸（NAD+）存在下，用市售的黄嘌呤脱氢酶（XDH） （EC 1.17.1.4）偶联反应实现实时检测。反应效率高，在HGPRT浓度为1 U mL-1时，循环速率常数约为60 min-1或更高。此外，我们还演示了xdh偶联酶循环反应在焦磷酸（PPi）测定中的初步应用。
Enzyme cycling method using hypoxanthine-guanine phosphoribosyltransferase: a highly sensitive assay for pyrophosphate†
We developed a novel enzyme cycling method using hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) from Hungateiclostridium thermocellum. This method exploits the reversible nature of the HGPRT reaction, catalyzing both forward and reverse reactions in the presence of excess inosine 5′-monophosphate (IMP) and guanine (Gua), similar to the established purine nucleoside phosphorylase (PNP)-based method. Real-time detection was achieved by coupling the reaction with commercially available xanthine dehydrogenase (XDH) (EC 1.17.1.4) in the presence of nicotinamide adenine dinucleotide (NAD+). The reaction exhibited high efficiency, with a cycling rate constant of approximately 60 min−1 or higher at an HGPRT concentration of 1 U mL−1. Additionally, we demonstrated a preliminary application of this XDH-coupled enzyme cycling reaction for the determination of pyrophosphate (PPi).
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