Mohamed K Al-Essa, Tamara Al-Qudah, Akram Kamal A Al Hadidi, Nida'a H Alshubbak
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In a second approach, we conjugated AF to the conserved RBCs and then exposed AF-labeled RBCs to trypsin. This was followed by directly measuring the fluorescence intensity (FI) in the reaction media to estimate the concentration of AF-labeled peptide fragments resulting from the enzyme's activity. <b>Results:</b> Show a concentration- and time-dependent increase in FIs, reflecting the activity of trypsin as a proteolytic enzyme. The FIs increased significantly by 4 to 5 folds in samples treated with different enzyme concentrations, and by over 11 folds after 2 h incubation in media containing a 50 <i>μ</i>L trypsin, as evidenced by CBQCA assays. <b>Conclusion:</b> These fast and affordable approaches could be applied with high reliability for the general estimation of protease activity in samples and customized for diagnostic purposes and prognostic evaluation in various diseases.</p>","PeriodicalId":9007,"journal":{"name":"BioMed Research International","volume":"2024 ","pages":"7919329"},"PeriodicalIF":2.6000,"publicationDate":"2024-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11452246/pdf/","citationCount":"0","resultStr":"{\"title\":\"Proteolysis Assays With Conserved or Aminofluorescein-Labeled Red Blood Cells.\",\"authors\":\"Mohamed K Al-Essa, Tamara Al-Qudah, Akram Kamal A Al Hadidi, Nida'a H Alshubbak\",\"doi\":\"10.1155/2024/7919329\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p><b>Backgrounds:</b> Various physiological functions and reaction cascades, as well as disease progression in the living systems, are controlled by the activity of specific proteolytic enzymes. 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引用次数: 0
摘要
背景:生命系统中的各种生理功能和反应级联以及疾病的进展都受特定蛋白水解酶活性的控制。我们进行了一项研究,通过评估反应介质中来自保守红细胞(RBC)或用氨基荧光素(AF)标记的红细胞(RBC)的肽片段来评估蛋白酶活性。研究方法在含有胰蛋白酶的培养基中培养红细胞。随后,用 3-(4-羧基苯甲酰基)喹啉-2-甲醛(CBQCA)作为胺反应型荧光试剂来估算反应介质中肽片段的浓度。在第二种方法中,我们将 AF 与保守红细胞连接,然后将 AF 标记的红细胞暴露于胰蛋白酶。然后直接测量反应介质中的荧光强度(FI),以估算酶活性产生的 AF 标记肽片段的浓度。结果显示 FIs 的增加与浓度和时间有关,反映了胰蛋白酶作为一种蛋白水解酶的活性。根据 CBQCA 检测结果,在使用不同浓度酶处理的样本中,FIs 明显增加了 4 至 5 倍,在含有 50 μL 胰蛋白酶的培养基中培养 2 小时后,FIs 增加了 11 倍以上。结论这些快速且经济实惠的方法可用于对样本中蛋白酶活性的一般估算,可靠性高,并可定制用于各种疾病的诊断和预后评估。
Proteolysis Assays With Conserved or Aminofluorescein-Labeled Red Blood Cells.
Backgrounds: Various physiological functions and reaction cascades, as well as disease progression in the living systems, are controlled by the activity of specific proteolytic enzymes. We conducted the study to evaluate protease activity by assessing peptide fragments from either conserved or labeled red blood cells (RBCs) with aminofluorescein (AF) in the reaction media. Methods: RBCs were incubated in media containing trypsin. Subsequently, the concentration of peptide fragments in the reaction media, resulted by the digestion with trypsin from conserved cells, was estimated by 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) as an amine-reactive fluorogenic reagent. In a second approach, we conjugated AF to the conserved RBCs and then exposed AF-labeled RBCs to trypsin. This was followed by directly measuring the fluorescence intensity (FI) in the reaction media to estimate the concentration of AF-labeled peptide fragments resulting from the enzyme's activity. Results: Show a concentration- and time-dependent increase in FIs, reflecting the activity of trypsin as a proteolytic enzyme. The FIs increased significantly by 4 to 5 folds in samples treated with different enzyme concentrations, and by over 11 folds after 2 h incubation in media containing a 50 μL trypsin, as evidenced by CBQCA assays. Conclusion: These fast and affordable approaches could be applied with high reliability for the general estimation of protease activity in samples and customized for diagnostic purposes and prognostic evaluation in various diseases.
期刊介绍:
BioMed Research International is a peer-reviewed, Open Access journal that publishes original research articles, review articles, and clinical studies covering a wide range of subjects in life sciences and medicine. The journal is divided into 55 subject areas.