Microplate assay for the quantification of catalase activity in biological samples

IF 3.6 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods Pub Date : 2026-02-01 Epub Date: 2025-12-19 DOI:10.1016/j.ymeth.2025.12.009

Unai Montejo , Gorane Beldarrain , Sheila Olza , Jon Ander Alart , Marc Chillida , Ignacio García-Alonso , Daniel Alonso-Alconada , Ana Alonso-Varona , Borja Herrero de la Parte

{"title":"Microplate assay for the quantification of catalase activity in biological samples","authors":"Unai Montejo , Gorane Beldarrain , Sheila Olza , Jon Ander Alart , Marc Chillida , Ignacio García-Alonso , Daniel Alonso-Alconada , Ana Alonso-Varona , Borja Herrero de la Parte","doi":"10.1016/j.ymeth.2025.12.009","DOIUrl":null,"url":null,"abstract":"<div><div>Catalase is a key antioxidant enzyme that protects cells from oxidative stress by decomposing hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>). Traditional methods for quantifying catalase activity, such as the Aebi UV method and the dichromate assay, are limited by low sensitivity, interference from biological molecules and require large sample and reagent volumes. Here, we present an optimized, microplate assay based on the oxidation of cobalt(II) to cobalt(III) by H<sub>2</sub>O<sub>2</sub>, followed by complexation with ethylenediamine-tetraacetic acid (EDTA) to form a highly colored Co(III)-EDTA complex measurable at 570 nm. This method demonstrates superior sensitivity and dynamic range (10–0.039 U/mL) compared to previous colorimetric assays, while minimizing reaction volume and avoiding interference from common biological substances. The assay has been validated using standard bovine liver catalase, liver homogenates, serum and cell lysates, showing strong linearity and specificity. This approach offers a rapid, cost-effective, and robust alternative for catalase activity measurement in biomedical research.</div></div>","PeriodicalId":390,"journal":{"name":"Methods","volume":"246 ","pages":"Pages 186-194"},"PeriodicalIF":3.6000,"publicationDate":"2026-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Methods","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S104620232500252X","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/12/19 0:00:00","PubModel":"Epub","JCR":"Q1","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}

引用次数: 0

Abstract

Catalase is a key antioxidant enzyme that protects cells from oxidative stress by decomposing hydrogen peroxide (H₂O₂). Traditional methods for quantifying catalase activity, such as the Aebi UV method and the dichromate assay, are limited by low sensitivity, interference from biological molecules and require large sample and reagent volumes. Here, we present an optimized, microplate assay based on the oxidation of cobalt(II) to cobalt(III) by H₂O₂, followed by complexation with ethylenediamine-tetraacetic acid (EDTA) to form a highly colored Co(III)-EDTA complex measurable at 570 nm. This method demonstrates superior sensitivity and dynamic range (10–0.039 U/mL) compared to previous colorimetric assays, while minimizing reaction volume and avoiding interference from common biological substances. The assay has been validated using standard bovine liver catalase, liver homogenates, serum and cell lysates, showing strong linearity and specificity. This approach offers a rapid, cost-effective, and robust alternative for catalase activity measurement in biomedical research.

Abstract Image

查看原文本刊更多论文

定量生物样品中过氧化氢酶活性的微孔板法。

过氧化氢酶是一种关键的抗氧化酶，通过分解过氧化氢（H2O2）来保护细胞免受氧化应激。传统的定量过氧化氢酶活性的方法，如Aebi紫外法和重铬酸盐法，受到灵敏度低、生物分子干扰和需要大量样品和试剂的限制。在这里，我们提出了一种优化的微孔板分析方法，该方法基于H2O2将钴（II）氧化为钴（III），然后与乙二胺-四乙酸（EDTA）络合形成高度着色的Co(III)-EDTA络合物，可在570 nm处测量。与以往的比色法相比，该方法具有更高的灵敏度和动态范围（10-0.039 U/mL），同时最大限度地减少了反应量，避免了常见生物物质的干扰。该方法已使用标准牛肝过氧化氢酶、肝脏匀浆、血清和细胞裂解液进行验证，显示出很强的线性和特异性。这种方法为生物医学研究中过氧化氢酶活性的测量提供了一种快速、经济、可靠的替代方法。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Methods 生物-生化研究方法

CiteScore

9.80

自引率

2.10%

发文量

222

审稿时长

11.3 weeks

期刊介绍： Methods focuses on rapidly developing techniques in the experimental biological and medical sciences. Each topical issue, organized by a guest editor who is an expert in the area covered, consists solely of invited quality articles by specialist authors, many of them reviews. Issues are devoted to specific technical approaches with emphasis on clear detailed descriptions of protocols that allow them to be reproduced easily. The background information provided enables researchers to understand the principles underlying the methods; other helpful sections include comparisons of alternative methods giving the advantages and disadvantages of particular methods, guidance on avoiding potential pitfalls, and suggestions for troubleshooting.