Pyridine Hemochromagen Assay for Determining the Concentration of Heme in Purified Protein Solutions.
Pyridine Hemochromagen Assay for Determining the Concentration of Heme in Purified Protein Solutions.
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DOI：
10.21769/bioprotoc.1594
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发表时间：
2015-09
期刊：
影响因子：
0.8
通讯作者：
Ian Barr;F. Guo
中科院分区：
文献类型：
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作者：
Ian Barr;F. Guo
血红素是蛋白质中常见的辅因子，存在于血红蛋白、肌红蛋白、细胞色素P450、DGCR 8和一氧化氮合酶等中。该方案描述了一种用于定量血红素的方法，该方法在纯化的蛋白质样品中效果最好。例如，该方案可用于确定给定的血红素结合蛋白是否完全被血红素占据，从而允许血红素含量与活性的相关性。这需要绝对血红素浓度和准确的蛋白质浓度。另一个用途是确定血红素结合蛋白的消光系数。如果操作正确，该测定快速、简单且可重复。
Heme is a common cofactor in proteins, found in hemoglobin, myoglobin, cytochrome P450, DGCR8, and nitric oxide synthase, among others. This protocol describes a method for quantifying heme that works best in purified protein samples. This protocol might be used to, for example, determine whether a given heme-binding protein is fully occupied by heme, thus allowing correlation of heme content with activity. This requires the absolute heme concentration and an accurate protein concentration. Another use is to determine the extinction coefficients of a heme-bound protein. This assay is fast, easy, and reproducible if done correctly.