Using S-adenosyl-L-homocysteine capture compounds to characterize S-adenosyl-L-methionine and S-adenosyl-L-homocysteine binding proteins

作者：Brown Lindsey J; Baranowski Matthias; Wang Yun; Schrey Anna K; Lenz Thomas; Taverna Sean D; Cole Philip A; Sefkow Michael^*

来源：Analytical Biochemistry, 2014, 467: 14-21.

DOI：10.1016/j.ab.2014.08.013

• Capture compound
• Fluorescence anisotropy
• S-Adenosyl-L-homocysteine
• S-Adenosyl-L-methionine

S-Adenosyl-L-methionine (SAM) is recognized as an important cofactor in a variety of biochemical reactions. As more proteins and pathways that require SAM are discovered, it is important to establish a method to quickly identify and characterize SAM binding proteins. The affinity of S-adenosyl-L-homocysteine (SAH) for SAM binding proteins was used to design two SAH-derived capture compounds (CCs). We demonstrate interactions of the proteins COMT and SAHH with SAH-CC with biotin used in conjunction with streptavidin-horseradish peroxidase. After demonstrating SAH-dependent photo-crosslinking of the CC to these proteins, we used a CC labeled with a fluorescein tag to measure binding affinity via fluorescence anisotropy. We then used this approach to show and characterize binding of SAM to the PR domain of PRDM2, a lysine methyltransferase with putative tumor suppressor activity. We calculated the K-d values for COMT, SAHH, and PRDM2 (24.1 +/- 2.2 mu M, 6.0 +/- 2.9 mu M, and 10.06 +/- 2.87 mu M, respectively) and found them to be close to previously established K-d values of other SAM binding proteins. Here, we present new methods to discover and characterize SAM and SAH binding proteins using fluorescent CCs.

• 出版日期2014-12-15