Histone Acetyltransferase Assay Services

Creative Biolabs provides a rapid and flexible histone acetyltransferase assay service for potential pharmaceutical inhibitors against histone acetyltransferases. To increase the effectiveness and reliability of our services, we also offer high-throughput screening along with useful techniques for assay optimization and validation.

Histone acetyltransferase (HAT) gene families are distinct, and acetyl-CoA serves as the acetyl donor, moving this group to the lysine side chain's epsilon amino group in histone proteins. Several acetyltransferases have been demonstrated to target non-histone proteins both in vivo and in vitro, despite their ability to acetylate histones. Some of these HATs could be better known as protein acetyltransferases (PATs), given the frequency of both histone and non-histone protein acetylation. Understanding the molecular mechanism of each gene family is important since histone/protein acetyltransferases are involved in a wide range of biological activities and because there is a significant diversity in the substrates and structures that they target. It is known that acetyl-CoA-dependent acetyltransferases employ one of two catalytic pathways. After attaching to and reacting with acetyl-CoA, one pathway involves the production of an intermediate acetylated enzyme. The ultimate acetylated protein product is produced by the intermediate reacting with the released product CoA and protein substrate. When using a ternary complex method, both substrates must bind together to create a ternary complex, which prevents the covalent enzyme intermediate from forming and enables the lysine to attack the bound acetyl-CoA directly.

Fig.1. Molecular mechanisms for acetyltransferase.¹

Targets of Histone Acetyltransferase Assay Services

For the acetyltransferase test, two common categories of spectrophotometric assays were developed:

Pyruvate dehydrogenase couples the synthesis of acetyl-CoA from pyruvate and CoA while lowering NAD+ to NADH in an enzyme-coupled assay. UV–Vis spectroscopy at 340 nm can be used to measure the quantity of NADH produced, which is directly proportional to the amount of acetylation.

The CPM used in the fluorescence detection test, which fluoresces when it reacts with the free sulfhydryl of CoA, is used to assess acetylation. Aliquots are taken out of the enzyme process and quenched in isopropanol. After that, CPM is introduced and left to react in the dark with the free sulfhydryls. Next, CPM is excited at 390 nm using a fluorescence plate reader or fluorimeter, and the emission is recorded at 469 nm. Creative Biolabs' histone acetyltransferase assays aim to achieve the following targets:

Creative Biolabs provides histone acetyltransferase assay services to study their roles in histone acetylation. Histone acetyltransferases are important for both normal cell homeostasis and pathological conditions because they are engaged in many vital physiological processes. Additionally, we focus on the clinical trials for medicines based on histone acetyltransferase inhibitors and activators. Please contact us for more information.

Reference

1. Berndsen, Christopher E., and John M. Denu. "Assays for mechanistic investigations of protein/histone acetyltransferases." Methods 36.4 (2005): 321-331.