The hexokinase activity assay is a crucial biochemical technique used to measure the activity of hexokinase, an enzyme that catalyzes the phosphorylation of glucose to glucose-6-phosphate, a key step in glycolysis.

This assay typically employs a coupled enzymatic reaction that involves glucose-6-phosphate dehydrogenase (G6PDH) and the coenzyme NAD+. The process begins with the conversion of glucose to glucose-6-phosphate by hexokinase, which concurrently converts ATP to ADP. The glucose-6-phosphate produced is then oxidized by G6PDH, resulting in the reduction of NAD+ to NADH. The increase in NADH can be quantitatively measured spectrophotometrically at 340 nm, providing a direct assessment of hexokinase activity.

The assay is sensitive and can be performed on various sample types, including tissue homogenates and cell extracts, making it valuable for both clinical diagnostics and research applications. It is particularly useful in studies related to metabolic diseases, cancer metabolism, and the effects of pharmaceuticals on glycolytic pathways. By measuring hexokinase activity, researchers can gain insights into cellular energy metabolism and the regulatory mechanisms that govern glycolysis, thus contributing to a better understanding of metabolic disorders and potential therapeutic targets.