Background: Tetrahydrobiopterin (BH4) is an essential cofactor for aromatic amino acid-hydroxylases and all isoforms of nitric oxide-synthase. In the cardiovascular system nitric oxide (NO) acts as a potent vasodilator and maintains endothelial function. Administration of BH4 can improve endothelial function in numerous cardiovascular pathologies, an effect that likely results from increased NO bioavailability and local antioxidant effects. GTP cyclohydrolase 1 (GCH1) is the rate-limiting enzyme for BH4 biosynthesis. GCH1 activity has been shown to be modified by both levels of phenylalanine and BH4 through allosteric regulation when bound to GCH1feedback regulatory protein (GFRP). Studies have shown that phenylalanine(L-phe) binds to and activates the GCH1-GFRP complex, whilst BH4 binding to the complex inhibits GCH1 activity. Our studies aim to investigate the significance of this protein-protein interaction on BH4 bioavailability. Methods: Human proteins GCH1 (both the full length and truncated forms) and GFRP, were recombinantly expressed, and the binding affinities were determined using surface plasmon resonance (SPR). In addition the activity of purified recombinant enzymes has been measured by two methods; using both a kinetic micro-plate assay and high performance liquid chromatography (HPLC). In order to investigate the regulation of BH4 levels in vivo via the GCH1-GFRP axis we conducted an oral challenge of L-phe (100mg/kg) in mice (n=8) and measured levels of L-phe and biopterin (a marker of BH4) in both tissue and plasma. Results: Soluble human truncated and full length GCH1 and GFRP have been successfully expressed (with the truncated form of GCH1 lacking the first 42 animo acids of the N-terminus). Initial SPR experiments determined a Kd of approximately 20-40nM for GCH1-GFRP. The activity of both truncated and full length GCH1 was measured by two methods and we found truncated GCH1 to be more active than full length GCH1. Oral L-phe challenge in mice ( n=8) was shown to significantly increase biopterin production in both plasma and tissue p<0.001 ANOVA (vs. saline control). Conclusion: The aim of this project is to investigate the protein-protein interaction between GCH1 and GFRP. Targeting BH4 bioavailability can provide a means to restore endothelial dysfunction in a range of cardiovascular disorders. Our studies suggest a regulatory role for the first 42 amino acids which may influence the interaction of GCH1 with GFRP. L-Phe oral challenge was shown to cause a rise in circulating and vascular biopterin in mice indicating the potential of pharmacological agents that can mimic the feed-forward allosteric changes to regulate BH4 levels. Further studies are required to understand the different factors that may affect the nature of the GCH1-GFRP interaction and how it may influence BH4 bioavailability.