Leucine enkephalin (1 mM) was reacted with mushroom tyrosinase under reductive conditions (ascorbic acid, 50 mM). Reaction products were isolated by high-performance liquid chromatography and identified using electrospray ionization mass spectrometry. The products of the reaction were found to be hydroxylated at the Tyr1 moiety of the peptide. The major product was a monohydroxylated derivative of leucine enkephalin ([HO-Tyr1]LE) and the minor product of the reaction was a dihydroxylated derivative ([(HO)2-Tyr1]LE). The affinity of [HO-Tyr1]LE to receptors in rat brain homogenate was compared to that of leucine enkephalin itself. Hydroxylation of LE was found to decrease receptor affinity to both μ and δ opioid receptor sites by a factor of about 20.
- Mass spectrometry
- Receptor affinity