Effect of Tris, HEPES, and TES buffers on binding at μ-, δ-, and κ-opioid sites in guinea pig brain

In homogenates of guinea-pig brain minus cerebellum, the δ-binding of 1.5 nM [³H]-[D-Pen²,D-Pen⁵]enkephalin is little affected by the type and concentration of the three buffers, Tris-HCl, HEPES-KOH, or TES-KOH (10–75 mM). However, the μ-binding of 1 nM [³H][D]A[a²,MePhe⁴,GIy-ol⁵]enkephalin or the κ-binding of 1.5 nM [³H]-U-69,593 is influenced by the choice of buffer. A suitable concentration of buffer for further analyses of opioid binding has been found to be 10 mM. At each site, the effects of MgCl₂ on binding are the same whether 10 mM Tris-HCl, HEPES-KOH, or TES-KOH are used but variations of the effects of NaCl confirm the view that μ- and κ-sites, but not δ-sites, are affected by choice of buffer. Furthermore, under some assay conditions the effects of NaCl and MgCl₂ at the κ-sites of guinea-pig cerebellum differ from their effects in brain minus cerebellum, indicating that these are differences of binding characteristics at the κ-sites of these tissues.