Non-radioactive localization of substance P binding sites in rat brain and spinal cord using peptides labeled with 1.4-nm gold particles

We present here a new method for non-radioactive labeling of substance P (SP) to demonstrate the distribution of its binding sites in histological sections. The peptide was labeled at the primary amino group with a 1.4-nm gold particle. In Western blots of membrane fractions of rat spinal cord, specific binding occurred at 38 and 58 KD. This binding was competitively suppressed by adding the native peptide. In addition, the SP-gold conjugate was able to displace the corresponding 125I-labeled peptide from binding proteins. In histological sections, binding sites could be shown in various parts of rat brain and spinal cord. The distribution patterns were comparable to those found in studies using autoradiographic methods. Adding the native peptide or a neurokinin 1 receptor agonist markedly reduced labeling of the tissue, whereas only a slight reduction was obtained after adding neurokinin A. Therefore, SP could be specifically labeled with a 1.4-nm gold particle to demonstrate its binding sites. This new method combines the advantages of receptor-ligand affinity binding with a non-radioactive detection system. It can be used for labeling peptides in general and therefore offers an alternative or addition to other methods used in study of the distribution of membrane receptors.