[A specific and quantitative determination of rat beta-endorphin. Combination of HPLC and RIA]

A specific and quantitative method for the determination of rat beta-endorphin by the combination of HPLC and RIA was developed. Rabbit antiserum against camel beta-endorphin (c beta-E) was raised and used for RIA at the final concentration of 1:10000. The quantitative range estimated from the displacement curve was 0.1-2.0 ng. Cross-reactivities with Met-Enk, Leu-Enk, alpha-MSH, alpha-endorphin, ACTH and human beta-E were less than 0.1, less than 0.1, less than 0.1, less than 0.1, 2 and 100%, respectively. These peptides were separated from each other by reversed phase HPLC with UV254 nm detection, and the minimum detectable dose of c beta-E was found to be 1 microgram. beta-E-like immunoreactivity (beta-ELIR) in the HPLC effluent was determined by RIA. The HPLC-RIA chromatogram of authentic c beta-E exhibited a single peak which coincided with the peak of c beta-E detected by UV, and 80% of the injected c beta-E (1-100 ng) was detected in the c beta-E fraction. The HPLC-RIA chromatogram of rat pituitary, hypothalamus, cerebrospinal fluid and plasma revealed the presence of 1-3 peaks, one of which was observed at the position of c beta-E. The HPLC elution of rat pituitary resolved the material into two peaks of biological activity, one of which coincided with the peak of beta-ELIR at the position of c beta-E. The HPLC-RIA chromatogram of the c beta-E fraction from pituitary obtained by gel-chromatography exhibited three peaks, one of which coincided with c beta-E. These results suggest that beta-ELIR in the c beta-E fraction of the HPLC elution may reflect rat beta-E accurately.