Peptides opioid, chromatography

Opiates Opioid peptides Ion-pair chromatography with TFA uBondapak C18 300x4 5mM TFA(pH 2.5) in linear gradient of 30-50 ACN(10 min, 2ml/min) lOmM HCOOH-MeOH(1 1) 5mM TFA-MeOH(l l) 49... 

GH Fridland, et al. Measurement of opioid peptides with combinations of reversed phase high performance liquid chromatography, radioimmunoassay, radiorecep-torassay, and mass spectrometry. Life Sci 41 809, 1987. 

Because of some of the problems with bioassays and immunoassays, liquid chromatography (LC)-based techniques are increasingly applied as an alternative. While modern LC-based assays have a comparable sensitivity to immunoassays, they oftentimes are characterized by a higher selectivity [18, 19]. Muller et ah, for example, used LC/mass spectrometry with matrix-assisted laser desorption ionization in ex vivo pharmacokinetic studies in combination with enzyme inhibition experiments to investigate the complex metabolism of dynorphin Al-13, a peptide with opioid activity, up to the fifth metabolite generation [20, 21]. 

Figure 7. Purification of proopiocortin from a single camel pituitary (from Kimura et al, 1979). Initial fractionation was carried out on Sephadex G-lOO (top panel). Fractions 21-30 were combined, concentrated under reduced pressure, and purified further on Sephadex G-75 (not shown). Chromatography of the peak fractions of opioid activity from the G-75 column was performed on a Lichrosorb RP-8 column (middle panel). Rechromatography of fraction 25 (middle panel) on the RP-8 column was performed under modified elution conditions (bottom panel). Fraction 50 (bottom panel) was shown to be homogeneous by gel electrophoresis, tryptic peptide mapping, and specific activity.

Figure 12. Chromatography of opioid peptides from bovine striatum (from Stern et al., 1980). A deproteinized extract was chromatographed on an Ultrasphere ODS column. Fractions were collected and tested for opioid activity with the radioreceptor assay. The column had been calibrated with the synthetic peptide markers as indicated.

High performance liquid chromatography has been used as a criterion of specificity when coupled with a bioassay or radioimmunoassay. With regard to the opioid peptides, brain extracts were chromatographed on an HPLC column that had previously been calibrated with known opioid peptides (Lewis et ai, 1979), and the collected fractions were assayed for activity (Stern et ai, 1980) (Fig. 12). The bioassay provides the quantitation and the HPLC provides the identity of each component. It would be interesting to use HPLC coupled with radioimmunoassay to confirm the identity of the many peptides that have been found in brain by immunohistochemical techniques. 

Figure 13. Tryptic peptides derived from adrenal opioid peptides F and I (see Fig. 7) (from Kimura et a/., 1980). Chromatography was carried out on an Ultrasphere octyl column using a gradient of n-propanol at pH 4.0. (A) Standard a, [Met ]enkephalin-Lys b, [Met ] enkephalin[Arg ] c, [Met ]enkephalin d, [Leu ]enkephalin e, [Met ]enkephalin-Arg -[Phe ]. (B) Tryptic digest of peptide F. (C) Tryptic digest of peptide I. Aliquots of the collected fractions were tested for opioid activity and hydrolyzed for amino acid analysis.

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