Vasopressin chromatography

Meienhofer J, du Vigneaud V. Preparation of lysine-vasopressin 116. through a crystalline protected nonapeptide intermediate and purification of the hormone by chromatography. J. Am. Chem. 

When the unusual bromine cleavage of oxytocin and vasopressin, reported by du Vigneaud and his associates, was repeated with a simplified tripeptide model, N-carbobenzyloxy-(S-benzyl-L-cysteinyl-L-tyrosyl-L-iso-leucine (CXV) (Ressler and du Vigneaud, 1957), bromine or NBS liberated a ninhydrin-reactive material in 40% yield. Paper chromatography proved isoleucine to be the sole ninhydrin-reactive substance present. The ultraviolet spectrum of the reaction mixture showed the characteristic 260 mM absorption for a dienone (CXVI). 

Figure 10.16 Separation of a peptide mixture by hydrophobic interaction chromatography (reproduced with permission from A.J. Alpert, J. Chromatogr., 444, 269 (1988)]. Conditions column, 20cm x 4.6mm i.d. stationary phase, polypropyl aspartamide 5pm mobile phase, 1mlmin of 2M ammonium sulfate with 0.025M sodium phosphate, pH 6.5-0.025M sodium phosphate pH 6.5, linear gradient as indicated detector, UV 220 nm. 1 = substance Ml—9) 2 = [Arg ]-vasopressin 3 = oxytocin 4 = substance P, free acid 5 —[Try ]-substance P, 6 = substance P,1 [Tyr ]-somatostatin 8 = somatostatin 9 = [Tyr ]-somatostatin.

Any chemist knows that the more steps there are in a chemical synthesis, the lower the final yield. For example, if each step in a 10-step synthesis furnishes a 90 percent yield of product, the yield of the final product will be only about 35 percent. That is why it is not possible to extend Du Vigneaud s masterful syntheses (see chapter 6) of the hormones oxytocin and vasopressin (9 amino acid residues each) to proteins, even small ones such as ribonuclease A (124 amino acid residues). In order to pursue this daunting challenge Robert Bruce Merrifield (1921-2006), at Rockefeller University, devised a new concept solid-phase synthesis. The idea is disarmingly simple covalently attach an amino acid to a macroscopic particle that can be exposed to the reaction, washed, and then separated by simple filtration. Each reaction step requires no chromatography and no crystallization, just washing and filtering. At the end, completed peptide chains are chemically released from the particles. 

The foremost advantage of the chemical assay is that the specificity of the analysis can be tested with the most rigid criteria. In the pituitary assay of oxytocin and vasopressin (Gruber et a/., 1976), specificity was demonstrated by the following four criteria (1) The oxytocin and vasopressin peaks were identified by their retention times. (2) Addition of synthetic oxytocin or vasopressin to the pituitary extract resulted in a quantitative increase in the height of the specific peak with no noticeable peak broadening. (3) After purification by reverse-phase chromatography, each of the nonapeptide fluorophors was collected, hydrolyzed in 6 N HCl, and then characterized... 

In Uppsala, J. Porath and P. Flodin developed an extremely important method for the separation of peptides and proteins gel permeation chromatography with crosslinked dextran (Sephadex). One of Porath s former associates, Ulf Ragnarsson contributed with innovative modifications to the solid phase method of peptide synthesis and in more recent years introduced the principle of complete protection of the amine function by diacylation. Another Porath student, Gunnar Lindeberg, explored the properties of vasopressin analogs. 

One of the major problems associated with the isolation of small relatively unstable polypeptides from biological tissue is that the peptides may break down or undergo other chemical transformation during the extraction procedures. Recently, a new approach to the study of the chemical nature of CRF has been developed [293] which overcomes this problem. Jones, Hillhouse and Burden [293] demonstrated that SJiydroxytryptamine selectively stimulates the release of CRF, but not vasopressin, from hypothalamic tissue in vitro and attempts are now being made to isolate and identify the CRF in the incubation medium. Preliminary separation by chromatography on Sephadex G-25 demonstrated two peaks of CRF activity, fraction A and B, with molecular weights of around 2500 and 1300 respectively. Both fractions evoked dose-related increases in pituitary ACTH release either when injected into basal hypothalamic-lesioned rats or when incubated with adenohypophysial tissue in vitro but Fraction A was the more potent in this respect. Neither fraction markedly influenced the release of... 

Two fractions were obtained by chromatography on a column of Sephadex G-75 The first emerged with the void volxane and contained protein with a molecular weight greater than 50,000 this protein appeared to lack affinity for either oxytocin or vasopressin. The second fraction, accounting for over 80 per cent of the protein recovered had a molecular weight of less than 50,000 and in dialysis experiments bound both hormones. 

Isolation of anti-([8-lysine]vasopressin) antibodies by affinity chromatography... 

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