Liquid chromatographic isolation of vincristine and vinblastine

A fundamentally new method is described for the separation of the dimeric indole alkaloids vincristine and vinblastine from monomeric indole alkaloid impurities. This method uses an RP-18 high-performance liquid chromatography column with a methanol-water mobile phase containing an inorganic acid and an unusually low concentration of inorganic buffer. By keeping the buffer concentration low, the elution of all indole alkaloids is retarded, but the dimeric ones are retarded more than the monomeric ones. A theoretical model developed to explain this behavior postulates that the anions of the buffer solubilize the protonated indole alkaloids by pairing with them. Lowering the buffer concentration reduces the availability of pairing ions and thus decreases the mobile phase affinity of protonated alkaloids, particularly those having a 2+ charge. A similar approach may be applicable in other situations where ionogenic organic compounds having a particular valence must be separated from related compounds having different valences, or from non-ionogenic compounds.     

A new perspective on beta-sheet structures using vibrational Raman optical activity: from poly(L-lysine) to the prion protein

The vibrational Raman optical activity (ROA) spectrum of a polypeptide in a model beta-sheet conformation, that of poly(l-lysine), was measured for the first time, and the alpha-helix --> beta-sheet transition monitored as a function of temperature in H(2)O and D(2)O. Although no significant population of a disordered backbone state was detected at intermediate temperatures, some side chain bands not present in either the alpha-helix or beta-sheet state were observed. The observation of ROA bands in the extended amide III region assigned to beta-turns suggests that, under our experimental conditions, beta-sheet poly(L-lysine) contains up-and-down antiparallel beta-sheets based on the hairpin motif. The ROA spectrum of beta-sheet poly(L-lysine) was compared with ROA data on a number of native proteins containing different types of beta-sheet. Amide I and amide II ROA band patterns observed in beta-sheet poly(L-lysine) are different from those observed in typical beta-sheet proteins and may be characteristic of an extended flat multistranded beta-sheet, which is unlike the more irregular and twisted beta-sheet found in most proteins. However, a reduced isoform of the truncated ovine prion protein PrP(94-233) that is rich in beta-sheet shows amide I and amide II ROA bands similar to those of beta-sheet poly(L-lysine), which suggests that the C-terminal domain of the prion protein is able to support unusually flat beta-sheets. A principal component analysis (PCA) that identifies protein structural types from ROA band patterns provides a useful representation of the structural relationships among the polypeptide and protein states considered in the study.     

A study of alpha-helix hydration in polypeptides, proteins, and viruses using vibrational raman optical activity

A vibrational Raman optical activity (ROA) study, supplemented by protein X-ray crystal structure data, of alpha-helices in polypeptides, proteins, and viruses has suggested that ROA bands in the extended amide III spectral region may be used to distinguish between two types of right-handed alpha-helix. One type, associated with a positive ROA band at approximately 1300 cm(-1), dominates in hydrophobic environments and appears to be unhydrated; the other, associated with a positive ROA band at approximately 1340 cm(-1), dominates in hydrophilic environments and appears to be hydrated. Evidence is presented to support the hypothesis that unhydrated alpha-helix corresponds to the canonical conformation alpha(c) and hydrated alpha-helix to a more open conformation alpha(o) stabilized by hydrogen bonding of a water molecule or a hydrophilic side chain to the peptide carbonyl. Alpha-helical poly(L-lysine) and poly(L-ornithine) in aqueous solution and poly(L-alanine) in dichloracetic acid display both bands, but alpha-helical poly(l-glutamic acid) in aqueous solution and poly(gamma-benzyl L-glutamate) in CHCl(3) display only the approximately 1340 cm(-1) band and so may exist purely as alpha(o) due to enhanced stabilization of this conformation by particular side chain characteristics. The ROA spectrum of poly(beta-benzyl L-aspartate) in CHCl(3) reveals that it exists in a single left-handed alpha-helical state more analogous to alpha(o) than to alpha(c).     

Analytical affinity chromatography. II. Rate theory and the measurement of biological binding kinetics

Affinity chromatography can be used to measure equilibrium constants and kinetics of biological interactions. The local-equilibrium theory presented in the preceding paper is extended to include mass transfer and kinetic effects. Solutions for both zonal and frontal elution are presented. For highly nonlinear isotherms, the frontal elution method is preferred. Experiments with bovine serum albumin binding to immobilized Reactive Blue show that the binding kinetics inside the porous gel are several orders of magnitude slower than typical biological binding reactions in solution. The temperature dependence of the kinetic constants indicate that the binding may still be diffusion-controlled.     

Field and pressure response of Yb compounds close to a quantum critical point

YbCu_5−x Al _x provides the possibility to tune ground state properties by a change of the valence due to the Cu/Al substitution, by pressure as well as by the application of a magnetic field. Near to the critical concentration x _cr≈1.5 non-Fermi-liquid properties (NFL) are obvious, obeying hyperscaling. If magnetic order sets in for x>1.5, the application of moderate magnetic fields quenches order and again NFL features become evident. Hyperscaling in this case indicates strongly interacting spin fluctuations.