Conformational changes in the reversed phase liquid chromatography of recombinant human growth hormone as a function of organic solvent: the molten globule state.

As a continuation of a previous paper on the retention behavior of recombinant human growth hormone (rhGH) in reversed phase chromatography at pH 6.5 (Oroszlan, P., et al. Anal. Chem. 1992, 64, 1623-1631) the effect of 1-propanol (1-PrOH) and acetonitrile on the conformation of rhGH at this pH has been investigated by circular dichroism (CD), second-derivative UV spectroscopy, fluorescence anisotropy, fluorescence quenching, and fluorescence lifetime measurements. Addition of 1-PrOH up to a concentration of 10% (v/v) does not cause any significant changes in protein structure. However, above this concentration, a transition from the native to a new state is observed; the transition is completed above 30% (v/v) of 1-PrOH, the composition for completion being dependent on temperature. This change in structure correlates with retention changes observed in reversed phase chromatography. The new rhGH conformation retains much of the alpha-helicity and possesses a slightly expanded hydrodynamic radius relative to native rhGH. Second-derivative UV spectroscopy suggests that the hydrogen bond between Trp 86 and Asp 169, spanning two alpha-helices, remains intact. On the other hand, the near-UV CD intensity changes from positive to negative in the Trp region of the spectrum, signaling an alteration in the Trp environment. In addition, fluorescence quenching measurements with trichloroethanol reveal greater accessibility to solvent of the Trp residue after the conformational transition has occurred. From the results, it is concluded that a molten globule state (compact state retaining much of the secondary structure of the native state but with a disrupted tertiary structure) is produced with the addition of > 30% (v/v) 1-PrOH.(ABSTRACT TRUNCATED AT 250 WORDS)

[Recent past years of the Network of Clinical Pharmacology-- contradictions, central measures, possibilities]. / A Klinikai Farmakológiai Hálózat elmúlt néhány éve--ellentmondások, központi intézkedések, lehetöségek.

Problems in connection with the activity of Clinical Pharmacological Network, the debate conferences of the recent past and measures taken recently put the Network i.e. the clinical pharmacological activity in the center of attention of the professional public opinion. It appeared therefore necessary that a short and possibly objective summary be prepared of the problems of the recent past and measures taken for their solution and last but not least of the present situation and ways planned for the future. Presenting these problems to the wide public is all the more worth as a high-level clinical pharmacology-activity in Hungary may be regarded as a key-issue of the pharmaceutical research. The development of the pharmaceutical industry in Hungary cannot be indifferent either from the viewpoint of economy or of a public health care on appropriate level.

Properties of the melanin binding of p-bromo-methylamphetamine (V-111).

The aim of the present study was to obtain detailed information on the binding properties of p-bromomethylamphetamine (V-111) to melanins under in vitro and in vivo conditions. The results obtained by the methods of both equilibrium and dynamic dialysis revealed that the binding of V-111 to bovine eye melanin was reversible, at least for the part of the binding sites, whereas the dissociation of V-111-melanin adduct was slow. The binding capacity of two different classes of binding sites of bovine eye melanin and dissociation constants of the drug-melanin complexes have been determined. The in vivo melanin binding of V-111-1-14C was studied by the method of whole body autoradiography. Extensive accumulation and retention was observed in the eyes of the pigmented mice while in the albino animal uptake was low in the corresponding tissues. In conclusion, V-111 may most probably be accumulated and retained for long periods in the pigmented cells of intact animals. Our data also imply that melanin in the pigmented cells serves as a depot, which gradually releases p-bromo-methylamphetamine resulting in a prolonged local effect of this compound.

Studies on the melanin affinity of selegiline (deprenyl) and other amphetamine derivatives.

The main aim of our study was to assess the melanin affinity of selegiline as well as the pharmacologic and pharmacokinetic aspects of its binding. The in vivo melanin binding of [14C] selegiline was studied by the method of whole body autoradiography. Extensive accumulation was observed in the pigmented mouse eye while in the albino animal uptake was low in the corresponding tissues. Our in vitro investigations demonstrated that the amphetamine derivatives tested can be taken up by melanins. Scatchard analysis of selegiline binding to the dopamine melanin (structurally similar to the neuromelanin) and beef eye melanin showed that more than one class of binding sites may be implicated. The total binding capacity of the beef-eye melanin was higher than that of the dopamine melanin. The selegiline inhibition of the [3H] MPP+ (the neurotoxic metabolite of MPTP) binding to dopamine melanin was also investigated. In the studied concentration range, the binding of [3H] MPP+ was depressed to about 70 per cent of the original maximum value. In conclusion, as a result of its melanin affinity, which is demonstrated in this study, selegiline may most probably accumulate in the pigmented nerve cells. The observed melanin affinity may contribute to the application of this compound for the treatment of Parkinson's disease or may play a role in its protective effect against MPTP neurotoxicity.