Micelles prepared from amphiphilic block copolymers in which a poly(styrene) segment is connected to a poly(ethylene oxide) block via a bis‐(2,2′:6′,2″‐terpyridine‐ruthenium) complex have been intensely studied. In most cases, the micelle populations were found to be strongly heterogeneous in size because of massive micelle/micelle aggregation. In the study reported in this article we tried to improve the homogeneity of the micelle population. The variant preparation procedure developed, which is described here, was used to prepare two “protomer”‐type micelles: PS20‐[Ru]‐PEO70 and PS20‐[Ru]‐PEO375. The dropwise addition of water to a solution of the compounds in dimethylformamide was replaced by the controlled addition of water by a syringe pump. The resulting micelles were characterized by sedimentation velocity and sedimentation equilibrium analyses in an analytical ultracentrifuge and by transmission electron microscopy of negatively stained samples. Sedimentation analysis showed virtually unimodal size distributions, in contrast to the findings on micelles prepared previously. PS20‐[Ru]‐PEO70 micelles were found to have an average molar mass of 318,000 g/mol (corresponding to 53 protomers per micelle, which is distinctly less than after micelle preparation by the standard method) and an average hydrodynamic diameter (dh) of 18 nm. For PS20‐[Ru]‐PEO375 micelles, the corresponding values were M = 603,000 g/mol (31 protomers per micelle) and dh = 34 nm. The latter particles were found to be identical to the “equilibrium” micelles prepared in pure water. Both micelle types had a very narrow molar mass distribution but a much broader distribution of s values and thus of hydrodynamic diameters. This indicates a conformational heterogeneity that is stable on the time scale of sedimentation velocity analysis. The findings from electron microscopy were in disagreement with those from the sedimentation analysis both in average micelle diameter and in the width of the distributions, apparently because of imperfections in the staining procedure. The preparation procedure described also may be useful in micelle formation from other types of protomers. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 4458–4465, 2004

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Human glutathione reductase (GR; which catalyzes the reaction NADPH + GSSG + H' -2 GSH + NADP') is an obligatory FAD-containing homodimer of known geometry. Native human GR, a potential target of antimalarial and cytostatic agents, cannot be dissociated by dilution or by means of subunitinterface mimetics, similarly to well-studied viral dimeric proteins. However, ab initio folding andlor dimerization of human GR can be inhibited by point mutations or by peptides corresponding to subunitinterface areas, for example synthetic peptide P I 1, which represents the intersubunit-contact helix HI 1. The structure of this peptide, which might assist inhibitor design, was solved by high-resolution NMR spectroscopy. Residues 440-453, were found to be a helical in the isolated peptide. To quantitate the efficacy of inhibitors such as P I 1, we developed the following unfolding/reactivation assay. The effects of various guanidine hydrochloride (Gdn/HCl) concentrations were studied by analytical ultracentrifugation. It was shown that human GR denatured by greater than 3 M Gdn/HCl is monomeric and free of FAD. Circular-dichroism experiments at 223 nm indicated a half-life of approximately 20 s at 20°C for the unfolding process. To optimize the reactivation yield, four parameters [protein concentration (x) in the range 0.3-10 pg/ml, cofactor supplementation, temperature (y; 0-32"C), and time (0-72 h)] were varied systematically, and a reactivation score z was given to each constellation of parameters. This type of analysis might be useful to optimize refolding and activation yields for other proteins. For human GR, the highest recovery was found not to occur at one of the corners of the x,y plain, but close to its center. Consequently, the optimal assay conditions for folding and dimerization inhibitors are as follows. The enzyme (at 300 pg/ml) is denatured by 5 M guanidine hydrochloride/5 mM dithiothreitol, then reactivated by dilution to 1 pg/ml at pH 6.9 and 20°C. In the absence of inhibitors, this procedure leads to 70% of the control activity within 8 h. Peptides representing the upper subunit interface (for instance residues 436-478) of human GR were found to inhibit refolding with EC,,,, values in the micromolar range, whereas fragments from other regions of the protein had no influence on this process. For peptide P11, the EC,,,, value was 20 pM. In conclusion, hGR, enzyme with a tight intersubunit contact area of 21 nm', appears to be suitable for studying protein folding, dimerization, and prosthetic-group complexation in the absence and presence of compounds that inhibit these processes. There is a shortage, at least for oligomeric enzymes of eukaryotes, of published systematic studies on protein (re)activation.Keywords: unfolding/reactivation ; human glutathione reductase; antiparasitic drug; protein-dimerization inhibitor; peptide-structure determination by NMR. Neuenheimer Feld 328, D-69120 Heidelberg, Germany. of glutathione, respectively ; Williams, 1992). Since each binding site for the substrate GSSG and each...

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