Analysis of protein self-association at constant concentration by fluorescence-energy transfer.

Fluorescence-resonance-energy transfer from subunits labelled with a fluorescence donor group to subunits labelled with a fluorescence acceptor group can be used for quantitative analysis of protein self-association. The present approach evaluates fluorescence measurements on mixtures of equimolar solutions of donor-labelled and acceptor-labelled protein composed by systematic variation of the volume ratio. Its attractive feature is that it allows the determination of equilibrium constants at fixed total concentration. Problems encountered by most other methods, which require the equilibria to be followed to high dilution, are avoided. Conditions to be fulfilled are that a reactive site is available on the protein for specific introduction of the labels and that labelling neither affects the conformation nor interferes with the intermolecular interactions. It is desirable that the Forster distance of the donor/acceptor pair complies with its separation. While dimerisation constants can be determined exclusively by fluorescence measurements, the analysis of more complex cases of self-association depends on additional independent information. This communication reports on an application of the approach to the association/dissociation equilibrium between insulin monomers and dimers. Labelling of insulin at the epsilon-amino group of LysB29 does not disturb the conformation nor does it affect dimerisation. 2-Aminobenzoyl and 3-nitrotyrosyl residues served as the donor/acceptor pairs. Because they are less bulky than most other fluorescence labels and are of balanced polarity they do not alter the chemical nature of the protein. Their Forster distance of 29 A matches their 32-A separation in the insulin dimer. Energy transfer was measured as a function of the molar fractions of donor-insulin and acceptor-insulin at constant total concentration. Evaluation of this dependence resulted in a dimerisation constant, K12, of 0.72x10(5) M(-1). Its agreement with values obtained with other methods demonstrates that the present approach is a reliable alternative.

Structure and rotational dynamics of fluorescently labeled insulin in aqueous solution and at the amphiphile-water interface of reversed micelles.

Insulin is a proteohormone with amphipathic three-dimensional structure and the ligand of a receptor, which itself spans the plasma membrane of glucose-metabolizing cells. In this study, the possible impact of amphiphiles on structural and dynamic properties of the hormone was investigated in reversed micelles mimicking the amphipathic nature of biological membranes. To make insulin susceptible to fluorescence measurements, two derivatives labeled with 2-aminobenzoic acid (Abz), N epsilon B29-Abz-insulin and [AbzB1]insulin, were prepared. First, the Abz-labeled insulins were shown by CD spectroscopy to exhibit conformational properties and self-association as well as the T-->R transition similar to the native hormone. By means of time-resolved fluorescence measurements, not only metal-ion induced hexamerization was observable in aqueous solution: The T-->R allosteric transition of the hexamer was shown to be accompanied by a diminution of its hydrodynamic radius. Second, structure and rotational dynamics of the labeled insulins were investigated in reversed micelles. In sodium bis(2-ethylhexyl)sulfosuccinate (AOT) reversed micelles, the main-chain conformation is similar to that in aqueous solution according to CD spectroscopy in the far-UV, whereas the weak circular dichroism in the near-UV is indicative of reduced aromatic contacts as well as of the absence of quaternary structure, and the CD spectra show the same shape as found for proteins in an intermediate state of folding referred to as the "molten globule". Fluorescence anisotropy decay measurements of N epsilon B29-Abz-insulin in reversed micelles of AOT, cetyltrimethylammonium bromide, and alpha-L-1,2-dioctanoylphosphatidylcholine showed that the internal mobility of the solubilizate is reduced compared to that in aqueous solution and that the rotational mobility of the labeled insulin decreases with decreasing micellar size. With respect to the immobilization, insulin interacts in a stronger way with the anionic than with the cationic or zwitterionic amphiphile; an integration into the amphiphile monolayer, however, could be ruled out in all cases. In conclusion, the results reveal an evident influence of amphiphiles on the structure and rotational dynamics of insulin. Further investigations should be focused on this finding also with regard to the possible importance of lipid-insulin interactions in vivo.

Proteolyses of a fluorogenic insulin derivative and native insulin in reversed micelles monitored by fluorescence emission, reversed-phase high-performance liquid chromatography, and capillary zone electrophoresis.

The preparation and substrate properties of the fluorogenic insulin derivative N alpha A1-aminobenzoyl-N epilson B29-Tyr(NO2)- insulin are described. This semisynthetic protein intramolecularly quenched by long-range resonance energy transfer between the donor/acceptor pair 2-aminobenzoic acid and 3-nitrotyrosine was used to prove the activity of serine proteases toward substrates of high molecular weight after incorporation in reversed micelles. The proteases investigated, trypsin and alpha-chymotrypsin, were shown to be hydrolytically active in reversed micellar solvent systems stabilized by cetyltrimethylammonium bromide or sodium-1,2-bis(2-ethylhexylcarbonyl)-1- ethane sulfonate. Apart from fluorometric enzyme assays, methods for monitoring proteolyses in reversed micelles were elaborated using either reversed-phase high-performance liquid chromatography or capillary zone electrophoresis. Enzymatic digestions of native insulin by the specific protease trypsin and the less specific protease alpha-chymotrypsin were performed. In contrast to aqueous solution, high but still variable specificity of alpha-chymotrypsin which was dependent on the micellar environment was observed. The results promise further insight into the influence of interfacial environments on enzyme action and a novel approach to enzyme-mediated protein modifications by the use of microstructured solvent systems.