Fluorine nuclear magnetic resonance studies of trifluoroacetylinsulin derivatives effects of salts and denaturants (original) (raw)
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The trifluoroacetylation of insulin
Biochimica Et Biophysica Acta - Proteins And Proteomics, 1973
A method has been described for the modification of the amino groups of insulin by means of trifluoroacetylation. In this procedure insulin hydrochloride is reacted with ethyl thioltrifluoroacetate in dimethylformamide to yield a product in which the terminal amino groups of the A and B chains and the e-amino group of lysine 2 9 have been modified with the trifluoroacetyl moiety. The physical and chemical properties of this derivative were studied by DEAE-Sephadex and Sephadex G-5o chromatography, amino acid analysis, deamination and trypsin digestion. The results indicated the formation of a homogeneous tritrifluoroacetylinsulin derivative in high yield possessing 7o% of the biological activity of native bovine insulin.
Preparation of several trifluoroacetyl insulin derivatives
Biochimica et biophysica acta, 1974
The selective modification of the amino groups in insulin has been effected using phenyltrifluoroacetate or ethylthioltrifluoroacetate affording several Irifluoroacetyl derivatives, where fluorine will serve as a reporter group for fluorine magnetic resonance studies. Insulin hydrochloride was reacted with one of the above reagents and imidazole in dimethyl-formamide. The resulting products were separated and purified using DEAE-Sephadex chromatography to yield 3 mono-and 3 di-trifluoroacetyl insulin products in addition to the tri-derivative. These derivatives were characterized by deamination, trypsin digestion and amino acid analysis. Radioimmunoassays indicated that these derivatives had immunoreactivities ranging from 60 to 89 % of native insulin. Glycine A l-trifluoroacetyl-insulin afforded a crystalline derivative in the presence of Zn 2+.
Magnetic Resonance in Chemistry, 2001
The conformational changes of free, monomeric glucagon-like peptide-1-(7-36)-amide (GLP-1) in aqueous solution with increasing concentrations of 2,2,2-trifluoroethanol (TFE) were monitored by NMR spectroscopy. It was found that GLP-1 gradually assumes a stable, single-stranded helical structure in water solution when the TFE concentration is increased from 0 to 35% (v/v). No further structural changes were observed at higher TFE concentrations. The structure of GLP-1 in 35% TFE was determined from 292 distance restraints and 44 angle restraints by distance geometry, simulating annealing and restrained energy minimization. The helical structure extends from T7 to K28, with a less well-defined region around G16 and a disordered six-residue N-terminal domain. The folding process of GLP-1 from random coil (in water) to helix (in 35% TFE) is initiated by the formation of the C-terminal segment of the helix that is extended gradually towards the N-terminus of the peptide with increasing concentration of TFE. The exchange rates of the slow exchanging amide protons indicate that the C-terminal part of the helix is more stable than the N-terminal part.
Synthesis and Characterization of Insulin−Fluorescein Derivatives for Bioanalytical Applications
Analytical Chemistry, 1997
Human insulin was labeled with fluorescein isothiocyanate (FITC) and fully characterized to yield four distinct insulin-FITC species. High-performance liquid chromatography and electrospray mass spectrometry were used to determine the extent and location of fluorescein conjugation. By changing the reaction conditions (i.e., pH, time, and FITC/insulin ratio) the selectivity of the fluorescein conjugation was altered, and all conjugates could be separated. The isolated species of insulin-FITC were labeled at the following residues: A1(Gly), B1(Phe), A1-(Gly)B1(Phe), and A1(Gly)B1(Phe)B29(Lys). All four insulin-FITC conjugates were then used to develop fluorescence polarization binding assays with monoclonal and polyclonal anti-insulin antibodies. The assay sensitivity differed between the conjugates depending on the site of modification (B1 > A1 > A1B1 > A1B1B29). Also, the type of antibody used had an important role in the binding of insulin-FITC conjugates. Finally, for the first time the biological activity of the four conjugates was demonstrated by an autophosphorylation assay. The positional substitution dramatically affected the biological activity, confirming insights into the residues responsible for the insulin binding region. The B1 conjugate was found to retain almost all biological activity while the A1 and A1B1 conjugates had ∼10 times lower activity. The trisubstituted species (labeled at A1, B1, and B29) was determined to be least active.
Biophysical Chemistry, 2004
The thermal denaturation of hen egg-white lysozyme was studied in the presence of 2,2,2-trifluoroethanol (TFE) at various pH values using micro differential scanning calorimetry. Quantitative thermodynamic parameters accompanying the thermal transitions were evaluated. It is observed that thermal unfolding of lysozyme in the presence of TFE upto a concentration of 4.0 mol dm follows a two-state denaturation mechanism as indicated by the equality y3 of van't Hoff and calorimetric enthalpies. The finer details of interaction were studied by measuring the partial molar volume of some constituent amino acids and glycine peptides from water to aqueous TFE at 298.15 K. The physicochemical properties of aqueous TFE: apparent molar heat capacities, apparent molar volumes and surface tension were measured to understand the intrinsic properties of the cosolvent as well. From the correlation among the thermal unfolding data on lysozyme in aqueous TFE, calculated preferential interaction parameters, physico chemical properties of aqueous TFE and partial molar volumes of transfer, it is concluded that both solvent mediated effect and direct interaction constitute the mechanism of TFE-protein interactions.
α-Helix Formation in Melittin and β-Lactoglobulin A Induced by Fluorinated Dialcohols
Journal of Physical Chemistry B, 2006
Extensive study of the effect of fluorinated alcohols on protein conformations, notably the induction of R-helix formation, is important because of its wide range of applications. Circular dichroism (CD) was used to show that the enhancement of helix induction in -lactoglobulin A and melittin by the fluorinated diols 2,2,3,3tetrafluoro-1,4-butanediol (TFBD), 2,2,3,3,4,4-hexafluoro-1,6-pentanediol (HFPD), and 2,2,3,3,4,4,5,5octafluoro-1,6-hexanediol (OFHD) increases in the order TFBD < HFPD < OFHD. For fluorinated diols and monoalcohols the effectiveness of helix induction was found to increase exponentially with increasing number of fluorine atoms per alcohol molecule, and OFHD was found to be more effective than any previously reported fluorinated alcohol. Formation of standard micelles was ruled out as the cause of the enhanced helix induction by the fluorinated diols. The negligible red-edge excitation shift in the fluorescence of melittin indicated that the fluorinated diol/water solvent shell surrounding the tryptophan chromophore is less immobilized than are molecules in a lamellar vesicle.
A Structural Study by 19F-Nuclear-Magnetic Resonance of the Binding of Sugars to Lysozyme
European Journal of Biochemistry, 1972
1. The 19F chemical shifts of several fluorinated monosaccharide inhibitors have been determined in the inhibitor/lysozyme complexes. 2 . The l9F shift of N-fluoroacetyl-a-D-glucosamine (aFAcG1uNH.J in lysozyme is different from the fi anomer and either a or fi anomers of methyl N-fluoroacetyl-D-ghcosamine (MeFAcGluNH,). 3. The l9F resonances of the inhibitors in the presence of lysozyme are broadened by addition of Gd(II1) and the Gd(II1)-F distances have been determined. 4. The distances for aFAcGluNH, and aMeFAcGluNH, are 0.56 nm while corresponding values are 0.50 nm. Comparison with crystal models suggests a preferred orientation of the -CH,F groups.
European journal of biochemistry / FEBS, 1979
The proton nuclear magnetic resonance (1H-NMR) spectra of glycophorin and its tryptic sialoglycopeptides were investigated. From the intensities of the assigned resonances it was concluded that all of the residues in the sialoglycopeptides are sufficiently mobile in conformation to give sharp resonances, while in glycophorin this is true for only approximately 80% of the peptide backbone. The resonances of the central sequence of some 20 of the hydrophobic residues are strongly broadened. This region is probably that of alpha-helical structure which is known to aggregate. The linewidths and intensities of the resonances are not, or only slightly, affected by changing the ionic strength, temperature or by carboxymethylation of the Met-81 residue in glycophorin. Glycophorin was found to bind about 100 mol sodium dodecylsulphate/mol protein as derived from studies on linebroadening of the latter's C-3 to C-11 methylene resonances. The bound dodecyl-sulphate probably increases the m...
Biochemistry, 2001
The structure and folding of a novel human insulin mutant, [Thr(B27) f Pro, Pro(B28) f Thr]insulin (PT insulin), in aqueous solution and in mixtures of water and 2,2,2-trifluoroethanol (TFE) have been studied by NMR spectroscopy. It was found that PT insulin has a highly flexible structure in pure water and is present in at least two different conformations, although with an overall tertiary structure similar to that of native insulin. Furthermore, the native helical structures are poorly defined. Surprisingly, the mutant has a biological activity about 50% higher than native insulin. In contrast, in TFE/water solution the mutant reveals a propensity of forming a well-defined structure at the secondary structure level, similar to monomeric native insulin. Thus, as shown by a detailed determination of the structure from 208 distance restraints and 52 torsion angle restraints by distance geometry, simulated annealing, and restrained energy minimization, the native insulin helices (A2-A7, A13-A19, and B10-B19) as well as the -turn (B20-B23) are formed in 35% TFE. However, the amount of tertiary structure is decreased significantly in TFE/water solution. The obtained results suggest that only an overall tertiary fold, as observed for PT insulin in pure water, is necessary for expressing the biological activity of insulin, as long as the molecule is flexible and retains the propensity to form the secondary structure required for its receptor binding. In contrast, a compact secondary structure, as found for native insulin in solution, is unnecessary for the biological activity. A model for the receptor binding of insulin is suggested that relates the increased bioactivity to the enhanced flexibility of the mutant. † This work was financially supported by the Danish Natural Science Research Council (J 9400351 and J 9801801), Direktør Ib Henriksens Fond, Carlsbergfondet, and Novo Nordisk Fonden. ‡ The 1 H chemical shifts of PT insulin in H2O and in 35% TFE and the 13 C chemical shifts of PT insulin in 35% TFE have been deposited in the BioMagResBank (http://www.bmrb.wisc.edu), BMRB accession number 4997. NMR-derived restraints have been deposited in the RCSB (Research Collaboratory for Structural Bioinformatics) and the Protein Data Bank, accession numbers PDB 1JCO and RCSB RCSB013628, together with the coordinates of the 25 refined structures with lowest energy.