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G Padron

Publications and source records attributed to G Padron.

6 recordsLinked to original sources
PubMed1992

Time-resolved fluorescence study of human recombinant interferon alpha 2. Association state of the protein, spatial proximity of the two tryptophan residues.

Human recombinant interferon alpha 2 belongs a to family of proteins active against a wide range of viruses. It contains two tryptophan residues located at positions 77 and 141 in the peptide sequence. The fluorescence emission spectrum of these tryptophan residues displays a maximum at 335 nm. The fluorescence intensity decay is described by one broad excited-state-lifetime population centered around a value of 1.7 ns (full width at half maximum, 1.5 ns). These observations suggest that in the native protein, both tryptophan residues emit from similar environments, not directly exposed to the surrounding solvent. The anisotropy decay is essentially biexponential. The correlation-time value characterizing the Brownian rotation of the protein varies linearly with the viscosity/temperature ratio. The calculated hydrodynamic volumes are compatible with the existence of a dimer and a tetramer, at pH 5.5 and 9.4, respectively. Addition of urea at pH 5.5 disrupts the dimer and modifies to some extent the excited-state-lifetime distribution which becomes more heterogeneous. Disulfide-bond reduction also dissociates the dimer and leads to a highly heterogeneous fluorescence-intensity decay with four excited-state-lifetime populations. An opening of the local structure in the Trp region of the protein is likely to occur in these conditions. The fast-anisotropy-decay components can be due to either fast rotation or energy transfer between the indoles. Close proximity of the two Trp residues (less than 1 nm) is suggested from steady-state and time-resolved fluorescence-anisotropy measurements in vitrified medium [95% (by mass) glycerol at -38 degrees C]. This suggestion is in agreement with the recently published three-dimensional structure of the homologous protein murine interferon beta [Senda, T., Shimazu, T., Matsuda, S. Kawano, G., Shimizu, H., Nakamura, K. T. & Mitsui, Y. (1992) EMBO J. 11, 3193-3201].

Fluorescence Polarization↗
PubMed1992

A method for determination of N-glycosylation sites in glycoproteins by collision-induced dissociation analysis in fast atom bombardment mass spectrometry: identification of the positions of carbohydrate-linked asparagine in recombinant alpha-amylase by treatment with peptide-N-glycosidase F in 18O-labeled water.

Previously, a combined use of fast atom bombardment (FAB) mass spectrometry and peptide N-glycosidase F, an enzyme that cleaves the beta-aspartylglycosylamine linkage of Asn-linked carbohydrates, was successfully applied to identification of N-glycosylation sites in a glycoprotein with the known or DNA-derived sequence (S. A. Carr and G. D. Roberts, 1986, Anal. Biochem. 157, 396-406). Here, we extended the method for easier identification of N-glycosylation sites in a glycoprotein even with unknown sequence. The glycoprotein is digested with peptide-N-glycosidase F in buffer containing 40 at% H2 18O, to yield a deglycosylated protein whose carbohydrate-linked Asn residues are converted to Asp partly labeled with 18O at their beta-carboxyl group during this digestion. The deglycosylated protein is further digested with proteolytic enzymes in an appropriate buffer prepared with normal water, and then peptides are separated on a reversed-phase column by HPLC. Peptides in which carbohydrate-linked Asn has been converted to Asp show a pair of signals ([M + 1]+ and [M + 3]+) in FAB mass spectra due to the partial incorporation of 18O into the beta-carboxyl groups of Asp residues, while the other peptides show normal isotopic ion distributions. Thus, both formally N-glycosylated peptides and, using collision-induced dissociation analysis, N-glycosylation sites can be identified. The application of the present method to the determination of N-glycosylation sites in a recombinant glycoprotein, Bacillus licheniformis alpha-amylase, is described.

Amidohydrolases↗
PubMed1992

Interaction of recombinant human epidermal growth factor with phospholipid vesicles. A steady-state and time-resolved fluorescence study of the bis-tryptophan sequence (Trp49-Trp50).

The interaction of recombinant human epidermal growth factor with small unilamellar phospholipid vesicles was studied by steady-state and time-resolved fluorescence of the bis-tryptophan sequence (Trp49-Trp50). Steady-state anisotropy measurements demonstrate that strong binding occurred with small unilamellar vesicles made up of acidic phospholipids at acidic pH only (pH < or = 4.7). An apparent stoichiometry for 1,2-dimyristoyl-sn-phosphoglycerol of about 12 phospholipid molecules per molecule of human epidermal growth factor was estimated. The binding appears to be more efficient at temperatures above the gel to liquid-crystalline phase transition. The conformation and the environment of the Trp-Trp sequence are not greatly modified after binding, as judged from the invariance of the excited state lifetime distribution and from that of the fast processes affecting the anisotropy decay. This suggests that the Trp-Trp sequence is not embedded within the bilayer, in contrast to the situation in surfactant micelles (Mayo et al. 1987; Kohda and Inigaki 1992).

Amino Acid Sequence↗
PubMed1990

Production and characterization of human gamma interferon from Escherichia coli.

The production of human gamma interferon as intracellular inclusion bodies in Escherichia coli, which simplified the purification process, is described. An expression plasmid carrying lipoprotein and the tryptophane promoters in tandem was used. Preparation of highly pure interferon was achieved using high resolution chromatography after denaturation and renaturation steps. Structural characteristics of this protein were verified by mass spectrometric analysis. Additional control tests have shown the suitability of the final product for clinical purposes.

Amino Acid Sequence↗