RESUMO
Glucose and fructose crystals undergo significant superheating during melting that allows one to study the kinetics of this process. Melting of both compounds has been studied by differential scanning calorimetry (DSC). The obtained data have been subjected to isoconversional kinetic analysis. The process has been determined to have unusually large values of the activation energy and preexponential factor that indicate that melting occurs by cooperatively breaking multiple hydrogen bonds. The experimentally determined activation energy of melting demonstrates a decrease with increasing temperature. The use of the nucleation and growth models has permitted deriving theoretical dependencies of the activation energy on temperature. Testing the theoretical dependencies against the experimental ones suggests that from either the statistical or physical viewpoint the melting kinetics should be parameterized by means of the growth model. This suggests that the mechanism of melting involves the growth of the stable melt nuclei that exist on the crystal surface below the equilibrium melting temperature.
RESUMO
Immunoglobulin G (IgG) is the main immunoglobulin in human serum, and its biological activity is modulated by glycosylation on its fragment crystallizable region. Glycosylation of IgGs has shown to be related to aging, disease progression, protein stability, and many other vital processes. A common approach to analyze IgG glycosylation involves the release of the N-glycans by PNGase F, which cleaves the linkage between the asparagine residue and the innermost N-acetylglucosamine (GlcNAc) of all N-glycans except those containing a 3-linked fucose attached to the core GlcNAc. The biological significance of these glycans necessitates the development of accurate methods for their characterization and quantification. Currently, researchers either perform PNGase F deglycosylation on intact or trypsin-digested IgGs. Those who perform PNGase F deglycosylation on trypsin-digested IgGs argue that proteolysis is needed to reduce steric hindrance, whereas the other group states that this step is not needed, and the proteolytic step only adds time. There is minimal experimental evidence supporting either assumption. The importance of obtaining complete glycan release for accurate quantitation led us to investigate the kinetics of this deglycosylation reaction for intact IgGs and IgG glycopeptides. Statistically significant differences in the rate of deglycosylation performed on intact IgGs and trypsin-digested IgGs were determined, and the rate of PNGase F deglycosylation on trypsin-digested IgGs was found to be 3- to 4-times faster than on intact IgG.
RESUMO
A current method to locate sites of N-linked glycosylation on a protein involves the identification of deamidated sites after releasing the glycans with peptide-N-glycosidase F (PNGase F). PNGase F deglycosylation converts glycosylated Asn residues into Asp. The 1-Da mass tag created by this process is observable by liquid chromatography-tandem mass spectrometry analysis. A potential interference to this method of N-glycosylation site mapping is the chemical deamidation of Asn residues, which occurs spontaneously and can result in false positives. Deamidation is a pH-dependent process that results in the formation of iso-Asp (i-Asp) and native Asp (n-Asp) by a succinimide intermediate, whereas PNGase F deglycosylation results in the conversion of the glycosylation Asn residue into n-Asp. N-linked glycosylation sites can thus be identified by the presence of a single chromatographic peak corresponding to an n-Asp residue within the consensus sequence Asn-X-Ser/Thr, whereas sites of deamidation led to 2 chromatographic peaks resulting from the presence of n-Asp and i-Asp. The intent of this study is to alert investigators in the field to the potential and unexpected errors resulting from this phenomenon and to suggest a strategy to overcome this pitfall and limit the number of false-positive identifications.