muramidase and glycine-amide

Many extracellular globular proteins have evolved to possess disulphide bonds in their native conformations, which aids in thermodynamic stabilisation. However, disulphide bond breakage by heating leads to irreversible protein denaturation through disulphide-thiol exchange reactions. In this study, we demonstrate that methanethiosulphonate (MTS) specifically suppresses the heat-induced disulphide-thiol exchange reaction, thus improving the heat-resistance of proteins. In the presence of MTS, small globular proteins that contain disulphides can spontaneously refold from heat-denatured states, maintaining wild-type disulphide pairing. Because the disulphide-thiol exchange reaction is triggered by the generation of catalytic amounts of perthiol or thiol, rapid and specific perthiol/thiol protection by MTS reagents prevents irreversible denaturation. Combining MTS reagents with another additive that suppresses chemical modifications, glycinamide, further enhanced protein stabilisation. In the presence of these additives, reliable remnant activities were observed even after autoclaving. However, immunoglobulin G and biotin-binding protein, which are both composed of tetrameric quaternary structures, failed to refold from heat-denatured states, presumably due to chaperon requirements. Elucidation of the chemical modifications involved in irreversible thermoinactivation is useful for the development of preservation buffers with optimum constitutions for specific proteins. In addition, the impact of disulphide bond breakage on the thermoinactivation of proteins can be evaluated using MTS reagents.

Topics: Animals; Carrier Proteins; Cattle; Chickens; Disulfides; Glycine; Hot Temperature; Humans; Hydrolysis; Immunoglobulin G; Lactalbumin; Mesylates; Muramidase; Protein Denaturation; Protein Refolding; Protein Stability; Protein Structure, Quaternary; Ribonuclease, Pancreatic; Solutions; Sulfhydryl Compounds

Glycine amide (GlyAd), a typically amidated amino acid, is a versatile additive that suppresses protein aggregation during refolding, heat treatment, and crystallization. In spite of its effectiveness, the exact mechanism by which GlyAd suppresses protein aggregation remains to be elucidated. Here, we show the crystal structure of the GlyAd-lysozyme complex by high resolution X-ray crystallographic analysis at a 1.05Å resolution. GlyAd bound to the lysozyme surface near aromatic residues and decreased the amount of bound waters and increased the mobility of protein. Arg and GlyAd molecules are different in binding sites and patterns from glycerol and related compounds, indicating that decreasing hydrophobic patches might be involved in suppression of protein aggregation.

Topics: Amino Acids, Aromatic; Animals; Arginine; Chickens; Crystallography, X-Ray; Egg Proteins; Enzyme Stability; Glycerol; Glycine; Hydrophobic and Hydrophilic Interactions; Molecular Conformation; Muramidase; Preservatives, Pharmaceutical; Protein Binding; Protein Denaturation; Solubility; Spectrometry, Fluorescence; Surface Properties; Water