Peptides denaturation

Using the more sensitive silver stain, however, Goldman and coworkers (G9) have been able to resolve more than 300 proteins and/or peptides (denatured sample) in csf using lEF/SDS-PAGE (Fig. 14). Twenty six of the proteins have been tentatively identified (Fig. 15), and comparison of the protein patterns with the corresponding plasma protein patterns has revealed several clusters of unidentified proteins that were more prominent in csf than in plasma and hence, may be of CNS origin. 

Large antigenic complex, polypeptide, peptide, denaturable, single epitope expressed, multiple epitopes expressed... 

The biologiccJ function of a protein or peptide is often intimately dependent upon the conformation(s) that the molecule can adopt. In contrast to most synthetic polymers where the individual molecules can adopt very different conformations, a protein usually exists in a single native state. These native states are found rmder conditions typically found in Uving cells (aqueous solvents near neutred pH at 20-40°C). Proteins can be unfolded (or denatured) using high-temperature, acidic or basic pH or certain non-aqueous solvents. However, this unfolding is often reversible cind so proteins can be folded back to their native structure in the laboratory. 

Bio-Rex 70 2.4 0.70 Weakly acidic cation exchanger with car-boxylate groups on a macroreticular acrylic matrix for separation and fractionation of proteins, peptides, enzymes, and amines, particularly high molecular weight solutes. Does not denature proteins as do styrene-based resins. 

Shifts in the SEC fractionation range are not new. It has been known for decades that adding chaotropes to mobile phases causes proteins to elute as if they were much larger molecules. Sodium dodecyl sulfate (SDS) (9) and guanidinium hydrochloride (Gd.HCl) (9-12) have been used for this purpose. It has not been clearly determined in every case if these shifts reflect effects of the chaotropes on the solutes or on the stationary phase. Proteins are denatured by chaotropes the loss of tertiary structure increases their hydrodynamic radius. However, a similar shift in elution times has been observed with SEC of peptides in 0.1% trifluoroacetic acid (TEA) (13-15) or 0.1 M formic acid (16), even if they were too small to have significant tertiary structure. Speculation as to the cause involved solvation effects that decreased the effective pore size of the... 

FIGURE 8.12 Effect of pore diameter on SEC of standards (nondenaturin > mobile phase). Nondenaturing" refers to the effect on the stationary phase. Most iarge proteins were in fact denatured by this mobile phase (which was optimized for use with peptides, not proteins). Accordingly, it was necessary to use polyacrylamide to demonstrate the approximate range and position of Vo under these conditions. The polyacryiamide standards both eiuted at V with the 300-A coiumn (not shown). Columns and flow rate Same as in Fig. 8.11. Mobile phase Same as in Fig. 8.1. Sample key (B) Ovalbumin (43,000 Da) 0) polyacrylamide (1,000,000 Da) (K) polyacrylamide (400,000 IDa) (L) low molecular weight impurity in the polyacrylamide standards. Other samples as in Fig. 8.11. 

Structure of GFP and its chromophore. To study the chro-mophore of GFP, a sample of GFP was denatured by heating it at 90°C. It was digested with papain, and then a peptide containing the fluorophore was isolated and purified from the digested mixture. The structural study of the peptide has indicated that the chromophore of GFP is an imidazolone derivative shown below (Shimomura, 1979). This chromophore structure was confirmed later by Cody etal. (1993) in a hexapeptide isolated from GFP. It is intriguing that the structure of the GFP chromophore is a part of the structure of coelenterazine. 

The loss of structure by a protein is called denaturation. This structural change may be a loss of quaternary, tertiary, or secondary structure it may also be degradation of the primary structure by cleavage of the peptide bonds. Even mild heating can cause irreversible denaturation. When we cook an egg, the protein called albumen denatures into a white mass. The permanent waving of hair, which consists primarily of long a helices of the protein keratin, is a result of partial denaturation. 

Few peptide bonds that are hydrolyzed by proteolytic enzymes are accessible without prior denaturation of dietary proteins (by heat in cooking and by the action of gastric acid). 

Korhonen, H., Pihlanto-Leppala, A., Rantamaki, P., and Tupasela, T. (1998). Impact of processing on bioactive proteins and peptides. Trends Food Sci. Technol. 9,307-319. Kunugi, S. and Tanaka, N. (2002). Cold denaturation of proteins under high pressure. Biochim. Biophys. Acta 1595, 329-344. 

One attempt to overcome these disadvantages has been to use multidimensional liquid chromatography (LC) followed directly by tandem mass spectrometry to separate, fragment and identify proteins (Link et al., 1999). In this process, a denatured and reduced protein mixture is digested with a protease to create a collection of peptides (Fig. 2.6). The peptide mixture is applied to a cation exchange column and a fraction of these peptides are eluted based on charge onto a reverse-phase column. The... 

Turning now to the chapters in this volume, a variety of complementary techniques and approaches have been used to characterize peptide and protein unfolding induced by temperature, pressure, and solvent. Our goal has been to assemble these complementary views within a single volume in order to develop a more complete picture of denatured peptides and proteins. The unifying observation in common to all chapters is the detection of preferred backbone conformations in experimentally accessible unfolded states. 

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