Analytical techniques used to study the degradation of proteins and peptides: chemical instability

The physical instability of proteins and peptides as well as the various analytical techniques used to study the various aspects of physical instability have been reviewed. Physical instability of proteins and peptides involve changes in secondary, tertiary and quaternary structures of these compounds. After a general introduction of the subject the literature data of these changes and their analytical aspects have been summarized in a Table.     

Accelerated chemical analysis of copper tellurides

Conclusions An accelerated method is proposed for the analysis of copper tellurides without prior separation of the elements: Copper is determined complexometrically in the presence of tellurium, when the amount of the latter is from 40 to 60%, while tellurium is determined by a bichromate method in the presence of copper, using a separate batch. The duration of the copper determination is 1.5 h and that of the tellurium determination, 4 h.The accuracy of the copper and tellurium determinations is ±0.02 and ±0.05%, respectively (at 0.95 probability confidence).Translated from Poroshkovaya Metallurgiya, No. 1 (85), pp. 69–70, January, 1970.     

Design and synthesis of amphipathic antimicrobial peptides

A large proportion of antimicrobial peptides share a common structural feature that is critical to their antimicrobial activity, i.e. amphipathic alpha-helices. The amphipathy of a polypeptide chain can be quantitated through the value of the hydrophobic moment. Generally, antimicrobial peptides are characterized by high hydrophobic moment and low hydrophobicity values. Using these criteria we have identified two short segments that possess hydrophobic moment properties associated with known antimicrobial peptides. Using in vitro assays the segment derived from the protein perforin displays no antifungal or antibacterial activity and, while showing no alpha-helicity in buffer or liposomes, exhibits a modest degree of alpha-helical structure in the presence of the alpha-helical inducer, 2,2,2-trifluoroethanol. However, rational modifications result in a derivative which assumes an alpha-helical conformation in the presence of liposomes, exhibits potent antifungal activity against plant fungal pathogens, has significant antibacterial activity, effects leakage of a fluorescent dye from acidic liposomes and is devoid of hemolytic activity. Results are also presented for a segment derived from the human immunodeficiency virus envelope protein. We suggest that the identification of putative amphipathic structures in proteins may provide a useful starting strategy in the design and synthesis of antimicrobial peptides.     

Glycosylasparaginase-catalyzed synthesis and hydrolysis of beta-aspartyl peptides

beta-Aspartyl di- and tripeptides are common constituents of mammalian metabolism, but their formation and catabolism are not fully understood. In this study we provide evidence that glycosylasparaginase (aspartylglucosaminidase), an N-terminal nucleophile hydrolase involved in the hydrolysis of the N-glycosidic bond in glycoproteins, catalyzes the hydrolysis of beta-aspartyl peptides to form L-aspartic acid and amino acids or peptides. The enzyme also effectively catalyzes the synthesis of beta-aspartyl peptides by transferring the beta-aspartyl moiety from other beta-aspartyl peptides or beta-aspartylglycosylamine to a variety of amino acids and peptides. Furthermore, the enzyme can use L-asparagine as the beta-aspartyl donor in the formation of beta-aspartyl peptides. The data show that synthesis and degradation of beta-aspartyl peptides are new, significant functions of glycosylasparaginase and suggest that the enzyme could have an important role in the metabolism of beta-aspartyl peptides.     

Ion beam tritium labeling of proteins and peptides

A general method for tritiating proteins, peptides, and other nonvolatile organic compounds has been developed. A carefully controlled particle beam composed of T3+ and T2+ ions and fast T2 molecules is accelerated into a sample target within a vacuum chamber. This beam method has been used to tritiate ribonuclease A, porcine pancreatic elastase, thermolysin, soybean trypsin inhibitor, alpha 1-protease inhibitor, and the peptide aldehydes leupeptin and antipain. After removal of all readily exchangeable tritium, the products were obtained in 32-83% yields with specific radioactivities of 18-856 Ci/mol. The products were carefully characterized, shown to be chemically pure, and to have complete biological activity. Simple tritium hydrogen exchange accounts for at least 82% of the reaction pathway with proteins and for 100% of the reaction with the peptide aldehydes. The ion beam method is a mild procedure for general tritium labeling of fragile protein macromolecules and other sensitive biological molecules.     

Design and synthesis of small semi-mimetic peptides with immunomodulatory activity based on myelin basic protein (MBP)

Bile reflux has been implicated in the pathogenesis and malignant degeneration of Barrett's esophagus, but clinical studies in patients with adenocarcinoma arising in Barrett's esophagus are lacking. Ambulatory esophageal measurement of acid and bile reflux was performed with the previously validated fiberoptic bilirubin monitoring system (Bilitec) combined with a pH probe in 20 asymptomatic volunteers, 19 patients with gastroesophageal reflux disease (GERD) but no mucosal injury, 45 patients with GERD and erosive esophagitis, 33 patients with GERD and Barrett's esophagus, and 14 patients with early adenocarcinoma arising in Barrett's esophagus. Repeat studies were done in 15 patients under medical acid suppression and 16 patients after laparoscopic Nissen fundoplication. The mean esophageal bile exposure time showed an exponential increase from GERD patients without esophagitis to those with erosive esophagitis and benign Barrett's esophagus and was highest in patients with early carcinoma in Barrett's esophagus (P <0.01). Pathologic esophageal bile exposure was documented in 18 (54.5%) of 33 patients with benign Barrett's esophagus and 11 (78.6%) of 14 patients with early adenocarcinoma in Barrett's esophagus. Nissen fundoplication but not medical acid suppression resulted in complete suppression of bile reflux. Bile reflux into the esophagus is particularly prevalent in patients with Barrett's esophagus and early cancer. Bile reflux into the esophagus can be completely suppressed by Nissen fundoplication but not medical acid suppression alone.     

Beta-turn induction by C60-based fulleroproline: synthesis and conformational characterization of Fpr/Pro small peptides

In this paper, we examined the induction of autoimmune-like histologic changes in the liver and other organs of mice undergoing graft-versus-host reaction (GVHR) with MHC class I disparity by the administration of bacterial lipopolysaccharide (LPS), on the assumption that stimulation with LPS could be an exacerbating factor. Spleen cells of C57BL/6 (B6) mice were injected twice into (B6 x bml) F1 recipient mice at an interval of 7 days to induce MHC class I GVHR and then challenged with 1 microg of LPS intravenously on the next day of the cell transfer. The hepatic lesions of the group of MHC class I GVHR mice challenged with LPS showed marked cellular infiltration at the portal area and focal necrosis was observed in the hepatic lobule. The major infiltrating cells were CD8+, and others including CD4+ cells being of minor populations. In addition, ductal lesions in extrahepatic organs, including the pancreas and salivary glands also showed marked cellular infiltration. Thus, we have demonstrated that LPS induced ductal lesions in mice with MHC class I disparity. CD8+ cells were detected at the destructive hepatic lesions, which might be effector cells. These findings indicate that LPS might be one of the potential factors which augment autoimmune-like lesions.     

Determination of sedimentation coefficients for small peptides

Direct fitting of sedimentation velocity data with numerical solutions of the Lamm equations has been exploited to obtain sedimentation coefficients for single solutes under conditions where solvent and solution plateaus are either not available or are transient. The calculated evolution was initialized with the first experimental scan and nonlinear regression was employed to obtain best-fit values for the sedimentation and diffusion coefficients. General properties of the Lamm equations as data analysis tools were examined. This method was applied to study a set of small peptides containing amphipathic heptad repeats with the general structure Ac-YS-(AKEAAKE)nGAR-NH2, n = 2, 3, or 4. Sedimentation velocity analysis indicated single sedimenting species with sedimentation coefficients (s(20,w) values) of 0.37, 0.45, and 0.52 S, respectively, in good agreement with sedimentation coefficients predicted by hydrodynamic theory. The described approach can be applied to synthetic boundary and conventional loading experiments, and can be extended to analyze sedimentation data for both large and small macromolecules in order to define shape, heterogeneity, and state of association.     

Helix propensities are identical in proteins and peptides

Our understanding of the factors stabilizing alpha-helical structure has been greatly enhanced by the study of model alpha-helical peptides. However, the relationship of these results to the folding of helices in intact proteins is not well characterized. Helix propensities measured in model peptides are not in good agreement with those from proteins. In order to address these questions, we have measured helix propensities in the alpha-helix of ribonuclease T1 and a helical peptide of identical sequence. We have previously demonstrated excellent agreement between peptide and protein for the nonpolar amino acids [Myers, J. K., Pace, C. N., and Scholtz, J. M. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 2833-2837]. Most other amino acids also show good agreement, although certain polar amino acids are exceptions. Helix propensities measured in the ribonuclease T1 peptide/protein are compared with those measured in other systems. Reasonable agreement is found between most systems; however, our propensities differ substantially from those measured in several model peptide systems. Alanine-based peptides overestimate the propensity differences by a factor of 2, and host/guest experiments underestimate them by a factor of 2-3.     

Purification of small peptides labeled with Bolton-Hunter reagent

A method utilizing high-voltage electrophoresis on paper is described whereby a pentapeptide (Asp-Ser-Asp-Pro-Arg) labeled with Bolton-Hunter reagent is separated from hydrolyzed reagent and unreacted peptide and is recovered from the electrophoretogram in high yield. The general applicability to other peptides is discussed.     

Semidry electroblotting of peptides and proteins from acid-urea polyacrylamide gels

BACKGROUND: The authors previously reported a statistically significant effect of psychosocial intervention on survival time of women with metastatic breast carcinoma. In this study, the authors investigated whether this effect could be explained by differences in the medical treatment patients received subsequent to their group participation or differences in causes of death. METHODS: Of the original 86 study participants, medical treatment charts for 61 and death certificates for 83 were available for further analysis. The authors reviewed the course of the medical treatment they received subsequent to their entry into the randomized psychotherapy trial. RESULTS: Although there were no statistically significant differences with regard to chemotherapy and hormone therapy between the control and treatment groups, women in the control group tended to have received more adrenalectomies, although this procedure did not account for the difference in survival time between the control group and the treatment group. Furthermore, women in the control group developed more bone and lung metastases than the women in the treatment group. CONCLUSIONS: Differences in disease course between the control and treatment groups appeared to be independent of any differences in medical treatment received.     

Chromatin proteins of large and small brain nuclei

A simple fluorometric assay for monoamine oxidase (MAO) [EC 1.4.3.4] activity towards beta-phenylethylamine (PEA) was devised. The procedure consists in measuring the disappearance of PEA fluorometrically. The disappearance of PEA was completely inhibited by pargyline, a potent inhibitor of MAO. MAO activity for PEA was linear with 10 mg to 100 mg of liver tissue in 3 ml of reaction mixture for up to 90 min of incubation. Using this method, the V max values and the apparent Km values of MAO for PEA in several rat tissues were determined, and compared with those for benzylamine and 5-hydroxytryptamine (5-HT).     

Design of peptides, proteins, and peptidomimetics in chi space

Peptide and protein biological activities depend on their three dimensionals structures in the free state and when interacting with their receptors/acceptors. The backbone conformations such as alpha-helix, beta-sheet, beta-turn, and so forth provide critical templates for the three-dimensional structure, but the overall shape and intrinsic stereoelectronic properties of the peptide or protein important for molecular recognition, signal transduction, enzymatic specificity, immunomodulation, and other biological effects depend on arrangement of the side chain groups in three-dimensional chi space (their chi 1, chi 2, etc. torsional angles). In this paper we explore approaches to the de novo design of polypeptides and peptidomimetics with biased or specific conformational/topographical properties in chi space. We consider computational and experimental methods that can be used to examine the effects of specific structural modifications in constraining side chain groups of amino acid residues and their similarities in chi space to the natural amino acids to evaluate what sort of mimetics are likely to mimic normal amino acids. We then examine some of the asymmetric synthetic methods that are being developed to obtain the amino acid mimetics. Finally, we consider selected examples in the literature where these specialized amino acids have been incorporated in biologically active peptides and the specific insights they have provided regarding the topographical requirements for bioactive peptide potency, selectivity, and other biochemical and pharmacological properties. Constraints in chi space show great promise as useful tools in peptide, protein, and peptidomimetic de novo design of structures and pharmacophores with specific stereostructural, biochemical and biological properties.     

Two-dimensional separations of peptides and proteins by comprehensive liquid chromatography-capillary electrophoresis

Analysis of biological samples is problematic because of their complex composition. Reversed phase liquid chromatography (RPLC), size exclusion chromatography (SEC), and, more recently, capillary zone electrophoresis (CZE) are routinely used for the analysis of these samples, but are eventually limited because they are one-dimensional (1-D) methods. As sample complexity increases, the separation efficiency necessary to resolve a large number of sample components in one dimension becomes prohibitively high. A solution to this problem has been to use a two-dimensional (2-D) approach. Each dimension in a 2-D separation relies on a different separating mechanism. By expanding the separation into two dimensions, sample components unresolved in the first dimension can often be separated in the second. This circumvents the requirement for extremely high efficiencies in either dimension. Two-dimensional slab gel electrophoresis has been used successfully in this area, but a more instrumental approach is desired. In this paper we describe three coupled-column approaches to 2-D separations. First, microcolumn SEC-CZE is explored as a means of 2-D protein analysis. Next, RPLC-CZE is investigated for analysis of peptides in tryptic maps. Finally, RPLC is coupled with fast CZE (FCZE), a unique form of CZE analysis, for fast 2-D analysis of peptides. Details of the instrumentation used in these 2-D systems will be presented along with the results of some typical 2-D analyses.     

A model for the interaction of trifluoroethanol with peptides and proteins

Scaphoid or longitudinal arch pads are frequently prescribed pedorthics for foot and ankle rehabilitation. These pedorthics are reported to be effective in mechanically supporting the medial longitudinal arch while reducing plantar and medial soft tissue strain. The objective of this study was to measure alterations in ambulatory plantar pressure metrics in a group of adults secondary to scaphoid pad application. The biomechanical rationale of this study was that the geometry of foot contact would be altered secondary to foot inversion. Ten adult male subjects with biomechanically normal feet were evaluated during multiple trials. A Holter type microprocessor-based portable in-shoe plantar pressure data acquisition system was used to record the dynamic data. Pressures were recorded from eight discrete plantar locations at the hindfoot, midfoot, and forefoot regions of the insole. Statistically significant (p < or = 0.05) increases in peak pressures were seen laterally with scaphoid pad application, while significant decreases in peak pressures with pad usage occurred at the hallux and the calcaneal region of the foot. At the medial longitudinal arch, peak pressures increased from near 0 to 115.3 kPa, contact durations increased from near 0 to 438 ms, and pressure-time integrals increased from near 0 to 33.4 kPa.s.     

Shock-induced chemical reactions and synthesis of nickel aluminides

Chemical reactions in Ni and Al powder mixtures, initiated by the passage of shock waves, are used for the synthesis of nickel aluminides. Mechanistic investigations reveal that the extent of these shock-induced chemical reactions and the type (stoichiometry) of shock-synthesized compound formed depend on shock-loading conditions and the initial powder particle morphology. More intense shock conditions and irregular powder morphology assist in attaining an intimately (mechanically) mixed and activated closed-packed mass, thereby favoring bulk chemical reactions and resulting in the synthesis of compounds. While the Ni₃Al compound is the preferred reaction product at lower shock conditions, more intense shock conditions favor the formation of the equiatomic B2-phase NiAl compound (having highest melting temperature and highest heat of reaction in contrast to other nickel aluminides), in spite of the starting powders mixed in a volumetric ratio corresponding to the Ni₃Al compound. This paper is based on a presentation made in the symposium “Reaction Synthesis of Materials” presented during the TMS Annual Meeting, New Orleans, LA, February 17–21, 1991, under the auspices of the TMS Powder Metallurgy Committee.