Comparison of total protein and enzyme levels in successive regenerations of venom from individual coralsnakes.

Coralsnakes produce highly potent neurotoxic venoms, but little is known about variations in specific enzyme components within a species or from one replenishment of venom to the next within the same animal. Since published studies are often conducted using venom pools from multiple snakes, individual differences are masked and variations among individual snakes and between subsequent venom regenerations from the same snake have rarely been documented. This study involves the analysis and comparison of four successive venom collections from each of nine individual coralsnakes in order to detect these differences. Significant variation was found within the successive re-synthesis of venom components. Even greater differences were observed between the venoms from similar individual snakes. Since studies of variation in enzymatic activity would be significant only if they were above these normal variations, it is important to be aware of these differences. These results suggest the importance of understanding the variations present within and between individuals of the same species when interpreting the potential significance of differences found as the result of genetic, environmental or ecological factors.

Comparison of total protein and phospholipase A(2) levels in individual coralsnake venoms.

Studies of differences or changes in venom protein levels or enzymatic activities have significance only if contrasted to the normal variations between individual snakes. This study involves the analysis and comparison of venom from 13 individual Texas coralsnakes (Micrurus tener tener) in order to detect differences in the volume, total protein concentration, electrophoretic profile, and PLA2 enzyme activity. A significant inverse correlation between venom volume and total protein concentration was found. Although the 13 venoms were indistinguishable from their electrophoretic protein profiles, phospholipase A2 enzymatic activities varied considerably.

Chemical signals of elephant musth: temporal aspects of microbially-mediated modifications.

Mature male African (Loxodonta africana) and Asian (Elephas maximus) elephants exhibit periodic episodes of musth, a state in which serum androgens are elevated, food intake typically decreases, aggressiveness often increases, and breeding success is enhanced. Urine is a common source of chemical signals in a variety of mammals. Elephants in musth dribble urine almost continuously for lengthy periods, suggesting that the chemicals in their urine may reveal their physiological condition to conspecifics. We investigated the volatile urinary chemicals in captive male elephants using automated solid phase dynamic extraction (SPDE) and gas chromatography-mass spectrometry (GC-MS). We found higher levels of alkan-2-ones, alkan-2-ols, and some aromatic compounds in urine from males in musth than in urine from non-musth males or from females. Levels of ketones and alcohols increased as the urine aged, likely due to microbial metabolism of fatty acids. Protein-derived aromatic metabolites also increased in abundance after urination, likely due to microbial hydrolysis of hydrophilic conjugates. We suggest that microbes may play an important role in timed release of urinary semiochemicals during elephant musth.

Adsorption of peanut (Arachis hypogaea, Leguminosae) proteins by activated charcoal.

The binding of peanut protein allergens to activated charcoal (AC), used medically for gastric decontamination following the ingestion of toxic substances, was investigated for potential clinical application. Crude peanut extract (CPE) or purified peanut protein allergens Ara h 1 and 2 were co-incubated with AC under a variety of conditions followed by centrifugation to remove the AC and adsorbed protein. The resulting supernatant solution was analyzed for unadsorbed protein by gel electrophoresis and quantitative protein assay. The extent of protein adsorption by a known amount of AC was determined. Protein binding to AC was rapid and irreversible. The extent of adsorption was unaffected by pH, but was optimal near physiological salt concentrations. Denatured proteins, or those of larger molecular weight, required more AC than smaller or native proteins. The extent of protein binding increased with temperature, supporting the concept that protein molecules diffuse into vacant pores of appropriate size on the charcoal surface.

Comparison of physiological and in vitro porcine gastric fluid digestion.

BACKGROUND: In previous studies, the major peanut allergen Ara h 1 was digested in vitro using pepsin and porcine gastric fluid. The results suggested that in vivo gastric digestion of allergen protein can be modeled accurately by peptic hydrolysis in vitro. In the current investigation, studies were designed to follow the gastrointestinal (GI) digestion of peanut allergens under true physiological conditions. In vitro digestion with porcine gastric fluid was compared with actual physiological digestion of peanut allergens in the porcine digestive tract in vivo. METHODS: Analysis of physiologic digestion was performed in piglets administered a 20-gram bolus of peanut meal followed by periodic sampling and analysis of GI contents. The pH was monitored, and digesta were analyzed by SDS-PAGE and immunoblot analysis. RESULTS: Peanut meal initially neutralized stomach contents to a pH of approximately 7, which was subsequently acidified by HCl secretion within 30 min. Acidification to pH 2-4 resulted in active pepsin digestion of soluble protein in the stomach. Soluble intact protein/allergens were rapidly degraded to pepsin-resistant peptides in the stomach followed by hydrolysis of these fragments in the small intestine. Particulate material was evident in both the stomach and small intestine that could contribute to continued release of peanut allergens Ara h 1, 2 and 3. CONCLUSIONS: Porcine gastric digestion of peanut proteins resembles true physiological digestion only under optimal physiologic conditions. Soluble proteins are rapidly digested and insoluble material continues to release IgE-reactive proteins throughout the GI tract. GI digestion of food allergens can play a prominent role when assessing allergens within the context of a food matrix or meal and during the sensitization phase of IgE-mediated allergy.

Peanut protein allergens: the effect of roasting on solubility and allergenicity.

BACKGROUND: A contributing factor to food allergen stability is heat resistance. Peanut allergens in particular are resistant to heat, which results in their decreased solubility upon routine extraction and may have a profound influence on their continued presence in the digestive tract. Although there have been a number of studies characterizing soluble extracts of raw and roasted proteins, the relative solubility of the insoluble material following routine extraction for residual allergen characterization has not been investigated. The effects of various treatments on the re-solubilization and subsequent allergenicity of this insoluble peanut protein material are presented here. METHODS: Various methods to resolubilize the insoluble protein material were used, including pH, proteases and glycosidases. Protease digestion of nonextractable peanut proteins with pepsin, chymotrypsin and trypsin was performed in appropriate buffers as previously optimized for peanut proteins. Glycosidase activity in the presence of protease inhibitors was performed at pH 2. Digested samples were then subjected to SDS-PAGE/Western blot analysis using serum IgE from peanut-sensitive individuals. RESULTS: Progressive roasting of peanuts resulted in a significant decrease in protein solubility. The acidic proteins were resolubilized moderately at high pH, with solubility decreasing as pH approached the pI of the protein. However, at pH 2 the solubility increased dramatically. More extensive resolubilzation was observed with amylase treatment, presumably due to cleavage of glycoside of glycoproteins. The protein released into solution had a high IgE-binding capacity. While amylase was effective at resolubilizing this material, digestive tract proteases were not. CONCLUSION: The presence of these insolubilized peanut proteins provides a continuous source of major allergens to the gastrointestinal mucosal immune system.

Peanut protein allergens: gastric digestion is carried out exclusively by pepsin.

BACKGROUND: A major characteristic of many food allergens, including Ara h 1, a major peanut allergen, is their resistance to gastric digestion. One estimate of the allergenic potential of a possible protein allergen is its stability under simulated gastric conditions. OBJECTIVE: Because the rate and extent of digestion of allergenic proteins will affect the severity of any subsequent allergic response, it is important to correlate protein allergen digestion in simulated gastric fluid with that in actual gastric fluid. METHODS: A major peanut allergen, Ara h 1, was digested in vitro by using both pepsin and porcine gastric fluid. Several comparisons between the 2 sets of proteolytic conditions were assessed including pH optima and the effect of temperature, denaturants, and specific enzyme inhibitors. RESULTS: In vitro digestion of Ara h 1 with pepsin and porcine gastric fluid resulted in virtually identical hydrolysis patterns as observed on SDS-PAGE. The protease activity of both pepsin and gastric fluid were inhibited at high pH and in the presence of pepstatin. However, both remained active in 4 mol/L urea and at 60 degrees C. CONCLUSIONS: Protein digestion in the porcine stomach is carried out by pepsin. In vivo gastric digestion is modeled accurately by peptic hydrolysis. Digestion conditions in vivo are comparable to experimental conditions in vitro provided that the acidic nature of the stomach contents is optimal for characterization of the allergen under standard pepsin digestion conditions. Additional experimentation using crude food extracts, both in the presence and absence of a complete meal, is needed to elucidate the complete physiologic nature of food allergen digestion.