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European Journal of Mass Spectrometry... Oct 2023Pepsin, because of its optimal activity at low acidic pH, has gained importance in mass spectrometric proteome research as a readily available and easy-to-handle...
Native and compactly folded conformers of pepsin are revealed and distinguished by mass spectrometric ITEM-TWO analyses of pepstatin A - pepsin complex binding strength differences.
Pepsin, because of its optimal activity at low acidic pH, has gained importance in mass spectrometric proteome research as a readily available and easy-to-handle protease. Pepsin has also been study object of protein higher-order structure analyses, but questions about how to best investigate pepsin conformers still remain. We first determined dependencies of pepsin ion charge structures on solvent pH which indicated the existence of (a) natively folded pepsin (N) which by nanoESI-MS analysis gave rise to a narrow charge state distribution with an 11-fold protonated most intense ion signal, (b) unfolded pepsin (U) with a rather broad ion charge state distribution whose highest ion signal carried 25 protons, and (c) a compactly folded pepsin conformer (C) with a narrow charge structure and a 12-fold protonated ion signal in the center of its charge state envelope. Because pepsin is a protease, unfolded pepsin became its own substrate in solution at pH 6.6 since at this pH some portion of pepsin maintained a compact/native fold which displayed enzymatic activity. Subsequent mass spectrometric ITEM-TWO analyses of pepstatin A - pepsin complex dissociation reactions in the gas phase confirmed a very strong binding of pepstatin A by natively folded pepsin (N). ITEM-TWO further revealed the existence of two compactly folded pepsin conformers (C and C) which also were able to bind pepstatin A. Binding strengths of the respective compactly folded pepsin conformer-containing complexes could be determined and apparent gas phase complex dissociation constants and reaction enthalpies differentiated these from each other and from the pepstatin A - pepsin complex which had been formed from natively folded pepsin. Thus, ITEM-TWO turned out to be well suited to pinpoint pepsin conformers by interrogating quantitative traits of pepstatin A - pepsin complexes in the gas phase.
Topics: Pepsin A; Pepstatins; Spectrometry, Mass, Electrospray Ionization
PubMed: 37259551
DOI: 10.1177/14690667231178999 -
International Journal of Biological... Apr 2022Among the matrices for enzyme immobilization, activated carbon has been standing out in immobilization processes due to its properties and to its characteristics that...
Among the matrices for enzyme immobilization, activated carbon has been standing out in immobilization processes due to its properties and to its characteristics that provide superficial modification by inserting new functional groups capable of binding the enzymes forming covalent bonds. In this study the effect of different modification methods of activated carbon (functionalization with genipin, metallization, metallization in the presence of chelating agent, and functionalization with glutaraldehyde) on efficiency of pepsin immobilization was evaluated. The effect of immobilization pH and the reaction medium on hydrolysis activity of bovine casein was also evaluated. The functionalization of activated carbon using iron ions allowed an immobilization capacity of 98.93 mg·g, with immobilization efficiency greater than 99%, and enzyme activity of 2.30 U, which was higher than the other modifications, and closer to the enzyme in the native form activity (3.32 U). In general, the carbon surface modifications were responsible for forming more stable bonds between support and enzyme, improving its proteolytic activity (from 1.84 to 2.30 U) when compared to traditional immobilization methods by adsorption and covalent binding using glutaraldehyde (from 1.04 to 1.1 U).
Topics: Adsorption; Animals; Cattle; Enzyme Stability; Enzymes, Immobilized; Glutaral; Hydrogen-Ion Concentration; Pepsin A
PubMed: 35090943
DOI: 10.1016/j.ijbiomac.2022.01.135 -
Medicine Aug 2021A rapid lateral flow test (Peptest) to detect pepsin in saliva/sputum has been considered as a valuable method for diagnosing laryngopharyngeal reflux (LPR) and... (Meta-Analysis)
Meta-Analysis
BACKGROUND
A rapid lateral flow test (Peptest) to detect pepsin in saliva/sputum has been considered as a valuable method for diagnosing laryngopharyngeal reflux (LPR) and gastroesophageal reflux disease (GERD). The aim of this meta-analysis is to analyze the utility of Peptest for diagnosis of LPR and GERD.
METHODS
PubMed, EMBASE, and the Cochran Library (from January 1980 to 26 January 2020) were searched for pepsin in saliva for LPR/GERD diagnosis. Sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, and area under the curve data were summarized to examine the accuracy.
RESULTS
A total of 16 articles that included 2401 patients and 897 controls were analyzed. The pooled sensitivity and specificity for the diagnosis of GERD/LPR with Peptest were 62% (95% confidence interval [CI] 49%-73%) and 74% (95% CI 50%-90%), respectively. The summarized diagnostic odds ratio and area under the curve were 5.0 (95% CI 2-19) and 0.70 (95% CI 0.66-0.74), respectively.
CONCLUSION
Peptest shows moderate diagnostic value for LPR and GERD. More studies with standard protocols should be done to verify its usefulness.
Topics: Biomarkers; Diagnosis, Differential; Gastroesophageal Reflux; Humans; Laryngopharyngeal Reflux; Pepsin A; ROC Curve; Saliva
PubMed: 34397878
DOI: 10.1097/MD.0000000000026756 -
Spectrochimica Acta. Part A, Molecular... Jan 2015Ligupurpuroside A is one of the major glycoside in Ku-Din-Cha, a type of Chinese functional tea. In order to better understand its digestion and metabolism in humans,...
Ligupurpuroside A is one of the major glycoside in Ku-Din-Cha, a type of Chinese functional tea. In order to better understand its digestion and metabolism in humans, the interaction between Ligupurpuroside A and pepsin has been investigated by fluorescence spectra, UV-vis absorption spectra and synchronous fluorescence spectra along with molecular docking method. The fluorescence experiments indicate that Ligupurpuroside A can effectively quench the intrinsic fluorescence of pepsin through a combined quenching way at the low concentration of Ligupurpuroside A, and a static quenching procedure at the high concentration. The binding constant, binding sites of Ligupurpuroside A with pepsin have been calculated. The thermodynamic analysis suggests that non-covalent reactions, including electrostatic force, hydrophobic interaction and hydrogen bond are the main forces stabilizing the complex. According to the Förster's non-radiation energy transfer theory, the binding distance between pepsin and Ligupurpuroside A was calculated to be 3.15 nm, which implies that energy transfer occurs between pepsin and Ligupurpuroside A. Conformation change of pepsin was observed from UV-vis absorption spectra and synchronous fluorescence spectra under experimental conditions. In addition, all these experimental results have been validated by the protein-ligand docking studies which show that Ligupurpuroside A is located in the cleft between the domains of pepsin.
Topics: Animals; Binding Sites; Energy Transfer; Glycosides; Kinetics; Molecular Conformation; Molecular Docking Simulation; Pepsin A; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Sus scrofa; Temperature
PubMed: 25078459
DOI: 10.1016/j.saa.2014.06.087 -
Acta Oto-laryngologica Aug 2021Laryngopharyngeal reflux (LPR) is a prevalent disease in the ENT outpatient department. Methods of LPR diagnosis differ much. Narrow-band imaging (NBI) and Salivary...
BACKGROUND
Laryngopharyngeal reflux (LPR) is a prevalent disease in the ENT outpatient department. Methods of LPR diagnosis differ much. Narrow-band imaging (NBI) and Salivary pepsin-level measurements have been considered as totally new ways for the diagnosis of LPR in recent years.
AIM
We aimed to identify the specific manifestation of LPR and assess the correlation between NBI views and pepsin levels in LPR diagnosing.
MATERIALS AND METHODS
130 Patients enrolled in our study were divided into LPR group and non-LPR group according to the scores of RSI and RFS. All individuals received endoscopic NBI tests and salivary pepsin measurements. NBI views of the larynx were divided into four grades based on the existing descriptive guidelines and our clinical observations, and the correlation between NBI grading and salivary pepsin levels was calculated.
RESULTS
NBI grading and pepsin levels are significantly correlated with RFS/RSI scores. The diagnostic concentration of salivary pepsin was 33 ng/ml. There was a significant correlation between NBI grading and salivary levels.
CONCLUSIONS AND SIGNIFICANCE
NBI grading system was proved to provide a better diagnostic value in the diagnosis of LPR. There was a possibility that a combination of these two methods might increase the diagnostic accuracy of this disease.
Topics: Adult; Female; Humans; Laryngopharyngeal Reflux; Laryngoscopy; Male; Narrow Band Imaging; Pepsin A; ROC Curve; Saliva; Sensitivity and Specificity
PubMed: 34296957
DOI: 10.1080/00016489.2021.1950929 -
Diseases of the Esophagus : Official... Mar 2023Gastroesophageal reflux disease (GERD) is primarily diagnosed based on symptoms and response to a proton-pump inhibitor (PPI) trial. Gold standard testing requires an...
Gastroesophageal reflux disease (GERD) is primarily diagnosed based on symptoms and response to a proton-pump inhibitor (PPI) trial. Gold standard testing requires an invasive endoscopic procedure, often with ambulatory pH monitoring. Salivary pepsin is a potential noninvasive modality for GERD diagnosis. This study aimed to assess diagnostic performance of salivary pepsin thresholds for GERD and determine optimal collection protocol of saliva in an external validation cohort. Over 10 months, adults with symptoms of GERD undergoing esophagogastroduodenoscopy with wireless pH-monitoring off PPI were enrolled. Saliva was self-collected by participants over 4 days across three different time points: fasting ante meridiem (AM), post-prandial, and bedtime (PM). Pepsin levels were calculated via Peptest. Pepsin variability and agreement were determined using linear mixed effects models and intraclass correlation. Validation of diagnostic threshold and performance characteristics were evaluated by receiver-operator curve analysis. Twenty participants enrolled in the study; 50% with physiologic acid exposure (acid exposure time < 4% no GERD) and 50% with elevated acid exposure (GERD). Mean pepsin concentrations were significantly lower in the AM (22.6 ± 25.2 ng/mL) compared to post-prandial (44.5 ± 36.7 ng/mL) and PM (55.4 ± 47.0 ng/mL). Agreement between pepsin concentrations across 3 days was substantial for AM samples (kappa 0.61), with lower agreement for post-prandial and PM samples. A single AM pepsin concentration of 25 ng/mL was 67% accurate for GERD with 56% sensitivity and 78% specificity. This validation study highlights fair accuracy and performance characteristics of a single fasting AM salivary pepsin concentration for the diagnosis of GERD.
Topics: Adult; Humans; Pepsin A; Sensitivity and Specificity; Gastroesophageal Reflux; Esophageal pH Monitoring; Saliva; Proton Pump Inhibitors
PubMed: 36148576
DOI: 10.1093/dote/doac063 -
The Laryngoscope Jul 2020The aim of this study was to compare the diagnostic accuracy of salivary pepsin with oropharyngeal pH monitoring using the Restech measurement system (Dx-pH) for the... (Comparative Study)
Comparative Study
OBJECTIVES
The aim of this study was to compare the diagnostic accuracy of salivary pepsin with oropharyngeal pH monitoring using the Restech measurement system (Dx-pH) for the diagnosis of laryngopharyngeal reflux (LPR).
STUDY DESIGN
Prospective cohort study.
METHODS
Seventy patients with primary symptoms related to LPR underwent gastroscopy, high-resolution manometry, pH throughout 24-hour monitoring (MII-pH), and barium esophagography between October 2015 and May 2018. In addition, an ear, nose, and throat examination was performed, including assessment of Belafsky Reflux Finding Score (RFS). Clinical symptoms were evaluated with the Belafsky Reflux Symptom Index (RSI) and the Gastrointestinal Quality of Life Index (GIQLI). Simultaneous to MII-pH, pepsin determination and Dx-pH were performed.
RESULTS
Of 70 patients, 41 (58.6%) subjects with a pathological DeMeester score showed higher mean values of pepsin (mean value: 216 ng/mL, 95% confidence interval [CI]: 172 to 260), compared to patients with a normal DeMeester score (mean value: 161 ng/mL, 95% CI: 115 to 207). Salivary pepsin showed a specificity of 86.2% and sensitivity of 41.5% for diagnosing LPR using the optimal cutoff value of 216 ng/mL. Furthermore, a significant correlation between the values of salivary pepsin and the RSI score was seen in patients with pathological results in MII-pH (r = 0.344; P = 0.046). However, elevated Dx-pH measurements showed no significant correlation with either MII-pH, RSI score, RFS score, or GIQLI score, or with the results of pepsin measurement.
CONCLUSION
Pepsin measurement in saliva could be an alternative tool to assist office-based diagnosis of LPR, whereas Dx-pH does not seem to be an adequate test.
LEVEL OF EVIDENCE
2B Laryngoscope, 130:1780-1786, 2020.
Topics: Esophageal pH Monitoring; Female; Follow-Up Studies; Humans; Hydrogen-Ion Concentration; Laryngopharyngeal Reflux; Male; Manometry; Middle Aged; Pepsin A; Pressure; Prospective Studies; Quality of Life; Reproducibility of Results; Saliva
PubMed: 31603541
DOI: 10.1002/lary.28320 -
Waste Management (New York, N.Y.) Feb 2020There is a growing search for alternative raw materials to obtain collagen and hydrolysates and processes that do not threaten the environment or human health. Thus,...
There is a growing search for alternative raw materials to obtain collagen and hydrolysates and processes that do not threaten the environment or human health. Thus, sheep slaughter residue, which doesn't yet have an adequate and sustainable destination, is an excellent source of study. The objective of this study was to investigate the technological properties of collagen extracted from sheep slaughter by-products. It was possible to produce and characterize collagens extracted from sheep slaughter by-products. The yield of collagen was 18.0% and 12.5% for lamb and sheep by-products, respectively, on a dry basis. Lamb and sheep collagens showed similar FTIR and digestibility spectra and increased solubility at acidic pH-value. Higher foaming capacity was found for lamb collagen, while the sheep collagen presented higher viscosity. The emulsifying power of the collagens was 59.1 and 69.6 m/g for lamb and sheep by-products, respectively. The collagens presented bands corresponding to α, α, and β chains, characteristic of collagen type I and a molecular weight (SDS-PAGE) between 100 and 5 kDa. The collagens of this study showed potential for application in food products, both for the technological improvement and nutrient enrichment, adding value and giving a sustainable destination to sheep slaughter by-products.
Topics: Animals; Collagen; Collagen Type I; Humans; Molecular Weight; Pepsin A; Sheep; Solubility
PubMed: 31835061
DOI: 10.1016/j.wasman.2019.12.004 -
Methods in Molecular Biology (Clifton,... 2018Tissue decellularization allows isolation of tissue extracellular matrix components, enabling bioengineering of native tissue microenvironments with minimal antigenic...
Tissue decellularization allows isolation of tissue extracellular matrix components, enabling bioengineering of native tissue microenvironments with minimal antigenic components. Here we describe a method to harvest decellularized cartilage tissue from donor trachea using a series of chemical and enzymatic washes and incorporating the extracellular matrix in gelatin methacrylate hydrogels. This decellularized cartilage tissue is easily incorporated into a variety of hydrogels to create a cartilage tissue scaffold.
Topics: Animals; Cartilage; Deoxyribonucleases; Detergents; Extracellular Matrix; Gelatin; Hydrogels; Methacrylates; Pepsin A; Swine; Tissue Engineering; Tissue Scaffolds; Trachea
PubMed: 28730534
DOI: 10.1007/7651_2017_52 -
Chemico-biological Interactions Apr 2018The interaction of nanoparticles (NPs) with proteins is a topic of high relevance for the medical application of nanomaterials. In the study, a comprehensive...
The interaction of nanoparticles (NPs) with proteins is a topic of high relevance for the medical application of nanomaterials. In the study, a comprehensive investigation was performed for the binding properties of silver nanoparticles (AgNPs) to pepsin. The results indicate that the binding of AgNPs to pepsin may be a static quenching mechanism. Thermodynamic analysis reveals that AgNPs binds to pepsin is synergistically driven by enthalpy and entropy, and the major driving forces are hydrophobic and electrostatic interactions. Synchronous fluorescence spectroscopy shows that AgNPs may induce microenvironmental changes of pepsin. The hydrophobicity of Trp is increased while the hydrophility of Tyr is increased. The adsorption of pepsin on AgNPs was analyzed by Langmuir and Freundlich models, suggesting that the equilibrium adsorption data fit well with Freundlich model. The equilibrium adsorption data were modeled using the pseudo-first-order and pseudo-second-order kinetic equations. The results indicate that pseudo-second-order kinetic equation better describes the adsorption kinetics. The study provides an accurate and full basic data for clarifying the binding mechanism, adsorption isotherms and kinetic behaviors of AgNPs with pepsin. These fundamental works will provide some new insights into the safe and effective application of AgNPs in biological and medical areas.
Topics: Adsorption; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Kinetics; Metal Nanoparticles; Pepsin A; Silver; Spectrometry, Fluorescence; Static Electricity; Temperature; Thermodynamics
PubMed: 29530510
DOI: 10.1016/j.cbi.2018.03.004