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Special Problems with Biological Fluids
Published in Joseph Chamberlain, The Analysis of Drugs in Biological Fluids, 2018
Protein can also be denatured using proteolytic enzymes, a procedure that should avoid the possibility of damage to the analyte using chemical-type denaturation. Such procedures are generally found in the preparation of tissue for drug analysis, but the enzyme subtilisin has been successfully used for the digestion of plasma proteins. Osselton82 showed that the better recoveries of drug from tissue using enzyme hydrolysis compared with direct extraction was also obtained in the analysis of whole blood and plasma. In Osselton’s procedure, 200 µl plasma is buffered to pH 10.5 with 50 µl Tris buffer, enzyme is added, and the mixture incubated at 55°C for 1 h before extraction with 50 µl butyl acetate. The organic phase is then analyzed by an appropriate method. Osselton points out that the enzyme hydrolysis is most useful for general screening procedures where it is not usually possible to optimize the normal extraction procedures for a specific compound. A number of other proteolytic enzymes have been proposed (Table 2.5), which presumably could be adapted for biological fluid assays with detriment to the cited analyte. The various methods of separating protein from solutions containing analytes are reviewed in Table 2.6.
Contact Urticaria, Dermatitis, and Respiratory Allergy Caused by Enzymes
Published in Ana M. Giménez-Arnau, Howard I. Maibach, Contact Urticaria Syndrome, 2014
Stanciu Monica, Denis Sasseville
In the early 1960s, the Danish company Novo Industri successfully produced a heat- and alkali-stable proteolytic enzyme obtained through fermentation of Bacillus subtilis. Named Alcalase, Maxatase, and Esperase among others, subtilisins soon found applications in the harsh environment of laundry detergents, and by the end of the decade, half of the detergents sold in Europe and North America were enzyme-laden.[85] In the years that followed, numerous reports described the occurrence of respiratory and cutaneous symptoms from enzymes in laundry detergents, mostly from occupational exposure.
Proteases as Biocatalysts for the Synthesis of Model Peptides
Published in Willi Kullmann, Enzymatic Peptide Synthesis, 1987
Nα-protected di- and tripeptide methyl esters and amino acid p-nitroanilides were used by Voyushina et al.61 as carboxyl and amine components, respectively, to prepare via subtilisin-catalysis a series of tri- and tetrapeptides. When Z-Ala-Ala-OMe and Z-Ala-Ala-Leu-OMe served as carboxyl components, the authors observed that the coupling efficiency was strongly influenced by the chemical nature of the amine components in the following order: H-Ala-pNA > H-Val-pNA > H-Leu-pNA > H-Phe-pNA. The product yields were signficantly reduced, if the above esterified acyl group donors were replaced by those having a free α-carboxyl group. So far, subtilisin elicits a behavior similar to that of chymotrypsin and trypsin (see above).
Icosapent ethyl: safely reducing cardiovascular risk in adults with elevated triglycerides
Published in Expert Opinion on Drug Safety, 2022
Manan Pareek, R. Preston Mason, Deepak L. Bhatt
The body of evidence supporting icosapent ethyl is rapidly growing, with several mechanistic studies underway. Elegant experiments assessing the differential effects of various omega-3 fatty acids on cell membrane preparations will provide further insights into molecular mechanisms of EPA that produce downstream beneficial effects, such as attenuating inflammation. It is already clear that the benefits of icosapent ethyl are mediated largely through EPA and are independent of baseline triglyceride levels. Thus, while the REDUCE-IT trial used elevated triglycerides to enrich the rate of ischemic events, the results likely apply to patients with lower triglyceride levels than what was studied, assuming such patients are otherwise at high cardiovascular risk. In conclusion, icosapent ethyl will have an increasingly important impact on cardiovascular risk reduction given the growing population of patients with hypertriglyceridemia and other associated risk factors. The evaluations of cost-effectiveness in both primary and secondary prevention have been quite favorable, particularly when compared with more expensive therapies such as the proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, alirocumab, and evolocumab [123,124]. Studies of the generalizability of REDUCE-IT have shown that tens of millions of people worldwide could derive benefit from this medication [125–127]. Implementation of this effective and safe therapy should be a priority in healthcare systems worldwide.
Development of an enzymatic method for the evaluation of protein deposition on contact lenses
Published in Biofouling, 2022
Enzymes cleansers have been used to clean contact lens protein deposits for many years (Begley et al. 1990; Jones et al. 2013; Rejisha and Murugan 2021). Some enzymes such as subtilisin and pancreatin caused allergic reactions in patients, while enzymes such as methyl trypsin caused minimal ocular irritation and have used as a lens care cleaner (Barton et al. 1988). The aim of this in vitro study was to establish an analytical tool for the identification and differentiation of proteins adsorbed onto and into contact lenses. Lysozyme, lactoferrin, albumin and secretory immunoglobulin A (sIgA) were chosen as they are major components of the tear film and there is a large body of information available in the literature regarding the deposition of lysozyme and albumin on contact lenses (Garrett and Milthorpe 1996; Knop and Knop 2005; Luensmann and Jones 2008; Maurya et al. 2014).
Protein amino-termini and how to identify them
Published in Expert Review of Proteomics, 2020
Annelies Bogaert, Kris Gevaert
Essential when directly enriching for N-terminal peptides are labeling methods that are highly selective for α-amines over ε- amines of lysine side-chains. One such labeling method was developed by the Wells lab, who engineered a ligase, termed subtiligase, a variant of the subtilisin protease, which exhibits absolute selectivity for primary (non-blocked) α-amines [69]. Proteomes are treated with subtiligase and a peptide glycolate ester substrate specifically tailored for N-terminomics, leading to the enzymatic biotinylation of protein N-termini. Subsequently, proteomes are digested and the biontinylated N-terminal peptides are captured by avidin beads. A tobacco etch virus (TEV) protease cleavage site present in the biotin-ligated peptides allows selective recovery of N-terminal peptides from the avidin beads (Figure 4). In the original protocol, these recovered N-terminal peptides contained an SY-dipeptide remnant after TEV protease cleavage, which readily allowed to differentiate between captured N-terminal peptides and co-eluted, contaminating peptides following LC-MS/MS data analysis [21]. In later versions of the method, the SY-dipeptide motif was changed for an aminobutyric acid residue, which led to less interference of the SY-tag fragment ions in MS/MS spectra and an MS-discernable, non-natural mass signature [70].