An optimized protocol to assess trypsin activity in biological samples

Hadwan, Mahmoud Hussein; Al-Obaidy, Saba S. M.; Al-Kawaz, Hawraa Saad; Almashhedy, Lamia A.; Kadhum, Mohammed A.; khudhair, Dunia Abbas; Hadwan, Asad M.; Hadwan, Muntadher M.

doi:10.1007/s00706-022-03028-1

Cited by 3 publications

(2 citation statements)

References 41 publications

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“…Cell-derived cytotoxicity studies, animal experiments, rat studies and laboratory studies have become frequently used in laboratories due to their ease of application and suitability for data 20,21 . The method used to evaluate cell viability and choosed in this study is the determination of lactate dehydrogenase (LDH) activity released from damaged/dead cells into the medium 22 . Lactate dehydrogenase is a cytoplasmic enzyme found in all cells.…”

Section: Discussionmentioning

confidence: 99%

The Cell Viability of Dental Materials by The Lactate Dehydrogenase Assay

Kamalak

2024

Int J Health Sci Res

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Objectives: the objective of this study is to search the cell membrane damage of resin composite materials on cultures of human gingival fibroblasts and neuron cells by the lactate dehydrogenase (LDH) test method. Methods: A range of thirteen resin composites sterilized under UV light for 2 hours were added to the culture medium of human gingival fibroblasts and neuron for 24 h and 72h. The samples were moved to 48- well-plates in a sterile cabin, one by one, in direct contact with the cells. After 24- and 72-hours incubation period, in the incubator at 37 oC with 5% CO2; Cell membrane damage was detected by the lactate dehydrogenase (LDH) test method. Results: In the lactate dehydrogenase (LDH) test method, cytotoxicity is determined by measuring lactate dehydrogenase, which is a cytosolic enzyme released in the event of membrane damage or cytolysis in the cell. A higher LDH value indicates that the toxicity of the material is greater. When the LDH results in Gingival fibroblast cells were examined after 24 hours, F25 was found to be the most toxic, and when the LDH results in neuron cells were examined, ISM and F25 were also found to be the most toxic. Significance: Dental restorative materials have some toxic effects on cell cultures. The LDH analysis supplies precious instructions about their toxic effects. Key words: Lactate dehydrogenase, fibroblasts, neuron cells, cytotoxicity

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Section: Discussionmentioning

confidence: 99%

The Cell Viability of Dental Materials by The Lactate Dehydrogenase Assay

Kamalak

2024

Int J Health Sci Res

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“…Trypsin digestion can be performed by several ways, e.g., in solution, via trypsin spin columns or on-line (immobilized trypsin reactor coupled with liquid chromatography or capillary electrophoresis systems) [26][27][28]. While digestion in solution requires the presence of a buffer (Tris-HCl), strict pH, and temperature control (pH = 7.8, temperature 37.0 °C) [29], and is very time consuming, digestion via spin column may be a suitable alternative, which is easy and fast [30].…”

Section: Introductionmentioning

confidence: 99%

The benefits of mixed-mode chromatography columns for separation of peptides and protein digests

Kadlecová,

Boudová,

Kalíková

2023

Monatsh Chem

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In this work, the evaluation and comparison of mixed-mode chromatography and reversed-phase chromatography for separation of peptides and protein digests have been performed. The effects of pH of aqueous part of mobile phase as well as the effects of organic modifier on retention, resolution, and peak shape were investigated on several columns including three mixed-mode columns possessing reversed-phase/anion-exchange mechanism, two reversed-phase octadecyl columns, and one column with mixed-mode reversed-phase/anion-exchange character only in defined pH range. The set of peptides varying in their polarity, length, amino acid sequence, and charge state, namely dipeptides, N-blocked dipeptides, and oligopeptides, was selected to describe the chromatographic behavior under different conditions properly. These measurements showed the potential of mixed-mode chromatography columns for analysis of differently charged peptides in a single run. The applicability of the tested conditions has been verified by the analysis of cytochrome C digested fragments. Two types of samples were analyzed and compared, i.e., commercial cytochrome C digested standard and cytochrome C digested via trypsin spin columns. The obtained results point to the necessity of using mass spectrometry detection because of large number of unknown peaks in cytochrome C digested standard, probably originating from chymotryptic and miscleavage activities. Graphical abstract

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NIR-Sensitive Squaraine Dye—Peptide Conjugate for Trypsin Fluorogenic Detection

Balyan,

Gupta,

Mavileti

et al. 2024

Biosensors

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Trypsin enzyme has gained recognition as a potential biomarker in several tumors, such as colorectal, gastric, and pancreatic cancer, highlighting its importance in disease diagnosis. In response to the demand for rapid, cost-effective, and real-time detection methods, we present an innovative strategy utilizing the design and synthesis of NIR-sensitive dye–peptide conjugate (SQ-3 PC) for the sensitive and selective monitoring of trypsin activity by fluorescence ON/OFF sensing. The current research deals with the design and synthesis of three unsymmetrical squaraine dyes SQ-1, SQ-2, and SQ-3 along with a dye–peptide conjugate SQ-3-PC as a trypsin-specific probe followed by their photophysical characterizations. The absorption spectral investigation conducted on both the dye alone and its corresponding dye–peptide conjugates in water, utilizing SQ-3 and SQ-3 PC respectively, reveals enhanced dye aggregation and pronounced fluorescence quenching compared to observations in DMSO solution. The absorption spectral investigation conducted on dye only and corresponding dye–peptide conjugates in water utilizing SQ-3 and SQ-3 PC, respectively, reveals not only the enhanced dye aggregation but also pronounced fluorescence quenching compared to that observed in the DMSO solution. The trypsin-specific probe SQ-3 PC demonstrated a fluorescence quenching efficiency of 61.8% in water attributed to the combined effect of aggregation-induced quenching (AIQ) and fluorescence resonance energy transfer (FRET). FRET was found to be dominant over AIQ. The trypsin-mediated hydrolysis of SQ-3 PC led to a rapid and efficient recovery of quenched fluorescence (5-fold increase in 30 min). Concentration-dependent changes in the fluorescence at the emission maximum of the dyes reveal that SQ-3 PC works as a trypsin enzyme-specific fluorescence biosensor with linearity up to 30 nM along with the limit of detection and limit of quantification of 1.07 nM and 3.25 nM, respectively.

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An optimized protocol to assess trypsin activity in biological samples

Cited by 3 publications

References 41 publications

The Cell Viability of Dental Materials by The Lactate Dehydrogenase Assay

The Cell Viability of Dental Materials by The Lactate Dehydrogenase Assay

The benefits of mixed-mode chromatography columns for separation of peptides and protein digests

NIR-Sensitive Squaraine Dye—Peptide Conjugate for Trypsin Fluorogenic Detection

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