Comparative study of the commonly used protein quantitation assays on different Hymenoptera venoms: A fundamental aspect of Hymenoptera venom proteome analysis.

Determination of protein concentration in Hymenoptera venoms requires an accurate and reproducible assay as the results will be used to support subsequent proteomic techniques employed in their analyses. However, all protein assay techniques have inherent strengths and weaknesses, demanding their assessment before selecting the most suitable platform for sample analysis. In this study, protein profiles of ant, honeybee, and wasp venoms, and bovine serum albumin (BSA) and hyaluronidase standards were qualitatively assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Their amino acid and protein concentration were quantitatively determined via Amino Acid Analysis (AAA). Amino acid concentration was determined via hydrolysis, derivatization, and chromatographic quantification. Protein concentration was estimated using four different protein concentration assays. The ratios of protein concentration in venom samples to protein standards were calculated, and the accuracy of the protein concentration assays was analysed relative to the concentration determined from AAA. SDS-PAGE analysis showed that BSA contained several protein bands, while hyaluronidase contained a mixture of peptide and protein bands. Ant and honeybee venoms contained a higher proportion of peptide bands, while wasp venom contained more protein bands. As determined by AAA, the ratio of protein concentration in Hymenoptera venoms varied between 1.01 and 1.11 to BSA, and between 0.96 and 1.06 to hyaluronidase. Overall, the Bradford assay was found to be the least accurate and the BCA assay was the most accurate in estimating protein concentration in Hymenoptera venoms. There was no significant advantage in using hyaluronidase as a standard or increasing incubation temperature of BCA assay when analysing Hymenoptera venoms. Diluent solutions containing phenol and human serum albumin interfered with Lowry-based assays.

Enhancing hyaluronidase enzyme activity: Insights from advancement in bovine and ovine testicular hyaluronidase purification.

This essay investigates the use of an affinity resin named Capto lentil lectin for the purification of bovine and ovine testicular hyaluronidase. Hyaluronidase, an enzyme that degrades hyaluronic acid, is used widely in medical fields like dermatology, orthopedics, and ophthalmology. The research highlights the importance of optimizing the purification process to increase enzyme activity and purity. A new purification method is proposed, which begins with ammonium sulfate precipitation, followed by Blue Sepharose and Capto Lentil Lectin chromatography. This novel approach significantly increases the yield, purity, and activity of the enzyme. This study paves the way for further research into improving the purification process. The study further discusses challenges in identifying hyaluronidase bands using SDS-PAGE and highlights the necessity of using Western blotting for precise results.

Hyaluronidase Availability in Emergency Rooms: A Statewide Analysis.

Objective: Authors sought to determine the immediate availability of hyaluronidase (HYAL) among emergency rooms (ERs) in California. Background: Hyaluronic acid (HA) fillers are regarded as a safe procedure; however, major ischemic complications do exist, notably blindness and tissue necrosis. The successful management of these vascular events relies on an injector's immediate HYAL, the enzymatic reversal agent for HA. Unfortunately, many barriers exist for injector sites to stock HYAL. As a result, ERs serve as unofficial safety nets in cases when providers encounter an ischemic complication and do not have HYAL in supply. Materials and Methods: Telephone survey inquiring about HYAL availability in all California ERs. Results: This study included 330 California ERs and achieved an 89.7% response rate (n = 296). 45.6% of the surveyed ERs did not have immediate access to HYAL. HYAL availability was positively associated with level I-III adult trauma center status, pediatric trauma center status, children's hospital status, higher bed counts, and regional geography (p < 0.05, all). Conclusions: HYAL availability is unreliable among Californian ERs, posing a potential risk to patient safety.

Haskap Berry Leaves (Lonicera caerulea L.)-The Favorable Potential of Medical Use.

The presented research evaluates the medical use potential of Lonicera caerulea leaves, which are waste plants in cultivating berries. The study's screening activity included the leaves of five varieties of Lonicera caerulea: Atut, Duet, Wojtek, Zojka, and Jugana. The microbiological analysis confirmed the safety of using Lonicera caerulea leaves without significant stabilization. Lonicera caerulea leaves standardization was carried out based on the results of the chromatographic analysis, and it showed differences in the contents of active compounds (loganic, chlorogenic and caffeic acids, and rutin), which are attributed to biological activity. For the Lonicera caerulea leaves varieties tested, the differences in the content of total polyphenol content, chlorophylls, and carotenoids were also confirmed. The screening of biological activity of five Lonicera caerulea leaf varieties was carried out concerning the possibility of inhibiting the activity of α-glucosidase, lipase, and hyaluronidase as well, and the antioxidant potential was determined. The defined profile of the biological activity of Lonicera caerulea leaves makes it possible to indicate this raw material as an essential material supporting the prevention and treatment of type II diabetes. However, this research showed that tested enzymes were strongly inhibited by the variety Jugana. The health-promoting potential of Lonicera caerulea leaves was correlated with the highest chlorogenic acid and rutin content in the variety Jugana.

Sergentomyia schwetzi: Salivary gland transcriptome, proteome and enzymatic activities in two lineages adapted to different blood sources.

During the blood feeding, sand fly females inject saliva containing immunomodulatory and anti-haemostatic molecules into their vertebrate hosts. The saliva composition is species-specific, likely due to an adaptation to particular haemostatic pathways of their preferred host. Research on sand fly saliva is limited to the representatives of two best-studied genera, Phlebotomus and Lutzomyia. Although the members of the genus Sergentomyia are highly abundant in many areas in the Old World, their role in human disease transmission remains uncertain. Most Sergentomyia spp. preferentially attack various species of reptiles, but feeding on warm-blooded vertebrates, including humans and domestic animals, has been repeatedly described, especially for Sergentomyia schwetzi, of which salivary gland transcriptome and proteome is analyzed in the current study. Illumina RNA sequencing and de novo assembly of the reads and their annotation revealed 17,293 sequences homologous to other arthropods' proteins. In the sialome, all proteins typical for sand fly saliva were identified-antigen 5-related, lufaxin, yellow-related, PpSP15-like, D7-related, ParSP25-like, and silk proteins, as well as less frequent salivary proteins included 71kDa-like, ParSP80-like, SP16-like, and ParSP17-like proteins. Salivary enzymes include apyrase, hyaluronidase, endonuclease, amylase, lipase A2, adenosine deaminase, pyrophosphatase, 5'nucleotidase, and ribonuclease. Proteomics analysis of salivary glands identified 631 proteins, 81 of which are likely secreted into the saliva. We also compared two S. schwetzi lineages derived from the same origin. These lineages were adapted for over 40 generations for blood feeding either on mice (S-M) or geckos (S-G), two vertebrate hosts with different haemostatic mechanisms. Altogether, 20 and 40 annotated salivary transcripts were up-regulated in the S-M and S-G lineage, respectively. Proteomic comparison revealed ten salivary proteins more abundant in the lineage S-M, whereas 66 salivary proteins were enriched in the lineage S-G. No difference between lineages was found for apyrase activity; contrarily the hyaluronidase activity was significantly higher in the lineage feeding on mice.

A hyaluronic acid fluorescent hydrogel based on fluorescence resonance energy transfer for sensitive detection of hyaluronidase.

Due to its important role in tumor development and treatment, hyaluronidase (HAase) has been widely investigated in vitro and in vivo. However, such investigation was limited by the absence of sensitive and in situ detection methods. Herein, a hyaluronic acid (HA) hydrogel based on the fluorescence resonance energy transfer (FRET) effect was constructed for the detection of HAase. FITC and AuNPs were covalently coupled with two HA derivatives respectively to form a fluorescent donor-acceptor pair. In the presence of HAase, the hydrogel established by cross-linking of HA derivatives was hydrolyzed specifically. The FRET effect in the hydrogel disappeared and the fluorescence intensity increased proportionally with the changes in the concentration of the HAase. Experiments proved that the HAase sensing system had a wide response range (0.5-100 U/mL), good anti-interference, and excellent biocompatibility. When the hydrogel was used for 3D culture of lung cancer cells, in situ fluorescent response could be achieved. Graphical abstract.

Efficient identification of Acetylcholinesterase and Hyaluronidase inhibitors from Paeonia parnassica extracts through a HeteroCovariance Approach.

ETHNOPHARMACOLOGICAL RELEVANCE: On the basis of the relevant reference in the poem Theriaca of the ancient Greek physician Nicander and its traditional use, Paeonia parnassica was selected for the evaluation of two extracts obtained from the roots and aerial parts to inhibit hydrolytic enzymes involved in snake envenomation. The secondary metabolites which contribute to these activities were detected through a novel HeteroCovariance NMR based approach. Afterwards these ingredients were isolated, identified and evaluated for their inhibitory potency. AIM OF THE STUDY: The identification of acetylcholinesterase and hyaluronidase inhibitors from Paeonia parnassica extracts was used as a case study for the introduction of a recently developed methodology to evaluate ethnopharmacological data and exploit them for the discovery of bioactive natural compounds. This process is based on the fractionation of the selected extracts and the simultaneous phytochemical analysis and biological assessment of the resulting fractions, which permits the rapid detection of the specified secondary metabolites prior to any laborious and time-consuming purification. MATERIALS AND METHODS: The roots and aerial parts of P. parnassica were extracted using methanol: water 50:50 and the two resulted extracts were fractionated by Centrifugal Partition Chromatography. The obtained fractions were evaluated in-vitro for their ability to inhibit acetylcholinesterase and hyaluronidase enzymes and their 1H NMR spectra were recorded. The biological activity was statistically correlated with the spectral data through the HeteroCovariance Approach (HetCA). Finally the purification, identification and biological evaluation of targeted secondary metabolites were carried out. RESULTS: The general chemical structures and some explicit secondary metabolites which contribute (e.g. gallotannins, gallic acid derivatives) or not (characteristic "cage-like" monoterpenes of the genus, glycosylated flavonoids) to the anti-acetylcholinesterase and anti-hyaluronidase activities were detected through HetCA. The consequent isolation and biological evaluation of targeted compounds were performed in order to validate the effectiveness and precision of the methodology. This procedure revealed the most active ingredients of both extracts obtained from roots and aerial parts against the above mentioned biological targets, as well as other compounds possessing moderate activity. CONCLUSIONS: The results of this study contributed to the verification of the ancient text Theriaca regarding the use of Paeonia parnassica to treat the snake bite symptoms. Furthermore, the ingredients of the Paeonia parnassica extracts, which were responsible for their anti-cholinesterase and anti-hyaluronidase activities, were determined applying a HetCA methodology before their isolation. Therefore, the current work provides clear evidence that HetCA could consist an efficient tool for the exploitation of traditional medicine information in order to discover bioactive natural compounds and develop new pharmacotherapies which serve the needs of contemporary medicine.

Investigation of the surface confinement effect of copper nanoclusters: construction of an ultrasensitive fluorescence turn-on bio-enzyme sensing platform.

Copper nanoclusters (CuNCs) have attracted considerable research interest due to their good physicochemical properties, ease of preparation, and low price. However, the low quantum yield and poor stability in aqueous solutions have greatly limited their applications. In order to improve the fluorescence properties and stability of CuNCs, in this paper, the surface confinement effect of CuNCs based on 2D layered double hydroxide (LDH) was proposed to prepare the fluorescent composites of glutathione protected CuNCs and LDH (GS-CuNCs/LDH) with excellent quantum yield and long fluorescence lifetime. Moreover, a novel, simple, and ultrasensitive fluorescence assay for the detection of hyaluronidase was proposed based on the surface confinement effect. The limit of detection for hyaluronidase was as low as 0.014 U mL-1. For the first time, this work developed a bio-enzyme sensing platform based on the surface confinement effect, which can serve as a promising candidate in biosensing.

Sensitive Hyaluronidase Biosensor Based on Target-Responsive Hydrogel Using Electronic Balance as Readout.

The development of simple but sensitive methods for hyaluronidase (HAase) detection has been paid a great deal of attention because HAase is a potential cancer marker. In this work, a novel system coupled with a controlled release system has been designed for HAase determination without complex analytical instruments and skilled technicians. Pt@SiO2 nanoparticles (NPs), which can catalyze the breakdown of H2O2 into O2 and H2O, was embedded in the hydrogel constructed by polyethylenimine (PEI) and hyaluronic acid (HA). In the presence of HAase, the hydrogel was broken down as HAase can catalyze the degradation of HA and hence the Pt@SiO2 NPs in the hydrogel was released. The released Pt@SiO2 NPs mixed with H2O2 solution in a drainage device, and then O2 was generated due to the decomposition of H2O2, resulting in an enhancement of pressure in the drainage device because of the low solubility of O2. A certain amount of H2O overflowed from the drainage device because the difference of the pressure between the inner and outer of the drainage device. The overflowed H2O was collected by a tube, and its amount was easily measured by an electronic balance. The weight of the H2O has a linear relationship with the HAase concentration in the range of 1-60 U/mL (120 min enzymatic hydrolysis time) and 0.2-10 U/mL (240 min enzymatic hydrolysis time). The developed system has been applied to detect the activity of HAase in urine samples with satisfied results.

Molecular composition of the paralyzing venom of three solitary wasps (Hymenoptera: Pompilidae) collected in southeast Mexico.

The chemical and biological characterization of peptide and protein components of the paralyzing venom from three Pompilidae solitary spider wasps (Pepsis mexicana, Pepsis terminata, and Anoplius nigritus) is described for the first time. The molecular masses of the most abundant peptides were determined. The N-terminal sequences of two cysteine-rich peptides were obtained from Pepsis. Metalloproteinase and hyaluronidase activities were identified in the venom of P. mexicana. A novel non-lethal method to collect venom is described.

Differentiation of Intracellular Hyaluronidase Isoform by Degradable Nanoassembly Coupled with RNA-Binding Fluorescence Amplification.

Hyaluronidase has two cruical isoforms, hyaluronidase-1 (Hyal-1) and hyaluronidase-2 (Hyal-2), which are essential for cellular hyaluronic acid (HA) catabolism to generate different-sized oligosaccharide fragments for performing different physiological functions. In particular, Hyal-1 is the major tumor-derived hyaluronidase. Thus, specific detection of one hyaluronidase isoform, especially Hyal-1, in live cells is of scientific significance but remains challenging. Herein, by use of differentiated tolerance capability of an amphiphilic HA-based nanoassembly to Hyal-1 and Hyal-2, we rationally design a Hyal-1 specific nanosensor, consisting of cholesterylamine-modified HA nanoassembly (CHA) and RNA-binding fluorophores (RBF). The RBF molecules were entrapped in CHA to switch off their fluorescence via aggregation caused quenching. However, CHA can be disassembled by Hyal-1 to release RBF, resulting in fluorescence activation. Moreover, the fluorescence of the released RBF is further enhanced by cytoplasm RNA. Owing to this cascade signal amplification, this nanosensor RBF@CHA displays a significant change of signal-to-background-noise ratio (120-fold) toward 16 µg/mL Hyal-1 in cellular lysates. In contrast, it is resistant to Hyal-2. By virtue of its selective and sensitive characteristics under a complicated matrix, RBF@CHA had been successfully applied for specifically visualizing Hyal-1 over Hyal-2 inside live cells for the first time, detecting a low level of intracellular Hyal-1 and distinguishing normal and cancer cells with different expressions of Hyal-1. This approach would be useful to better understand biological functions and related diseases of intracellular Hyal-1.

Analyzing Hyaluronidases in Biological Fluids.

Hyaluronidases are a group of enzymes responsible for the degradation of hyaluronan. They seem to be associated with a plethora of pathological conditions, as it has been showcased by numerous studies over the past years. The emerging role of hyaluronidases in various pathological states, especially cancer, is of a great interest. Thus, they are considered as important research targets.In this chapter the popular assay for hyaluronidase analysis in biological fluids is presented and discussed in detail. The assay is divided into two steps; the first is zymography that aims mainly to detect different hyaluronidase enzymes in a biological sample, and the second is the direct quantitative measurement of enzymatic activity by a microtiter plate assay. Both steps are characterized by high sensitivity, simplicity, and limited time consumption.

Naja annulifera Snake: New insights into the venom components and pathogenesis of envenomation.

BACKGROUND: Naja annulifera is a medically important venomous snake occurring in some of the countries in Sub-Saharan Africa. Accidental bites result in severe coagulation disturbances, systemic inflammation and heart damage, as reported in dogs, and death, by respiratory arrest, in humans. Despite the medical importance of N. annulifera, little is known about its venom composition and the pathogenesis of envenomation. In this paper, the toxic, inflammatory and immunogenic properties of N. annulifera venom were analyzed. METHODOLOGY/PRINCIPAL FINDINGS: Venom proteomic analysis identified 79 different proteins, including Three Finger Toxins, Cysteine Rich Secretory Proteins, Metalloproteinases, Phospholipases A2 (PLA2), Hyaluronidase, L-amino-acid oxidase, Cobra Venom Factor and Serine Proteinase. The presence of PLA2, hyaluronidase, fibrinogenolytic and anticoagulant activities was detected using functional assays. The venom was cytotoxic to human keratinocytes. In an experimental murine model of envenomation, it was found that the venom induced local changes, such as swelling, which was controlled by anti-inflammatory drugs. Moreover, the venom caused death, which was preceded by systemic inflammation and pulmonary hemorrhage. The venom was shown to be immunogenic, inducing a strong humoral immune response, with the production of antibodies able to recognize venom components with high molecular weight and to neutralize its lethal activity. CONCLUSIONS/SIGNIFICANCE: The results obtained in this study demonstrate that N. annulifera venom contains toxins able to induce local and systemic inflammation, which can contribute to lung damage and death. Moreover, the venom is immunogenic, an important feature that must be considered during the production of a therapeutic anti-N. annulifera antivenom.

Proteoglycan-substrate gel zymography for the detection of chondroitin sulfate-degrading enzymes.

Chondroitin sulfate (CS), a linear polysaccharide, is a major component of the cartilage matrix. Although CS plays various roles in several biological and pathological processes, most details regarding its metabolism are still poorly understood. Some CS-degrading enzymes have been identified in mammals, but their expression patterns and localizations remain unclear. Here we present a simple zymography procedure to detect CS-degrading enzymes using salmon nasal cartilage proteoglycans as substrates. This method should be useful to explore CS-degrading enzymes.

Transcriptomic and Proteomic Analyses Reveal the Diversity of Venom Components from the Vaejovid Scorpion Serradigitus gertschi.

To understand the diversity of scorpion venom, RNA from venomous glands from a sawfinger scorpion, Serradigitus gertschi, of the family Vaejovidae, was extracted and used for transcriptomic analysis. A total of 84,835 transcripts were assembled after Illumina sequencing. From those, 119 transcripts were annotated and found to putatively code for peptides or proteins that share sequence similarities with the previously reported venom components of other species. In accordance with sequence similarity, the transcripts were classified as potentially coding for 37 ion channel toxins; 17 host defense peptides; 28 enzymes, including phospholipases, hyaluronidases, metalloproteases, and serine proteases; nine protease inhibitor-like peptides; 10 peptides of the cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 protein superfamily; seven La1-like peptides; and 11 sequences classified as "other venom components". A mass fingerprint performed by mass spectrometry identified 204 components with molecular masses varying from 444.26 Da to 12,432.80 Da, plus several higher molecular weight proteins whose precise masses were not determined. The LC-MS/MS analysis of a tryptic digestion of the soluble venom resulted in the de novo determination of 16,840 peptide sequences, 24 of which matched sequences predicted from the translated transcriptome. The database presented here increases our general knowledge of the biodiversity of venom components from neglected non-buthid scorpions.

Affinity adsorption of bovine hyaluronidase with ligands targeting to active site.

Four affinity ligands were designed from 6-chloromethyluracil and 2-aminobenzimidazole and simulated for the interaction with bovine hyaluronidase-1. Regarding sequence alignment, bovine hyaluronidase-1 precursor showed circa 83.6% similarity with human hyaluronidase-1. Regarding structural modeling and molecular docking, bovine hyaluronidase-1 interacted with ligands in the active site. Using epichlorohydrin, 1,3-propanediamine and cyanuric chloride as spacers, 6-chloromethyluracil and 2-aminobenzimidazole were composed to Sepharose beads. The modified Sepharose beads were then subjected to adsorption analysis with bovine hyaluronidase. After one step of affinity adsorption, the samples extracted from bovine testes were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and activity assay. As calculated, the densities of four ligands on sorbents (entitled as L-1, L-2, L-3 and L-4) were 37.7 ±â€¯2.3, 36.4 ±â€¯3.2, 42.4 ±â€¯4.2 and 33.7 ±â€¯2.3 µmol/g wet gel; the theoretical maximum adsorption (Qmax) of bovine hyaluronidase on the four sorbents were 63.6 ±â€¯1.6, 72.0 ±â€¯0.7, 111.0 ±â€¯4.1 and 121.7 ±â€¯2.3 mg/g wet gel, respectively; the dissociation constants (Kd) of the four sorbents were 18.5 ±â€¯0.8, 48.1 ±â€¯4.3, 35.0 ±â€¯3.0, 40.6 ±â€¯2.7 µg/g wet gel, respectively. After optimization, the proteins captured by sorbents attaching 2-aminobenzimidazole based ligands (L-3 and L-4) revealed the main single band at approximately 50 kDa, and the purities were about 85.2 and 96.4%; the bioactivity recoveries were 83.5 and 89.4%. In addition, the bands on SDS-PAGE gel were also extracted and confirmed with linear trap quadropole mass spectrometry (LTQ-MS) analysis.

6% Hydroxyethyl starch (HES 130/0.4) diminishes glycocalyx degradation and decreases vascular permeability during systemic and pulmonary inflammation in mice.

BACKGROUND: Increased vascular permeability is a pathophysiological hallmark of sepsis and results in increased transcapillary leakage of plasma fluid, hypovolemia, and interstitial edema formation. 6% hydroxyethyl starch (HES 130/0.4) is commonly used to treat hypovolemia to maintain adequate organ perfusion and oxygen delivery. The present study was designed to investigate the effects of 6% HES 130/0.4 on glycocalyx integrity and vascular permeability in lipopolysaccharide (LPS)-induced pulmonary inflammation and systemic inflammation in mice. METHODS: 6% HES 130/0.4 or a balanced electrolyte solution (20 ml/kg) was administered intravenously 1 h after cecal ligation and puncture (CLP) or LPS inhalation. Sham-treated animals receiving 6% HES 130/0.4 or the electrolyte solution served as controls. The thickness of the endovascular glycocalyx was visualized by intravital microscopy in lung (LPS inhalation model) or cremaster muscle (CLP model). Syndecan-1, hyaluronic acid, and heparanase levels were measured in blood samples. Vascular permeability in the lungs, liver, kidney, and brain was measured by Evans blue extravasation. RESULTS: Both CLP induction and LPS inhalation resulted in increased vascular permeability in the lung, liver, kidney, and brain. 6% HES 130/0.4 infusion led to significantly reduced plasma levels of syndecan-1, heparanase, and hyaluronic acid, which was accompanied by a preservation of the glycocalyx thickness in postcapillary venules of the cremaster (0.78 ± 0.09 µm vs. 1.39 ± 0.10 µm) and lung capillaries (0.81 ± 0.09 µm vs. 1.49 ± 0.12 µm). CONCLUSIONS: These data suggest that 6% HES 130/0.4 exerts protective effects on glycocalyx integrity and attenuates the increase of vascular permeability during systemic inflammation.

Hyaluronic Acid-Modified Porous Silicon Films for the Electrochemical Sensing of Bacterial Hyaluronidase.

The development of enzyme-responsive hyaluronic acid methacrylate (HYAMA)-coated porous silicon (pSi) films and their application in electrochemical diagnostic devices for the in situ detection of the enzyme hyaluronidase (hyal), which is secreted by Staphylococcus aureus (S. aureus) bacteria, are reported. The approach relies on a HYAMA-pSi electrode made of thermally hydrocarbonized pSi (pSi-THC) that is impregnated with crosslinked HYAMA/polyethylene glycol diacrylate (PEGDA) hydrogels. The enzymatic degradation of HYAMA by bacterial hyal is monitored by differential pulse voltammetry (DPV) utilizing pSi-THC as a working electrode and ferro/ferricyanide (FF) as external redox probe. The degradation of HYAMA results in reduced diffusion of the redox probe through the partially charged film, thereby enabling the detection of hyal by DPV. In addition to the determination of the concentration-dependent response in NaOAc buffer (pH 5.2), the detection of hyal as indicator for the presence of S. aureus bacteria above a threshold level in bacterial supernatants and artificial wound fluid is highlighted.

Use of chromatographic and electrophoretic tools for assaying elastase, collagenase, hyaluronidase, and tyrosinase activity.

Elastase, collagenase, hyaluronidase and tyrosinase, are very interesting enzymes due to their direct implication in skin aging and as therapeutic hits. Different techniques can be used to study these enzymes and to evaluate the influence of effectors on their kinetics. Nowadays, analytical techniques have become frequently used tools for miniaturizing enzyme assays. The main intention of this article is to review chromatographic and electrophoretic tools that study the four enzymes above mentioned. More specifically, the use of high-performance liquid chromatography and capillary electrophoresis and their derivative techniques for monitoring these enzymes will be investigated. The advantages and limitations of these assays will also be discussed. The original use of microscale thermophoresis and thin layer chromatography in this domain will also be covered.

Cationic Carbon Dots for Modification-Free Detection of Hyaluronidase via an Electrostatic-Controlled Ratiometric Fluorescence Assay.

Carbon dots (CDs) emerge as excellent fluorescent nanomaterials, but the full exploitation and application of their exceptional properties in the development of fluorescence assay are still rare. In this work, cationic carbon dots (C-CDs) covered with plenty of positive charges on the surface were synthesized through a facile ultrasonic method. Negatively charged hyaluronic acid (HA) caused the aggregation of positively charged C-CDs and neutral red (NR) along its linear chain via electrostatic adsorption, leading to a remarkable Förster resonance energy transfer (FRET) from C-CDs to NR. However, the presence of hyaluronidase (HAase) resulted in the enzymolysis of HA, as well as the liberation of C-CDs and NR. The corresponding change of fluorescence color from red to green-yellow afforded a reliable ratiometric assay for HAase. Also the ratio of fluorescence intensity for C-CDs (I525) to that for NR (I630) was used for quantitative detection of HAase. The proposed sensing system was easily operated in aqueous media with a detection limit of 0.05 U/mL. This strategy provides a new approach for the wider application of some special CDs in detecting biomolecules.