Positive cooperativity in substrate binding of human prostatic acid phosphatase entrapped in AOT–isooctane–water reverse micelles

Luchter-Wasylewska, Ewa; Iciek, Małgorzata

doi:10.1016/j.jcis.2004.01.075

Cited by 8 publications

(2 citation statements)

References 43 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…At low degrees of hydration, the micelles have too small an internal cavity to accommodate the optimal amount of substrate and FRET agent (R6G). The catalitic paramenter obtained using the new FRET approach are consistent with the literature data obtained by other methods [31,37].…”

Section: Acid Phosphatase Activity In Buffer Solution and In Aot-octa...supporting

confidence: 88%

FRET Probes as a Technique for Determining the Catalytic Activity of Enzymes in the Model Biomembranes Systems and Further in the Living Cells

Zlotnikov,

Ezhov,

Kudryashova

2024

Preprint

View full text Add to dashboard Cite

There are several well-known methods for determining enzyme catalytic activity, including spectral techniques and electrochemical approaches. However, at present, there is no definitive solution to the challenges associated with the fluorescent approach in, for instance, systems of reverse micelles, non-aqueous media, and model biomembranes. We propose the use of the fluorescent resonant energy transfer (FRET) technique, which can enhance the selectivity of fluorescent detection of enzyme activity by shifting the maximum emission wavelength to the longer-wavelength range and reducion the influence of medium components. As fluorescence donors, we have used derivatives of 4-methylumbelliferone (MUmb) and 7-amino-4-methylcoumarin (AMC), which also serve as substrates for hydrolases such as chymotrypsin, phosphatase, and asparaginase. During the catalytic depletion of substrates, an accumulation of a highly fluorescent product is observed. This product can be specifically detected using the fluorescent marker rhodamine 6G (R6G), which emits light in the red region of the spectrum (550–600 nm) with high quantum yield. A significant FRET effect is observed when forming AOT reverse micelles. This is due to the proximity of fluorophores within the hydrophobic core of the micelle, which is shorter than in a buffer solution. This approach have practical implications for visualizing intracellular processes. The FRET technique can be used to determine intracellular enzymatic activity by detection with confocal laser scanning microcopy (CLSM), as demonstrated with L-asparaginase, a clinically important anti-leukemia medication. The approach suggested will allow researchers to study enzyme activity within cells. This has the potential to revolutionize medicine by improving drug development and shedding light on previously unknown aspects of cellular metabolism.

show abstract

Section: Acid Phosphatase Activity In Buffer Solution and In Aot-octa...supporting

confidence: 88%

FRET Probes as a Technique for Determining the Catalytic Activity of Enzymes in the Model Biomembranes Systems and Further in the Living Cells

Zlotnikov,

Ezhov,

Kudryashova

2024

Preprint

View full text Add to dashboard Cite

show abstract

“…[25][26][27] The reverse micelles of AOT provide a controlled water environment in an apolar medium, and this feature can be used to mimic the water-biomembrane interface and to probe membrane interaction with short biologically active peptides, proteins and enzymes. [28][29][30] The interactions of AOT micelles with serum proteins in aqueous media have not been investigated yet. Although Oliveira, Jr, et al have examined the interactions of AOT with BSA at the air-solution interface.…”

Section: Introductionmentioning

confidence: 99%

Twin-tailed surfactant induced conformational changes in bovine serum albumin: a detailed spectroscopic and physicochemical study

Kaur

Mahajan

2014

RSC Adv.

View full text Add to dashboard Cite

The interactions of a twin-tailed anionic surfactant sodium bis-2-ethylhexyl sulfosuccinate (AOT) and cationic surfactant ditetradecyldimethylammonium bromide (DTDAB) with bovine serum albumin (BSA) has been examined using various spectroscopic and physicochemical techniques such as tensiometry, steady-state fluorescence, potentiometry and isothermal titration calorimetry (ITC). The interactional behavior of the surfactants with BSA at the air-solution interface which depends upon the nature of the surfactant was examined and is discussed in detail. Steady-state fluorescence, and potentiometry combined with ITC measurements provide vital insights into the binding mechanism of the surfactants with BSA. An enhancement in the intrinsic fluorescence intensity and a strong exothermic enthalpy change at low concentrations of AOT demonstrated the crosslinking of AOT monomers between a group of non-polar residues and a positively charged residue located on different helical loops of BSA.The quenching of the intrinsic fluorescence intensity and endothermic enthalpy changes were observed over the whole of the concentration range of DTDAB that was studied indicating powerful interactions between them. The synchronous fluorescence spectra revealed the conformational changes in the peptide backbone of BSA and the altered environment of tryptophan and tyrosine residues. The binding isotherms obtained from intrinsic fluorescence spectroscopy as well as from potentiometry were analyzed using Scatchard plots to obtain insights into the binding mechanism and to evaluate the binding constants and the number of binding sites.

show abstract

Micro and Nano Systems in Biomedicine and Drug Delivery

Hasırcı

Nanomaterials and Nanosystems for Biomedical Applications

View full text Add to dashboard Cite

Positive cooperativity in substrate binding of human prostatic acid phosphatase entrapped in AOT–isooctane–water reverse micelles

Cited by 8 publications

References 43 publications

FRET Probes as a Technique for Determining the Catalytic Activity of Enzymes in the Model Biomembranes Systems and Further in the Living Cells

FRET Probes as a Technique for Determining the Catalytic Activity of Enzymes in the Model Biomembranes Systems and Further in the Living Cells

Twin-tailed surfactant induced conformational changes in bovine serum albumin: a detailed spectroscopic and physicochemical study

Micro and Nano Systems in Biomedicine and Drug Delivery

Contact Info

Product

Resources

About