Fluorescence and FTIR study of pressure‐induced structural modifications of horse liver alcohol dehydrogenase (HLADH)

Trovaslet, Marie; Dallet‐Choisy, Sandrine; Meersman, Filip; Heremans, Karel; Balny, Claude; Legoy, Marie‐Dominique

doi:10.1046/j.1432-1033.2003.03370.x

Cited by 19 publications

(15 citation statements)

References 59 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This fluorescence emission maximum is characteristic of tryptophan (Trp) placed in relatively hydrophobic environment [45]. Olofsson [46] have found that TbADH possesses four tryptophan residues, partially exposed on the surface Trp14 and Trp110 and buried in the subunit Trp90 and Trp281.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Gold and silver nanoparticles for biomolecule immobilization and enzymatic catalysis

et al. 2012

View full text Add to dashboard Cite

In this work, a simple method for alcohol synthesis with high enantiomeric purity was proposed. For this, colloidal gold and silver surface modifications with 3-mercaptopropanoic acid and cysteamine were used to generate carboxyl and amine functionalized gold and silver nanoparticles of 15 and 45 nm, respectively. Alcohol dehydrogenase from Thermoanaerobium brockii (TbADH) and its cofactor (NADPH) were physical and covalent (through direct adsorption and using cross-linker) immobilized on nanoparticles' surface. In contrast to the physical and covalent immobilizations that led to a loss of 90% of the initial enzyme activity and 98% immobilization, the use of a cross-linker in immobilization process promoted a loss to 30% of the initial enzyme activity and >92% immobilization. The yield of NADPH immobilization was about 80%. The best results in terms of activity were obtained with Ag-citr nanoparticle functionalized with carboxyl groups (Ag-COOH), Au-COOH(CTAB), and Au-citr functionalized with amine groups and stabilized with CTAB (Au-NH2(CTAB)) nanoparticles treated with 0.7% and 1.0% glutaraldehyde. Enzyme conformation upon immobilization was studied using fluorescence and circular dichroism spectroscopies. Shift in ellipticity at 222 nm with about 4 to 7 nm and significant decreasing in fluorescence emission for all bioconjugates were observed by binding of TbADH to silver/gold nanoparticles. Emission redshifting of 5 nm only for Ag-COOH-TbADH bioconjugate demonstrated change in the microenvironment of TbADH. Enzyme immobilization on glutaraldehyde-treated Au-NH2(CTAB) nanoparticles promotes an additional stabilization preserving about 50% of enzyme activity after 15 days storage. Nanoparticles attached-TbADH-NADPH systems were used for enantioselective (ee > 99%) synthesis of (S)-7-hydroxy-2-tetralol.

show abstract

Section: Resultsmentioning

confidence: 99%

“…Observed changes in fluorescence intensity and λ max can be attributed to the change in Trp environment, which alter enzyme conformation. Such an alteration was associated with a new conformational state (unfolded state) [45]. …”

Section: Resultsmentioning

confidence: 99%

Gold and silver nanoparticles for biomolecule immobilization and enzymatic catalysis

et al. 2012

View full text Add to dashboard Cite

show abstract

“…[17,18] In one study, the enzyme alcohol dehydrogenase was reported to lose activity in the HP range of 100-400 MPa. [30] Effect of PPO and HP/PPO on IgE binding Figure 4 shows the inhibition of IgE binding to the plate by peanut extracts treated at 3 min with PPO and HP/PPO, respectively, in ELISA. IgE binding was reduced by the PPO-treated extract with an IC 50 (inhibitor concentration at 50% inhibition) value of 0.9 μg/mL, compared to 0.5 μg/mL for the control, which means the reduction was almost twofold (i.e.…”

Section: Effect Of Ppo Versus a Combined Hp/ppomentioning

confidence: 99%

Reducing peanut allergens by high pressure combined with polyphenol oxidase

Chung

Houška

Reed

2013

High Pressure Research

View full text Add to dashboard Cite

Polyphenol oxidase (PPO) has been shown to reduce major peanut allergens. Since high pressure (HP) can increase enzyme activity, we postulated that further reduction of peanut allergens can be achieved through HP combined with PPO. Peanut extracts containing caffeic acid were treated with each of the following: (1) HP; (2) HP + PPO; (3) PPO; and (4) none. HP was conducted at 300 and 500 MPa, each for 3 and 10 min, 37 • C. After treatment, SDS-PAGE was performed and allergenic capacity (IgE binding) was determined colorimetrically in inhibition enzyme-linked immunosorbent assay and Western blots, using a pooled plasma from peanut-allergic patients. Data showed that HP alone had no effect on major peanut allergens. However, HP at 500 MPa combined with PPO (HP500/PPO) induced a higher (approximately twofold) reduction of major peanut allergens and IgE binding than PPO alone or HP300/PPO. There was no difference between treatment times. We concluded that HP500/PPO at 3-min enhanced a twofold reduction of the allergenic capacity of peanut extracts, as compared to PPO itself.

show abstract

“…3B, solid line) shows a two‐state transition, accompanied by an increase in ANS‐binding fluorescence and a loss of enzymatic activity [25], indicating exposure of the hydrophobic core to the solvent; further conformational change induced by higher pressures of 500–700 MPa is not complete, even at 700 MPa. Such a complex pressure‐induced transition has been observed and interpreted as a reflection of ‘multiple molten globule‐like state transitions’[26–29].…”

Section: Structural Properties Of Mature Form (Cpy)mentioning

confidence: 99%

The propeptide in the precursor form of carboxypeptidase Y ensures cooperative unfolding and the carbohydrate moiety exerts a protective effect against heat and pressure

Kato¹,

Sato²,

Shirai³

et al. 2003

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

The heat-and pressure-induced unfolding of the glycosylated and unglycosylated forms of mature carboxypeptidase Y and the precursor procarboxypeptidase Y were analysed by differential scanning calorimetry and/or by their intrinsic fluorescence in the temperature range of 20-75°C or the pressure range of 0.1-700 MPa. Under all conditions, the precursor form showed a clear two-state transition from a folded to an unfolded state, regardless of the presence of the carbohydrate moiety. In contrast, the mature form, which lacks the propeptide composed of 91 amino acid residues, showed more complex behaviour: differential scanning calorimetry and pressure-induced changes in fluorescence were consistent with a three-step transition. These results show that carboxypeptidase Y is composed of two structural domains, which unfold independently but that procarboxypeptidase Y behaves as a single domain, thus ensuring cooperative unfolding. The carbohydrate moiety has a slightly protective role in heat-induced unfolding and a highly protective role in pressure-induced unfolding.Keywords: carboxypeptidase Y; fluorescence spectrometry; pressure unfolding; procarboxypeptidase Y; thermal unfolding.Carboxypeptidase Y (CPY), a member of the serine carboxypeptidase family, is a 61-kDa vacuolar enzyme obtained from Saccharomyces cerevisiae [1]. This enzyme is synthesized in the form of procarboxypeptidase Y (pro-CPY) and sorted to the vacuole via the Golgi apparatus where it undergoes carbohydrate modification. ProCPY has an N-terminal extension (propeptide) of 91 residues [2,3], compared to the mature CPY. This propeptide structure is essential for folding both in vivo and in vitro, as well as for maintaining CPY in an inactive form [4][5][6]. The mature and precursor forms are glycoproteins [7], which contain % 16% carbohydrates [8]: the four carbohydrate chains are of similar sizes and are bound to asparagine residues at Asn-Xaa-Thr glycosylation sites [9][10][11][12]. The genetic replacement of these asparagine residues by alanine residues produces unglycosylated (Dgly) CPY [13] and proCPY with no change in their activities.The reason why the presence of the propeptide is important for the correct folding of CPY and role that the large amount of carbohydrate moiety plays on the stability and function of CPY has not been fully clarified. To answer these questions, we examined the folding/unfolding of mature and precursor CPY as well as their unglycosylated forms using temperature and pressure as the structural perturbant. Compared to heat, pressure studies have been used to obtain complementary information concerning protein-solvent interactions [14,15], the unfolded states of proteins [16,17], and protein folding pathways [18].Our analytical techniques involved the use of differential scanning calorimetry (DSC) and protein fluorescence as a function of temperature and pressure. The intrinsic fluorescence of CPY is due mainly to tryptophan and, to a lesser extent, tyrosine residues [19]. The shape and the wavelength of the emission...

show abstract

Fluorescence and FTIR study of pressure‐induced structural modifications of horse liver alcohol dehydrogenase (HLADH)

Cited by 19 publications

References 59 publications

Gold and silver nanoparticles for biomolecule immobilization and enzymatic catalysis

Gold and silver nanoparticles for biomolecule immobilization and enzymatic catalysis

Reducing peanut allergens by high pressure combined with polyphenol oxidase

The propeptide in the precursor form of carboxypeptidase Y ensures cooperative unfolding and the carbohydrate moiety exerts a protective effect against heat and pressure

Contact Info

Product

Resources

About