Gul Low Urea Paper (original) (raw)
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Effect of Physiological Concentration of Urea on the Conformation of Human Serum Albumin
Journal of Biochemistry, 2006
We report that the presence of very low concentrations (50.1 M) of urea, a widely used chemical denaturant, induces structure formation in the water-soluble globular protein human serum albumin (HSA) at pH 7. We have presented results suggesting an almost 8% and 5% increase in a-helix in the presence of 10 mM urea (U) and 20 mM monomethylurea (MMU), respectively. Far and near-UV circular dichroism studies along with tryptophan fluorescence and 1-anilino-8-naphthalenesulphonicacid (ANS) binding support our view. We hypothesize that both U and MMU, at such low concentrations, modify the solvent structure, increase the dielectric constant and consequently increase hydrophobic forces resulting in enhanced a-helical content. The implications of these results of the lower urea regime are significant because the physiological blood urea ranges from 2.5 to 7.5 mM.
Brazilian Journal of Physics, 2004
The influence that urea has on the conformation of water-soluble globular protein, bovine serum albumin (BSA), exposed directly to the aqueous solution as compared to the condition where the macromolecule is confined in the Aerosol-OT (AOT -sodium bis-2-ethylhexyl sulfosuccinate)/n-hexane/water reverse micelle (RM) is addressed. Small angle X-ray scattering (SAXS), tryptophan (Trp) fluorescence emission and circular dichroism (CD) spectra of aqueous BSA solution in the absence and in the presence of urea (3M and 5M) confirm the known denaturating effect of urea in proteins. The loss of the globular native structure is observed by the increase in the protein maximum dimension and gyration radius, through the Trp emission increase and maximum red-shift as well as the decrease in α-helix content. In RMs, the Trp fluorescence and CD spectra show that BSA is mainly located in its interfacial region independently of the micellar size. Addition of urea in this BSA/RM system also causes changes in the Trp fluorescence (emission decrease and maximum red-shift) and in the BSA CD spectra (decrease in α-helix content), which are compatible with the denaturation of the protein and Trp exposition to a more apolar environment in the RM. The fact that urea causes changes in the protein structure when it is located in the interfacial region (evidenced by CD) is interpreted as an indication that the direct interaction of urea with the protein is the major factor to explain its denaturating effect.
Materials Today: Proceedings, 2020
Human Serum Albumin (HSA) is a prominent protein in plasma. Protein binds with different types of ligands such as fatty acids, drugs and surfactants. Interaction of serum albumin and bile salts is studied, due to its important application in drug delivery and biopharmaceuticals. The bile salts are amphibilic molecules, synthesized by liver and undergo aggregation in aqueous media. In the present work, sodium cholate (NaC) is used as model bile salt. The interaction of NaC and HSA is monitored using intrinsic Trp-214 fluorescence. Urea (U) ([U] = 0-9.6 M) is added to the pre-formed HSA-NaC system to study. The association of NaC with Trp 214, which is buried within the hydrophobic core of sub domain IIA (Sudlow site I) of HSA, shows a prominent nature of maintaining the protein in the native conformation and hence there is very minimal change in photophysical properties of HSA-NaC system. The denaturation study carried out on HSA-NaC system by using chemical denaturant urea, shows very minimal change in the photophysical property of Trp 214, up to a 6 M concentration of U. Whereas, from 7.2 to 9.6 M [U] the fluorescence intensity show a decrease along with a 3 nm red shift.
The effect of sodium dodecyl sulfate (SDS) on human, bovine, porcine, rabbit and sheep serum albumins were investigated at pH 3.5 by using various spectroscopic techniques like circular dichroism (CD), intrinsic fluorescence and dynamic light scattering (DLS). In the presence of 4.0 mM SDS the secondary structure of all the albumins were not affected as measured by CD but fluorescence spectra revealed 8.0 nm blue shift in emission maxima. We further checked the stability of albumins in the absence and presence of 4.0 mM SDS by urea and temperature at pH 3.5. In the absence of SDS, urea starts unfolding both secondary as well as tertiary structural elements of the all the albumins at 2.0 Murea but in the presence of 4.0 mM SDS, urea was unable to unfold even up to 9.0 M. The albumins were thermally less stable at pH 3.5 with decrease in T m but in the presence of 4.0 mM SDS, the T was increased. From this study, it was concluded that SDS is showing a protective effect against urea as well as thermal denaturation of albumins. This behavior may be due to electrostatic as well as the hydrophobic interaction of SDS with albumins. Further, we have proposed the mechanism of action of urea. It was found that urea interacted with proteins directly when proteins are in charged form. Indirect interaction may be taking place when the environment is more hydrophobic.
Urea induced unfolding of F isomer of human serum albumin: A case study using multiple probes
Archives of biochemistry and biophysics, 2005
The human serum albumin is known to undergo N F (neutral to fast moving) isomerization between pH 7 and 3.5. The N F isomerization involves unfolding and separation of domain III from rest of the molecule. The urea denaturation of N isomer of HSA shows two step three state transition with accumulation of an intermediate state around 4.8-5.2 M urea concentration. While urea induced unfolding transition of F isomer of HSA does not show the intermediate state observed during unfolding of N isomer. Therefore, it provides direct evidence that the formation of intermediate in the unfolding transition of HSA involves unfolding of domain III. Although urea induced unfolding of F isomer of HSA appears to be an one step process, but no coincidence between the equilibrium transitions monitored by tryptophanyl fluorescence, tyrosyl fluorescence, far-UV CD and near-UV CD spectroscopic techniques provides decisive evidence that unfolding of F isomer of HSA is not a two state process. An intermediate state that retained significant amount of secondary structure but no tertiary structure has been identified (around 4.4 M urea) in the unfolding pathway of F isomer. The emission of Trp-214 (located in domain II) and its mode of quenching by acrylamide and binding of chloroform indicate that unfolding of F isomer start from domain II (from 0.4 M urea). But at higher urea concentration (above 1.6 M) both the domain unfold simultaneously and the protein acquire random coil structure around 8.0 M urea. Further much higher K sv of NATA (17.2) than completely denatured F isomer (5.45) of HSA (8.0 M urea) suggests the existence of residual tertiary contacts within local regions in random coil conformation (probably around lone Trp-214).
Latin American Applied Research - An international journal
The conformational stability of bovine serum albumin (BSA) against urea denaturation was investigated in aqueous solutions both in the absence and presence of buffers. Various buffers differing in polar and nonpolar characters such as sodium phosphate, Tris-HCl, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) HEPES and [3-(N-morpholino)propanesulfonic acid] MOPS buffers were used in this study. Urea-induced structural changes were analyzed using different probes, i.e., intrinsic fluorescence, ANS fluorescence and UV-difference spectral signal. Presence of different buffers in the incubation medium offered different degrees of resistance to the protein against urea-induced structural changes compared to those obtained in water (in the absence of buffers). A similar trend of buffer-induced structural resistance was noticed with three different probes. The stabilizing effect of these buffers followed the order: MOPS > HEPES > sodium phosphate > Tris-HCl > water. As fo...
Biochemical and biophysical …, 2004
The urea-induced unfolding of 'N' isomer (occurring at pH 7.0) and 'B' isomer (occurring at pH 9.0) of human serum albumin was studied by fluorescence and circular dichroism spectroscopic measurements. Urea-induced destabilization in different domains of both the isomers was monitored by using domain specific ligands, hemin (domain-I), chloroform, bilirubin (domain-II), and diazepam (domain-III). Urea-induced denaturation of N and B isomers of HSA showed a two-step, three-state transition with accumulation of intermediates around 4.8-5.2 M and 3.0-3.4 M urea concentrations, respectively. During first transition (0-4.8 M urea for N isomer and 0-3.0 M urea for B isomer) a continuous decrease in diazepam binding suggested major conformational changes in domain-III prior to intermediate formation. On the other hand, binding of hemin, a ligand for domain-IB and chloroform, whose binding site is located in domain-IIA remains unchanged up to 5.0 M urea for N isomer and 3.0 M urea for B isomer. Similarly, fluorescence intensity of Trp-214 that resides in domain-IIA remained unchanged up to the above-said urea concentrations and decreased thereafter. Absence of any decrease in hemin binding, chloroform binding, and Trp-214 fluorescence suggested the non-involvement of domain-IB and domain-IIA in intermediate formation. A significant increase in bilirubin binding prior to intermediate formation showed favorable conformational rearrangement in bilirubin binding cavity formed by loop 4 of domain-IB and loop 3 of domain-IIA. Further, a nearly complete abolishment of bilirubin binding to both isomers around 7.0 M and 6.0 M urea concentrations, respectively, indicated complete separation of domain-I from domain-II from each other. From these observations it can be concluded that N to B transition of human serum albumin shifted the intermediate formation towards lower urea concentration (3.0-3.4 M urea for B isomer as against 4.8-5.2 M urea for N isomer). Further both the intermediates were found to possess similar a-helical ($39%) content and ligand binding properties.
1982
The mechanism of the unfolding of human serum albumin by urea was studied using d.c. polarography. It was found that this reaction is a complex process which cannot be described in terms of a two-state transition model. As well as the BrdiEka catalytic current we have also studied the reduction current of disulfide groups in native and denatured human serum albumin. The number of cystine residues accessible for electrode reduction in native and denatured protein was calculated. On the basis of these results a scheme for the unfolding of human serum albumin by urea is proposed.
Characterisation of molten globule-like state of sheep serum albumin at physiological pH
Sheep serum albumin (SSA) is a 583 amino acid residues long multidomain monomeric protein which is rich in cysteine and low in tryptophan content. The serum albumins (from human, bovine and sheep) play a vital role among all proteins investigated until now, as they are the most copious circulatory proteins. We have purified SSA from sheep kidneys by a simple and efficient two-step purification procedure. Further, we have studied urea-induced denaturation of SSA by monitoring changes in the difference absorption coefficient at 287 nm ( ε 287 ), intrinsic fluorescence emission intensity at 347 nm (F 347 ) and mean residue ellipticity at 222 nm ([Â] 222 ) at pH 7.4 and 25 • C. The coincidence of denaturation curves of these optical properties suggests that urea-induced denaturation is a bi-phasic process (native (N) state ↔ intermediate (X) state ↔ denatured (D) state) with a stable intermediate populated around 4.2-4.7 M urea. The intermediate (X) state was further characterized by the far-UV and near-UV CD, dynamic light scattering (DLS) and fluorescence using 1-anilinonaphthalene-8-sulfonic acid (ANS) binding method. All denaturation curves were analyzed for Gibbs free energy changes associated with the equilibria, N state ↔ X state and X state ↔ D state in the absence of urea.