Elucidation of the local dynamics of domain-III of human serum albumin over the ps–μs time regime using a new fluorescent label

2016 ◽  
Vol 18 (41) ◽  
pp. 28548-28555 ◽  
Author(s):  
Bhaswati Sengupta ◽  
Arusha Acharyya ◽  
Pratik Sen
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Fluorescent Label ◽  
Local Dynamics ◽  
Fluorescent Marker ◽  
Time Regime ◽  
Domain Iii

The ps–μs dynamics of domain-III of human serum albumin (HSA) has been investigated using a new fluorescent marker selectively labeled to the Tyr-411 residue.

scholarly journals Extending the Serum Half-Life of G-CSF via Fusion with the Domain III of Human Serum Albumin

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Shuqiang Zhao ◽  
Yu Zhang ◽  
Hong Tian ◽  
Xiaofei Chen ◽  
Di Cai ◽  
...  
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Half Life ◽  
Fusion Gene ◽  
Recombinant Expression ◽  
Pharmacokinetic Studies ◽  
Protein Fusion ◽  
Reading Frame ◽  
Domain Iii

Protein fusion technology is one of the most commonly used methods to extend the half-life of therapeutic proteins. In this study, in order to prolong the half-life of Granulocyte colony stimulating factor (G-CSF), the domain III of human serum albumin (3DHSA) was genetically fused to the N-terminal of G-CSF. The 3DHSA-G-CSF fusion gene was cloned intopPICZαA along with the open reading frame of theα-factor signal under the control of the AOX1 promoter. The recombinant expression vector was transformed intoPichia pastoris GS115, and the recombinant strains were screened by SDS-PAGE. As expected, the 3DHSA-G-CSF showed high binding affinity with HSA antibody and G-CSF antibody, and the natural N-terminal of 3DHSA was detected by N-terminal sequencing. The bioactivity and pharmacokinetic studies of 3DHSA-G-CSF were respectively determined using neutropenia model mice and human G-CSF ELISA kit. The results demonstrated that 3DHSA-G-CSF has the ability to increase the peripheral white blood cell (WBC) counts of neutropenia model mice, and the half-life of 3DHSA-G-CSF is longer than that of native G-CSF. In conclusion, 3DHSA can be used to extend the half-life of G-CSF.

scholarly journals Comparative binding character of two general anaesthetics for sites on human serum albumin

2004 ◽  
Vol 380 (1) ◽  
pp. 147-152 ◽  
Author(s):  
Renyu LIU ◽  
Qingcheng MENG ◽  
Jin XI ◽  
Jinsheng YANG ◽  
Chung-Eun HA ◽  
...  
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Hydrogen Exchange ◽  
Van Der Waals Interactions ◽  
Titration Calorimetry ◽  
Hydrogen Bond Formation ◽  
Binding Characteristics ◽  
Solution Studies ◽  
Domain Iii

Propofol and halothane are clinically used general anaesthetics, which are transported primarily by HSA (human serum albumin) in the blood. Binding characteristics are therefore of interest for both the pharmacokinetics and pharmacodynamics of these drugs. We characterized anaesthetic–HSA interactions in solution using elution chromatography, ITC (isothermal titration calorimetry), hydrogen-exchange experiments and geometric analyses of high-resolution structures. Binding affinity of propofol to HSA was determined to have a Kd of 65 µM and a stoichiometry of approx. 2, whereas the binding of halothane to HSA showed a Kd of 1.6 mM and a stoichiometry of approx. 7. Anaesthetic–HSA interactions are exothermic, with propofol having a larger negative enthalpy change relative to halothane. Hydrogen-exchange studies in isolated recombinant domains of HSA showed that propofol-binding sites are primarily found in domain III, whereas halothane sites are more widely distributed. Both location and stoichiometry from these solution studies agree with data derived from X-ray crystal-structure studies, and further analyses of the architecture of sites from these structures suggested that greater hydrophobic contacts, van der Waals interactions and hydrogen-bond formation account for the stronger binding of propofol as compared with the less potent anaesthetic, halothane.

Investigation of interaction of fluorescent marker Bengal Rose with human serum albumin at various values of pH

2012 ◽  
Vol 1016 ◽  
pp. 1-7 ◽  
Author(s):  
Irina M. Vlasova ◽  
Anna A. Kuleshova ◽  
Andrey I. Panchishin ◽  
Alexander A. Vlasov
Keyword(s):  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Fluorescent Marker ◽  
Bengal Rose

Domain-Specific Stabilization of Structural and Dynamic Responses of Human Serum Albumin by Sucrose

2019 ◽  
Vol 26 (4) ◽  
pp. 287-300
Author(s):  
Vaisakh Mohan ◽  
Bhaswati Sengupta ◽  
Nilimesh Das ◽  
Indrani Banerjee ◽  
Pratik Sen
Keyword(s):  
Steady State ◽  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Intermediate State ◽  
Domain Specific ◽  
External Agents ◽  
Domain Iii ◽  
Domain I ◽  
Circular Dichroïsm

Background: Human Serum Albumin (HSA) is the most abundant protein present in human blood plasma. It is a large multi-domain protein with 585 amino acid residues. Due to its importance in human body, studies on the interaction of HSA with different external agent is of vital interest. The denaturation and renaturation of HSA in presence of external agents are of particular interest as they affect the biological activity of the protein. Objective: The objective of this work is to study the domain-specific and overall structural and dynamical changes occurring to HSA in the presence of a denaturing agent, urea and a renaturing agent, sucrose. Methods: In order to carry out the domain-specific studies, HSA has been tagged using N-(7- dimethylamino-4-methylcoumarin-3-yl) iodoacetamide (DACIA) at Cys-34 of domain-I and pnitrophenyl coumarin ester (NPCE) at Tyr-411 site in domain-III, separately. Steady-state absorption, emission and solvation dynamic measurements have been carried out in order to monitor the domain-specific alteration of HSA caused by the external agents. The overall structural change of HSA have been monitored using circular dichroism spectroscopy. Results: The α-helicity of HSA was found to decrease from 65% to 11% in presence of urea and was found to further increase to 25% when sucrose is added, manifesting the denaturing and renaturing effects of urea and sucrose, respectively. The steady state studies show that domain-III is more labile towards denaturation as compared to domain-I. The presence of an intermediate state is observed during the denaturation process. The stabilization of this intermediate state in presence of sucrose is attributed as the reason for the stabilization of HSA by sucrose. From solvation dynamics studies, it could be seen that the solvation time of DACIA inside domain-I of HSA decreases and increases regularly with increasing concentrations of urea and sucrose, respectively, while in the case of NPCE-tagged domain-III, the effect of sucrose on solvation time is evident only at high concentrations of urea. Conclusion: The denaturing and renaturing effects of urea and sucrose could be clearly seen from the steady state studies and circular dichroism spectroscopy measurements. A regular change in solvation time could only be observed in the case of domain-I and not in domain-III. The results indicate that the renaturing effect of sucrose on domain-III is not very evident when protein is in its native state, but is evident in when the protein is denatured.</P>

Efficient identification of photolabelled amino acid residues by combining immunoaffinity purification with MS: revealing the semotiadil-binding site and its relevance to binding sites for myristates in domain III of human serum albumin

2002 ◽  
Vol 363 (2) ◽  
pp. 223-232 ◽  
Author(s):  
Kohichi KAWAHARA ◽  
Akihiko KUNIYASU ◽  
Katsuyoshi MASUDA ◽  
Masaji ISHIGURO ◽  
Hitoshi NAKAYAMA
Keyword(s):  
Amino Acid ◽  
Human Serum Albumin ◽  
Serum Albumin ◽  
Human Serum ◽  
Amino Acid Residue ◽  
Binding Sites ◽  
Immunoaffinity Purification ◽  
Ms Analysis ◽  
Domain Iii ◽  
Tof Ms

To identify photoaffinity-labelled amino acid residue(s), we devised an effective method utilizing immunoaffinity purification of photolabelled fragments, followed by matrix-assisted laser-desorption ionization-time of flight (MALDI-TOF) MS and nanoelectrospray ionization tandem MS (nano-ESI-MS/MS) analysis. Human serum albumin (HSA) was photolabelled with an azidophenyl derivative of semotiadil, FNAK {(+)-(R)-3,4-dihydro-2-[5-methoxy-2-[3-[N-methyl-N-[2-(3-azidophenoxy)-ethyl]amino]propoxyl]phenyl]-4-methyl-2H-1,4-benzothiazin-3-(4H)-one}, since HSA is a major binding protein for semotiadil in serum. After lysyl endopeptidase digestion, photolabelled HSA fragments were adsorbed selectively on to Sepharose beads on which an anti-semotiadil antibody was immobilized, and fractions were eluted quantitatively by 50% acetonitrile/10mM HCl. MALDI-TOF MS analysis of the eluted fraction showed that it contained two photolabelled fragments of m/z 2557.54 (major) and 1322.44 (minor), corresponding to Lys-414—Lys-432 and Ala-539—Lys-545, respectively. Further nano-ESI-MS/MS analysis revealed that Lys-414 was the photolabelled amino acid residue in fragment 414–432 and Lys-541 was a likely candidate in fragment 539–545. Based on the photolabelling results, we constructed a three-dimensional model of the FNAK—HSA complex, revealing that FNAK resides in a pocket that overlaps considerably with myristate (Myr)-binding sites, Myr-3 and −4, by comparison with crystallographic data of HSA—Myr complexes described in Curry, Mandelkow, Brick and Franks (1998) Nat. Struct. Biol. 5, 827–835. Moreover, addition of Myr increased photo-incorporation into Lys-414, whereas incorporation into Lys-541 decreased under conditions of [Myr]/[HSA]<1. Further addition of Myr, however, uniformly decreased photo-incorporation into both Lys residues. These results indicate that FNAK labelling can also be used to monitor Myr binding in domain III. An interpretation for the concomitant local conformational change of HSA is provided.

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