From adsorption to crystallization of proteins: Evidence for interface-assisted nucleation.

Protein crystallization is among the key processes in biomolecular research, but the underlying mechanisms are still elusive. Here, we address the role of inevitable interfaces for the nucleation process. Quartz crystal microbalance with dissipation monitoring (QCM-D) with simultaneously optical microscopy, confocal microscopy, and grazing-incidence small angle X-rays scattering (GISAXS) were employed to investigate the temporal behavior from the initial stage of protein adsorption to crystallization. Here we studied the crystallization of the Human Serum Albumin (HSA), the most abundant blood protein, in the presence of a charged surface and a trivalent salt. We found evidence for interface-assisted nucleation of crystals. The kinetic stages involved are initial adsorption followed by enhanced adsorption after longer times, subsequent nucleation, and finally crystal growth. The results highlight the importance of interfaces for protein phase behavior and in particular for nucleation.

Corrigendum: HSA-nanobinders crafted from bioresponsive prodrugs for combined cancer chemoimmunotherapy-an in vitro exploration.

[This corrects the article DOI: 10.3389/fchem.2024.1378233.].

The mechanism of interaction between tri-para-cresyl phosphate and human serum protein: a multispectroscopic and in-silico study.

Organophosphate flame retardants (OPFRs) pose the significant risks to the environment and human health and have become a serious public health issue. Tricresyl phosphates (TCPs), a group of aryl OPFRs, exhibit neurotoxicity and endocrine disrupting toxicity. However, the binding mechanisms between TCPs and human serum albumin (HSA) remain unknown. In this study, through fluorescence and ultraviolet-visible (UV-vis) absorption spectroscopy, molecular docking and molecular dynamics (MD), tri-para-cresyl phosphate (TpCP) was selected to explore potential interactions between HSA and TCPs. The results of the fluorescence spectroscopy demonstrated that a decrease the fluorescence intensity of HSA and a blue shift were observed with the increasing concentrations of TpCP. The binding constant (Ka) was 2.575 × 104 L/mol, 4.701 × 104 L/mol, 5.684 × 104 L/mol and 9.482 × 104 L/mol at 293 K, 298 K, 303 K, and 310 K, respectively. The fluorescence process between HSA and TpCP involved a mix of static and dynamic quenching mechanism. The gibbs free energy (ΔG0) of HSA-TpCP system was -24.452, -25.907, 27.363, and 29.401 kJ/mol at 293 K, 298 K, 303 K, and 310 K, respectively, suggesting that the HSA-TpCP reaction was spontaneous. The enthalpy change (ΔH0) and thermodynamic entropy change (ΔS0) of the HSA-TpCP system were 291.08 J/K mol and 60.83 kJ/mol, respectively, indicating that hydrophobic force was the major driving forces in the HSA-TpCP complex. Furthermore, multispectral analysis also revealed that TpCP could alter the microenvironment of tryptophan residue and the secondary structure of HSA and bind with the active site I of HSA. Molecular docking and MD simulations confirmed that TpCP could spontaneously form a stable complex with HSA, which was consistent with the fluorescence experimental results. This study provides novel insights into the mechanisms of underlying the transportation and distribution of OFPRs in humans.

Recombinant production of interleukin-1 receptor antagonist in fusion to albumin binding domain with potential affinity to human serum albumin.

Background and purpose: Anakinra must be injected daily due to its short half-life and this leads to lower patient compliance. Therefore, the aim of this study was to produce an interleukin-1 receptor antagonist (IL-1Ra) with albumin binding domain (ABD) as a novel fusion protein and evaluate its binding ability to albumin and its biological effects. Experimental approach: The three-dimensional structure of IL-1Ra-ABD was predicted by MODELLER software and its interaction with IL-1R was evaluated by the HADDOCK server. The expression of IL-1Ra-ABD was performed in E. coli in fusion with intein 1 of pTWIN1 in soluble form and then purified. The affinity of IL-1Ra-ABD to human serum albumin (HSA) was determined on native-PAGE, and its release percent toward time was evaluated. Moreover, an MTT assay was used to determine the antagonizing properties of recombinant IL-1Ra-ABD against IL-1ß in A375 and HEK293 cell lines. Findings/Results: The stable complex of IL-1Ra-ABD with IL-1R established the absence of steric hindrance due to the addition of ABD to IL-1Ra. The expression induction of intein 1-IL-1Ra-ABD using 0.1 mM IPTG at 15 °C, and its cleavage represented bands approximately in 50 and 23 kDa. Furthermore, about 78% of IL-1Ra-ABD was attached to the HSA after 2 h of incubation, and the MTT assay showed no significant differences between the effects of IL-1Ra-ABD and native IL-1Ra in cell survival. Conclusions and implications: The production of soluble IL-1Ra-ABD with no significant differences in IL-1Ra antagonizing effects was successfully performed. IL-1Ra-ABD showed suitable interaction with HSA and was released over time. However, the half-life of IL-1Ra-ABD in vivo must be determined in the subsequent investigations.

Mass spectrometry detection of organophosphorus pesticide adducts on butyrylcholinesterase and albumin.

This study established a method to prepare and detect OPs adducts on butyrylcholinesterase (BChE) and human serum albumin (HSA). OPs (methyl paraoxon, ethyl paraoxon, methyl parathion, parathion) were incubated with BChE or HSA in vitro, and the adducts of OPs-BChE or OPs-HSA were prepared and qualitatively analyzed by ultra-performance liquid chromatography data-dependent high-resolution tandem mass spectrometry (UPLC-ddHRMS/MS). The amounts of BChE and HSA in the incubating systems were varied and the resulting amounts of the adducts were determined using linear regression. OPs-BChE in the blood were isolated by immunomagnetic separation (IMS), and then digested into the OPs-nonapeptide adduct by pepsin. The proteins in the remaining blood plasma were precipitated and digested by pronase to OPs-tyrosines(OPs-Tyr), which were quantified by UPLC-ddHRMS/MS. 4 OPs-nonapeptides and 4 OPs-Tyr adducts were obtained through the process above. The relative mass deviation of incubated adducts between the actual and theoretical exact masses was less than 10 ppm, and further confirmed by fragmentation mass spectra analysis. Calibration curves were linear for all adducts with a coefficient of determination value (R2) ≥0.995. The limits of detection (LOD) and limits of quantification (LOQ) for adducts detected by MS ranged from 0.05 to 1.0 ng/mL, and from 0.1 to 2.0 ng/mL, respectively. The recovery percentages for adducts ranged from 76.1 % to 107.1 %, matrix effects ranged from 83.4 % to 102.1 %. The inter-day and intra-day precision were 6.1-10.1 % and 6.9-12.9 % for adducts. This study provides a new reference method for the detection of organophosphorus pesticide poisoning. In addition, two blood samples with organophosphorus poisoning were tested by the designed method, and the corresponding adducts were detected in both samples.

Spectroscopic study on volasertib: Highly stable complexes with albumin and encapsulation into alginate/montmorillonite bionanocomposites.

In the present work, we study different physicochemical properties related to LADME processes of volasertib, a Polo-like kinase 1 inhibitor in advanced clinical trials. Firstly, the protonation equilibria, the extent of ionization at the physiological pH and pKa values of this drug are studied combining spectroscopic techniques and computational calculations. Secondly, the binding process of volasertib to the human serum albumin (HSA) protein is analyzed by fluorescence spectroscopy. We report a high binding constant to HSA (Ka = 4.10 × 106 M-1) and their pharmacokinetic implications are discussed accordingly. The negative enthalpy and entropy (ΔH0 = -54.49 kJ/mol; ΔS0 = -58.90 J K-1 mol-1) determined for the binding process suggests the implication of hydrogen bonds and van der Waals interactions in the formation of the HSA-volasertib complex. Additionally, volasertib is encapsulated in an alginate/montmorillonite bionanocomposite as a proof of concept for an oral delivery nanocarrier. The physical properties of that nanocomposite as well as volasertib delivery kinetics are analyzed.

Multispectroscopic and Theoretical Investigation on the Binding Interaction of a Neurodegenerative Drug, Lobeline with Human Serum Albumin: Perturbation in Protein Conformation and Hydrophobic-Hydrophilic Surface.

Lobeline (LOB), a naturally occurring alkaloid, has a broad spectrum of pharmacological activities and therapeutic potential, including applications in central nervous system disorders, drug misuse, multidrug resistance, smoking cessation, depression, and epilepsy. LOB represents a promising compound for developing treatments in various medical fields. However, despite extensive pharmacological profiling, the biophysical interaction between the LOB and proteins remains largely unexplored. In the current article, a range of complementary photophysical and cheminformatics methodologies were applied to study the interaction mechanism between LOB and the carrier protein HSA. Steady-state fluorescence and fluorescence lifetime experiments confirmed the static-quenching mechanisms in the HSA-LOB system. "K" (binding constant) of the HSA-LOB system was determined to be 105 M-1, with a single preferable binding site in HSA. The forces governing the HSA-LOB stable complex were analyzed by thermodynamic parameters and electrostatic contribution. The research also investigated how various metal ions affect complex binding. Site-specific binding studies depict Site I as probable binding in HSA by LOB. We conducted synchronous fluorescence, 3D fluorescence, and circular dichroism studies to explore the structural alteration occurring in the microenvironment of amino acids. To understand the robustness of the HSA-LOB complex, we used theoretical approaches, including molecular docking and MD simulations, and analyzed the principal component analysis and free energy landscape. These comprehensive studies of the structural features of biomolecules in ligand binding are of paramount importance for designing targeted drugs and delivery systems.

Binding studies of promethazine and its metabolites with human serum albumin by high-performance affinity chromatography and molecular docking in the presence of codeine.

"Purple Drank", a soft drink containing promethazine (PMZ) and codeine (COD), has gained global popularity for its hallucinogenic effects. Consuming large amounts of this combination can lead to potentially fatal events. The binding of these drugs to plasma proteins can exacerbate the issue by increasing the risk of drug interactions, side effects, and/or toxicity. Herein, the binding affinity to human serum albumin (HSA) of PMZ and its primary metabolites [N-desmethyl promethazine (DMPMZ) and promethazine sulphoxide (PMZSO)], along with COD, was investigated by high-performance affinity chromatography (HPAC) though zonal approach. PMZ and its metabolites exhibited a notable binding affinity for HSA (%b values higher than 80%), while COD exhibited a %b value of 65%. To discern the specific sites of HSA to which these compounds were bound, displacement experiments were performed using warfarin and (S)-ibuprofen as probes for sites I and II, respectively, which revealed that all analytes were bound to both sites. Molecular docking studies corroborated the experimental results, reinforcing the insights gained from the empirical data. The in silico data also suggested that competition between PMZ and its metabolites with COD can occur in both sites of HSA, but mainly in site II. As the target compounds are chiral, the enantioselectivity for HSA binding was also explored, showing that the binding for these compounds was not enantioselective.

Development of a screening platform for the formulation of poorly water-soluble drugs as albumin-stabilized nanosuspensions using nab™ technology.

The nanoparticle albumin bound™ (nab™) technology generally offers great potential for the formulation of poorly water-soluble drugs as albumin-stabilized nanosuspensions for intravenous use while avoiding solubilizers and cross-linking agents. The nab™ technology is a three-step process consisting of emulsification, high-pressure homogenization and solvent evaporation Within this work, a screening approach was developed to predict whether active pharmaceutical ingredients are suitable for nab™ formulations. A design of experiments approach was used to investigate the effects of ultrasonic homogenization on an albumin-stabilized itraconazole nanosuspension. Based on this, a screening platform was developed, and subsequently evaluated and applied to a selection of poorly water-soluble drugs. The screening process to produce albumin-stabilized nanosuspensions consists of two process steps: Ultrasonic treatment, which combined emulsification and homogenization, followed by solvent evaporation. The results of the screening process were fully transferable to the standard three-step process of nab™ technology. In addition, based on drug screening, drug properties were highlighted that are important for the development of nab™ formulations. All in all, the nab™ technology is a promising but not universal formulation platform for poorly water-soluble drugs. Nevertheless, for some poorly soluble drugs it offers a valuable approach for the formulation of nanosuspensions for intravenous use.

Characterization of the structural and molecular interactions of Ferulic acid ethyl ester with human serum albumin and Lysozyme through multi-methods.

Ferulic acid ethyl ester (FAEE) is an essential raw material for the formulation of drugs for cardiovascular and cerebrovascular diseases and leukopenia. It is also used as a fixed aroma agent for food production due to its high pharmacological activity. In this study, the interaction of FAEE with Human serum albumin (HSA) and Lysozyme (LZM) was characterized by multi-spectrum and molecular dynamics simulations at four different temperatures. Additionally, the quenching mechanism of FAEE-HSA and FAEE-LZM were explored. Meanwhile, the binding constants, binding sites, thermodynamic parameters, molecular dynamics, molecular docking binding energy, and the influence of metal ions in the system were evaluated. The results of Synchronous fluorescence spectroscopy, UV-vis spectroscopy, CD, three-dimensional fluorescence spectrum, and resonance light scattering showed that the microenvironment of HSA and LZM and the protein conformation changed in the presence of FAEE. Furthermore, the effects of some common metal ions on the binding constants of FAEE-HSA and FAEE-LZM were investigated. Overall, the experimental results provide a theoretical basis for promoting the application of FAEE in the cosmetics, food, and pharmaceutical industries and significant guidance for food safety, drug design, and development.

The First In Vivo Study Shows That Gyrophoric Acid Changes Behavior of Healthy Laboratory Rats.

Gyrophoric acid (GA), a lichen secondary metabolite, has attracted more attention during the last years because of its potential biological effects. Until now, its effect in vivo has not yet been demonstrated. The aim of our study was to evaluate the basic physicochemical and pharmacokinetic properties of GA, which are directly associated with its biological activities. The stability of the GA in various pH was assessed by conducting repeated UV-VIS spectral measurements. Microsomal stability in rat liver microsomes was performed using Ultra-Performance LC/MS. Binding to human serum albumin (HSA) was assessed using synchronous fluorescence spectra, and molecular docking analysis was used to reveal the binding site of GA to HSA. In the in vivo experiment, 24 Sprague-Dawley rats (Velaz, Únetice, Czech Republic) were used. The animals were divided as follows. The first group (n = 6) included healthy males as control intact rats (♂INT), and the second group (n = 6) included healthy females as controls (♀INT). Groups three and four (♂GA/n = 6 and ♀GA/n = 6) consisted of animals with daily administered GA (10 mg/kg body weight) in an ethanol-water solution per os for a one-month period. We found that GA remained stable under various pH and temperature conditions. It bonded to human serum albumin with the binding constant 1.788 × 106 dm3mol-1 to reach the target tissue via this mechanism. In vivo, GA did not influence body mass gain, food, or fluid intake during the experiment. No liver toxicity was observed. However, GA increased the rearing frequency in behavioral tests (p < 0.01) and center crossings in the elevated plus-maze (p < 0.01 and p < 0.001, respectively). In addition, the time spent in the open arm was prolonged (p < 0.01 and p < 0.001, respectively). Notably, GA was able to pass through the blood-brain barrier, indicating its ability to permeate into the brain and to stimulate neurogenesis in the hilus and subgranular zone of the hippocampus. These observations highlight the potential role of GA in influencing brain function and neurogenesis.

Predictability of combining Technetium-99m-galactosyl human serum albumin single-photon emission computed tomography/computed tomography and indocyanine green clearance test for posthepatectomy liver failure.

PURPOSE: To evaluate the predictive ability of combining Technetium-99m-galactosyl human serum albumin (99mTc­GSA) single-photon emission computed tomography (SPECT)/computed tomography (CT) volume and plasma clearance rate of indocyanine green (ICGK) for posthepatectomy liver failure (PHLF). MATERIALS AND METHODS: Fifty patients who underwent 99mTc-GSA scintigraphy as a preoperative examination for segmentectomy or more from July 2021 to June 2023 were evaluated prospectively. Patients were divided into two groups according to the presence or absence of posthepatectomy liver failure (PHLF). Total functional liver volume (t-FLV) and remnant FLV (r-FLV) were measured from 99mTc-GSA SPECT/CT image. Future liver remnant ICGK (ICGK-F) was calculated by ICGK and remnant liver volume from CT. Area under the curve (AUC) of ICGK-F, r-FLV, r-FLV/t-FLV, ICGK × r-FLV, ICGK × r-FLV/t-FLV was calculated to evaluate predictive ability of each parameter for PHLF. RESULTS: PHLF was occurred in 7 patients. AUC of ICGK × r-FLV was significantly higher than that of ICGK-F (0.99; 95% confidence interval [CI]: 0.96-1 vs 0.82; 95%CI: 0.64-0.96; p = 0.036). There was no significant difference between the AUC of r-FLV, r-FLV/t-FLV, ICGK × r-FLV/t-FLV and that of ICGK-F, respectively. CONCLUSION: The combination of 99mTc­GSA SPECT/CT volume and ICGK can predict PHLF more accurately than ICGK-F.

Carbapenem-resistant Acinetobacter baumannii (CRAB): metabolic adaptation and transcriptional response to human urine (HU).

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a major human pathogen and a research priority for developing new antimicrobial agents. CRAB is a causative agent of a variety of infections in different body sites. One of the manifestations is catheter-associated urinary tract infection, which exposes the bacteria to the host's urine, creating a particular environment. Exposure of two CRAB clinical isolates, AB5075 and AMA40, to human urine (HU) resulted in the differential expression levels of 264 and 455 genes, respectively, of which 112 were common to both strains. Genes within this group play roles in metabolic pathways such as phenylacetic acid (PAA) catabolism, the Hut system, the tricarboxylic acid (TCA) cycle, and other processes like quorum sensing and biofilm formation. These results indicate that the presence of HU induces numerous adaptive changes in gene expression of the infecting bacteria. These modifications presumably help bacteria establish and thrive in the hostile conditions in the urinary tract. These analyses advance our understanding of CRAB's metabolic adaptations to human fluids, as well as expanding knowledge on bacterial responses to distinct human fluids containing different concentrations of human serum albumin (HSA).

Biomimetic gold nanocages incorporating copper-human serum albumin for tumor immunotherapy via cuproptosis-lactate regulation.

Cancer immunotherapy remains a significant challenge due to insufficient proliferation of immune cells and the sturdy immunosuppressive tumor microenvironment. Herein, we proposed the hypothesis of cuproptosis-lactate regulation to provoke cuproptosis and enhance anti-tumor immunity. For this purpose, copper-human serum albumin nanocomplex loaded gold nanocages with bacterial membrane coating (BAu-CuNCs) were developed. The targeted delivery and disassembly of BAu-CuNCs in tumor cells initiated a cascade of reactions. Under near infrared (NIR) laser irradiation, the release of copper-human serum albumin (Cu-HSA) was enhanced that reacted with intratumoral glutathione (GSH) via a disulfide exchange reaction to liberate Cu2+ ions and exert cuproptosis. Subsequently, the cuproptosis effect triggered immunogenic cell death (ICD) in tumor by the release of damage associated molecular patterns (DAMPs) to realize anti-tumor immunity via robust production of cytotoxic T cells (CD8+) and helper T cells (CD4+). Meanwhile, under NIR irradiation, gold nanocages (AuNCs) promoted excessive reactive oxygen species (ROS) generation that played a primary role in inhibiting glycolysis, reducing the lactate and ATP level. The combine action of lower lactate level, ATP reduction and GSH depletion further sensitized the tumor cells to cuproptosis. Also, the lower lactate production led to the significant blockage of immunosuppressive T regulatory cells (Tregs) and boosted the anti-tumor immunity. Additionally, the effective inhibition of breast cancer metastasis to the lungs enhanced the anti-tumor therapeutic impact of BAu-CuNCs + NIR treatment. Hence, BAu-CuNCs + NIR concurrently induced cuproptosis, ICD and hindered lactate production, leading to the inhibition of tumor growth, remodeling of the immunosuppressive tumor microenvironment and suppression of lung metastasis. Therefore, leveraging cuproptosis-lactate regulation, this approach presents a novel strategy for enhanced tumor immunotherapy.

Revolutionizing Alzheimer's treatment: Harnessing human serum albumin for targeted drug delivery and therapy advancements.

Alzheimer's disease (AD) is a neurodegenerative disorder initiated by amyloid-beta (Aß) accumulation, leading to impaired cognitive function. Several delivery approaches have been improved for AD management. Among them, human serum albumin (HSA) is broadly employed for drug delivery and targeting the Aß in AD owing to its biocompatibility, Aß inhibitory effect, and nanoform, which showed blood-brain barrier (BBB) crossing ability via glycoprotein 60 (gp60) receptor and secreted protein acidic and rich in cysteine (SPARC) protein to transfer the drug molecules in the brain. Thus far, there is no previous review focusing on HSA and its drug delivery system in AD. Hence, the reviewed article aimed to critically compile the HSA therapeutic as well as drug delivery role in AD management. It also delivers information on how HSA-incorporated nanoparticles with surfaced embedded ligands such as TAT, GM1, and so on, not only improve BBB permeability but also increase neuron cell targetability in AD brain. Additionally, Aß and tau pathology, including various metabolic markers likely BACE1 and BACE2, etc., are discussed. Besides, the molecular interaction of HSA with Aß and its distinctive forms are critically reviewed that HSA can segregate Zn(II) and Cu(II) metal ions from Aß owing to high affinity. Furthermore, the BBB drug delivery challenges in AD are addressed. Finally, the clinical formulation of HSA for the management of AD is critically discussed on how the HSA inhibits Aß oligomer and fibril, while glycated HSA participates in amyloid plaque formation, i.e., ß-structure sheet formation. This review report provides theoretical background on HSA-based AD drug delivery and makes suggestions for future prospect-related work.

Extension of the circulatory half-life of recombinant ecallantide via albumin fusion without loss of anti-kallikrein activity.

Ecallantide comprises Kunitz Domain 1 of Tissue Factor Pathway Inhibitor, mutated at seven amino acid positions to inhibit plasma kallikrein (PK). It is used to treat acute hereditary angioedema (HAE). We appended hexahistidine tags to the N- or C-terminus of recombinant Ecallantide (rEcall) and expressed and purified the resulting proteins, with or without fusion to human serum albumin (HSA), using Pichia pastoris. The inhibitory constant (Ki) of rEcall-H6 or H6-rEcall for PK was not increased by albumin fusion. When 125I-labelled rEcall proteins were injected intravenously into mice, the area under the clearance curve (AUC) was significantly increased, 3.4- and 3.6-fold, for fusion proteins H6-rEcall-HSA and HSA-rEcall-H6 versus their unfused counterparts but remained 2- to 3-fold less than that of HSA-H6. The terminal half-life of H6-rEcall-HSA and HSA-H6 did not differ, although that of HSA-rEcall-H6 was significantly shorter than either other protein. Receptor Associated Protein (RAP), a Low-density lipoprotein Receptor-related Protein (LRP1) antagonist, competed H6-rEcall-HSA clearance more effectively than intravenous immunoglobulin (IVIg), a neonatal Fc receptor (FcRn) antagonist. HSA fusion decreases rEcall clearance in vivo, but LRP1-mediated clearance remains more important than FcRn-mediated recycling for rEcall fusion proteins. The properties of H6-rEcall-HSA warrant investigation in a murine model of HAE.

The multi-dimensional impact of captopril modification on human serum albumin.

Captopril is a thiol drug, widely used for the management of hypertension and cardiovascular diseases. Reactive thiols are found to covalently modify the cysteines of plasma proteins and affect their structure and function. Human serum albumin (HSA) is prone to undergo modification by various low molecular weight compounds, including drugs. Cysteine34 (Cys34) in HSA has a free thiol group with antioxidant properties, considered to be the most redox-sensitive amino acid in plasma. Through mass-spectrometric analysis, we demonstrate for the first time that captopril forms a disulfide adduct at Cys34 residue and increases the protease susceptibility of HSA to trypsin. As evidenced by our biophysical and electron microscopy studies, HSA undergoes structural alteration, aggregation and morphological changes when treated with different captopril concentrations. Molecular dynamics studies further revealed the regions of secondary structural changes in HSA due to disulfide adduct formation by captopril at Cys34. It also elucidated the residues involved in the noncovalent interactions with captopril. It is envisaged that structural change in HSA may influence the efficacy of drug delivery as well as its own biological function. These findings may thus provide significant insights into the field of pharmacology intriguing further investigation into the effects of long-term captopril treatment.

Exploring the binding mechanism and esterase-like activity of human serum albumin with levofloxacin and its choline based conjugates: A biophysical approach.

Human serum albumin (HSA) effectively binds to compounds having different molecular weight and thus facilitates their distribution in the living organisms. Thus, the binding interactions between a potential antibacterial drug (levofloxacin) and synthesized choline based levofloxacinate conjugates with HSA have been explored. The binding efficacy and mechanism were explored by utilizing different spectroscopic techniques; UV-Visible, steady state fluorescence, time resolved fluorescence and esterase-like activity. The interactions between the ligands and protein were electrostatic as well as hydrophobic in nature. The influence of different ligands having different alkyl chain shows quenching of the fluorescence emission of HSA. The spontaneous binding/quenching of HSA with ligands was static in nature, validated by steady state and time resolved fluorescence spectroscopy. Also, the impact of these ligands on the conformation of the native HSA structure was evaluated by using circular dichroism spectroscopy. In combination to the structural change study, the native protein functionality was observed (in terms of 'esterase-like activity') which has been found to be on lower side due to ligand binding. Further, we have performed the reverse study to check the impact of HSA on the fluorescent fluoroquinolone drug. The current study may prove helpful in elucidating the chemico-biological interactions which may prove useful in the pharmaceuticals, pharmacology, and different biochemistry fields.

Transport of Zinc-Phthalocyanine to Cancer Cells using Myoglobin-Albumin Fusion Protein for Photodynamic Therapy.

Photodynamic therapy (PDT) is a noninvasive approach to cancer treatment, wherein cell death is initiated by singlet oxygen (1O2) production via energy transfer from excited photosensitizers to ground-state O2. Effective clinical photosensitizers necessitate water solubility for in vivo administration. Hydrophobic dyes, such as phthalocyanines, cannot be used directly as photosensitizers. Herein, we synthesized a myoglobin-(human serum albumin) fusion protein reconstituted with zinc-phthalocyanine (ZnPc), termed ZnPcMb-HSA. The photophysical properties of ZnPcMb-HSA closely resemble those of ZnPc-substituted Mb. Notably, ZnPc dissociates from ZnPcMb-HSA and selectively accumulates within cancer cells, while the protein components remain extracellular. Treatment of four distinct cell lines with ZnPcMb-HSA, followed by red-light irradiation, effectively induced apoptosis. The half-maximal inhibitory concentrations (IC50) against these cancer cell lines ranged between 0.1-0.5 µM. Reconstituted Mb-HSA emerges as a promising carrier for transporting various water-insoluble porphyrinoid photosensitizer to target cancer cells in PDT applications.

Preserved structure and function of human serum albumin self-folded in the oxidative cytoplasm of Escherichia coli.

Human serum albumin (HSA), a polypeptide featuring 17 disulfide bonds, acts as a crucial transport protein in human blood plasma. Its extended circulation half-life, mediated by FcRn (neonatal Fc receptor)-facilitated recycling, positions HSA as an excellent carrier for long-acting drug delivery. However, the conventional method of obtaining HSA from human blood faces limitations due to availability and potential contamination risks, such as blood-borne diseases. This study introduced SHuffle, an oxidative Escherichia coli (E. coli) expression system, for the production of recombinant HSA (rHSA) that spontaneously self-folds into its native conformation. This system ensures precise disulfide bond formation and correct folding of cysteine-rich rHSA, eliminating the need for chaperone co-expression or domain fusion of a folding enhancer. The purified rHSA underwent thorough physicochemical characterization, including mass spectrometry, circular dichroism spectroscopy, intrinsic fluorescence spectroscopy, esterase-like activity assay, and size exclusion chromatography, to assess critical quality attributes. Importantly, rHSA maintained native binding affinity to FcRn and the albumin-binding domain. Collectively, our analyses demonstrated a high comparability between rHSA and plasma-derived HSA. The expression of rHSA in E. coli with an oxidizing cytosol provides a secure and cost-effective approach, enhancing the potential of rHSA for diverse medical applications.