DOCSLIB.ORG

Use of Cysteine As a Spectroscopic Probe for Determination of Heme-Scavenging Capacity of Serum Proteins and Whole Human Serum

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Use of Cysteine As a Spectroscopic Probe for Determination of Heme-Scavenging Capacity of Serum Proteins and Whole Human Serum Use of cysteine as a spectroscopic probe for determination of heme-scavenging capacity of serum proteins and whole human serum Remi Noe, Nina Bozinovic, Maxime Lecerf, Sébastien Lacroix-Desmazes, Jordan Dimitrov To cite this version: Remi Noe, Nina Bozinovic, Maxime Lecerf, Sébastien Lacroix-Desmazes, Jordan Dimitrov. Use of cysteine as a spectroscopic probe for determination of heme-scavenging capacity of serum proteins and whole human serum. Journal of Pharmaceutical and Biomedical Analysis, Elsevier, 2019, 172, pp.311-319. 10.1016/j.jpba.2019.05.013. hal-02127294 HAL Id: hal-02127294 https://hal.archives-ouvertes.fr/hal-02127294 Submitted on 13 May 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Journal of Pharmaceutical and Biomedical Analysis 172 (2019) 311–319 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis j ournal homepage: www.elsevier.com/locate/jpba Use of cysteine as a spectroscopic probe for determination of heme-scavenging capacity of serum proteins and whole human serum 1 1 Rémi Noé , Nina Bozinovic , Maxime Lecerf, Sébastien Lacroix-Desmazes, ∗ Jordan D. Dimitrov Centre de Recherche des Cordeliers, INSERM, Sorbonne Université, USPC, Université Paris Descartes, Université Paris Diderot, F-75006 Paris, France a r t i c l e i n f o a b s t r a c t Article history: Heme serves as a prosthetic group of numerous proteins involved in the oxidative metabolism. As result of Received 19 December 2018 various pathological conditions associated with hemolysis or tissue damage, large quantities of hemopro- Received in revised form 3 May 2019 teins and heme can be released extracellularly. Extracellular heme has pronounced pathogenic effects Accepted 5 May 2019 in hemolytic diseases, mediated by its pro-oxidative and pro-inflammatory activities. The pathogenic Available online 7 May 2019 potential of heme is mostly expressed when the molecule is in protein unbound form. The pathological relevance of free heme deems it necessary to develop reliable approaches for its assessment. Here we Keywords: developed a technique based on UV–vis absorbance spectroscopy, where cysteine was used as a spec- Heme Hemolysis troscopy probe to distinguish between heme-bound to plasma proteins or hemoglobin from free heme. This technique allowed estimation of the heme-binding capacity of human serum, of particular heme Heme-binding proteins Human serum scavenging proteins (albumin, hemopexin) or of immunoglobulins. The main advantage of the proposed Absorbance spectroscopy approach is that it can distinguish free heme from heme associated with proteins with a wide range of affinities. The described strategy can be used for evaluation of heme-binding capacity of human plasma or serum following intravascular hemolysis or for estimation of stoichiometry of interaction of heme with a given protein. © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction hemoglobin. As a result of its oxidative potential and prominent hydrophobicity, free heme is inherently toxic molecule. Likewise, Heme (Fe-protoporphyrin IX) is a macrocyclic compound heme is considered as endogenous danger signal (alarmin) that that serves as a prosthetic group of many proteins involved activates different types of immune cells and endothelia, increases in the aerobic metabolism. By transiently interacting with a vascular permeability, triggers complement cascade activation and number of intracellular or membrane-associated proteins, heme dysregulates coagulation [3,4]. Accordingly, extracellular hemo- also participates in the cell signalling and regulation of cellu- proteins and heme contribute to the pathogenesis of diseases such lar functions [1]. Under physiological conditions most of heme is as malaria, sickle cell disease, sepsis, rhabdomyolysis and other. intracellularly sequestered. However, release of large quantities In the clinical practice the assessment of hemolysis is performed of heme-containing proteins (hemoproteins), such as hemoglobin by measurement of the plasma scavenger of hemoglobin, i.e. hap- and myoglobin can occur as consequence of diverse patholo- toglobin [5]. Thus, a decrease in the plasma concentration of this gies. Thus, damage of erythrocytes due to genetic abnormalities protein signifies recent hemolytic events. Extensive hemolysis may of hemoglobin, infections, trauma, or autoimmunity can result also result in overwhelming of scavenging capacity of hemopexin, in intravascular hemolysis and liberation of massive quantities the plasma protein that binds heme with high affinity [6]. In this of extracellular hemoglobin [2]. In extracellular compartment case, the extracellular heme can associate with lower affinity to and upon oxidation, heme relatively easily dissociates from other plasma constituents, including albumin, and lipoproteins [6,7]. However, as albumin has a slow exchange rate (half-life of ca. 20 days), recurrent and extensive hemolysis may also result in ∗ saturation of the heme-binding capacity of this abundant plasma Corresponding author at: INSERM UMRS 1138, Centre de Recherche des Corde- protein. Since the pathologically-relevant form of heme is the one liers, 75006 Paris, France. that is loosely bound to proteins (referred to as free heme), its esti- E-mail addresses: [email protected], [email protected] (J.D. Dimitrov). mation is of utmost clinical importance [3]. However, to the best 1 These authors contributed equally to the work. https://doi.org/10.1016/j.jpba.2019.05.013 0731-7085/© 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4. 0/). 312 R. Noé et al. / Journal of Pharmaceutical and Biomedical Analysis 172 (2019) 311–319 of knowledge, there is no estimation of the total heme-scavenging was used directly. After recording the spectra of the protein solu- capacity of human serum. There are commercialized techniques for tions, potassium cyanide, glutathione or cysteine were added to measurement of concentration of total heme in plasma that have the protein solutions at final concentration of 10 mM. Following, been applied in different studies [8–10]. These approaches relay on vigorous homogenization, the spectra were measured as described colorimetric detection of heme or on the pseudo-peroxidase activ- above. ity of heme in the presence of hydrogen peroxide. Nonetheless, these techniques fail to differentiate the hemoprotein-bound from 2.2.3. Determination of cysteine concentration for heme-cysteine free heme. spectral measurements Here we describe a simple method that relays on characteris- Cysteine was diluted in PBS to concentrations of 0.156, 0.312, tic spectral changes of protein-free heme upon interaction with a 0.625, 1.25, 2.5, 5, 10 and 20 mM. The UV–vis spectra of each thiol-containing compound (cysteine). We demonstrated that this concentration of cysteine in the presence of 20 ␮M hemin were technique could be reliably used for estimation of the free extra- measured as described above. To obtain differential spectra, the cellular heme. The method can also be applied for evaluation of the absorbance spectrum in the range 300–700 nm of hemin at 20 ␮M heme-binding capacity of different proteins and human serum as was subtracted from the spectra of hemin in the presence of each well as for assessment of quality of heme-binding proteins used in concentration of cysteine. therapy or as blood substituents. 2.2.4. Building of a calibration curve for heme-cysteine Hemin was diluted in PBS to concentrations of 0.312, 0.625, 1.25, 2. Material and methods 2.5, 5, 10, 20, and 40 ␮M. The UV–vis spectra of each concentration in the presence of excess of cysteine (10 mM final concentration) 2.1. Materials were measured as described above. To obtain differential spec- tra, the absorbance in the range 300–700 nm of heme at a given Hemin was obtained from Frontier Scientific, Inc. (Logan, UT). concentration was subtracted from the spectrum of hemin in the Cysteine, reduced glutathione, potassium cyanide, DMSO, phenyl- presence of cysteine. The obtained differential spectra have promi- hydrazine hydrochloride, 5,5 -dithiobis(2-nitrobenzoic acid), 2,2 - nent absorbance maximum at 364 nm. The values of the differential azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), human absorbance at this maximum were plotted versus the concentration hemopexin and human hemoglobin were obtained from Sigma- of hemin. Aldrich (St. Louis, MO). All chemicals were with the highest available purity. Human serum albumin (LFB, Les Ulis, France) and 2.2.5. Evaluation of heme-binding capacity of human plasma human pooled immunoglobulin G (IVIg, Endobulin, Baxter USA) proteins were thoroughly dialyzed against PBS and stored before use at − ◦ Human serum albumin, and human pooled IgG were diluted in 20 C at concentrations of 200 mg/ml and 80 mg/ml, respectively. PBS at concentrations ranging from 1.25–320 ␮M (dilution by fac- Human AB serum obtained from healthy donor was purchased from tor of 2). Alternatively, human hemopexin and human monoclonal Etablissement franc¸ ais du sang, Paris, France, (ethical authorization ◦ IgG1 (identified in our previous studies to interact with heme) were N 12/EFS/079). All stock solutions were freshly prepared and used diluted in PBS in concentrations ranging 0.312–40 ␮M. Next, 20 within 24 h. Hemin was dissolved in DMSO to final concentration ␮M final concentration of hemin was added to each protein dilu- of 2 mM. The stock solutions of the other chemicals and proteins tion and UV–vis spectra were recorded as described above.
Load more

Recommended publications
  • Plasma Protein Binding Structure Activity Relationship Related to the N-Terminus of Daptomycin
    Monash Institute of Pharmaceutical P1318 Sciences ECCMID 2017, Plasma protein binding structure activity relationship related to the N-terminus of daptomycin 381 Royal Parade, Parkville, VIC 3052 Melbourne, Australia Vienna 1 2 3 2 4 1 Tel: +61 3 9903 9539 Elena K. Schneider , Johnny X. Huang , Vincenzo Carbone , Matthew A. Cooper , Jian Li *, Tony Velkov * [email protected] 1Drug Development and Innovation, Drug Delivery, Disposition and Dynamics. Monash Institute of Pharmaceutical Sciences, Monash University, Australia. 2Institute for Molecular Bioscience, The University of Queensland St Lucia QLD 4072. 3Animal Nutrition and Health, Ag Research Limited, Grasslands Research Centre, Palmerston North, New Zealand. 4Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University, Australia. Abstract Methods Results Background: Daptomycin is a lipopeptide antibiotic that is highly bound to plasma proteins. To date the plasma components and • Flourometric binding assay: • SPR: structure-activity relationships responsible for the plasma protein binding profile of daptomycin remain uncharacterized. We used the site selective fluorescent probes . Native daptomycin binds in order of decreasing Methods: dansylamide (site 1) and dansylsarcosine affinity to: HSA >> α-1-antitrypsin > low density (site 2) on HSA. As the control we employed lipoprotein (LDL) ≥ haemoglobin > sex hormone • In the present study we have employed surface plasmon resonance (SPR) and fluorescence displacement assay to Nifedipine for site 1 and ibuprofen
    [Show full text]
  • Detailed Structure and Pathophysiological Roles of the Iga-Albumin Complex in Multiple Myeloma
    International Journal of Molecular Sciences Article Detailed Structure and Pathophysiological Roles of the IgA-Albumin Complex in Multiple Myeloma Yuki Kawata 1, Hisashi Hirano 1, Ren Takahashi 1, Yukari Miyano 1, Ayuko Kimura 1, Natsumi Sato 1, Yukio Morita 2, Hirokazu Kimura 1,* and Kiyotaka Fujita 1 1 Department of Health Sciences, Gunma Paz University Graduate School of Health Sciences, 1-7-1, Tonyamachi, Takasaki-shi, Gunma 370-0006, Japan; [email protected] (Y.K.); [email protected] (H.H.); [email protected] (R.T.); [email protected] (Y.M.); [email protected] (A.K.); [email protected] (N.S.); [email protected] (K.F.) 2 Laboratory of Public Health II, Azabu University School of Veterinary Medicine, 1-17-71, Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5201, Japan; [email protected] * Correspondence: [email protected]; Tel.: +81-27-365-3366; Fax: +81-27-388-0386 Abstract: Immunoglobulin A (IgA)-albumin complexes may be associated with pathophysiology of multiple myeloma, although the etiology is not clear. Detailed structural analyses of these protein– protein complexes may contribute to our understanding of the pathophysiology of this disease. We analyzed the structure of the IgA-albumin complex using various electrophoresis, mass spectrom- etry, and in silico techniques. The data based on the electrophoresis and mass spectrometry showed that IgA in the sera of patients was dimeric, linked via the J chain. Only dimeric IgA can bind to albumin molecules leading to IgA-albumin complexes, although both monomeric and dimeric forms of IgA were present in the sera.
    [Show full text]
  • Efficacy and Safety of Human Serum Albumin–Cisplatin Complex In
    International Journal of Molecular Sciences Article Efficacy and Safety of Human Serum Albumin–Cisplatin Complex in U87MG Xenograft Mouse Models Cho Rong Park 1,2,3, Hyo Young Kim 1,2, Myung Geun Song 1,2,4,*, Yun-Sang Lee 1,5, Hyewon Youn 1,2,6, June-Key Chung 1,2,3,4, Gi Jeong Cheon 1,2,4,5,7 and Keon Wook Kang 1,2,3,4,5,6,8,* 1 Department of Nuclear Medicine, Seoul National University Hospital, 101 Daehak-ro, Jongno-gu, Seoul 03080, Korea; siofl[email protected] (C.R.P.); [email protected] (H.Y.K.); [email protected] (Y.-S.L.); [email protected] (H.Y.); [email protected] (J.-K.C.); [email protected] (G.J.C.) 2 Laboratory of Molecular Imaging and Therapy, Cancer Research Institute, Seoul 03080, Korea 3 Department of Biomedical Sciences, Seoul National University Graduate School, Biomedical Science Building, Seoul 03080, Korea 4 Biomedical Research Institute, Seoul National University Hospital, Seoul 03080, Korea 5 Radiation Research Institute, Seoul National University College of Medicine, Seoul 03080, Korea 6 Cancer Imaging Center, Seoul National University Hospital, Seoul 03080, Korea 7 Institute on Aging, Seoul National University, Seoul 08826, Korea 8 Bio-MAX Institute, Seoul National University, Seoul 08826, Korea * Correspondence: [email protected] (M.G.S.); [email protected] (K.W.K.); Tel.: +82-2-2072-2803 (K.W.K.) Received: 18 September 2020; Accepted: 22 October 2020; Published: 26 October 2020 Abstract: Cisplatin (cis-diamminedichloroplatinum (II), CDDP) is a chemotherapeutic drug widely used against many solid tumors.
    [Show full text]
  • The Influence of Oxidative Stress on Serum Albumin Structure As A
    pharmaceuticals Article The Influence of Oxidative Stress on Serum Albumin Structure as a Carrier of Selected Diazaphenothiazine with Potential Anticancer Activity Małgorzata Maci ˛azek-Jurczyk˙ 1,* , Beata Morak-Młodawska 2 , Małgorzata Jele ´n 2 , Wiktoria Kope´c 1, Agnieszka Szkudlarek 1 , Aleksandra Owczarzy 1, Karolina Kulig 1, Wojciech Rogóz˙ 1 and Jadwiga Pozycka˙ 1 1 Department of Physical Pharmacy, Faculty of Pharmaceutical Sciences in Sosnowiec, Medical University of Silesia in Katowice, 40-055 Katowice, Poland; [email protected] (W.K.); [email protected] (A.S.); [email protected] (A.O.); [email protected] (K.K.); [email protected] (W.R.); [email protected] (J.P.) 2 Department of Organic Chemistry, Faculty of Pharmaceutical Sciences in Sosnowiec, Medical University of Silesia in Katowice, 40-055 Katowice, Poland; [email protected] (B.M.-M.); [email protected] (M.J.) * Correspondence: [email protected]; Tel.: +48-32-364-15-80 Abstract: Albumin is one of the most important proteins in human blood. Among its multiple functions, drug binding is crucial in terms of drug distribution in human body. This protein undergoes many modifications that are certain to influence protein activity and affect its structure. One such reaction is albumin oxidation. Chloramine T is a strong oxidant. Solutions of human serum albumin, both non-modified and modified by chloramine T, were examined with the use of fluorescence, ˙ Citation: Maci ˛azek-Jurczyk, M.; absorption and circular dichroism (CD) spectroscopy. 10H-3,6-diazaphenothiazine (DAPT) has Morak-Młodawska, B.; Jele´n,M.; anticancer activity and it has been studied for the first time in terms of binding with human serum Kope´c,W.; Szkudlarek, A.; Owczarzy, albumin—its potential as a transporting protein.
    [Show full text]
  • Detection of Food Proteins in Human Serum Using Mass Spectrometry Methods
    University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Dissertations, Theses, & Student Research in Food Science and Technology Food Science and Technology Department 8-2020 DETECTION OF FOOD PROTEINS IN HUMAN SERUM USING MASS SPECTROMETRY METHODS Abigail S. Burrows University of Nebraska-Lincoln, [email protected] Follow this and additional works at: https://digitalcommons.unl.edu/foodscidiss Part of the Food Science Commons Burrows, Abigail S., "DETECTION OF FOOD PROTEINS IN HUMAN SERUM USING MASS SPECTROMETRY METHODS" (2020). Dissertations, Theses, & Student Research in Food Science and Technology. 109. https://digitalcommons.unl.edu/foodscidiss/109 This Article is brought to you for free and open access by the Food Science and Technology Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Dissertations, Theses, & Student Research in Food Science and Technology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. DETECTION OF FOOD PROTEINS IN HUMAN SERUM USING MASS SPECTROMETRY METHODS by Abigail S. Burrows A DISSERTATION Presented to the Faculty of The Graduate College of the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy Major: Food Science and Technology Under the Supervision of Professor Philip E. Johnson Lincoln, Nebraska August, 2020 DETECTION OF FOOD PROTEINS IN HUMAN SERUM USING MASS SPECTROMETRY METHODS Abigail S. Burrows, Ph.D. University of Nebraska, 2020 Advisor: Philip E. Johnson Allergenic peanut proteins are highly resistant to digestion and are detectable by immunoassays after gastrointestinal digestion. The application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for in vivo detection of peptides originating from allergenic food proteins has not been thoroughly studied.
    [Show full text]
  • By Transferrin (Prostate Cancer/Tumor Metastasis/Growth Factors) MARCELA CHACKAL Rossi* and BRUCE R
    Proc. Natl. Acad. Sci. USA Vol. 89, pp. 6197-6201, July 1992 Medical Sciences Selective stimulation of prostatic carcinoma cell proliferation by transferrin (prostate cancer/tumor metastasis/growth factors) MARCELA CHACKAL RossI* AND BRUCE R. ZETTERtt *Department of Biological Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139; and tDepartment of Surgery and Department of Cellular and Molecular Physiology, Children's Hospital and Harvard Medical School, Boston, MA 02115 Communicated by Judah Folkman, March 20, 1992 ABSTRACT Aggressive prostatic carcinomas most fre- stimulate prostatic carcinoma cell growth, but none had quently metastasize to the skeletal system. We have previously substantial activity (7). shown that cultured human prostatic carcinoma cells are highly In the present study, we describe the purification of a responsive to growth factors found in human bone marrow. To mitogenic factor for human prostatic carcinoma cells from identify the factor(s) responsible for the increased prostatic human bone marrow. Our results reveal that the purified carcinoma cell proliferation, we fractionated crude bone mar- activity resides in transferrin (Tf), an iron-transporting mol- row preparations by using hydroxylapatite HPLC. The major ecule found in high concentration in bone marrow. In addi- activity peak contained two high molecular weight bands (Mr tion, prostatic carcinoma cells show an increased respon- = 80,000 and 69,000) that cross-reacted with antibodies to siveness to the growth-promoting activity of Tf relative
    [Show full text]
  • Serum Albumin
    Entry Serum Albumin Daria A. Belinskaia 1,*, Polina A. Voronina 1, Anastasia A. Batalova 1 and Nikolay V. Goncharov 1,2 1 Sechenov Institute of Evolutionary Physiology and Biochemistry, Russian Academy of Sciences, pr. Torez 44, 194223 St. Petersburg, Russia; [email protected] (P.A.V.); [email protected] (A.A.B.); [email protected] (N.V.G.) 2 Research Institute of Hygiene, Occupational Pathology and Human Ecology, p/o Kuzmolovsky, 188663 Leningrad Region, Russia * Correspondence: [email protected] Definition: Being one of the most abundant proteins in human and other mammals, albumin plays a crucial role in transporting various endogenous and exogenous molecules and maintaining of colloid osmotic pressure of the blood. It is not only the passive but also the active participant of the pharmacokinetic and toxicokinetic processes possessing a number of enzymatic activities. A free thiol group of the albumin molecule determines the participation of the protein in redox reactions. Its activity is not limited to interaction with other molecules entering the blood: of great physiological importance is its interaction with the cells of blood, blood vessels and also outside the vascular bed. This entry contains data on the enzymatic, inflammatory and antioxidant properties of serum albumin. Keywords: albumin; blood plasma; enzymatic activities; oxidative stress 1. Introduction: Physico-Chemical, Evolutionary and Genetic Aspects Albumin is a family of globular proteins, the most common of which are the serum albumins. All the proteins of the albumin family are water-soluble and moderately soluble Citation: Belinskaia, D.A.; Voronina, in concentrated salt solutions. The key qualities of albumin are those of an acidic, highly P.A.; Batalova, A.A.; Goncharov, N.V.
    [Show full text]
  • Alpha-2-Macroglobulin
    Albumin Modular P Application Note Guideline for Determination of Albumin in Serum/Plasma on Modular P General information Intended use The Application Note is intended for the quantitative determination of albumin in human sample material by turbidimetry on Modular P (1, 2). Measuring range Approximately 7.7-61 g/L depending on the specific lot of the calibrator. In case of post-concentration or -dilution the range can be expanded to 4-70 g/L. Reference interval 35-52 g/L (3). It is recommended to determine the reference interval for the local population. Instrument settings Instrument programming is performed according to “Instrument Settings” on page 3. Code No. Name Reagents Antibody Q 0328 DakoCytomation Polyclonal Rabbit Anti-Human Serum Albumin Reaction buffer S 2006 DakoCytomation Reaction Buffer 3 Diluent S 2005 DakoCytomation Dilution Buffer 1 Calibrator X 0908 DakoCytomation Human Serum Protein Calibrator Controls X 0939 DakoCytomation Human Serum Protein Low Control Samples Human serum, heparin-plasma or EDTA-plasma. Stable for 7 days at 2-8 ºC. Stable for 3 months at –20 ºC (if frozen only once). It is recommended to thaw frozen samples at 37 °C. Mix well before analysis. Calibrator Predilute the DakoCytomation Human Serum Protein Calibrator, code No. X 0908, 1:26.0 (e.g. 100 µL calibrator + 2500 µL diluent). The final dilution of standards is performed automatically by the instrument. Reaction buffer (R1) The reaction buffer is ready for use. On board stability is 28 days. Antibody (R3) Predilute the antibody 1:16.0 (e.g. 1200 µL antibody + 18000 µL diluent).
    [Show full text]
  • Fragmentation of Human Albumin with Proteolytic Enzymes and Its Antigenicity with Special Reference to Human-Specificity KAORU S
    Tohoku J. exp. Med., 1978, 125, 115-120 Fragmentation of Human Albumin with Proteolytic Enzymes and Its Antigenicity with Special Reference to Human-Specificity KAORUSAGISAKA, YOKO SUGIYAMAand NOBORUTSUGAWA Department of Legal Medicine, Gifu University School of Medicine, Gifu 500 SAGISAKA, K., SUGIYAMA, Y. and TSUGAWA, N. Fragmentation of Human Albumin with Proteolytic Enzymes and Its Antigenicity with Special Reference to Human-Specificity. Tohoku J. exp. Med., 1978, 125 (2), 115-120 Human albumin prepared by the trichloroacetic acid method was treated with bromelin, ficin, papain, pronase or trypsin. Pronase or trypsin-treated albumin showed four fragments on polyacrylamide disc electrophoresis. When rabbit antiserum to pronase-treated albumin was adsorbed with monkey albumin, antibody with human specificity was completely abolished, whereas anti-albumin serum adsorbed with pronase-treated albumin retained the antibody to human albumin. It was considered that antigenic site with human specificity in albumin structure was inactivated by pronase.„Ÿalbumin fragmentation; human-specificity The species identification of blood stains found on the scene has been one of the most important problem in forensic science. Nowadays, it has been most solved by immunoassay with anti-human hemoglobin. On the other hand, human specificity of blood proteins other than hemoglobin has been investigated to determine species-specificity, indicating that human alpha 2-macroglobulin and albumin can be distinguished serologically from those of the other mammals (Tsugawa et al. 1971; Hara 1974; Bauer 1974). Recently, complete amino acid sequence of human albumin and antigenicity of trypsin fragments of bovine albumin have been studied (Habeeb et al. 1974; Behrens et al. 1975; Meloun et al.
    [Show full text]
  • Fetal Bovine Serum Albumin Inhibits Antimicrobial Peptide Activity and Binds Drug Only in Complex with Α1‑Antitrypsin Wen‑Hung Tang, Chiu‑Feng Wang & You‑Di Liao*
    www.nature.com/scientificreports OPEN Fetal bovine serum albumin inhibits antimicrobial peptide activity and binds drug only in complex with α1‑antitrypsin Wen‑Hung Tang, Chiu‑Feng Wang & You‑Di Liao* Several antimicrobial peptides (AMPs) have been developed for the treatment of infections caused by antibiotic‑resistant microbes, but their applications are primarily limited to topical infections because in circulation they are bound and inhibited by serum proteins. Here we have found that some AMPs, such as TP4 from fsh tilapia, and drugs, such as antipyretic ibuprofen, were bound by bovine serum albumin only in complex with α1‑antitrypsin which is linked by disulfde bond. They existed in dimeric complex (2 albumin ‑2 α1‑antitrypsin) in the bovine serum only at fetal stage, but not after birth. The hydrophobic residues of TP4 were responsible for its binding to the complex. Since bovine serum is a major supplement in most cell culture media, therefore the existence and depletion of active albumin/ α1‑antitrypsin complex are very important for the assay and production of biomolecules. Te widespread use of antibiotics in both medicine and agriculture has contributed to the emergence of drug- resistant bacteria1,2. Tus, development of new antimicrobials with unique targets and mechanism of action that are diferent from those of conventional antibiotics is urgently needed. Natural antimicrobial peptides (AMPs) have been isolated from multiple sources. Tey possess amphipathic structure (cationic and hydrophobic) and consist of 10–50 amino acid residues in length. Tey are able to disrupt the membrane integrity of bacteria in few minutes even for antibiotic-resistant bacteria and not prone to inducing drug-resistance2–4.
    [Show full text]
  • Binding of Bovine Serum Albumin to Heparin Determined by Turbidimetric Titration and Frontal Analysis Continuous Capillary Electrophoresis
    Analytical Biochemistry 295, 158–167 (2001) doi:10.1006/abio.2001.5129, available online at http://www.idealibrary.com on Binding of Bovine Serum Albumin to Heparin Determined by Turbidimetric Titration and Frontal Analysis Continuous Capillary Electrophoresis Toshiaki Hattori,1 Kozue Kimura, Emek Seyrek, and Paul L. Dubin2 Department of Chemistry, Indiana University–Purdue University, Indianapolis, Indiana 46202 Received October 23, 2000; published online July 19, 2001 The binding of proteins to proteoglycans (PGs)3 is an The association of proteins with glycosaminogly- important biological phenomenon. For example, the cans is a subject of growing interest, but few tech- interaction between chondroitin sulfate or keratan sul- niques exist for elucidating this interaction quantita- fate proteoglycans and collagen fibrils in the cartilage tively. Here we demonstrate the application of matrix defines the final shape of the tissue and pro- capillary electrophoresis to the system of serum albu- vides the ability to withstand compressive load (1). min (SA) and heparin (Hp). These two species form Heparan sulfate proteoglycans (HSPGs) interact with soluble complexes, the interaction increasing with re- fibronectin–collagen complexes in plasma membranes duction in pH and/or ionic strength (I). The acid–base to assist cell adhesion (1). HSPGs in the glomerular property of Hp was characterized by potentiometric basement membrane (GBM) also provide selective fil- titration of ion-exchanged Hp. Conditions for complex tration of proteins (1, 2). These interactions appear to formation with SA were qualitatively determined by have a strong electrostatic component, arising from the turbidimetry, which revealed points of incipient bind- interactions between charges on the proteins and the ing (pHc) and phase separation (pH␾), both of which intense negative charge on glycosaminoglycans (GAGs) depend on I.
    [Show full text]
  • Molecular Docking of Amitriptyline to Ceruloplasmin, Retinol-Binding Protein, and Serum Albumin
    Online - 2455-3891 Vol 11, Issue 2, 2018 Print - 0974-2441 Research Article MOLECULAR DOCKING OF AMITRIPTYLINE TO CERULOPLASMIN, RETINOL-BINDING PROTEIN, AND SERUM ALBUMIN RAMCHANDER MERUGU1, KALPANA V SINGH2* 1Department of Biochemistry, University College of Science, Mahatma Gandhi University, Nalgonda, Telangana, India. 2Department of Chemistry and Pharmaceutical Chemistry, Government Madhav Science Postgraduate College, Ujjain, Madhya Pradesh, India. Email: [email protected] Received: 22 August 2017, Revised and Accepted: 07 November 2017 ABSTRACT Objective: A drug’s efficiency depends on the binding capacity of the drug with the particular plasma protein. The less bound drug can be easily diffused through cell membranes. The present study deals with in silico studies of amitriptyline binding to three plasma proteins human ceruloplasmin (HCP), cellular retinol-binding protein (CRBP), and human serum albumin (HSA) and tries to establish the binding capacity behavior with the frontier molecular orbital approach. Methods: Amitriptyline is selected as legend and docked with three plasma proteins HCP, HCP PDB ID 1KCW, CRBP PDB ID 5LJC, and HSA. Docking calculations were carried out using docking server. frontier molecular orbital calculations are performed through web-based computational chemistry interface WEBMO version 17.0.012e using server Buchhner.chem.hope.edu. on computational engine MOPAC. Results: HCP and HSA predominantly show polar and hydrophobic interactions, whereas CRBP forms hydrogen bond apart from polar and hydrophobic interactions. Favorable values of inhibition constant, Ki, is obtained which is equal to 1.13 µM for CRBP, 6.00 µM for HCP, and 2.00 µM for has. Conclusion: A studies prove that amitriptyline can bind to all three plasma proteins, namely, HCP, CRBP, and HSA.
    [Show full text]