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1.
Summary : We present the O2 binding properties of recombinant human serum albumin (rHSA) mutants complexed with an iron(II) protoporphyrin IX as a prosthetic heme group. Iron(III) protoporphyrin IX (hemin) is bound within subdomain IB of HSA with weak axial coordination by Tyr-161. In order to confer O2 binding capability to this naturally occurring hemoprotein: (i) a proximal histidine was introduced into position Ile-142; and (ii) the coordinated Tyr-161 was replaced with hydrophobic Leu using site-directed mutagenesis. It provided a recombinant HSA double-mutant [rHSA(I142H/Y161L) = rHSA(HL)]. The rHSA(HL)–heme formed a ferrous five-coordinate high-spin complex with axial ligation of His-142 under an Ar atmosphere. This artificial hemoprotein binds O2 at room temperature. Laser flash photolysis experiments demonstrated that O2 rebinidng to rHSA(HL)–heme displays monophasic kinetics, whereas the CO recombination process obeyed a double-exponential pattern. This might be attributable to the two different geometries of the axial imidazole coordination arising from the two orientations of the porphyrin plane in the heme pocket. The O2 binding affinity of rHSA(HL)–heme was considerably lower than those of R-state hemoglobin (Hb) and myoglobin (Mb), principally because of the high O2 dissociation rate constant. The third mutations have been introduced into the distal side of the heme (at position Leu-185 or Arg-186) to increase the O2 binidng affinity. The rHSA(HL/L185N)–heme showed high O2 binding affinity ( : 1 Torr), which is 18-fold greater than that of the original double mutant rHSA(HL)–heme and which is rather close to those of Hb (R-state) and Mb. Furthermore, replacement of polar Arg-186 with Leu or Phe adjusted the O2 binding affinity ( ) to 10 Torr, which is almost equivalent to value for human red blood cells. 相似文献
2.
3.
The simple one-pot reaction of protoporphyrin IX and omega-(N-imidazolyl)alkylamine or O-methyl-L-histidyl-glycine with benzotriazol-1-yl-oxytris(dimethylamino)phosphonium hexafluorophosphate at room temperature produced a series of protoporphyrin IX species with a covalently linked proximal base at the propionate side-chain. The central iron was inserted by the general FeCl2 method, converting the free-base porphyrins to the corresponding protoheme IX derivatives. Mesoporphyrin IX and diacetyldeuteroporphyrin IX analogues were also prepared by the same procedure. The Fe(II) complexes formed dioxygen (O2) adducts in dimethylformamide at 25 degrees C. Some of them were incorporated into the hydrophobic domain of recombinant human serum albumin (rHSA), providing albumin-heme hybrids (rHSA-heme), which can bind and release O2 in aqueous media (pH 7.3, 25 degrees C). The oxidation process of converting the dioxygenated heme in rHSA to the inactive Fe(III) state obeyed first-order kinetics, indicating that the mu-oxo dimer formation was prevented by the immobilization of heme in the albumin scaffold. The rHSA-heme, in which the histidylglycil tail coordinates to the Fe(II) center, showed the most stable O2 adduct complexes. 相似文献
4.
It has long been known that homologous blood transfusion will result in a lot ofserious problems such as viral infections,for example AIDS,hepatitis,antigenicsensitization and GVHD;therefore aggressive testing of donor blood has beenadopted[1 ,2 ] .Even after this introduction,which is time-consuming and expensive,wecould not eliminate all the risks. In the wake of these kinds of pitfalls,production andclinical use of the blood substitutes have emerged.The essential aim of blood substitutei… 相似文献
5.
Komatsu T Nakagawa A Zunszain PA Curry S Tsuchida E 《Journal of the American Chemical Society》2007,129(36):11286-11295
Complexing an iron protoporphyrin IX into a genetically engineered heme pocket of recombinant human serum albumin (rHSA) generates an artificial hemoprotein, which can bind O2 in much the same way as hemoglobin (Hb). We previously demonstrated a pair of mutations that are required to enable the prosthetic heme group to bind O2 reversibly: (i) Ile-142-->His, which is axially coordinated to the central Fe2+ ion of the heme, and (ii) Tyr-161-->Phe or Leu, which makes the sixth coordinate position available for ligand interactions [I142H/Y161F (HF) or I142H/Y161L (HL)]. Here we describe additional new mutations designed to manipulate the architecture of the heme pocket in rHSA-heme complexes by specifically altering distal amino acids. We show that introduction of a third mutation on the distal side of the heme (at position Leu-185, Leu-182, or Arg-186) can modulate the O2 binding equilibrium. The coordination structures and ligand (O2 and CO) binding properties of nine rHSA(triple mutant)-heme complexes have been physicochemically and kinetically characterized. Several substitutions were severely detrimental to O2 binding: for example, Gln-185, His-185, and His-182 all generated a weak six-coordinate heme, while the rHSA(HF/R186H)-heme complex possessed a typical bis-histidyl hemochrome that was immediately autoxidized by O2. In marked contrast, HSA(HL/L185N)-heme showed very high O2 binding affinity (P1/2O2 1 Torr, 22 degrees C), which is 18-fold greater than that of the original double mutant rHSA(HL)-heme and very close to the affinities exhibited by myoglobin and the high-affinity form of Hb. Introduction of Asn at position 185 enhances O2 binding primarily by reducing the O2 dissociation rate constant. Replacement of polar Arg-186 with Leu or Phe increased the hydrophobicity of the distal environment, yielded a complex with reduced O2 binding affinity (P1/2O2 9-10 Torr, 22 degrees C), which nevertheless is almost the same as that of human red blood cells and therefore better tuned to a role in O2 transport. 相似文献
6.
Komatsu T Ohmichi N Nakagawa A Zunszain PA Curry S Tsuchida E 《Journal of the American Chemical Society》2005,127(45):15933-15942
The binding properties of O2 and CO to recombinant human serum albumin (rHSA) mutants with a prosthetic heme group have been physicochemically and kinetically characterized. Iron(III) protoporphyrin IX (hemin) is bound in subdomain IB of wild-type rHSA [rHSA(wt)] with weak axial coordination by Tyr-161. The reduced ferrous rHSA(wt)-heme under an Ar atmosphere exists in an unusual mixture of four- and five-coordinate complexes and is immediately autoxidized by O2. To confer O2 binding capability on this naturally occurring hemoprotein, a proximal histidine was introduced into position Ile-142 or Leu-185 by site-directed mutagenesis. A single mutant (I142H) and three double mutants (I142H/Y161L, I142H/Y161F, and Y161L/L185H) were prepared. Both rHSA(I142H/Y161L)-heme and rHSA(I142H/Y161F)-heme formed ferrous five-N-coordinate high-spin complexes with axial ligation of His-142 under an Ar atmosphere. These artificial hemoproteins bind O2 at room temperature. Mutation at the other side of the porphyrin, Y161L/L185H, also allowed O2 binding to the heme. In contrast, the single mutant rHSA(I142H)-heme could not bind O2, suggesting that removal of Y161 is necessary to confer reversible O2 binding. Laser flash photolysis experiments showed that the kinetics of CO recombination with the rHSA(mutant)-heme were biphasic, whereas O2 rebinding exhibited monophasic kinetics. This could be due to the two different geometries of the axial imidazole coordination arising from the two orientations of the porphyrin plane in the heme pocket. The O2 binding affinities of the rHSA(mutant)-heme were significantly lower than those of hemoglobin and myoglobin, principally due to the high O2 dissociation rates. Changing Leu-161 to Phe-161 at the distal side increased the association rates of both O2 and CO, which resulted in enhanced binding affinity. 相似文献
7.
基因工程技术已经成为研究和生产重组人血清白蛋白(rHSA)替代人血清白蛋白(HSA)的重点技术,而白蛋白的纯化则是该技术的关键。本文主要介绍了从转基因猪血中纯化rHSA的一种新方法,即热乙醇沉淀与多级色谱分离相结合的rHSA纯化方法。热乙醇沉淀法可从猪血浆中获得rHSA粗提取液,此时rHSA的纯度可达69.5%,回收率达51.3%。进一步采用多级色谱分离法,即阴离子交换色谱和反相色谱法进一步纯化,得到rHSA的最终纯度约为100.0%,总回收率为41.1%。该方法为从转基因猪血浆中大规模纯化用于临床和生化研究的高纯度rHSA提供可能,同时也为rHSA替代HSA奠定了基础。 相似文献
8.
Prof. Dr. Kasper P. Kepp 《Chemphyschem》2013,14(15):3551-3558
This paper reports the computed O2 binding to heme, which for the first time explains experimental enthalpies for this process of central importance to bioinorganic chemistry. All four spin states along the relaxed Fe? O2‐binding curves were optimized using the full heme system with dispersion, thermodynamic, and scalar‐relativistic corrections, applying several density functionals. When including all these physical terms, the experimental enthalpy of O2 binding (?59 kJ mol?1) is closely reproduced by TPSSh‐D3 (?66 kJ mol?1). Dispersion changes the potential energy surfaces and leads to the correct electronic singlet and heptet states for bound and dissociated O2. The experimental activation enthalpy of dissociation (~82 kJ mol?1) was also accurately computed (~75 kJ mol?1) with an actual barrier height of ~60 kJ mol?1 plus a vibrational component of ~10 and ~5 kJ mol?1 due to the spin‐forbidden nature of the process, explaining the experimentally observed difference of ~20 kJ mol?1 in enthalpies of binding and activation. Most importantly, the work shows how the nearly degenerate singlet and triplet states increase crossover probability up to ~0.5 and accelerate binding by ~100 times, explaining why the spin‐forbidden binding of O2 to heme, so fundamental to higher life forms, is fast and reversible. 相似文献
9.
Komatsu T Wang RM Zunszain PA Curry S Tsuchida E 《Journal of the American Chemical Society》2006,128(50):16297-16301
We present the photophysical properties of complexes of recombinant human serum albumin (rHSA) with Zn(II)-protoporphyrin IX (ZnPP) and their activities in the photosensitized reduction of water to hydrogen (H2) using methyl viologen (MV2+) as an electron relay. The ZnPP is bound in subdomain IB of wild-type rHSA [rHSA(wt] by an axial coordination of Tyr-161 and, in the rHSA(I142H/Y161L) mutant [rHSA(His], by a His-142 coordination. Both the rHSA(wt)-ZnPP and rHSA(His)-ZnPP complexes showed a long-lived photoexcited triplet state with lifetimes (tauT) of 11 and 2.5 ms, respectively. The accommodation of ZnPP into the protein matrix efficiently eliminated the collisional triplet self-quenching process. The addition of a water-soluble electron acceptor, MV2+, resulted in a significant decrease in the triplet lifetime. The transition absorption spectrum revealed the oxidative quenching of rHSA-3ZnPP* by MV2+. The quenching rate constant (kq) and backward electron transfer rate constant (kb) were determined to be 1.4 x 10(7) and 4.7 x 10(8) M(-1) s(-1) for rHSA(wt)-ZnPP. In the presence of the colloidal PVA-Pt as a catalyst and triethanolamine (TEOA) as a sacrificial electron donor, the photosensitized reduction of water to H2 takes place. The efficiency of the photoproduction of H2 was greater than that of the system using the well-known organic chromophore, tetrakis(1-methylpyridinium-4-yl)porphinatozinc(II) (ZnTMPyP4+), under the same conditions. 相似文献
10.
Recombinant human serum albumin (rHSA) incorporating the tetraphenylporphinatoiron(II) derivative with a covalently linked proximal base (FeP) [albumin-heme (rHSA-FeP)] is a synthetic hemoprotein, which can bind and release dioxygen (O2) reversibly under physiological conditions. The coordination structure and spin-state of the active site in rHSA-FeP with O2 and carbon monoxide (CO) were revealed by magnetic circular dichroism (MCD), resonance Raman (RR), and infrared (IR) spectroscopy. Under an N2 atmosphere, the MCD spectrum of rHSA-FeP showed the formation of the five-coordinate ferrous high-spin complex of FeP. Upon exposure of this solution to O2 or CO, the spectral pattern immediately changed to that of a six-coordinate ferrous low-spin species. The vibration stretching frequencies of the coordinated O2 (νO2) and CO (νCO) were observed at 1158 cm−1 and 1964 cm−1, respectively. The electronic structures of the O2- and CO-adduct complexes of FeP in the hydrophobic pocket of albumin are both identical to those for FeP itself in toluene solution. 相似文献
11.
Oxygen‐binding Protein Fiber and Microgel: Supramolecular Myoglobin–Poly(acrylate) Conjugates
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Dr. Toshikazu Ono Yasushi Hisaoka Dr. Akira Onoda Dr. Koji Oohora Prof. Dr. Takashi Hayashi 《化学:亚洲杂志》2016,11(7):1036-1042
A supramolecular conjugate of myoglobin (Mb) and water‐soluble poly(acrylate), (PA5k and PA25k, where 5k and 25k represent the molecular weight of the polymers, respectively), is constructed on the basis of a noncovalent heme‐heme pocket interaction. The modified heme with an amino group linked to the terminus of one of the heme‐propionates is coupled to the side‐chain carboxyl groups of poly(acrylate) activated by N‐hydroxysuccinimide. The ratios of the heme‐modified monomer unit and the unmodified monomer unit (m:n) in the polymer chains of Heme‐PA5k and Heme‐PA25k were determined to be 4.5:95.5 and 3.1:96.9, respectively. Subsequent addition of apoMb to the conjugated polymers provides Mb‐connected fibrous nanostructures confirmed by atomic force microscopy. A mixture of the heme‐modified polymer and dimeric apomyoglobin as a cross‐linker forms a microgel in which the reconstituted myoglobin retains its native exogenous ligand binding activity. 相似文献
12.
Bourhim M Rajendran A Ramos Y Srikrishnan T Sulkowski E 《Journal of chromatographic science》2008,46(6):574-576
An investigation of the binding of native and recombinant human serum albumin and bovine serum albumin on three thiophilic gels, PyS, 2S, and 3S was performed. In addition to these proteins, we studied serum albumins from several species such as goat, rabbit, guinea pig, rat, hamster, baboon, and pig. Our results reveal that recombinant human serum albumin (rHSA) binds completely to PyS whereas native human serum albumin and bovine serum albumin bind only partially to PyS. The binding affinities of rHSA, human serum albumin and bovine serum albumin to 2S and 3S gels are less than their binding to PyS. Serum albumins from goat, rabbit, guinea pig, rat, hamster, baboon, and pig bind much stronger to 3S gel than human and bovine serum albumins. The binding of pig and hamster serum albumins is stronger than that of rat, goat, baboon, and rabbit. 相似文献
13.
Characterization of recombinant human serum albumin using matrix-assisted laser desorption ionization time-of-flight mass spectrometry 总被引:1,自引:0,他引:1
Chromatographically purified recombinant human serum albumin (rHSA), produced in genetically transformed yeast cells, was characterized using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS techniques. The molecular mass of the intact protein was determined to be 66671, in good agreement with that of purified HSA which was used as a standard. The identity of rHSA to its natural counterpart was established with high precision using peptide mass fingerprinting of tryptic peptides. Partial amino acid sequence data for rHSA were obtained using Ettan CAF MALDI Sequencing Kit and post-source decay on the tryptic peptides. The results achieved provide strong evidence that MALDI-TOF-MS is an important analytical technique for characterising gene products and for establishing the identity and bio-compatibility of recombinant proteins relative to their natural counterparts. 相似文献
14.
Electrospray ionization mass spectrometry (ESI‐MS) was employed to monitor the heme release and the conformational changes of myoglobin (Mb) under different solvent conditions, and to observe ligand bindings of Mb. ESI‐MS, complemented by circular dichroism and fluorescence spectroscopy, was used to study the mechanism of acid‐ and organic solvent‐induced denaturation by probing the changes in the secondary and the tertiary structure of Mb. The results obtained show that complete disruption of the heme–protein interactions occurs when Mb is subjected to one of the following solution conditions: pH 3.2–3.6, or solution containing 20–30% acetonitrile or 40–50% methanol. Outside these ranges, Mb is present entirely in its native state (binding with a heme group) or as apomyoglobin (i.e. without the heme). Spectroscopic data demonstrate that the denaturation mechanism of Mb induced by acid may be significantly different from that by the organic solvent. Low pH reduces helices in Mb, whereas certain organic content level in solution results in the loss of the tertiary structure. ESI‐MS conditions were established to observe the H2O‐ and CO‐bound Mb complexes, respectively. H2O binding to metmyoglobin (17 585 Da), where the heme iron is in the ferric oxidation state, is observed in ESI‐MS. CO binding to Mb (17 595 Da), on the other hand, can be only observed after the heme iron is reduced to the ferrous form. Therefore, ESI‐MS combined with spectroscopic techniques provides a useful means for probing the formation of ligand‐binding complexes and characterizing protein conformational changes. Copyright © 2010 John Wiley & Sons, Ltd. 相似文献
15.
Direct Electron Transfer and Bioelectrocatalysis by a Hexameric,Heme Protein at Nanostructured Electrodes
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Johannes Tanne Jae‐Hun Jeoung Lei Peng Aysu Yarman Birgit Dietzel Burkhard Schulz Daniel Schad Holger Dobbek Ulla Wollenberger Frank F. Bier Frieder W. Scheller 《Electroanalysis》2015,27(10):2262-2267
A nanohybrid consisting of poly(3‐aminobenzenesulfonic acid‐co‐aniline) and multiwalled carbon nanotubes [MWCNT‐P(ABS‐A)]) on a gold electrode was used to immobilize the hexameric tyrosine‐coordinated heme protein (HTHP). The enzyme showed direct electron transfer between the heme group of the protein and the nanostructured surface. Desorption of the noncovalently bound heme from the protein could be excluded by control measurements with adsorbed hemin on aminohexanthiol‐modified electrodes. The nanostructuring and the optimised charge characteristics resulted in a higher protein coverage as compared with MUA/MU modified electrodes. The adsorbed enzyme shows catalytic activity for the cathodic H2O2 reduction and oxidation of NADH. 相似文献
16.
Cytochromeb5(Cytb5)isfoundbothasacompo nentofthemicrosomalmembranesandasasolubleforminerythrocytes .Itplaysanimportantroleinbiologicalsystems ,inwhichCytb5functionsasanelectroncarrier,participatinginaseriesofelectron transferprocesses ,in cludingreductionof… 相似文献
17.
Thermal properties of tetrabutylammonium bromotrichloro-, tribromochloro-and tetrabromoferrates(III)
D. Wyrzykowski T. Maniecki Maria Gazda E. Styczeń Z. Warnke 《Journal of Thermal Analysis and Calorimetry》2007,90(3):893-897
Apparent molar volume and enthalpy changes for mixing NaCl (aq.) with albumin from human serum (aq.) are experimentally determined
(25°C). Calorimetric experiments were carried out in an LKB 10700-2 calorimeter, whereas volumetric measurements were realized
using an Anton Paar 60/602 densimeter. The density measurements were made after 1 and 24 h of the dissolution in the buffer
(pH 4.2). The relation between the changes of the enthalpy and apparent molar volumes vs. molality of NaCl were determined. The obtained data are discussed together with data obtained previously for bovine albumin
and hen egg lysozyme solutions with NaCl, Li2SO4 and (NH4)2SO4 salts of various concentration. As results correlations between the changes of enthalpy of salting and apparent molar volumes
vs. molality of salts were made. 相似文献
18.
The solubility property of the ZnCl2-Leu-H2O(Leu=L-a-leucine) system at 298.15K in the whole concentration range was investigatey by the semimicro-phase equilibrium method.The corresponding solubility diagram and refractive index diagram were constructed.The results indicated that there was one complex formed in this system.namely,Zn(Leu)Cl2.The complex is congruently soluble in water.Based on Phase equilibrium data,the complex was prepared.Its composition and properties were characterized by chemical analysis,elemental analysis,IR spectra,and TG-DTG.The thermochemical properties of coordination reaction of zinc chloride with L-a-leucine were investigated by a microcalorimeter.The enthalpies of solution of L-a-leucine in water and its zinc complex at infinite dilution and the enthalpy change of solid-liquid reaction wrer determined at 298.15K.The enthalpy change of soild phase reaction and the standard enthalpy of formation of zinc complex were claculated.On the basis of experimental and calculated results,three thermodynamic parameters(the activation enthalpy,the activation entropy and the activation free energy),the rate constant and three kinetic parameters(the activation energy,the preexponential constant and the reaction order) of the reaction,and the standard enthalpy of formation of Zn(Leu)^2 (aq) were obtained.The results showed that the title reaction took place easily at studied temperature. 相似文献
19.
Dr. Takashi Matsuo Kazuki Fukumoto Takuro Watanabe Prof. Dr. Takashi Hayashi 《化学:亚洲杂志》2011,6(9):2491-2499
H64D myoglobin mutant was reconstituted with two different types of synthetic hemes that have aromatic rings and a carboxylate‐based cluster attached to the terminus of one or both of the heme‐propionate moieties, thereby forming a “single‐winged cofactor” and “double‐winged cofactor,” respectively. The reconstituted mutant myoglobins have smaller Km values with respect to 2‐methoxyphenol oxidation activity relative to the parent mutant with native heme. This suggests that the attached moiety functions as a substrate‐binding domain. However, the kcat value of the mutant myoglobin with the double‐winged cofactor is much lower than that of the mutant with the native heme. In contrast, the mutant reconstituted with the single‐winged cofactor has a larger kcat value, thereby resulting in overall catalytic activity that is essentially equivalent to that of the native horseradish peroxidase. Enhanced peroxygenase activity was also observed for the mutant myoglobin with the single‐winged cofactor, thus indicating that introduction of an artificial substrate‐binding domain at only one of the heme propionates in the H64D mutant is the optimal engineering strategy for improving the peroxidase activity of myoglobin. 相似文献
20.
3‐Nitro‐1,2,4‐triazol‐5‐one (NTO) europium complex of [Eu(NTO)3(H2O)5]·5H2O was synthesized by mixing the aqueous solution of lithium 3‐nitro‐1,2,4‐triazol‐5‐onate and the dilute nitric acid solution of europium oxide. The title complex was characterized by elemental analysis and IR spectra. The single crystal structure was determined by a four‐circle x‐ray diffractometer. The title complex is monoclinic with space group P21/n and unit cell parameters of a = 1.8720(2) nm, b = 0.6548(3) nm, c = 1.9323(3) nm and β = 95.33(1)°. The coordination geometry around the europium ion is a distorted dodecahedron and there are five crystalline water molecules to form the stable structure of the crystal. From measurements of the enthalpy of solution in water at 298.15 K, the standard enthalpy of formation, lattice enthalpy and lattice energy have been determined as ‐(3798.6 ± 3.7), ?4488.4 and ?4452.4 kJ·mol?;1, respectively. 相似文献