Albumin clusters: structurally defined protein tetramer and oxygen carrier including thirty-two iron(II) porphyrins

Recombinant human serum albumin (rHSA) clusters have been synthesized and physicochemically characterized. Cross-linking between the Lys groups of the core albumin and a unique Cys-34 of the shell albumins with an N-succinimidyl-6-[3'-(2-pyridyldithio)propionamido]hexanoate produced the structurally defined rHSA trimer and tetramer. MALDI-TOF-MS showed a single peak with the triple and quadruple masses of rHSA. Their molar ellipticities and the isoelectric points (pI = 4.8) are all identical to those of the monomer, suggesting that the essential structures of the albumin units were intact. TEM observations demonstrated a uniform morphology of the rHSA tetramer with a diameter of 20-30 nm. The circulation half-life (tau1/2) of the 125I-labeled rHSA tetramer in rat (5.5 h) was significantly longer than that of the monomer (2.3 h) due to the low ratio of the distribution phase (alpha-phase). A total of 24 and 32 molecules of the synthetic iron(II) porphyrins (FePs) are incorporated into the hydrophobic cavities of the rHSA trimer and tetramer, respectively, producing huge artificial hemoproteins. These albumin-heme clusters can reversibly bind and release O2 under physiological conditions (37 degrees C, pH 7.3) and showed similar O2-binding properties (O2-binding affinity, association and dissociation rate constants) to those of the corresponding monomer. A large volume of O2 can be chemically dissolved into the albumin-heme cluster solutions relative to the monomeric rHSA-FeP when the molar concentration of the albumin scaffold is identical.     

Human serum albumin incorporating Tetrakis(o-pivalamido) phenylporphinatoiron(II) derivative as a totally synthetic O2-carrying hemoprotein.

2-[8-{N-(2-Methylimidazolyl)}octanoyloxymethyl]-5,10,15, 20-tetrakis(o-pivalamido)phenylporphinatoiron(II)s (FePs) were incorporated into hydrophobic cavities of recombinant human serum albumin (rHSA), providing a totally synthetic O(2)-carrying hemoprotein (rHSA-FeP). An rHSA host absorbs maximally eight FeP molecules. Solution properties of the obtained albumin hybrid [[rHSA] = 5 wt %; FeP/HSA = 1-8 (mol/mol)] are almost identical to those of the rHSA itself; the specific gravity is 1.013 and the viscosity is 1.1 cP. Circular dichroism spectroscopy and isoelectric focusing measurement revealed that the second-order structure and surface charge distribution of rHSA were always constant independent of the binding numbers of FeP. Hydrophobic interaction is probably a major molecular force of the incorporation of this synthetic heme. rHSA-FeP can bind and release dioxygen reversibly under physiological conditions (in aqueous media, pH 7.3, 37 degrees C) like hemoglobin and myoglobin. Its O(2)-coordination structure was evaluated by resonance Raman spectroscopy. The O(2) rebinding after the laser flash photolysis showed three-phases decay, which were analyzed by triple-exponential kinetics. The O(2)-binding affinity and O(2)-association and -dissociation rate constants of rHSA-FeP satisfy the initial clinical requirements for O(2) infusion as a red cell substitute.     

Human serum albumin bearing covalently attached iron(II) porphyrins as O2-coordination sites

Tetrakis{(alpha,alpha,alpha,alpha-o-pivalamido)phenyl}porphinatoiron(II) with a bifunctional tail possessing an axially coordinated imidazolyl group and a protein attachable succinimidyl(glutamyl) group (FeP-GluSu) has been synthesized. It can efficiently react with the lysine residues of recombinant human serum albumin (rHSA), giving a new albumin-heme conjugate [rHSA(FeP-Glu)]. MALDI-TOFMS showed a distinct molecular ion peak at m/z 70 643, which indicates that three FeP-Glu molecules were covalently linked to the rHSA scaffold. The binding number of FeP-Glu is approximately three (mol/mol) and independent of the mixing ratio. The CD spectrum and Native PAGE revealed that the albumin structure remained unaltered after the covalent bonding of the hemes. This rHSA(FeP-Glu) conjugate can bind and release O2 reversibly under physiological conditions (pH 7.3, 37 degrees C) in the same manner as hemoglobin and myoglobin. The O2-adduct complex had a remarkably long lifetime (tau(1/2): 5 h). The O2-binding affinity [P(1/2)O2: 27 Torr] was identical to that of human red cells. Laser flash photolysis experiments gave the O2- and CO-association rate constants and suggested that there are two different geometries of the imidazole binding to the central ion.     

Site-directed mutagenesis studies of human serum albumin define tryptophan at amino acid position 214 as the principal site for nitrosation

The patterns of nitric oxide (NO) release from nitrosated bovine serum albumin (BSA), human serum albumin (HSA) and a number of recombinant HSA mutants were compared. All albumin species were nitrosated by incubation with acidified NO(2)(-). The pattern of NO release from BSA nitrosated with acidified NO(2)(-) was in agreement with previous reports which indicated that Cys-34 is the primary target for nitrosation in BSA. In contrast, the pattern of NO release from HSA nitrosated with acidified NO(2)(-) indicated that the primary nitrosation target was an amino acid residue other than Cys-34. Based on our initial findings and a previous report that tryptophan is a potential target for nitrosation by acidified NO(2)(-), several recombinant HSA mutants were synthesized in the yeast species Pichia pastoris. The following recombinant HSA species were produced: wild-type, C34S, W214L, W214E and W214L/Y411W HSA. Nitrosation of these mutants using acidified NO(2)(-) showed that Trp-214 is the primary nitrosation target in HSA. Mutation of Trp-214 led to an increase in Cys-34 nitrosation, indicating possible competition between these two residues for reaction with N(2)O(3), the reactive nitrosating species formed in aqueous acidified NO(2)(-) solutions.     

Effect of heme structure on O(2)-binding properties of human serum albumin-heme hybrids: intramolecular histidine coordination provides a stable O(2)-adduct complex

5,10,15,20-Tetrakis[(alpha,alpha,alpha,alpha-o-pivaloylamino)phenyl]porphinatoiron(II) and 5,10,15,20-tetrakis([alpha,alpha,alpha,alpha-o-(1-methylcyclohexanoylamino)]phenyl)porphinatoiron(II) complexes bearing a covalently bound 8-(2-methyl-1-imidazolyl)octanoyloxymethyl or 4-(methyl-L-histidinamido)butanoyloxymethyl side-chain [FeRP(B) series: R = piv or cyc, B = Im or His] have been synthesized. The histidine-bound derivatives [FepivP(His), FecycP(His)] formed five N-coordinated high-spin iron(II) complexes in organic solvents under an N(2) atmosphere and showed large O(2)-binding affinities in comparison to those of the 2-methylimidazole-bound analogues [FepivP(Im), FecycP(Im)] due to the low O(2)-dissociation rate constants. On the contrary, the difference in the fence groups around the O(2)-coordination site (pivaloyl or 1-methylhexanoyl) did not significantly influence to the O(2)-binding parameters. These four porphinatoiron(II)s were efficiently incorporated into recombinant human serum albumin (rHSA), thus providing the synthetic hemoprotein, the albumin-heme hybrid [rHSA-FeRP(B)]. An rHSA host absorbs a maximum of eight FeRP(B) molecules in each case. The obtained rHSA-FeRP(B) can reversibly bind and release O(2) under physiological conditions (in aqueous media, pH 7.3, 37 degrees C) like hemoglobin and myoglobin. As in organic solutions, the difference in the fence groups did not affect their O(2)-binding parameters, but the axial histidine coordination significantly increased the O(2)-binding affinity, which is again ascribed to the low O(2)-dissociation rates. The most remarkable effect of the heme structure appeared in the half-life (tau(1/2)) of the O(2)-adduct complex. The dioxygenated rHSA-FecycP(His) showed an unusually long lifetime (tau(1/2): 25 h at 37 degrees C) which is ca. 13-fold longer than that of rHSA-FepivP(Im).     

重组人血白蛋白临床研究进展

人血白蛋白是人血浆中最丰富的蛋白质,具有许多重要的生理特性,用途广泛。目前主要以毕赤酵母作为宿主表达的重组人血白蛋白,开发了重组人血白蛋白的纯化技术,同时对重组人血白蛋白结构进行了分析,结果表明与人血浆白蛋白基本一致。临床研究结果表明重组人血白蛋白与人血浆白蛋白有着几乎相同的疗效和安全性。综述了重组人血白蛋白的性质结构分析及酵母表达系统;重点介绍了重组人血白蛋白在临床方面研究进展。     

Synthesis and physicochemical characterization of a series of hemoglobin-based oxygen carriers: objective comparison between cellular and acellular types

A series of hemoglobin (Hb)-based O(2) carriers, acellular and cellular types, were synthesized and their physicochemical characteristics were compared. The acellular type includes intramolecularly cross-linked Hb (XLHb), polyoxyethylene (POE)-conjugated pyridoxalated Hb (POE-PLP-Hb), hydroxyethylstarch-conjugated Hb (HES-XLHb), and glutaraldehyde-polymerized XLHb (Poly-XLHb). The cellular type is Hb-vesicles (HbV) of which the surface is modified with POE (POE-HbV). Their particle diameters are 7 +/- 2, 22 +/- 2, 47 +/- 17, 68 +/- 24, and 224 +/- 76 nm, respectively, thus all the materials penetrate across membrane filters with 0.4 microm pore size, though only the POE-HbV cannot penetrate across the filter with 0.2 microm pore size. These characteristics of permeability are important to consider an optimal particle size in microcirculation in vivo. POE-PLP-Hb ([Hb] = 5 g/dL) showed viscosity of 6.1 cP at 332 s(-1) and colloid osmotic pressure (COP) of 70.2 Torr, which are beyond the physiological conditions (human blood, viscosity = 3-4 cP, COP = ca. 25 Torr). XLHb and Poly-XLHb showed viscosities of 1.0 and 1.5 cp, respectively, which are significantly lower than that of blood. COP of POE-HbV is regulated to 20 Torr in 5% human serum albumin (HSA). HES-XLHb and POE-HbV/HSA showed comparable viscosity with human blood. Microscopic observation of human red blood cells (RBC) after mixing blood with POE-PLP-Hb or HES-XLHb disclosed aggregates of RBC, a kind of sludge, indicating a strong interaction with RBC, which is anticipated to modify peripheral blood flow in vivo. On the other hand, XLHb and POE-HbV showed no rouleaux or aggregates of RBC. The acellular Hbs (P(50) = 14-32 Torr) have their specific O(2) affinities determined by their structures, while that of the cellular POE-HbV is regulated by coencapsulating an appropriate amount of an allosteric effector (e.g., P(50) = 18, 32 Torr). These differences in physicochemical characteristics between the acellular and cellular types indicate the advantages of the cellular type from the physiological points of view.     

Kinetics of CO and O(2) binding to human serum albumin-heme hybrid

The kinetics of the CO and O(2) binding to the synthetic hemoprotein, recombinant human serum albumin (rHSA) incorporating eight 2-[8-?N-(2-methylimidazolyl)?octanoyloxymethyl]-5,10,15, 20-tetrakis(o-pivalamido)phenylporphinatoiron(II)s (FePs) [rHSA-FeP(8)] have been investigated by laser flash photolysis. Time dependence of the absorption change accompanied the CO rebinding to rHSA-FeP(8) was composed of three phases. The fastest component was the axial base elimination, and the long-lived biphasic decay corresponds to the direct recombination of CO to the five-N-coordinated FePs in rHSA. The rate constants of the fast and slow phases of the CO association [(fast), (slow)] were determined to be 4.9 x 10(6) M(-)(1) s(-)(1) and 6.7 x 10(5) M(-)(1) s(-)(1), respectively. The initial amplitude after the laser pulse gave the concentration ratio of the fast and slow phases (n = 3); (i) two of the eight FePs exhibited the slow rate constants and (ii) they are presumably accommodated in the second and fifth binding sites of FeP in the albumin structure. The absorption decay following the O(2) photodissociation of rHSA-FeP(8) also showed the same behavior. Thermodynamically, the large DeltaG() of the slow phase of the CO rebinding, which mainly comes from the enthalpic factor, suggests the appearance of additional steric hindrance on the central metal iron of FeP. Furthermore, orientation of the porphyrin plane in rHSA was predicted by molecular simulation, which supports the experimental data from the kinetic observations.     

Characterization of deltoid, a chimeric protein containing the oligomerization site of hepatitis delta antigen

Both forms of the hepatitis delta antigen (HDAg) encoded by hepatitis delta virus are active only as oligomers. Previous studies showed that quadrin, a synthetic 50-residue peptide containing residues 12-60 from the N-terminus of HDAg, interferes with HDAg oligomerization, forms an alpha-helical coiled coil in solution, and forms a novel square octamer in the crystal consisting of four antiparallel coiled-coil dimers joined at the corners by hydrophobic binding of oligomerization sites located at each end of the dimers. We designed and synthesized deltoid (CH3CO-[Cys23]HDAg-(12-27)-seryl-tRNA synthetae-(59-65)-[Cys42]HDAg-(34-60)-Tyr-NH2), a chimeric protein that structurally resembles one end of the quadrin dimer and contains a single oligomerization site. The 51-residue chain of deltoid contains a seven-residue alpha-hairpin loop in place of the remainder of the quadrin dimer plus Cys12 and Cys31 for forming an intrachain disulfide bridge. Reduced, unbridged deltoid (Tm=61 degrees C, DeltaG(H2O)=-1.7 kcal mol(-1)) was less stable to denaturation by heat or guanidine HCl than oxidized, intrachain disulfide-bridged deltoid (Tm>80 degrees C, DeltaG(H2O)=-2.6 kcal mol(-1)). Each form is an alpha-helical dimer that reversibly dissociates into two monomers (Kd=80 microM).     

Generation of a transgenic silkworm that secretes recombinant proteins in the sericin layer of cocoon: production of recombinant human serum albumin

In this study we produced germline transgenic silkworms that spin cocoons containing recombinant human serum albumin (rHSA) in the sericin layer. A piggyBac-based transformation vector was constructed that carried HSA cDNA driven by sericin-1 gene promoter, viral enhancer hr3, and gene encoding viral trans-activator IE1. Isolated silk glands were bombarded with the vector and transplanted into host larvae. Three days later, the transplants were immunohistochemically analyzed, which showed that middle silk gland (MSG) cells expressed rHSA and secreted it into the MSG lumen. Then, silkworm eggs were injected with the vector and developed to larvae. The obtained transgenic silkworms spun silk threads whose sericin layers contained rHSA at 3.0microg/mg of cocoons. Most (83%) of the rHSA in cocoons was extracted with phosphate buffered saline, which was then subjected to ammonium sulfate precipitation and affinity chromatography. Finally, we obtained 2.8mg of 99%-pure rHSA from 2g of cocoons. Measurements of circular dichroism spectra of rHSA, and equilibrium dissociation constants of rHSA to warfarin and naproxen indicated that rHSA was conformationally and functionally identical to natural plasma HSA. Germline transgenic silkworms will be useful for producing various recombinant proteins in the sericin layer of cocoons.     

Effects of maternal identity and incubation temperature on snapping turtle (Chelydra serpentina) metabolism

Individual variation in physiological traits may have important consequences for offspring survivorship and adult fitness. Variance in offspring phenotypes is due to interindividual differences in genotype, environment, and/or maternal effects. This study examined the contributions of incubation environment, maternal effects, and clutch identity to individual variation in metabolic rates in the common snapping turtle, Chelydra serpentina. We measured standard metabolic rate, as determined by oxygen consumption, for 246 individuals representing 24 clutches at 15 degrees and 25 degrees C, and we measured standard metabolic rates additionally for 34 individuals at 20 degrees and 30 degrees C. Standard metabolic rate for 34 snapping turtles measured at 15 degrees, 20 degrees, 25 degrees, and 30 degrees C increased with increasing temperature. Mean standard metabolic rate for 246 individuals was 0.247 microL O(2) min(-1) g(-1) at 15 degrees C and 0.919 microL O(2) min(-1) g(-1) at 25 degrees C. At 15 degrees C, mass at hatching, individual mass, and egg mass had no significant effects on metabolic rate, but at 25 degrees C, mass at hatching, individual mass, and egg mass did have significant effects on metabolic rate. Incubation temperature had no significant effect on metabolic rate at 15 degrees, but it did have a significant effect at 25 degrees C. Clutch identity had a significant effect on metabolic rate at both 15 degrees and 25 degrees C. Interindividual variation in standard metabolic rate due to incubation temperature, and especially clutch identity, could have large effects on energy budgets. Results suggest that there were both environmental and genetic effects on standard metabolic rate.     

Formation of albumin dimers induced by exposure to peroxides in human plasma: a possible biomarker for oxidative stress

Human serum albumin (HSA) has one free thiol residue at Cys-34 that is likely oxidized by various reactive oxygen species (ROS). We attempted to identify the oxidation product of Cys-34 of HSA following exposure of plasma to ROS. Oxidation induced by tert-butyl hydroperoxide (t-BuOOH) of this free cysteine residue in HSA was observed in detail. Analysis of oxidized albumin in a partially purified fraction obtained by affinity column chromatography clearly revealed the formation of albumin disulfide dimers following t-BuOOH exposure. Albumin disulfide dimer formation was observed in normal plasma following treatment with various peroxides, as well as in untreated plasma from patients on hemodialysis using SDS-PAGE and Western blot analysis. The present results indicate that albumin dimers are oxidative products derived from peroxides, and that their presence in plasma might be a marker of oxidative stress as secondary metabolites of peroxidation.     

Differences in local conformation around cysteine residues in alpha alpha, alpha beta, and beta beta rabbit skeletal tropomyosin

The ability of 5,5'-dithiobis-2-nitrobenzoate (Nbs2) to form a disulfide crosslink between the Cys-190s of the alpha alpha and alpha beta molecular components of rabbit skeletal tropomyosin (Tm) and the Cys-36s and Cys-190s of purified beta beta was studied in separate experiments, as a function of urea concentration in 0.5 M NaCl, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.4, 15 degrees C. In the absence of urea, complete reaction of the Cys-190s of Tm with Nbs2 as well as with 2- and 4-pyridine disulfide quantitatively produced two crosslinked species, alpha-alpha and alpha-beta, in a 60/40 ratio, respectively, visualized as bands on sodium dodecyl sulfate-polyacrylamide gels; similar reactions of beta beta produced both doubly (at Cys-36 and Cys-190) and singly crosslinked species (at Cys-190 as identified by amino acid analysis of separated tryptic peptides). In the presence of 4 M urea where the chains were unfolded and separated, only Nbs-blocked uncrosslinked species were obtained after complete reaction with Nbs2. The loss of Nbs2-crosslinking at increasing [urea] showed that the relative stability of the Cys-containing regions of the three species of Tm, alpha alpha, alpha beta, and beta beta increases in the order Cys-36 of beta beta, Cys-190 of alpha beta, Cys-190 of alpha alpha.     

Elimination of disulfide bonds affects assembly and secretion of the human chorionic gonadotropin beta subunit

Human chorionic gonadotropin (hCG) consists of two noncovalently joined alpha and beta subunits similar to the other glycoprotein hormones. To study the function of the individual disulfide bonds in subunit assembly and secretion, site-directed mutagenesis was used to convert the 12 cysteine (Cys) residues in the beta subunit of hCG to either alanine or serine. Both cysteines of proposed disulfide pairs were also mutated. These mutant hCG beta genes were transfected alone or together with the wild-type alpha gene into Chinese hamster ovary cells. Only 3-10% assembly could be achieved with derivatives containing single Cys mutations at positions 26, 110, 72, and 90, whereas no assembly was detected with the other 8 mutants. However, double mutations of pairs 26-110 or 23-72 showed increased dimer formation (11 and 36%, respectively). The secretion rate of individual mutants varied significantly. Whereas the Cys-23 and 72 mutants were secreted normally (t1/2 = 140-190 min), the Cys-26 mutant was secreted faster (t1/2 = 70 min), and the other 9 mutants were secreted slower (t1/2 = 280-440 min); mutations of both Cys at 26 and 110 caused much faster secretion (t1/2 = 34 min). Although the secretion rate of these mutants differed, they were quantitatively recovered in the medium except for mutant Cys-88, Cys-23-72, and Cys-34-88 (40, 55, and 10% secreted, respectively). Thus, interruption of any disulfide bond in the hCG beta subunit alters the structure sufficiently to block dimer formation and in some cases slow secretion, although the stability for most of the mutant hCG beta subunits is not greatly affected. The data indicate that interruption of any hCG beta disulfide bond generates different structural forms that are unable to assemble with the alpha subunit, and that the structural requirements for stability and assembly are different.     

The binuclear iron site of the membrane-bound methane hydroxylase from Methylococcus capsulatus (strain M)

The particulate membrane-bound methane hydroxylase (pMMOH) was isolated from methane-oxidizing cells of Methylococcus capsulatus (strain M). At SDS PAGE, pMMOH displays three bands: 47 (alpha), 27 (beta), and 25 kDa (gamma). The ESR spectrum of pMMOH incubated with hydrogen peroxide (final concentration 20 mM) at 4 degrees C exhibited, along with the copper signal of type I with g = 2.05, signals of cytochrome with g = 3.0 and of high-spin ferriheme with g = 6.00. After incubation at -30 degrees C, additional signals with g 8.5 and 13.5 were observed. These signals, which have not been recorded previously in pMMOH preparations, are due to an intermediate of the pMMOH active site, which arises in the reaction of hydrogen peroxide with pMMOH at -30 degrees C. It was established that this intermediate is a high-spin dimer [Fe(IlI)-Fe(IV)] with S = 9/2 and different degree of rhombic distortion of structure (it is responsible for both signals). Presumably, the signal with g = 8.5 also arises from the same dimer [Fe(III)-Fe(IV)], but with S = 7/2. The presence of the intermediate [Fe(lII)-Fe(IV)] in pMMOH preparations suggests that the original state of the pMMOH active site is the dimer [Fe(III)-Fe(III)] which is located in the beta-subunit and cannot be detected by ESR. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http:// www.maik.ru.     

Formation of an active dimer during storage of interleukin-1 receptor antagonist in aqueous solution.

The degradation products of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) formed during storage at 30 degrees C in aqueous solution were characterized. Cationic exchange chromatography of the stored sample showed two major, new peaks eluting before (P1) and after (L2) the native protein, which were interconvertible. Size-exclusion chromatography and electrophoresis documented that both the P1 and L2 fractions were irreversible dimers, formed by noncovalent interactions. A competition assay with interleukin-1 indicated that on a per monomer basis the P1 and L2 dimers retained about two-thirds of the activity of the native monomer. Infrared and far-UV circular dichroism spectroscopies showed that only minor alterations in secondary structure arose upon the formation of the P1 dimer. However, alteration in the near-UV circular dichroism spectrum suggested the presence of disulfide bonds in the P1 dimer, which are absent in the native protein. Mass spectroscopy and tryptic mapping, before and after carboxymethylation, demonstrated that the P1 dimer contained an intramolecular disulfide bond between Cys-66 and Cys-69. Although conversion of native protein to the P1 dimer was irreversible in buffer alone, the native monomer could be regained by denaturing the P1 dimer with guanidine hydrochloride and renaturing it by dialysis, suggesting that the intramolecular disulfide bond does not interfere with refolding. Analysis of the time course of P1 formation during storage at 30 degrees C indicated that the process followed first-order, and not second-order, kinetics, suggesting that the rate-limiting step was not dimerization. It is proposed that a conformational change in the monomer is the rate-limiting step in the formation of the P1 dimer degradation product. Sucrose stabilized the native monomer against this process. This result can be explained by the general stabilization mechanism for this additive, which is due to its preferential exclusion from the protein surface.     

Selectivity and stereospecificity of the reactions of dichlorodiammineplatinum(II) with three purified plasma proteins

The reactions of cis- and trans-dichlorodiammineplatinum(II) (cis- and trans-DDP) with albumin and two plasma proteinase inhibitors were compared. Reaction with alpha 2-macroglobulin (alpha 2M) resulted in subunit crosslinking and loss of proteinase binding activity. The reaction also modified a receptor recognition site present on each alpha 2M subunit. While more trans-DDP was incorporated into alpha 2M than cis-DDP, cis-DDP was more effective at blocking receptor recognition, alpha 1-proteinase inhibitor was also inactivated by reaction with either cis- or trans-DDP. These reactions resulted in binding of platinum to methionine-358 at the reactive center of this inhibitor. Trans-DDP, however, was less selective and also bound to the single cysteine residue (Cys-232) of alpha 1PI. Reaction of albumin with cis-DDP resulted in incorporation of about 1 mol platinum per mol protein, and this platinum modified the single cysteine (Cys-34) in the molecule. Albumin incorporated twice as much trans-DDP, but the binding did not involve cysteine-34. In general, reactions of cis-DDP with proteins appear to be more selective than those observed for modification with the trans isomer.     

Equation for osmotic pressure of serum protein (fractions).

The colloid or protein osmotic pressure (Pi) is a function of protein molarity (linear) and of Donnan and other effects. Albumin is the major osmotic protein, but also globulins influence Pi. Equations based on concentrations of albumin and nonalbumin (globulin concentration + fibrinogen concentration) protein approximate Pi better than albumin alone. Globulins have a wide range of molecular weights, and a 1956 diagram indicated that Pi of globulin fractions decreased in the order alpha1-, alpha2-, beta-, and gamma-globulin. The molecular weight of the serum protein fractions had been extrapolated, so van't Hoff's law and nonlinear regression analysis of the curves permitted expression of the diagram as an equation: product Pi(s,Ott,2 degrees C,cmH2O)=x(alb)(0.338C(tot)+0.00339C(tot)(2))+x(alpha1)(0.518C(tot)+0.0107C(tot)(2))+x(alpha2)(0.203C(tot)+0.00155C(tot)(2))+x(beta)(0.187C(tot)+0.000577C(tot)(2))+x(gamma)(0.161C(tot)+0.000223C(tot)(2)), where Pi(s,Ott,2 degrees C,cmH2O) is Pi of serum at 2 degrees C (in cmH2O) computed from the 1956 diagram, C(tot) is the concentration (g/l) of total protein in serum, and x(alb), x(alpha1), x(alpha2), x(beta), and x(gamma) are the fractions of albumin, alpha1-, alpha2-, beta-, and gamma-globulin, respectively. At one and the same concentration of fractions, Pi("Ott") decreases in the order alpha1-globulin, albumin, alpha2-globulin, beta-globulin, and gamma-globulin.     

Hydrogen peroxide triggers the formation of a disulfide dimer of muscle acylphosphatase and modifies some functional properties of the enzyme

Acylphosphatase is expressed in vertebrates as two molecular forms, the organ common and the muscle types. The former does not contain cysteine residues, whereas the latter contains a single conserved cysteine (Cys-21). We demonstrated that H(2)O(2) at micromolar levels induces, in vitro, the formation of a disulfide dimer of muscle acylphosphatase, which displays properties differing from those of the reduced enzyme. In particular, we observed changes in the kinetic behavior of its intrinsic ATPase activity, whereas the kinetic behavior of its benzoyl phosphatase activity does not change. Moreover, the disulfide dimer is capable of interacting with some polynucleotides such as poly(G), poly(C), and poly(T) but not with poly(A), whereas the reduced enzyme does not bind polynucleotides. Experiments performed with H(2)O(2) in the presence of increasing SDS concentrations demonstrated that disulfide dimer formation is prevented by SDS concentrations higher than 300 microm, suggesting that a non-covalently-linked dimer is present in non-denaturing solvents. Light-induced cross-linking experiments performed on the Cys-21 --> Ser mutant in the pH range 3.8-9.0 have demonstrated that a non-covalently-linked dimer is in fact present in non-denaturing solutions and that an enzyme group with a pK(a) of 6.4 influences the monomer-dimer equilibrium.     

Reaction of nitric oxide with synthetic hemoprotein, human serum albumin incorporating tetraphenylporphinatoiron(II) derivatives

The reaction of nitric oxide (NO) with a synthetic hemoprotein, the recombinant human serum albumin (rHSA) incorporating eight tetraphenylporphinatoiron(II) derivatives bearing a covalently linked axial base (FeP) [rHSA-FeP], has been investigated. The UV--vis absorption spectrum of the phosphate buffer solution (pH 7.3) of rHSA-FeP showed maxima at 425 and 546 nm upon the addition of NO. The carbonyl rHSA-FeP, in which FePs are six-coordinate CO-adducts, also moved to the same species after bubbling with NO gas. ESR spectroscopy revealed that the incorporated FePs in the albumin formed six-coordinate nitrosyl complexes; the proximal imidazole moiety does not dissociate from the central iron when NO binds to the trans side. The NO-binding affinity of rHSA-FeP (P(1/2)(NO), 1.7 x 10(-6) Torr, pH 7.3, 298 K) was significantly lower than that of FeP itself (P(1/2)(NO), 1.8 x 10(-8) Torr in toluene). Kinetically, this arises from the decreased association rate constant (k(on)(NO), 8.9 x 10(8) M(-1) s(-1) --> 1.5 x 10(7) M(-1) s(-1)). Since NO-association is diffusion controlled, incorporation of the synthetic heme into the albumin matrix appears to restrict the NO access to the central iron(II).