Hemes and hemoproteins. 3. The reaction of microperoxidase-8 with cyanide: comparison with aquocobalamin and hemoproteins

The monomeric heme octapeptide from cytochrome c, microperoxidase-8, (MP-8), coordinates CN- with log K = 7.55 +/- 0.04 at 25 degrees C in 20% (v/v) aqueous methanol. Log K values are independent of pH between 6 and 9. A spectrophotometric titration of cyanoMP-8 between pH 5.5 and 13.8 gave a single pKa greater than or equal to 13.5 ascribed to ionization of the proximal His ligand. A study of the kinetics of the reaction of MP-8 with cyanide between pH 5.5 and 12, at 25 degrees C and mu = 0.1, indicates that formation of cyanoMP-8 occurs via three routes: attack of CN- on Fe(III) (k1 = 6.0 +/- 0.3 X 10(5) M-1 sec-1); attack of HCN on Fe(III) (k2 = 4.8 +/- 2.0 X 10(3) M-1 sec-1), followed by deprotonation and isomerization to form the C-bound species; and displacement of OH- by CN- when the proximal His ligand is ionized (k5 = 1.8 +/- 0.1 X 10(5) M-1 sec-1). These results are compared with available data for the reaction of cyanide with aquocobalamin and with various hemoproteins.     

Characterization of cDNAs, mRNAs, and proteins related to human liver microsomal cytochrome P-450 (S)-mephenytoin 4'-hydroxylase

A cytochrome P-450 (P-450) multigene family codes for several related human liver enzymes, including the P-450 responsible for (S)-mephenytoin 4'-hydroxylation. This enzyme activity has previously been shown to be associated with a genetic polymorphism. Genomic (Southern) blot analysis using non-overlapping 5' and 3' portions of a cDNA clone suggests that approximately seven related sequences are present in this gene family. In this study four cDNA clones, all nearly full-length, were isolated from a bacteriophage lambda gt11 library prepared from a single human liver. These clones can be grouped into two categories that are approximately 85% identical at the level of DNA sequence. The cDNA clones in one category (MP-4, MP-8) both match the N-terminal sequences of the P-450MP-1 and P-450MP-2 proteins, which had previously been shown to be catalytically active in (S)-mephenytoin 4'-hydroxylation. These two cDNAs, MP-4 and MP-8, differ in only two bases in the coding region but are quite distinct in their 3' noncoding regions. Another protein (P-450MP-3) was isolated on the basis of its immunochemical similarity to P-450MP-1 but was found to be catalytically inactive; amino acid sequencing of tryptic peptides of P-450MP-3 showed a correspondence to the second category of cDNA clones (MP-12, MP-20), which differ from each other in only four (nonsilent) base changes. Oligonucleotides specific for the two groups of cDNA clones were used as probes of human liver mRNAs--individual liver samples examined expressed both types of mRNAs but no correlation was observed between the abundance levels of any mRNA and catalytic activity. Further, oligonucleotide probes indicated that mRNAs corresponding to both the MP-4 and MP-8 clones were apparently present in individual liver samples. A monoclonal antibody was isolated that recognized P-450MP-1 but not P-450MP-2 or P-450MP-3; the amount of protein detected by the antibody in different liver samples was not correlated with the mephenytoin 4'-hydroxylase activity. These results indicate that several closely related P-450 genes are all expressed in individual human livers. The MP-4/MP-8 gene products are proposed to be the ones most likely involved in mephenytoin 4'-hydroxylation, and much of the variation in catalytic activity among individuals is not a result of differences in levels of P-450MP-1 or mRNA but may be due to base differences in the structural gene(s).     

H NMR investigation of the influence of interacting sites on the dynamics and thermodynamics of substrate and ligand binding to horseradish peroxidase.

The influence of substrate benzhydroxamic acid (BHA) and iron ligand (cyanide) on the thermodynamics and dynamics of each of the two binding sites of horseradish peroxidase (HRP) isozyme C has been investigated by 1H NMR spectroscopy. A combination of line-width analysis and saturation transfer spectroscopy has allowed the direct determination of the off-rate of substrate and ligand in the absence or presence of the other. These off-rates, together with available dissociation constants obtained by optical spectroscopy (Schonbaum, 1973), provide estimates for kon. The dissociation constant for cyanide binding to the BHA.HRP complex was also directly determined by NMR. In all cases the 1H NMR determined dynamic and thermodynamic data agree well with those values available in the literature. BHA binding leads to a 200-fold decrease in CN- affinity that arises from a factor greater than 10 decrease in koff(CN-) and greater than 2 x 10(3) decrease in kon(CN-). While a portion of the decrease in kon(CN-) can be rationalized by water coordination of the iron in the BHA.HRP complex, the additional decrease in kon(CN-) and that in koff(CN-) indicates that BHA in the binding pocket blocks the CN- ligation channel and serves as a "gate" to CN- exchange. This view is supported by observing a factor greater than 4 decrease in distal His labile proton exchange with bulk water in HRP-CN upon BHA binding. The ternary complex BHA.HRP-CN is shown to be heterogeneous. While the thermodynamics of BHA and CN- binding appear similar in the two ternary complexes, the BHA on- and off-rates for the two complexes differ by a factor of approximately 10. The two heterogeneous forms interconvert at 25 degrees C at approximately 2 x 10(2) s-1, precluding the determination of any difference in the CN- binding rates by saturation transfer. The greater lability of one of the two ternary complexes is attributed to an alternate orientation of some distal residue that blocks the substrate binding channel in one of the forms. Transferred nuclear Overhauser effects from the heme to BHA in the ternary complex reveal that the BHA substrate is in contact not only with the heme pyrrole D substituents but also with the distal His 42, indicating that the polar side chain of BHA extends well into the distal heme pocket.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of heme and myristate binding to human serum albumin by anti-HIV drugs. An optical and NMR spectroscopic study

Human serum albumin (HSA) has an extraordinary ligand-binding capacity, and transports Fe(III)heme and medium- and long-chain fatty acids. In human immunodeficiency virus-infected patients the administered drugs bind to HSA and act as allosteric effectors. Here, the binding of Fe(III)heme to HSA in the presence of three representative anti-HIV drugs and myristate is investigated. Values of the dissociation equilibrium constant K(d) for Fe(III)heme binding to HSA were determined at different myristate concentrations, in the absence and presence of anti-HIV drugs. Nuclear magnetic relaxation dispersion profiles of HSA-Fe(III)heme were measured, at different myristate concentrations, in the absence and presence of anti-HIV drugs. Structural bases for anti-HIV drug binding to HSA are provided by automatic docking simulation. Abacavir and nevirapine bind to HSA with K(d) values of 1 x 10(-6) and 2 x 10(-6) M, respectively. Therefore, at concentrations used in therapy (in the 1-5 x 10(-6) M range) abacavir and nevirapine bind to HSA and increase the affinity of heme for HSA. In the presence of abacavir or nevirapine, the affinity is not lowered by myristate. FA7 should therefore be intended as a secondary binding site for abacavir and nevirapine. Binding of atazanavir is limited by the large size of the drug, although preferential binding may be envisaged to a site positively coupled with FA1 and FA2, and negatively coupled to FA7. As a whole, these results provide a foundation for the comprehension of the complex network of links modulating HSA-binding properties.     

Low spin states of microperoxidases produced by inter- and intra-peptide chain sixth ligands: effect of pH and the oligopeptide type

The low spin states of microperoxidases (MP)-8, -9 and -9 N-acetylated (N-Ac) were characterized using UV-visible, circular dichroism, and electron paramagnetic resonance spectroscopies over the 6.0-12.0 pH range. The first MP-8 alkaline transition (pK(a)=8.53) produced hemepeptide aggregates in the low spin state in which a water molecule was replaced by the peptide chain N-terminal group of a neighboring MP-8 molecule. Higher pH led to the deprotonation of the MP-8 histidine imidazole ring (pK(a)=10.37) at the fifth coordination position. This MP-8 species was in equilibrium with a high spin state aggregate in which OH(-) replaced histidinate, the histidinate becoming the heme iron sixth ligand in a neighboring MP-8 molecule. In a similar way to the N-AcMP-8, the low spin state of N-AcMP-9 was produced by the deprotonation of the water molecule (pK(a)=9.6) situated at the sixth coordination position of the heme iron. Up to pH 8.5, the low spin states of MP-9 were aggregates in which the alpha-amino group of Lys13 replaced water at the sixth coordination position of a neighboring MP-9 molecule. Above pH 8.5, the epsilon-amino groups of Lys13 established intra-chain coordination and impaired the formation of aggregates. Such intra-chain interaction in MP9 was supported by molecular dynamics simulation. These MP-9 monomers might also exhibit OH(-) or histidinate at the fifth coordination position.     

A new drug binding subsite on human serum albumin and drug-drug interaction studied by X-ray crystallography

3'-Azido-3'-deoxythymidine (AZT) is the first clinically effective drug for the treatment of human immunodeficiency virus infection. The drug interaction with human serum albumin (HSA) has been an important component in understanding its mechanism of action, especially in drug distribution and in drug-drug interaction on HSA in the case of multi-drug therapy. We present here crystal structures of a ternary HSA-Myr-AZT complex and a quaternary HSA-Myr-AZT-SAL complex (Myr, myristate; SAL, salicylic acid). From this study, a new drug binding subsite on HSA Sudlow site 1 was identified. The presence of fatty acid is needed for the creation of this subsite due to fatty acid induced conformational changes of HSA. Thus, the Sudlow site 1 of HSA can be divided into three non-overlapped subsites: a SAL subsite, an indomethacin subsite and an AZT subsite. Binding of a drug to HSA often influences simultaneous binding of other drugs. From the HSA-Myr-AZT-SAL complex structure, we observed the coexistence of two drugs (AZT and SAL) in Sudlow site 1 and the competition between these two drugs in subdomain IB. These results provide new structural information on HSA-drug interaction and drug-drug interaction on HSA.     

Binding and relaxometric properties of heme complexes with cyanogen bromide fragments of human serum albumin

The spectroscopic and reactivity properties of hemin complexes formed with cyanogen bromide fragments B (residues 1-123), C (124-298), A (299-585), and D (1-298) of human serum albumin (HSA) have been investigated. The complex hemin-D exhibits binding, spectral, circular dichroism, and reactivity characteristics very similar to those of hemin-HSA, indicating that fragment D contains the entire HSA domain involved in heme binding. The characteristics of the other hemin complexes are different, and a detailed investigation of the properties of hemin-C has been carried out because this fragment contains the HSA binding region of several important drugs. Hemin-C contains a low-spin Fe(III) center, with two imidazole ligands, but the complex undergoes a reversible structural transition at basic pH leading to a high-spin, five-coordinated Fe(III) species. This change determines a marked increase in the relaxation rate of water protons. Limited proteolysis experiments and mass spectral analysis carried out on fragment C and hemin-C show that the region encompassing residues Glu-208 to Trp-214 is protected from activity of proteases in the complex and, therefore, is involved in the interaction with hemin. A structural model of fragment C enables us to propose that His-242 and His-288 are the axial ligands for the Fe(III) center.     

Escherichia coli sulfite reductase hemoprotein subunit. Prosthetic groups, catalytic parameters, and ligand complexes

Escherichia coli NADPH-sulfite reductase can be dissociated into an oligomeric flavoprotein and a monomeric hemoprotein (HP) subunit in 4 M urea. HP catalyzes stoichiometric 6-electron reductions of SO32- (to S2-) and of NO2-, as well as 2-electron reduction of NH2OH, with reduced methyl viologen (MV+) as reductant. While Vmax values are highest with the nitrogenous substrates, Km for SO32- is 2 to 3 orders of magnitude less than the Km for NO2- or NH2OH. EPR spectroscopic and chemical analyses show that HP contains one siroheme and one Fe4S4 center per polypeptide. The heme is in the high spin Fe3+ state in HP as isolated. Near-quantitative reduction of the Fe4S4 center to a state yielding a g = 1.94 type of EPR spectrum by S2O42- and/or MV+ could be achieved if HP was converted to either the CN- or CO complex or treated with 80% dimethyl sulfoxide. HP binds one SO32- or CN- per peptide. Binding of these ligands, as well as CO, appears to be mutually exclusive and to involve the heme. The heme Fe3+/Fe2+ potential is shifted from -340 mV in the free HP to -155 mV in the HP-CN- complex. The potential of the Fe4S4 center is approximately 70 mV more negative in the CN- as opposed to the CO-ligated HP (-420 mV), a result which indicates the presence of heme-Fe4S4-ligand interaction in the HP complexes.     

4-Aminobenzoic acid hydrazide inhibition of microperoxidase-11: catalytic inhibition by reactive metabolites

Inhibition of human peroxidase enzymes such as myeloperoxidase or eosinophil peroxidase represents a novel therapeutic area, for which there are no current clinical therapeutics. We utilized 4-aminobenzoic acid hydrazide which was reported to be a potent irreversible inhibitor of myeloperoxidase to gain insight into the role of reactive metabolites in catalytic inhibition. In order to carry out detailed studies, we used a model peroxidase, microperoxidase-11 (MP-11). We investigated the heme spectrum of MP-11 in the presence of 4-ABAH and found that heme bleaching occurred that was irreversible. This coincided with an absence of catalytic activity. The spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was able to significantly prevent inactivation of peroxidase activity, therefore, we performed ESR spin trapping studies and detected a carbonyl carbon-centered radical of 4-ABAH. In order to determine if the free radical metabolites became bound to MP-11, we performed high-resolution MALDI with elemental analysis to determine the change in elemental composition that occurred in these reactions. These masses were assigned to free radical metabolites of 4-ABAH and were not observed in reactions containing DMPO. We conclude that the 4-ABAH free radical metabolites which were bound to MP-11 were involved in the catalytic inhibition and were scavenged by DMPO.     

Human serum albumin hybrid incorporating tailed porphyrinatoiron(II) in the alpha,alpha,alpha,beta-conformer as an O2-binding site

We have found that recombinant human serum albumin (HSA) incorporating tailed porphyrinatoiron(II) in the alpha,alpha,alpha,beta-conformer can reversibly bind and release O2 under physiological conditions (pH 7.3, 37 degrees C) like hemoglobin and myoglobin. beta-2-Methylimidazolyl-tailed porphyrinatoirons (6a, 6b) are synthesized via four steps from the atropisomers of tetrakis(o-aminophenyl)porphyrin. The stereochemistry of the alpha,alpha,alpha,beta-conformer has been determined by NMR spectroscopy. 6a and 6b form stable O2-adduct complexes in toluene solution at room temperature. The association rate constants of O2 are 3.1- and 1.9-fold lower than those of the corresponding alpha,alpha,alpha,alpha-conformers (1a, 1b), indicating that the three substituents (cyclohexanamide or pivalamide groups) are close to each other on the porphyrin platform and construct a narrow encumbrance around the O2-coordination site. Although 6a and 6b are incorporated into the hydrophobic domains of HSA to produce the albumin-heme hybrid, only HSA-6a can bind O2 in aqueous medium. The cyclohexanamide fences are necessary for the tailed porphyrinatoiron to form a stable O2-adduct complex under physiological conditions. The O2-binding affinity (P(1/2)) of HSA-6a is 45 Torr (37 degrees C), and the O2 transporting efficiency between lungs and muscle tissues in the human body is estimated to be identical to that of human red blood cells. The HSA-6a solution will become one of the most promising materials for red blood cell substitutes, which can be manufactured on an industrial scale.     

Hemes and hemeproteins, 2: The pH-dependent equilibria of microperoxidase-8 and characterization of the coordination sphere of Fe(III)

Titration of the monomeric heme octapeptide from horse heart cytochrome c, microperoxidase-8 (MP-8) from pH 1 to pH 13 in 20% (v/v) methanol-water solutions, mu = 0.1, at 25 degrees C shows three reversible concentration-independent pKs (4.43 +/- 0.09; 8.90 +/- 0.03; 10.48 +/- 0.09) which are ascribed to successive proton loss from the conjugate acid of His (and its coordination to Fe(III)), bound H2O, and from bound His to form an imidazolate complex, respectively. The equilibrium constant for coordination of imidazole between pH 5.5 and 7.0 is independent of pH (logK = 4.45) which proves that His-18 is coordinated to Fe(III) in aqueous solution.     

Use of Heme-Protein Complexes by the Yersinia enterocolitica HemR Receptor: Histidine Residues Are Essential for Receptor Function

The abilities of two bacterial active heme transporters, HmbR of Neisseria meningitidis and HemR of Yersinia enterocolitica, to use different heme sources were compared. While HmbR-expressing cells used only hemoglobin (Hb) and heme, HemR-expressing bacteria were able to grow on Hb, heme, myoglobin, hemopexin, catalase, human and bovine serum albumin-heme, and haptoglobin-hemoglobin complexes as sources of iron. Expression of functional HemR allowed Escherichia coli cells to respond to heme-containing peptides, microperoxidases MP-8, MP-9, and MP-11, suggesting the ability of HemR to transport heme covalently linked to other molecules. Comparison of HemR with other heme receptors identified several highly conserved histidine residues as well as two conserved amino acid motifs, the FRAP and NPNL boxes. A site-directed mutagenesis approach was used to investigate the roles of His128, His192, His352, and His461 residues in HemR function. The HemR receptor with histidine changed to lysine at position 128 (HemR(H128K)), HemR(H461L), HemR(H461A), and HemR(H128A,H461A) mutant receptors were unable to use Hb, human serum albumin-heme, and myoglobin as sources of porphyrin and iron. Utilization of free heme was also severely affected, with some residual heme uptake in cells expressing HemR(H128K), HemR(H461A), and HemR(H461L). Conversely, the HemR(H192T), HemR(H352A), HemR(H352K), and HemR(H192T,H352K) mutant receptors were fully functional. All mutant HemR proteins were expressed in the outer membrane at levels similar to that of the wild-type HemR receptor. Nonfunctional HemRs were able to bind heme- and Hb-agarose. A hypothetical model of the HemR function in which two conserved histidine residues, His128 and His461, participate in the transport of heme through the receptor pore is postulated.     

ZOO-FISH Suggests a Complete Homology between Human and Capuchin Monkey (Platyrrhini) Euchromatin

Chromosome comparisons usingin situhybridization of all human chromosome-specific libraries on Capuchin monkey (Cebus capucinus,Cebidae, Platyrrhini) metaphases were performed with a new technique simultaneously revealing a G-banding and chromosome “painting.” A complete homology between human (HSA) andC. capucinus(CCA) chromosomes was demonstrated, except for constitutive heterochromatin. ElevenC. capucinuschromosomes are homologous to 11 human chromosomes: CCA 2 = HSA 4; CCA 3 = HSA 6; CCA 12 = HSA 9; CCA 16 = HSA 11; CCA 10 = HSA 12; CCA 11 = HSA 13; CCA 20 = HSA 17; CCA 8 = HSA 19; CCA 23 = HSA 20; CCA 24 = HSA 22; and CCA X = HSA X. TenC. capucinuschromosomes are homologous to parts of human chromosomes: CCA 13 = HSA 8q; CCA 14 = HSA 2q; CCA 15 = HSA 1p + 1q proximal; CCA 17 = HSA 7 part; CCA 18 and 19 = HSA 3 part; CCA 21 and 22 = HSA 1q distal; CCA 25 = HSA 10p; and CCA 26 = HSA 15q part. SixC. capucinuschromosomes are homologous to parts of two human chromosomes: CCA 1 = HSA 5 + 7 part; CCA 4 = HSA 2p + q proximal + 16q; CCA 5 = HSA 10q + 16p; CCA 6 = HSA 14 + 15 part; CCA 7 = HSA 8p + 18; and CCA 9 = HSA 3 part + 21. Many previous banding comparisons were confirmed but several cryptic or complex rearrangements could be identified. With theC. capucinuskaryotype having been shown to be fairly ancestral, this comparison opens the possibility to compare human chromosomes to most Cebidae species.     

A Comparative Study of Interaction of Tetracycline with Several Proteins Using Time Resolved Anisotropy,Phosphorescence, Docking and FRET

A comparative study of the interaction of an antibiotic Tetracycline hydrochloride (TC) with two albumins, Human serum albumin (HSA) and Bovine serum albumin (BSA) along with Escherichia Coli Alkaline Phosphatase (AP) has been presented exploiting the enhanced emission and anisotropy of the bound drug. The association constant at 298 K is found to be two orders of magnitude lower in BSA/HSA compared to that in AP with number of binding site being one in each case. Fluorescence resonance energy transfer (FRET) and molecular docking studies have been employed for the systems containing HSA and BSA to find out the particular tryptophan (Trp) residue and the other residues in the proteins involved in the binding process. Rotational correlation time (θ_c) of the bound TC obtained from time resolved anisotropy of TC in all the protein-TC complexes has been compared to understand the binding mechanism. Low temperature (77 K) phosphorescence (LTP) spectra of Trp residues in the free proteins (HSA/BSA) and in the complexes of HSA/BSA have been used to specify the role of Trp residues in FRET and in the binding process. The results have been compared with those obtained for the complex of AP with TC. The photophysical behaviour (viz., emission maximum, quantum yield, lifetime and θ_c) of TC in various protic and aprotic polar solvents has been determined to address the nature of the microenvironment of TC in the protein-drug complexes.     

Localization of subtelomeric sequences of human chromosomes 1q, 11p, 13q, and 16q in the higher primates

Relative phylogenetic divergence of the members of the Pongidae family has been based on genetic evidence. The recent isolation of subtelomeric probes specific for human (HSA) chromosomes 1q, 11p, 13q, and 16q has prompted us to cross hybridize these to the chromosomes of the chimpanzee (Pan troglodytes, PTR), gorilla (Gorilla gorilla, GGO), and orangutan (Pongo pygmaeus, PPY) to search for their equivalent locations in the great apes. Hybridization signals to the 1q subtelomeric DNA sequence probe were observed at the termini of human (HSA) 1q, PTR 1q, GGO 1q, PPY 1q, while the fluorescent signals to the 11p subtelomeric DNA sequence probe were observed at the termini of HSA 11p, PTR 9p, GGO 9p, and PPY 8p. Fluorescent signals to the 13q subtelomeric DNA sequence probe were observed at the termini of HSA 13q, PTR 14q, GGO 14q, and PPY 14q, and positive signals to the 16p subtelomeric DNA sequence probe were observed at the termini of HSA 16q, PTR 18q, GGO 17q, and PPY 19q. These findings apparently suggest sequence homology of these DNA families in the ape chromosomes. Obviously, analogous subtelomeric sequences exist in apes' chromosomes that apparently have been conserved through the course of differentiation of the hominoid species. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of cis-, trans-diamminedichloroplatinum(II) and DBP on human serum albumin

Both isomers of diamminedichloroplatinum(II) bind to albumin and induce the formation of the albumin dimer (MW approximately 140 kDa). The trans isomer exhibits a much greater tendency to induce a protein dimerization than the cis isomer. Under similar experimental conditions, the phosphonic derivative of diammineplatinum(II) (DBP) does not induce any dimer formation. The amount of bound complex per mol of human serum albumin (HSA, for an incubation time of 7 days) was found to be 6, 10.5 and 1 mol for cis-, trans-DDP and DBP, respectively. The relative fluorescence intensity of platinum-bound HSA decreases to about 55% for cis-DDP, 45% for trans-DDP and to 85% for DBP when compared to the complex-free protein, suggesting that the binding occurs in the proximity of the Trp214 residue. The structural studies (CD) have shown that only DDP-isomers cause the distinct modification of HSA native structure (alpha-helical content). Pt(II) complexes binding to HSA affect the affinity of HSA towards heme and bilirubin. High excess of DDP prevents the heme and bilirubin binding, while DBP affects this binding much less effectively due to the low amount of the protein-bound complex. Reactions of platinum complexes with albumin are believed to play an important role in the metabolism of this anticancer drug. The minor effect of DBP on HSA may indicate that the toxicity of the phosphonate analog is much lower than toxicities of DDP isomers, most likely due to kinetic reasons.     

Voltammetric investigation on interaction of protein with chromotrope 2R and its analytical application

The electrochemical behaviors of the interaction of chromotrope 2R (CH2R) with human serum albumin (HSA) are investigated on the hanging mercury drop electrode with linear sweep voltammetry. In the acidic buffer solution (pH 2.5) CH2R has a well-defined voltammetric reductive wave at −0.34 V (SCE). On the addition of HSA into the CH2R solution, the reductive peak current of CH2R decreases with little movement of the peak potential. The voltammetric study shows that the electrochemical parameters of interaction solution do not change and a new electrochemically non-active complex is formed via interaction of CH2R with HSA, which cannot be reduced on the Hg electrode and results in the decrease of the free concentration of CH2R. The decrease of reductive peak current is proportional to HSA concentration and further used for protein detection. The binding ratio and the binding constant are further calculated with the experimental voltammetric data.     

Paclitaxel-HSA interaction. Binding sites on HSA molecule

Paclitaxel (trade name Taxol) is one of the world's most effective anticancer drugs. It is used to treat several cancers including tumours of the breast, ovary and lung. In the present work the interaction of paclitaxel with human serum albumin (HSA) in aqueous solution at physiological pH has been investigated through CD, fluorescence spectroscopy and by the antibody precipitate test. Binding of paclitaxel to albumin impact on protein structure and it influences considerably albumin binding of other molecules like warfarin, heme or bilirubin. The paclitaxel-HSA interaction causes the conformational changes with the loss of helical stability of protein and local perturbation in the domain IIA binding pocket. The relative fluorescence intensity of the paclitaxel-bound HSA decreased, suggesting that perturbation around the Trp 214 residue took place. This was confirmed by the destabilization of the warfarin binding site, which includes Trp 214, and high affinity bilirubin binding site located in subdomain IIA.     

Tyrosine residues play an important role in heme detoxification by serum albumin

Background

Serum albumin binds avidly to heme to form heme–serum albumin complex, also called methemalbumin, and this binding is thought to protect against the potentially toxic effects of heme. However, the mechanism of detoxification has not been fully elucidated.

Methods

SDS-PAGE and Western blot were used to determine the efficiency of methemalbumin on catalyzing protein carbonylation and nitration. HPLC was used to test the formation of heme to protein cross-linked methemalbumin.

Results

The peroxidase activity of heme increased upon human serum albumin (HSA) binding. Methemalbumin showed higher efficiency in catalyzing tyrosine oxidation than free heme in the presence of H₂O₂. Methemalbumin catalyzed self-nitration and significantly promoted the nitration of tyrosine in coexistent protein, but decreased the carbonylation of coexistent protein compared with heme. The heme to protein cross-linked form of methemalbumin suggested that HSA trapped the free radical accompanied by the formation of ferryl heme. When tyrosine residues in HSA were modified by iodination, HSA lost of protection effect on protein carbonylation. The low concentration of glutathione could effectively inhibit tyrosine nitration, but had no effect on protein carbonylation.

Conclusion

HSA protects against the toxic effect of heme by transferring the free radical to tyrosine residues in HSA, therefore protecting surrounding proteins from irreversible oxidation, rather than by direct inhibiting the peroxidase activity. The increased tyrosine radicals can be reduced by endogenic antioxidants such as GSH.

General significance

This investigation indicated the important role of tyrosine residues in heme detoxification by HSA and suggested a possible novel mechanism.     

Parallel RH mapping of BTA1 with HSA3 and HSA21

The development of radiation hybrid (RH) panels has elevated comparative gene mapping to a new level of resolution. In this study, we have constructed parallel RH maps defining rearrangements of gene order within conserved segments of bovine Chromosome (Chr) 1 (BTA1) and human Chrs 3 and 21 (HSA3, HSA21). Six new markers, including one gene, have been added to the bovine map, and 11 human genes were ordered with the human G3 panel. BTA1 is clearly a composite of genetic material conserved on these two human chromosomes with HSA21 homologs at each end of BTA1 flanking a large segment homologous to HSA3. Each of the three conserved segments of BTA1 contains rearrangements of gene order relative to their human counterparts. Received: 31 March 1999 / Accepted: 12 July 1999