A study of the properties of two porphyringlobin species formed in the reaction of protoporphyrin IX with human globin

Globin was prepared from the main (A(0)) component of human haemoglobin and reacted with protoporphyrin IX; the product, when subjected to chromatography on CM-Sephadex, separated into fast- and slow-moving species. These were isolated for examination. The dissociation constant for the tetramer-dimer equilibrium of fast-moving porphyringlobin was determined at 2.8x10(-6)m; this is to be compared with values of 2.2x10(-6)m and 8x10(-8)m determined for oxyhaemoglobin and the slow-moving porphyringlobin respectively. It was also shown that the thiol groups of fast-moving porphyringlobin react with 4,4'dithiodipyridine at an identical rate with those of oxyhaemoglobin; in comparison, the rates of reaction of deoxyhaemoglobin and porphyringlobin are much slower but are again identical with one another. The quenching of porphyringlobin fluorescence by I(-) ions was also studied. The quenching could not be represented by a simple Stern-Volmer relationship (whereas that of porphyrin-apomyoglobin is), but was represented by a model in which the fluorescence of fast-moving porphyringlobin was more accessible to the quencher than that of the slow-moving component. Similarly, fast-moving porphyringlobin was photodecomposed more rapidly by oxygen than the slow-moving species.     

The kinetics of binding of oxygen and carbon monoxide to Gastrophilus haemoglobin

The dimeric haemoglobin in the tracheal cells of the Gastrophilus larva was extracted and purified, and the spectral properties of its oxy- and carbon monoxide adducts are recorded. In dilute solutions the kinetic parameters of binding of oxygen and carbon monoxide were determined. In solutions between 0.1 and 50mum for oxygen k(on) is 1x10(7)m(-1).s(-1) and k(off) is 1s(-1); for carbon monoxide l(on) is 6.5x10(5)m(-1).s(-1) and l(off) is 0.14s(-1). These values are in agreement with previous equilibrium results on oxygen binding and carbon monoxide/oxygen partition. These results are discussed and compared with the known values for other monomeric protohaem proteins.     

Structure and function of haemoglobin philly (Tyr C1 (35) β→Phe)

We have purified haemoglobin Philly by isoelectric focusing on polyacrylamide gel, and studied its oxygen equilibrium, proton nuclear magnetic resonance spectra, mechanical stability, and pH-dependent u.v. difference spectrum. Stripped haemoglobin Philly binds oxygen non-co-operatively with high affinity. Inorganic phosphate and 2,3-diphosphoglycerate have little effect on the equilibrium curve, but inositol hexaphosphate lowers the affinity and induces co-operativity. These properties are explained by the nuclear magnetic resonance spectra which show that stripped deoxyhaemoglobin Philly has the quaternary oxy structure and that inositol hexaphosphate converts it to the deoxy structure. An exchangeable proton resonance at ?8.3 p.p.m. from water, which is present in oxy- and deoxyhaemoglobin A, is absent in both these derivatives of haemoglobin Philly and can therefore be assigned to one of the hydrogen bonds made by tyrosine C1-(35)β, probably the one to aspartate H8(126)α at the α₁β₁ contact. Haemoglobin Philly shows the same pH-dependent u.v. difference spectrum as haemoglobin A, only weaker, so that a tyrosine other than 35β must be mainly responsible for this.     

Quenching of fluorescence of pyrene-substituted lecithin by tetracyanoquinodimethane in liposomes.

In this work we have applied a kinetic scheme derived from fluorescence kinetics of pyrene-labeled phosphatidylcholine in phosphatidylcholine membrane to explain the fluorescence quenching of 1-palmitoyl-2-(10-[pyrenl-yl]-sn-glycerol-3-phosphatidylchol ine (PPDPC) liposomes by tetracyanoquinodimethane (TCNQ). The scheme was also found to be applicable to neat PPDPC and the effect of the quencher could be attributed to certain steps of the proposed mechanism. The TCNQ molecules influence the fluorescence of pyrene moieties in PPDPC liposome in two ways. Firstly, an interaction between the quencher molecule and the pyrene monomer in the excited state quenches monomer fluorescence and effectively prevents the diffusional formation of the excimer. Secondly, an interaction between the quencher molecule and the excited dimer quenches the excimer fluorescence. The TCNQ molecule does not prevent the formation of the excimer in pyrene moieties aggregated in such a way that they require only a small rotational motion to attain excimer configuration. The diffusional quenching rate constant is calculated to be 1.0 x 10(8) M-1 s-1 for the pyrene monomer quenching and 1.3 x 10(7) M-1 s-1 for the pyrene excimer quenching. The diffusion constant of TCNQ is 1.5 x 10(-7) cm2 s-1 for the interaction radii of 0.8-0.9 nm. The TCNQ molecules are practically totally partitioned in the membrane phase.     

Effect of polyanionic polymers on haemoglobin oxygen-binding properties: application to the synthesis of covalent conjugates with low oxygen affinity

Polyanionic water-soluble polymers containing sulphate, phosphate and polycarboxylate groups were synthesized. These compounds, when simply added to haemoglobin solutions, were shown to lower the affinity of the protein for oxygen. Their influence on oxygen affinity was regarded as the result of a specific interaction of the polymer anionic groups inside the 2,3-diphosphoglycerate-binding site of deoxyhaemoglobin. On the other hand, these polymers were linked to deoxyhaemoglobin to give covalent conjugates also exhibiting an oxygen affinity lower than that of free haemoglobin in the presence of 2,3-diphosphoglycerate, its natural effector, which means that after fixation, the polyanionic polymers are still acting as effectors.     

Characterization of the interactions of NADH with the dimeric and tetrameric states of methaemoglobin

The binding of NADH to the dimeric (αβ) and tetrameric (α₂β₂) states of human aquomethaemoglobin has been characterized by sedimentation equilibrium studies of the effect of the concentration of free ligand on the macromolecular state of the haemoprotein. Both macromolecular states of aquomethaemoglobin exhibit a single binding site for NADH, which interacts approximately tenfold more strongly (6000 cf. 700 M⁻¹) with the tetramer under the conditions studied (pH 6.0, I 0.10). Because the structure of aquomethaemoglobin resembles that of the deoxy state of haemoglobin, there is a high probability that organic phosphates also bind to dimeric deoxyhaemoglobin, a phenomenon which is not considered in thermodynamic treatments of the interplay between oxygen binding and its allosteric inhibition by 2,3-bisphosphoglycerate. Fortunately, the equilibrium constant for deoxyhaemoglobin self-association is so large that neglect of the interaction between allosteric inhibitor and dimeric haemoglobin is an oversight that should have no deleterious implications in the resultant thermodynamic analysis of the interplay between the preferential interactions of oxygen and organic phosphate with the various macromolecular states of deoxyhaemoglobin.     

Effect of organic co-solvents on the dimer/tetramer equilibrium of human haemoglobin

We have studied the effect of organic co-solvents (monohydric alcohols and formamide) on the dimer-tetramer equilibrium of human haemoglobin by measuring the dependence of oxygen affinity upon haemoglobin concentration over a 100-fold concentration range and analysing the data with a modified Monod-Wyman-Changeux model in which the equilibrium (tetramer) in equilibrium with (dimer) + (dimer) was taken into account. This procedure enabled us to obtain the dimer-tetramer equilibrium constant and to find its dependence upon co-solvent concentration. Then by following the procedure already reported for the tense----relaxed state transition of haemoglobin, we separated the co-solvent effects into bulk electrostatic and non-bulk electrostatic (hydrophobic) contributions. We believe that our results demonstrate that during haemoglobin dissociation, just as we have already shown for the tense to relaxed state transition, both charged groups and hydrophobic surfaces became exposed to the solvent.     

脱落酸对低温下雷公藤幼苗光合作用及叶绿素荧光的影响

以1年生雷公藤扦插苗为试材,研究低温胁迫下不同浓度外源脱落酸（ABA,0、5、10、15、20、25 mg·L^-1)叶面喷施处理对雷公藤叶片光合作用及叶绿素荧光参数的影响.结果表明：喷施20 mg·L^-1的ABA能显著提高雷公藤幼苗的抗冷性,减缓低温下雷公藤叶片净光合速率(P_n)、蒸腾速率(T_r)、气孔导度(g_s)、胞间CO₂浓度(C_i)的下降幅度,提高幼苗叶片的光合能力.低温处理6 d后,随着ABA浓度上升,雷公藤叶片的初始荧光(F_o)下降,最大荧光(F_m)和PSII最大光化学效率(F_v/F_m)上升,PSII实际光化学量子产量(Φ_PSⅡ)、光化学猝灭系数(q_P)先下降后上升,而非光化学猝灭系数(q_N)呈下降－上升－下降趋势.P_n、g_s、q_P、F_m和F_v/F_m均在20 mg·L^-1ABA处理时达到峰值.不同浓度ABA的相对电子传递速率(rETR)随着光化光强度增加呈先上升后下降的趋势,当光化光强度(PAR)达到395 μmol·m^-2s^-1时,各处理的rETR达到最高值,其中25 mg·L^-1和20 mg·L^-1ABA处理分别比对照高17.1%和5.2%.雷公藤叶片Φ_PSⅡ的光响应曲线均随光化光强度升高而下降,q_N的光响应曲线则呈相反趋势.     

Stereochemical mechanism of oxygen transport by haemoglobin

Spectroscopic and chemical evidence speak in favour of the iron-oxygen bond being polar. X-ray analysis shows that the oxygen molecule is inclined at an angle of about 115 degrees to the haem plane. Cooperative binding of oxygen by haemoglobin is due to an equilibrium between two alternative structures, which differ in oxygen affinity by the equivalent of 3-3.5 kcal/mol. I proposed that in the low affinity structure the globin opposes the movement of the iron atom from its five-coordinated pyramidal geometry in the haem of deoxyhaemoglobin to its six-coordinated planar geometry in the haem of oxyhaemoglobin, while in the high affinity structure this restraint is absent. Recent evidence supporting this mechanism is described.     

Redox properties of protein disulfide isomerase (DsbA) from Escherichia coli.

The redox properties of periplasmic protein disulfide isomerase (DsbA) from Escherichia coli were analyzed by measuring the equilibrium constant of the oxidation of reduced DsbA by oxidized glutathione. The experiments are based on the finding that the intrinsic tryptophan fluorescence of DsbA increases about threefold upon reduction of the enzyme, which can be explained by the catalytic disulfide bridge quenching the fluorescence of a neighboring tryptophan residue. From the specific fluorescence of DsbA equilibrated in the presence of different ratios of reduced and oxidized glutathione at pH 7, an equilibrium constant of 1.2 x 10(-4) M was determined, corresponding to a standard redox potential (E'0) of DsbA of -0.089 V. Thus, DsbA is a significantly stronger oxidant than cytoplasmic thioredoxins and its redox properties are similar to those of eukaryotic protein disulfide isomerase. The equilibrium constants for the DsbA/glutathione equilibrium were found to be strongly dependent on pH and varied from 2.5 x 10(-3) M to 3.9 x 10(-5) M between pH 4 and 8.5. The redox state-dependent fluorescence properties of DsbA should allow detailed physicochemical studies of the enzyme as well as the quantitative determination of the oxidized protein by fluorescence titration with dithiothreitol and open the possibility to observe bacterial protein disulfide isomerase "at work" during catalysis of oxidative protein folding.     

Interaction of lipoproteins in blood serum with steroid hormones

Interaction of human and serum lipoproteins with steroid hormones (corticosterone and cortisol) was studied. Methods of fluorescence quenching titration and equilibrium dialysis were used for quantitative evaluation of VLDL, LDL and HDL glucocorticoid-binding ability. Association constants were found to be 0.6-2.0 x 10(6) M-1 for corticosterone and 4.0-8.0 x 10(6) M-1 for cortisol. The number of binding sites varied from 3 to 300 for different classes of lipoproteins.     

Singlet oxygen quenching and the redox properties of hydroxycinnamic acids.

The singlet oxygen quenching rate constants (kq) for a range of hydroxycinnamic acids in acetonitrile and D2O solutions were measured using time resolved near infrared phosphorescence in order to establish their antioxidant activity. The magnitude of kq observed depends on both the nature of the substituent groups and solvent polarity. The variations in kq depend on the energy of the hydroxycinnamic acid/molecular oxygen charge transfer states, (O2delta- ...HCAdelta+). In D2O the values of kq range from 4x10(7) M(-1) s(-1) to 4x10(6) M(-1) s(-1) for caffeic acid and o-coumaric acid respectively. In acetonitrile, the charge transfer energy levels are raised and this is reflected in lower singlet oxygen quenching rate constants with a kq value of 5x10(6) M(-1) s(-1) for caffeic acid. The phenoxyl radical spectra derived from the hydroxycinnamic acids were determined using pulse radiolysis of aqueous solutions and the reduction potentials were found to range from 534 to 596 mV. A linear correlation is observed between reduction potential, and hence free energy for electron transfer, and log kq. These correlations suggest a charge transfer mechanism for the quenching of singlet oxygen by the hydroxycinnamic acids.     

Purification of heparin,dermatan sulfate and chondroitin sulfate from mixtures by sequential precipitation with various organic solvents

Heparin, dermatan sulfate and chondroitin sulfate in mixtures were fractionated by sequential precipitation with methanol, ethanol and propanol. The recovered fractions from 0.1 to 2.0 volumes of various solvents were analyzed by agarose-gel electrophoresis and densitometric analysis. Heparins with different relative percentages of slow-moving and fast-moving components were precipitated from 0.5 to 0.7 volumes of methanol, and in this range of volumes, the amount of slow-moving component of heparin decreases and that of the fast-moving species increases. From 0.8 to 1.6 volumes of methanol, mixtures with different percentages of the fast-moving component, dermatan sulfate and chondroitin sulfate are precipitated. Heparin was precipitated from mixtures in the range of 0.1 to 0.4 volumes of ethanol, and from 0.5 to 0.8 volumes mixtures with different relative percentages of dermatan sulfate and chondroitin sulfate were precipitated. From 1.0 to 2.0 volumes of ethanol, high purity (about 100%) chondroitin sulfate can be precipitated. Propanol induces the precipitation of heparin from 0.3 to 0.4 volumes, whilst dermatan sulfate with a purity greater than 85% is precipitated at 0.5 and 0.6 volumes of propanol. 100% chondroitin sulfate is obtained with volumes greater than 0.8. Heparin and chondroitin sulfate from a bovine lung extract of glycosaminoglycans were purified by sequential precipitation with ethanol. The fraction precipitated with 0.4 volumes of ethanol shows greater than 90% heparin and that recovered from 0.9 to 2.0 volumes is composed of 100% chondroitin sulfate.     

Nanosecond time-resolved absorption studies of human oxyhemoglobin photolysis intermediates.

The time-resolved spectra of photoproducts from ligand photodissociation of oxyhemoglobin are measured in the Soret spectral region for times from 10 ns to 320 microseconds after laser photolysis. Four processes are detected at a heme concentration of 80 microM: a 38-ns geminate recombination, a 137-ns tertiary relaxation, and two bimolecular processes for rebinding of molecular oxygen. The pseudo-first-order rate constants for rebinding to the alpha and beta subunits of hemoglobin are 3.2 x 10(4) s-1 (31 microseconds lifetime) and 9.4 x 10(4) s-1 (11 microseconds lifetime), respectively. The significance of kinetic measurements made at different heme concentrations is discussed in terms of the equilibrium compositions of hemoglobin tetramer and dimer mixtures. The rebinding rate constants for alpha and beta chains are observed to be about two times slower in the dimer than in the tetramer, a finding that appears to support the observation of quaternary enhancement in equilibrium ligand binding by hemoglobin tetramers.     

Asymmetric (deoxy dimer/azido-met dimer) hemoglobin hybrids dissociate within seconds.

Double mixing stopped-flow experiments have been performed to study the stability of asymmetric hemoglobin (Hb) hybrids, consisting of a deoxy and a liganded dimer. The doubly liganded [deoxy/cyano-met] hybrid (species 21) was reported to have an enhanced stability, with tetramer to dimer dissociation requiring over 100 seconds, based on a method that required an incubation of over two days. However, kinetic experiments revealed rapid ligand binding to species 21, as for triply liganded tetramers, which dissociate within a few seconds. For the present study, [deoxy dimer/azido-met dimer] hybrids are formed within 200 ms by stopped-flow mixing of dithionite with a solution containing oxyHb and azido-metHb. The dithionite scavenges oxygen, thus transforming oxyHb to deoxyHb, and the [oxy dimer/azido-met dimer] hybrid to the asymmetric [deoxy/azido-met] hybrid (species 21). After a variable aging time of the asymmetric hybrids, their allosteric state is probed by CO binding in a second mixing. As previously observed the freshly produced asymmetric hybrids bind CO rapidly as for R-state Hb. As the hybrids are aged from 0.1 to 10 seconds, the fraction of slow CO binding increases, consistent with a dissociation of the asymmetric hybrid to form the more stable deoxy Hb tetramer which reacts slowly with CO. Control experiments showed a predominantly slow phase for deoxy Hb, and fast rebinding for the symmetric hybrids.The kinetic data can be simulated with a tetramer to dimer dissociation rate for species 21 of 1.5/second at 100 mM NaCl (pH 7.2) and 1.9/second at 180 mM NaCl (pH 7.4). These values are similar to those reported for liganded Hb, as opposed to deoxy (T-state) tetramers which dissociate over four orders of magnitude more slowly. As expected from simulations of dimer exchange, the observed transition rate depends on the initial fractions of oxy- and metHb; this effect is not consistent with a slow R to T transition. These results, showing a lifetime of about one second for species 21, do not support the symmetry rule which is based on an enhanced stability of the asymmetric hybrid.     

A study on apoenzyme from Rhodotorula gracilis D-amino acid oxidase.

The apoenzyme of D-amino acid oxidase from Rhodotorula gracilis was obtained at pH 7.5 by dialyzing the holoenzyme against 2 M KBr in 0.25 M potassium phosphate, 0.3 mM EDTA, 5 mM 2-mercaptoethanol and 20% glycerol. To recover a reconstitutable and highly stable apoprotein, it is essential that phosphate ions and glycerol be present at high concentrations. Apo-D-amino acid oxidase is entirely present as a monomeric protein, while the reconstituted holoenzyme is a dimer of 79 kDa. The equilibrium binding of FAD to apoprotein was measured from the quenching of flavin fluorescence and by differential spectroscopy: a Kd of 2.0 x 10(-8) M was calculated. The kinetics of formation of the apoprotein-FAD complex were studied by the quenching of protein and flavin fluorescence, by differential spectroscopy and by activity measurements. In all cases a two-stage process was shown to be present with a fairly rapid first phase, followed by a slow secondary change which represents only 4-6% of the total recombination process. In no conditions was a lag in the recovery of maximum catalytic activity observed. The process of FAD binding to yeast D-amino acid oxidase appears to be of the type Apo + FAD in equilibrium holoenzyme, even though the existence of a transient intermediate not detectable under our conditions cannot be ruled out.     

DNA topology of the ordered chromatin domain 5'' to the human c-myc gene.

DNA restriction fragments located 5' to the human c-myc gene display anomalous electrophoretic mobility on polyacrylamide gels. Computer modeling of the c-myc flanking DNA suggests that the slow-moving DNA fragments spanning nucleotides -1690 to -1054 (relative to c-myc promoter P1) and -718 to -452 form large left handed superhelices or curved structures while the fast-moving DNA fragment spanning nucleotides -407 to +78 has an unusually straight structure. These analyses also predict a periodic array of localized regions of bending through the superhelical domains. Micrococcal nuclease digestion of isolated nuclei reveals that the slow-moving DNA fragments exist in an ordered chromatin structure stable to nuclease, whereas the digestion pattern of the fast-moving DNA fragment suggests a less ordered array of nucleosomes or a non-nucleosomal chromatin structure.     

Predissociated dimers and molten globule monomers in the equilibrium unfolding of yeast glutathione reductase

The equilibrium unfolding of dimeric yeast glutathione reductase (GR) by guanidine hydrochloride (GdnHCl) was investigated. Unfolding was monitored by a variety of techniques, including intrinsic fluorescence emission, anisotropy and iodide quenching measurements, far-ultraviolet circular dichroism and thiol reactivity measurements. At 1 M GdnHCl, one thiol group of GR became accessible to modification with 5,5′-dithiobis-(2-nitrobenzoic) acid (DTNB), whereas no changes could be detected in the spectroscopic properties (fluorescence, circular dichroism) of the protein. Between 2 and 3 M GdnHCl, two partially folded intermediate states possessing flexible tertiary structures (revealed by fluorescence data) but compact secondary structures (as indicated by circular dichroism measurements) were identified. The quaternary structure of GR in the presence of GdnHCl was also investigated by size-exclusion liquid chromatography. These results indicated the presence of an expanded predissociated dimer at 2.5 M GdnHCl and partially folded monomers at 3 M GdnHCl. Taken together, these results suggest the existence of two molten-globule-like intermediate species (one dimeric and one monomeric) in the unfolding of GR. The results are discussed in terms of the mechanism of GR folding and dimerization.     

Oxidation of human haemoglobin by copper. Mechanism and suggested role of the thiol group of residue beta-93.

Addition of Cu(II) ions to human oxyhaemoglobin caused the rapid oxidation of the haem groups of the beta-chain. Oxidation required binding of Cu(II) to sites involving the thiol group of beta-93 residues and was prevented when these groups were blocked with iodoacetamide or N-ethylmaleimide. Equilibrium-dialysis studies showed three pairs of binding sites, two pairs with high affinity for Cu(II) and one pair with lower affinity. It was the second pair of high-affinity sites that were blocked with iodoacetamide and were involved in haem oxidation. Cu(II) oxidized deoxyhaemoglobin at least ten times as fast as oxyhaemoglobin, and analysis of rates suggested that binding rather than electron transfer was the rate-determining step. No thiol-group oxidation to disulphides occurred during the period of haem oxidation, although it did occur subsequently in the presence of oxygen, or when Cu(II) was added to methaemoglobin. It is proposed that thiol oxidation did not occur because there exists a pathway of electron transfer between the haem group and copper bound to the beta-93 thiol groups. The route for this electron transfer is discussed, as well as the implications as to the function of the beta-93 cysteine in the haemoglobin molecule.     

The chemistry of flavins and flavoproteins. Aerobic photochemistry

1. When a mixture of FMN and a reducing substrate (e.g. unprotonated amine) is illuminated oxygen is consumed. 2. The rate of oxygen uptake increases as oxygen concentration falls with some substrates (type I reaction), but with other substrates (typically aromatic compounds) the rate falls as the oxygen concentration falls (type II reaction). 3. The kinetics of type I reactions with EDTA, dl-alpha-phenylglycine and diethanolamine are all consistent with a mechanism in which the rate-determining step, hydrogen abstraction by the FMN triplet, is followed by rapid reoxidation of reduced FMN by oxygen. The reaction is faster at low oxygen concentrations because oxygen quenches the triplet. 4. The sensitivity of reaction rates to substituents in dl-alpha-phenylglycine can be described by a Hammett rho value of -0.6. 5. Individual rate constants for quenching and reaction of the FMN triplet with substrate were calculated (2.4x10(8) and 2.1x10(7)m(-1)s(-1) respectively for EDTA) on the assumption that oxygen quenches the triplet in a diffusion-controlled reaction. 6. The pH-dependences of oxygen uptake rates with six natural amino acids as substrates were measured. 7. Photoinactivations of l-glutamate dehydrogenase and d-amino acid oxidase by FMN were demonstrated.