Pathway and Mechanism of pH Dependent Human Hemoglobin Tetramer-Dimer-Monomer Dissociations

Hemoglobin dissociation is of great interest in protein process and clinical medicine as well as in artificial blood research. However, the pathway and mechanisms of pH-dependent human Hb dissociation are not clear, whether Hb would really dissociate into monomers is still a question. Therefore, we have conducted a multi-technique investigation on the structure and function of human Hb versus pH. Here we demonstrate that tetramer hemoglobin can easily dissociate into dimer in abnormal pH and the tetramer → dimer dissociation is reversible if pH returns to normal physiological value. When the environmental pH becomes more acidic (<6.5) or alkaline (>8.0), Hb can further dissociate from dimer to monomer. The proportion of monomers increases while the fraction of dimers decreases as pH declines from 6.2 to 5.4. The dimer → monomer dissociation is accompanied with series changes of protein structure thus it is an irreversible process. The structural changes in the dissociated Hbs result in some loss of their functions. Both the Hb dimer and monomer cannot adequately carry and release oxygen to the tissues in circulation. These findings provide a comprehensive understanding on the pH-dependent protein transitions of human Hb, give guideline to explain complex protein processes and the means to control protein dissociation or re-association reaction. They are also of practical value in clinical medicine, blood preservation and blood substitute development.     

Hemoglobins Austin and Waco: two hemoglobins with substitutions in the alpha 1 beta 2 contact region.

Hemoglobins (Hbs) Austin and Waco were detected by their electrophoretic migration on cellulose acetate (pH 8.4) and citrate agar (pH 6.2). By these methods, both variants migrated between Hbs A and F. Globin chain analysis at pH 8.6 indicated that the mutant β chain of Hb Austin was faster moving than the β^A chain; however, the mutant chain of Hb Waco was indistinguishable from the β^A chain by this technique. The two variants were isolated by ion-exchange column chromatography. Sequence studies demonstrated a substitution of serine (Hb Austin) and lysine (Hb Waco) for arginine at position 40 in the β chain. These mutations involve an amino acid residue in the α₁β₂ contact region, which, before this report, had been considered invariant in all hemoglobin sequences. Hb Austin was found to exist as dimers when oxygenated and as tetramers when deoxygenated. The equilibrium constant (K_d) for the tetramer to dimer transition was approximately 300 × 10^?6m, as calculated from sedimentation velocity studies. This variant also had high oxygen affinity, a much reduced heme-heme interaction, and a normal Bohr effect. The functional properties of Hb Waco were similar to those of Hb A.     

Biochemical and Stability Properties of Recombinant Human MnSOD

The light absorption spectral properties of recornbinant human MnSOD. which contains an N-terminal additional methionyl residue, were investigated as a function of pH in the range 4.5–10.5. Whereas the extinction coefficient, ?_M at the UV maximum (282 nm) was essentially independent of pH, the ?_M values of the visible spectrum maximum (482 nm) displayed a bell-shaped dependence with a plateau between pH 6.5 and B. Those spectral changes were reversible and the enzymatic activity was not affected by exposure to buffered solutions at 25°C in the pH range 5–10.5. The stability of MnSOD was determined between 25 and 60°C at two different pH: 6.5 and 8.2. The enzyme was found to be considerably more stable at pH 6.5 than at pH 8.2, both toward aggregation and degradation. The gel permeation properties of MnSOD were investigated: the enzyme is a tetramer, with a subunit of 22.2 kD; however. it elutes from a Superose 12 column (Pharmacia) with an apparent molecular weight of ～60kD. Under dissociative conditions (such as guanidine-HCI). molecular weights corresponding to the dimer and monomer could also be demonstrated. It thus appears that the tetramer adopts a non-globular shape. which causes the deviation from the Stokes radius corresponding to its molecular weight.     

Effect of 2,3-diphosphoglycerate on the Bohr effect of human adult hemoglobin

The effect of 2,3-diphosphoglycerate (DPG) on the Bohr effect of human hemoglobin has been studied by means of hydrogen ion titration techniques. The results indicate a) that both the acid and the alkaline Bohr effect are equally affected, b) that the DPG binding to deoxyhemoglobin (Hb) is much stronger than to carboxyhemoglobin (HbCO) and c) that Hb binds effectively one DPG molecule. The effect on the Bohr effect can roughly be described by assuming that upon binding two groups per tetramer change their pK from 6.8 to 7.8 and two others from 6.8 to 5.8. These groups very probably are the imidazole groups of the two histidines H21 (143)β and the two phosphate groups of DPG (second dissociation). From the experiments a value for the dissociation constant K of the Hb-DPG complex of about 10⁻⁵ M⁻¹ could be estimated at pH 6.2 and pH 7.5.     

Electrochemical nitrite biosensor based on the immobilization of hemoglobin on an electrode modified by multiwall carbon nanotubes and positively charged gold nanoparticle

The report is on an electrochemical biosensor with remarkably improved sensitivity toward nitrite. In this strategy, positively charged gold nanoparticle (PCNA) is used in combination with multiwall carbon nanotubes (MWCNT) by electrostatic adsorption for fabricating PCNA/MWCNT films. Then hemoglobin (Hb) biocatalyst will easily be attached to the surface of the combination films aforementioned. After that, the Hb/PCNA films are immobilized onto the Hb/PCNA/MWCNT films through layer-by-layer assembly technique. The (Hb/PCNA)₂/MWNT/GC electrode thus prepared exhibits enhanced electrocatalytic behavior to the reduction of nitrite at −0.10 V versus SCE in 0.05 M H₂SO₄ solution_. On condition of the low detecting potential and low pH, interference caused by direct electrochemical oxidation or oxidizable substances can be prevented. Therefore, the modified electrode shows fast response time, very high sensitivity, good selectivity and stability. The current response of the sensor increases linearly with nitrite concentration from a range of 3.6 × 10⁻⁶ to 3.0 × 10⁻³ M with a detection limit(S /N = 3) of 9.6 × 10⁻⁷ M.     

An Irreversible and Kinetically Controlled Process: Thermal Induced Denaturation of <Emphasis Type="SmallCaps">L</Emphasis>-2-Hydroxyisocaproate Dehydrogenase from <Emphasis Type="Italic">Lactobacillus confusus</Emphasis>

The thermal denaturation of Lactobacillus confusus l-2-Hydroxyisocaproate Dehydrogenase (l-HicDH) has been studied by Differential Scanning Calorimetry (DSC). The stability of this enzyme has been investigated at different pH conditions. The results of this study indicate that the thermal denaturation of this enzyme is irreversible and the T _m is dependent on the scan-rate, which suggests that the denaturation process of l-HicDH is kinetically determined. The heat capacity function of l-HicDH shows a single peak with the T _m values between 52.14°C and 55.89°C at pH 7.0 at different scan rates. These results indicate that the whole l-HicDH could unfold as a single cooperative unit, and intersubunit interactions of this homotetrameric enzyme must play a significant role in the stabilization of the whole enzyme. The rate constant of the unfolding is analyzed as a first order kinetic constant with the Arrhenius equation, and the activation energy has been calculated. The variation of the activation energy values obtained with different methods does not support the validity of the one-step irreversible model. The denaturation pathway was described by a three-state model, N → U → F, in which the dissociation of the tetramer takes place as an irreversible step before the irreversible unfolding of the monomers. The calorimetric enthalpy associated with the irreversible dissociation and the calorimetric enthalpy associated with the unfolding of the monomer were obtained from the best fitting procedure. Thermal unfolding of l-HicDH was also studied using Circular Dichroism (CD) spectroscopy. Both methods yielded comparable values.     

Equilibrium,kinetic and structural properties of hemoglobin Cranston,an elongated β chain variant

Hemoglobin Cranston has an elongated β subunit owing to a frame shift mutation. Oxygen equilibrium measurements of stripped Hb Cranston³ at 20 °C in the absence of phosphate revealed a high affinity (P₅₀ = 0·2 mm Hg at pH 7), non-co-operative hemoglobin variant with markedly reduced Böhr effect ($\text{?Δ}\text{log}\text{P}{}_{50}\text{Δ}\text{pH}{}_{\mathrm{7–8}}\text{= 0·2}$). The addition of inositol hexaphosphate resulted in an overall decrease in oxygen affinity (P₅₀ = 0·7 mm Hg at pH 7), as well as an increase in co-operativity and Böhr effect ($\text{?Δ}\text{log}\text{P}{}_{50}\text{Δ}\text{pH}{}_{\mathrm{7–8}}\text{= 0·2}$). Rapid mixing and flash photolysis experiments reflected the equilibrium results. Over a pH range from 6 to 9 in the absence of phosphate, the rate of combination of carbon monoxide with Hb Cranston measured by a stopped-flow technique and following full or partial flash photolysis was extremely rapid (l′, l′₄, of ～ 6 × 10⁶m^?1s^?1). In rapid kinetic experiments the addition of inositol hexaphosphate lowered the value of l′ to ～ 0·5 × 10⁶m^?1s^?1 only after prior incubation with the deoxygenated protein. Inositol hexaphosphate had no effect on the rate of recombination of carbon monoxide following either full or partial flash photolysis. Overall oxygen dissociation and oxygen dissociation with carbon monoxide replacement, were measured and found to be slow (k, k₄～ 11 s^?1), consistent with a high affinity hemoglobin. Sedimentation equilibrium experiments revealed that Hb Cranston, at concentrations used in the functional studies, is somewhat less tetrameric than Hb A but nonetheless does not exist solely as a non-co-operative dimer. These kinetic and centrifugational findings in conjunction with X-ray diffraction evidence suggested that a high affinity tetramer of Hb Cranston exists which may equilibrate slowly with inositol hexaphosphate. Oxygen equilibrium measurements, ligand binding kinetics and X-ray diffraction studies on equivalent mixtures of Hb Cranston and Hb A revealed an interaction between these two hemoglobins in vitro that most probably exists in vivo. The presence of asymmetric hybrid molecules, α₂β^Aβ^Cranston, in the difference Fourier maps indicated that the hydrophobic tail of Hb Cranston is accommodated in the central cavity of the hybrid molecule between the two β chains and is relatively protected from the water environment, thus aiding in the stability of Hb Cranston in the red cell.     

1H-Nuclear magnetic resonance study on imidazole–imidazole interaction in L-histidyl-L-histidine and D-histidyl-L-histidine: Analysis including microscopic dissociation series

The ¹H-nmr titration curves of chemical shifts versus pH were observed for the protons of _D,L-histidyl-_D,L-histidine as representative of cases with two or more ionizable groups with similar pK_a values. The titration curves of L -histidyl-L -histidine and D -histidyl-L -histidine were individually analyzed according to two mathematical models: one of a macroscopic dissociation series and one of a microscopic dissociation series. Most-probable values and standard deviations were obtained for pK_a values and intrinsic chemical shifts. An analysis including the microscopic dissociation series yielded an electrostatic interaction between twoimidazole rings of about 0.3 pH units for L -histidyl-L -histidine and about 0.7 pH units for D -histidyl-L -histidine. The difference of the magnitude of imidazole-imidazole interactions between L -histidyl-L -histidyne and D -histidyl-L -histidine was interpreted in terms of the spatial arrangement of two imidazole rings in each molecule based on the solution conformation estimated from Gd(III)-induced relaxation enhancements.     

Studies on the interaction between nanodiamond and human hemoglobin by surface tension measurement and spectroscopy methods

In this study, a novel method to probe molecular interactions and binding of human hemoglobin (Hb) with nanodiamond (ND) was introduced based on the surface tension measurement. This method complements conventional techniques, which are basically done by zeta potential and dynamic light scattering (DLS) measurements, near and far circular dichroism (CD) spectroscopy, intrinsic and extrinsic fluorescence spectroscopy. Addition of ND to Hb solution increased the surface tension value of Hb–ND complex relative to those of Hb and ND molecules. The zeta potential values reveled that Hb and ND provide identical charge distribution at pH 7.5. DLS measurements demonstrated that Hb, ND, and ND–Hb complex have hydrodynamic radiuses of 98.37 ± 4.57, 122.07 ± 7.88 nm and 62.27 ± 3.70 at pH of 7.5 respectively. Far and near UV-CD results indicated the loss of α-helix structure and conformational changes of Hb, respectively. Intrinsic fluorescence data demonstrated that the fluorescence quenching of Hb by ND was the result of the static quenching. The hydrophobic interaction plays a pivotal role in the interaction of ND with Hb. Fluorescence intensity changes over time revealed conformational change of Hb continues after the mixing of the components (Hb–ND) till 15 min, which is indicative of the denaturation of the Hb relative to the protein control. Extrinsic fluorescence data showed a considerable enhancement of the ANS fluorescence intensity of Hb–ND system relative to the Hb till 60 nM of ND, likely persuaded by greater exposure of nonpolar residues of Hb hydrophobic pocket. The remarkable decrease in T_m value of Hb in Hb–ND complex exhibits interaction of Hb with ND conducts to conformational changes of Hb. This study offers consequential discrimination into the interaction of ND with proteins, which may be of significance for further appeal of these nanoparticles in biotechnology prosecution.     

Dynamic light scattering studies of internal motions in DNA. II. Clean viral DNAs

Internal Brownian motions of clean ?29 and λ-DNAs have been studied using photon-correlation techniques at both visible (λ₀ = 632.8 nm) and uv (λ₀ = 363.8 nm) wavelengths. The present dynamic light scattering data, which extend to K² = 19 × 10¹⁰ cm^?2, can in every case be satisfactorily simulated by a Rouse-Zimm model polymer with an appropriate choice of the three model parameters. The effects of pH, salt concentration, single-strand breaks, and molecular weight on those model parameters have also been investigated. Intact clean DNAs exhibit surprisingly little variation with pH from 7.85 to 10.25, with salt concentration from 0.01 NaCl to 5.4M NH₄Cl, or with molecular weight or GC content. The single-strand breaks have no effect at pH 9.46, but produce dramatic changes in the model parameters at pH 10.0 and 10.25, indicating the introduction of titratable joints at those pHs. The failure of either single-strand breaks or a large change in GC content to alter the model parameters in the neutral pH range is a strong indication that local denaturation is not required for those flexions and torsions that dominate the relaxation of fluctuations in the scattered light. The Langevin relaxation time for the slowest internal mode of a particular Rouse-Zimm model derived from the dynamic light scattering data is compared with pertinent literature data extrapolated to the same molecular weight. The present algorithm for determining model parameters from the light-scattering D_app vs K² curve actually yields a Langevin time in fairly good agreement with the literature value. For unknown reasons the light-scattering D₀ values generally exceed those obtained from the molecular weight and sedimentation coefficient by about 20%.     

Synthesis and characterization of an ionizable polyhexapeptide collagen model

Poly(Lys(HBr)-Gly-Pro-Pro-Gly-Pro) has been synthesized and studied by circular dichroism (CD) spectroscopy. It is apparently the first polyhexapeptide collagen model reported with an ionizable side chain. The monomer (ε-(p-nitrobenzyloxycarbonyl)-Lys-Gly-Pro-Pro-Gly-Pro-p-nitrophenyl-ester) was prepared by a stepwise strategy employing active esters. Polymerization in N,N-dimethyl formamide, followed by removal of the Lys side chain protection with HBr/acetic acid, gave a polydisperse product. Fractionation was accomplished by gel filtration chromatography. The polydisperse material had a molecular weight (M_r = 5–17,000). High molecular weight fractions from triple helices under concentrated conditions at 2°C. The triple helical structure gives a CD pattern very similar to that of collagen and its triple helical analogs. However, unlike collagen, the polyhexapeptide undergoes spontaneous dissociation at temperatures substantially below the melting temperature from a triple helical form to single chains. This process is promoted at low concentrations, high temperature, neutral pH, and low molecular weight, and is apparently due, in large part, to unfavorable ionic side-chain interactions. In addition to this relatively slow “ionic” dissociation the triple helical polypeptide may be thermally dissociated in a manner similar to collagen. The thermal denaturation is a relatively fast process compared with ionic dissociation. A high molecular weight fraction (3 × M_r = 48,000) was found to melt at 42°C at neutral pH but increased to 54°C at pH 12 where the lysyl side chains are predominantly deprotonated. Furthermore, reconstitution of triple helices appeared to be more readily achieved at high pH. Thus it is concluded that ionic repulsion between side chains causes destabilization of the triple helix and hinders reconstitution.     

Regulation of the aggregation state of maize phosphoenolpyruvate carboxylase: evidence from dynamic light-scattering measurements

The molecular weights of different aggregational states of phosphoenolpyruvate carboxylase purified from the leaves of Zea mays have been determined by measurement of the molecular diameter using a Malvern dynamic light scattering spectrometer. Using these data to identify the monomer, dimer, tetramer, and larger aggregate(s) the effect of pH and various ligands on the aggregational equilibria of this enzyme have been determined. At neutral pH the enzyme favored the tetrameric form. At both low and high pH the tetramer dissociated, followed by aggregation to a "large" inactive form. The order of dissociation at least at low pH appeared to be two-step: from tetramer to dimers followed by dimer to monomers. The monomers then aggregate to a large aggregate, which is inactive. The presence of EDTA at pH 8 protected the enzyme against both inactivation and large aggregate formation. Dilution of the enzyme at pH 7 at room temperature results in driving the equilibrium from tetramer to dimer. The presence of malate with EDTA stabilizes the dimer as the predominant form at low protein concentrations. The presence of the substrate phosphoenolpyruvate alone and with magnesium and bicarbonate induced formation of the tetramer, and decreased the dissociation constant (Kd) of the tetrameric form. The inhibitor malate, however, induced dissociation of the tetramer as evidenced by an increase in the Kd of the tetramer.     

Numerical simulation of aldolase tetramer stability

A theoretical study of aldolase tetramer stability, conducted by finite difference Poisson-Boltzmann (FDPB) and modified Tanford-Kirkwood (MTK) techniques using the atomic coordinates of human aldolase, is described. A method for calculating the interaction energy between subunits is proposed. An analysis of the contribution of different energy terms to the stability and oligomeric equilibria (monomer ⇔ dimer ⇔ tetramer) of aldolase is made. It is shown that the loss of solvation energy and electrostatic interactions at very high and low pH-s destabilise the oligomers. These energy terms are compensated over a wide pH range by the stabilization energy due to hydrophobic interactions. It is shown that the aldolase tetramer is energetically more preferable than other oligomers in the pH range from 5 to 11. Subunit-subunit interactions within the tetramer suggest one dimeric form to be the most stable of the possible sub-parts. For this reason the tetramer can be thought of as a “dimer of dimers”. A comparison between our theoretical results and available experimental data shows that the dissociation of the aldolase tetramer below pH 3–4 cooperatively leads to acid denaturation. A second dissociation is predicted to occur at high pH (>12) in addition to the well known acidic dissociation. The analysis suggests that a mutation of His20 or Arg257 to a neutral residue could decrease the pH of the acidic dissociation by approximately 1 pH unit. Received: 16 February 1998 / Revised version: 8 April 1998 / Accepted: 19 April 1998     

Studying the interaction between three synthesized heterocyclic sulfonamide compounds with hemoglobin by spectroscopy and molecular modeling techniques

The interaction between synthesized heterocyclic benzene sulfonamide compounds, N-(7-benzyl-56-biphenyl-2m-tolyl-7H-pyrrolo[23-d]pyrimidine–4–yl)-benzene sulfonamide (HBS₁), N-(7-benzyl-56-biphenyl-2-m-tolyl-7H-pyrrolo[23-d] pyrimidine-4-yl)-4-methyl- benzene sulfonamide (HBS₂), and N-(7-benzyl-56-biphenyl-2-m-tolyl-7H-pyrrolo[23-d]pyrimidine-4-yl)-4-chloro-benzene sulfonamide (HBS₃) with Hb was studied by fluorescence quenching, zeta potentional, circular dichroism, and molecular modeling techniques. The fluorescence spectroscopy experiments were performed in order to study the conformational changes, possibly due to a discrete reorganization of Trp residues during binding between HBS derivatives and Hb. The variation of the K_SV value suggested that hydrophobic and electrostatic interactions were the predominant intermolecular forces stabilizing the complex. The K_SV1 ans K_SV2 values of HBS derivatives with Hb are .6 × 10¹³ and 3 × 10¹³ M^?1 for Hb–HBS₁, 1 × 10¹³ and 4 × 10¹³ M^?1 for Hb–HBS₂, .9 × 10¹³, and 6 × 10¹³ M^?1 for Hb–HBS₃, respectively. The molecular distances between Hb and HBS derivatives in binary and ternary systems were estimated according to Förster’s theory of dipole–dipole non-radiation energy transfer. The quantitative analysis data of circular dichroism spectra demonstrated that the binding of the three HBS derivatives to Hb induced conformational changes in Hb. Changes in the zeta potential of the Hb–HBS derivatives complexes demonstrated a hydrophobic adsorption of the anionic ligand onto the surface of Hb as well as both electrostatic and hydrophobic adsorption in the case of the complex. The modeling data thus confirmed the experimental results. This study is expected to provide important insight into the interaction of Hb with three HBS derivatives to use in various toxicological and therapeutic processes.     

Kinetics of subunit dissociation of partially oxygenated hemoglobin determined by haptoglobin binding

Human haptoglobin 1-1 binds very rapidly to hemoglobin dimers but not to tetramers. We have studied the binding kinetics of partially oxygenated Hb A to haptoglobin 1-1. Under the oxygenation conditions used for the measurement of the K₁ of oxygenation ($\text{Hb O}{}_2\text{Hb}\text{≤ 1\%}$, pO₂ ≤ 0.5 mm Hg), the dissociation kinetics were found to be 50 times faster than that of deoxy Hb A. This result suggested that the binding of one molecule of oxygen to hemoglobin tetramer changed the quaternary structure of the intersubunit α₁β₂ contact surface.     

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.     

Isolation and characterization of light- and dithiothreitol-modulatable glucose-6-phosphate dehydrogenase from pea chloroplasts

Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP oxidoreductase, EC 1.1.1.49) has been purified to electrophoretic homogeneity from pea chloroplasts. The enzyme, which has a Stokes radius of 52 Å, is a tetramer made up of four 56000 Da monomers. The pH optimum is around 8.2. The enzyme is absolutely specific for NADP. The apparent K_m(NADP) is 2.4 ± 0.1 μM. NADPH inhibition of the enzyme is competitive with respect to NADP (mean K_i, 18 ± 5 μM) and is mixed (K_p >K_m, V_max >V_p) with respect to glucose 6-phosphate (mean crossover point, 0.5 ± 0.1 mM). The apparent K_m(glucose 6-phosphate) is 0.37 ± 0.01 mM. The purified enzyme is inactivated in the light in the presence of dilute stroma and washed thylakoids, and by dithiothreitol. Enzyme which has been partially inactivated by treatment with dithiothreitol can be further inactivated in the light in the presence of dilute stroma and washed thylakoids and reactivated in the dark, but only to the extent of the reverse of light inactivation. Dithiothreitol-inactivated enzyme is not reactivated further by addition of crude stroma or oxidized thioredoxin. Dithiothreitol-dependent inactivation of the enzyme follows pseudo-first-order kinetics and shows rate saturation. The enzyme which has been partially inactivated by treatment with dithiothreitol does not differ from the untreated control with respect to thermal and tryptic inactivation. However, enzyme which has been partially light inactivated shows different thermal and tryptic inactivation patterns as compared to the dark control. These observations suggest that the changes in the enzyme brought about by light modulation are not necessarily identical with those brought about by dithiothreitol inactivation.     

A circadian rhythm of the luciferin binding protein fromGonyaulax polyedra

Summary It has previously been shown that a protein extracted fromGonyaulax polyedra strongly and specifically binds luciferin, the substrate of the bioluminescent reaction. This binding is markedly dependent on pH with tight binding at pH 8.0 and almost no binding at pH 6.5, as measured by two independent methods. A procedure for the determination of the dissociation constant (K_d) of the luciferin binding protein (LBP) is presented, and K_d is estimated to be7×10^–9 M at pH 8.0, assuming an overall quantum yield of 0.1 for the bioluminescent reaction. With cells grown in a 12 h light — 12 h dark cycle, 5 to 10 times more LBP activity can be extracted from dark phase cells than from light phase cells. This rhythm persists in a circadian fashion in cultures maintained in constant dim light.Supported in part by a grant from the National Institutes of Health to J.W.H. (GM 19536)     

Double-stranded helix of xanthan: Rigidity in 0.01M aqueous sodium chloride containing 0.01 N hydrochloric acid

Six samples of Na xanthan in 0.01M aqueous NaCl containing 0.01 N HCl (pH = 2) were studied by light scattering and viscosity. This study was motivated by the finding that the intrinsic viscosity [η] fairly sharply decreased when the pH of the solvent was lowered from about 6 to 2 by adding HCl to 0.01M aqueous NaCl in which Na xanthan dissolves as rigid dimers having a double-helical structure. The data for weight-average molecular weight, radius of gyration, and [η] showed that Na xanthan at pH = 2 remains a dimer behaving as a semiflexible chain. Data analysis in terms of known theories for unperturbed wormlike chains yielded 0.47 ± 0.02, 2.0 ± 0.6, and 68 ± 7 nm for the contour length h per main-chain residue, diameter d, and persistence length q of the dimer, respectively. these h and d values agreed with the pitch per main-chain residue and the diameter of the double helix of Na xanthan in 0.01 or 0.1M aqueous NaCl. However, the q value, which was close to the intrinsic persistence length q₀ ( = q in the absence of electrostatic interaction) of Na xanthan at pH = 2, was much smaller than the q₀ (106 nm) of this helix. We concluded that the xanthan dimer at pH = 2 assumes a double-helical structure, which is geometrically the same as, but is more flexible than, that at neutral pH.