Reversible acidic-alkaline transition of the carbon monoxide complex of cytochrome c peroxidase

The Soret absorption band of the ferrous carbon monoxide (CO) complex of cytochrome c peroxidase exhibited a blue shift from 423.7 to 420 nm upon an increase in pH from 6.5 to 8.5. The spectral change was reversible with an isosbestic point at 422 nm. The pH dependence of this spectral change gave a sigmoidal curve fitted well to a theoretical curve of a cooperative release of two protons with a pK value of 7.5, indicating the existence of the acidic and alkaline forms of the ferrous CO enzyme. Upon irradiation of light flash (100 J of power and 30-microseconds), the heme-bound CO was readily dissociated in both acidic and alkaline forms with a quantum yield of approximately unity. On the other hand, the rate of recombination of the dissociated CO with the heme iron was significantly different between these two forms; the recombination rate constants were 1.1 X 10(3) and 3.0 X 10(4) M-1 S-1 at 25 degrees C for the acidic and alkaline forms, respectively. At intermediate pH values, kinetics of recombination were biphasic, consisting of the slow and fast processes with the appropriate rate constants mentioned above. When the fraction of the fast process was plotted against pH, the pH profile coincided with the spectrophotometric pH titration curve described above. Thus, it was concluded that the acidic and alkaline forms of the enzyme were responsible for the slow and fast processes, respectively. In infrared spectroscopy, the acidic form showed a narrow CO stretching band at 1922 cm-1 with a half-band width of 12.5 cm-1, while the alkaline form exhibited a broad CO-stretching band at 1948 cm-1 with a half-band width of 33 cm-1. Significance of these results are discussed in relation to the structure of the heme vicinity on the CO complex of cytochrome c peroxidase.     

Regulatory subunit of type II cAMP-dependent protein kinase as substrate and inhibitor of protein phosphatase-1 and -2A

The dissociated regulatory subunit (RII) of autophosphorylated cAMP-dependent protein kinase II was dephosphorylated by the catalytic subunits of protein phosphatase-1 and -2A (phosphatase-1c and -2Ac) and by a high-Mr polycation-dependent form of phosphatase-2A (2Ao) with Km values of 5, 0.3 and 1 microM, respectively. Dissociation of protein kinase by cAMP preferentially increased the dephosphorylation of RII by phosphatase-1c, whereas polycations (histone Hl or polybrene) markedly stimulated phosphatase-2Ac and -2Ao even in the absence of cAMP. Thiophosphorylated RII inhibited the dephosphorylation of phosphorylase a by these phosphatases with half-maximum inhibitory concentrations of 0.1-0.36 microM.     

Activation of acyl condensation reaction of monomeric 6-hydroxymellein synthase,a multifunctional polyketide biosynthetic enzyme,by free coenzyme A

6-Hydroxymellein (6HM) synthase is a multifunctional polyketide enzyme induced in carrot cells, whose fully active homodimer catalyzes condensation of acyl-CoAs and the NADPH-dependent ketoreduction of the enzyme-bound intermediate. 6HM-forming activity of the synthase was markedly decreased when the reaction mixture pH was adjusted from 7.5 to 6.0. However, under these slightly acidic conditions, the acyl condensation catalyzed by the dissociated monomer enzyme was appreciably stimulated by addition of free coenzyme A (CoA). In contrast, the condensation reaction at pH 6.0 was significantly inhibited in the presence of CoA when the reaction was carried out with the NADPH-omitted dimer synthase. Among the kinetic parameters of the acyl condensation, velocity of the monomer-catalyzing reaction at the acidic pH was appreciably increased upon addition of CoA while K(m)s did not show any significant change in the presence and absence of the compound. These results suggest that CoA associates with a specific site in the dissociated monomeric form of 6HM synthase, and the velocity of the acyl condensation reaction catalyzed by the CoA-synthase complex appreciably increases in acidic conditions.     

Reaction of PHMBHCl with acidic polysaccharides, and its application to the purification of xanthan gum

Poly(iminocarbonimidoyliminocarbonimidoylimino-1,6-hexanediyl hydrochloride) [PHMBH⁺Cl⁻] reacts with acidic polysaccharides to form white, insoluble salts. The PHMBH⁺ salts of sulphated polysaccharides can only be dissociated at or below pH 0·2. The salts of polysaccharides containing only carboxylate groups as their acidic functions are dissociated at or below pH 1·6, and by strong electrolytes above a critical electrolyte concentration.

The acidic polysaccharide xanthan may be recovered from a dispersion of its PHMBH⁺ salt in aqueous potassium chloride by treatment with 2-propanol. This forms the basis of a method for the recovery of xanthan, in purified form, from Xanthomonas campestris fermentation broths. The reaction of PHMBH⁺Cl⁻ with nucleic acids and proteins is also discussed.     

The interrelation between high- and low-molecular-weight forms of GM1-beta-galactosidase purified from porcine spleen

1) Two forms of acid beta-galactosidase [EC 3.1.23] with different molecular weights catalyzing the hydrolysis of GM1-ganglioside and p-nitrophenyl-beta-D-galactoside were separated and purified from porcine spleen. 2) The apparent molecular weights were 400,000-600,000 and 70,000-74,000 for the high (termed Am form) and low (termed A1 form) molecular weight forms, respectively. 3) On examination by sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis, both forms of the enzyme had a common protein band of molecular weight 63,000, and the Am form showed three additional protein bands with molecular weights of 31,000, 21,000, and 20,000. 4) Both forms of the enzyme had similar catalytic functions with regard to pH-optimum, Km, substrate specificity and sensitivity to substrate analogues and other substances such as detergents, bovine serum albumin (BSA) and NaCl. 5) Both forms of the enzyme were fairly stable upon preincubation at 45 degrees C at acidic pH (pH 4.5), but lost their activities at neutral pH (pH 7.0). 6) The A1 form was a monomer at neutral pH (pH 7.0) and formed a dimer at acidic pH (pH 4.5). However, most of the Am form could not be converted to a dimeric form on gel filtration at acidic pH.     

Carbon monoxide complex of cytochrome b(5) at acidic pH

CO complex of cyt b(5) generated at acidic pH is investigated by absorption, resonance Raman (RR), and far UV CD measurements. The Soret maximum wavelength blue-shifted to 420 nm with other absorption bands observed around 540 and 570 nm for reduced cyt b(5) upon interaction with CO at acidic pH (pH 3.1-3.5). Under this condition, the iron-carbon stretching RR band was observed at 529 cm(-1) (520 cm(-1) for C(18)O), which indicated formation of a heme&bond;CO adduct with a histidine as an axial ligand. Heme dissociated from the reduced cyt b(5) protein at pH approximately 3.5, whereas its rate decreased under CO atmosphere compared with N(2) atmosphere, due to formation of a heme&bond;CO adduct with a histidine as an axial ligand.     

Partially folded conformations of bovine liver glutamate dehydrogenase induced by mild acidic conditions

The acid-induced unfolding of bovine liver glutamate dehydrogenase (GDH) was studied using various spectroscopic methods such as far- and near-UV circular dichroism (CD), intrinsic and 1-anilino naphthalene-8-sulphonate (ANS) extrinsic fluorescence spectroscopy, light scattering and fluorescence quenching in 20 mM mixed buffer at various pHs. CD spectra show that at pH 3.5, GDH retains its secondary structure substantially, whereas its tertiary structure content is reduced considerably. Intrinsic fluorescence of GDH and ANS binding suggest that, at pH 3.5, the hydrophobic surface of enzyme is more exposed in comparison to the native form. Acrylamide quenching indicates more exposure of tryptophan residues of enzyme at pH 3.5 in comparison to pH 7.5. Another partially unfolded intermediate was detected at pH 5.0, which with its ANS binding capacity lies between the pH 3.5 intermediate and the native form of the enzyme. Gel filtration results revealed that the enzyme at pH 3.5 is dissociated into trimeric species whereas it exists as hexamer at pH 7.5 and 5.0. All the data taken together suggest the existence of two partially unfolded states of GDH at moderate acidic pHs which may be considered as molten and pre-molten globule-like states.     

Thermal stability of pyrrolidone carboxyl peptidases from the hyperthermophilic Archaeon, Pyrococcus furiosus.

The temperature adaptation of pyrrolidone carboxyl peptidase (PCP) from a hyperthermophile, Pyrococcus furiosus (Pf PCP), was characterized in the context of an assembly form of the protein which is a homotetramer at neutral pH. The Pf PCP exhibited maximal catalytic activity at 90-95 degrees C and its activity was higher in the temperature range 30-100 degrees C than its counterpart from the mesophilic Bacillus amyloliquefaciens (BaPCP). Thermal stability was monitored by differential scanning calorimetry (DSC). Two clearly separated peaks appeared on the DSC curves for Pf PCP at alkaline and acidic pH. Using the oxidized Pf PCP and two mutant proteins (Pf C188S and Pf C142/188S), it was found that the peaks on the high and low temperature sides of the DSC curve of Pf PCP were produced by the forms with an intersubunit disulfide bridge between the two subunits and without the bridge, respectively, indicating the stabilization effect of intersubunit disulfide bridges. The denaturation temperature (Td) of Pf PCP with intersubunit disulfide bridges was higher by 53 degrees C at pH 9.0 than that of BaPCP. An analysis of the equilibrium ultracentrifugation patterns showed that the tetrameric Pf C142/188S dissociated into dimers with decreasing pH in the acidic region and became monomer subunits at pH 2.5. The heat denaturation of Pf PCP and its two Cys mutants was highly reversible in the dimeric forms, but completely irreversible in the tetrameric form. The Td of Pf C142/188S decreased as the enzyme became dissociated, but the monomeric form of the protein was still folded at pH 2.5, although BaPCP was completely denatured at acidic pH. These results indicate that subunit interaction plays an important role in stabilizing PCP from P. furiosus in addition to the intrinsic enhanced stability of its monomer.     

Electrodissociation of high molecular weight complexes of interferon alpha during recycling isoelectric focusing

Natural human interferon alpha has been separated by selective ultrafiltration into low molecular weight components and the molecules exceeding 100K daltons. Interferon associated with a higher molecular weight fraction showed partial pH sensitivity and resisted dissociation after treatment with urea, mercaptoethanol, sodium chloride or significant changes in pH. However, interferon activity was released from high molecular weight components during recycling isoelectric focusing. Electrodissociation was carried out in 1% ampholytes for 574 watt-hours. The interferon activity was concentrated in a pH range of 6-6.5, whereas, the majority of proteins were generally found in a more acidic position. The dissociated interferon was neutralized by polyclonal antibody to human interferon alpha (IFN alpha) and showed no presence of pH labile form. A pH sensitivity of high molecular weight interferon (HMW-IFN) may reflect an aggregation phenomenon rather than intrinsic structural differences.     

Anthrax protective antigen: prepore-to-pore conversion.

PA(63), the active 63 kDa form of anthrax protective antigen, forms a heptameric ring-shaped oligomer that is believed to represent a precursor of the membrane pore formed by this protein. When maintained at pH >/=8.0, this "prepore" dissociated to monomeric subunits upon treatment with SDS at room temperature, but treatment at pH 相似文献   

Structural Basis of pH Dependence of Neoculin,a Sweet Taste-Modifying Protein

Among proteins utilized as sweeteners, neoculin and miraculin are taste-modifying proteins that exhibit pH-dependent sweetness. Several experiments on neoculin have shown that His11 of neoculin is responsible for pH dependence. We investigated the molecular mechanism of the pH dependence of neoculin by molecular dynamics (MD) calculations. The MD calculations for the dimeric structures of neoculin and His11 mutants showed no significant structural changes for each monomer at neutral and acidic pH levels. The dimeric structure of neoculin dissociated to form isolated monomers under acidic conditions but was maintained at neutral pH. The dimeric structure of the His11Ala mutant, which is sweet at both neutral and acidic pH, showed dissociation at both pH 3 and 7. The His11 residue is located at the interface of the dimer in close proximity to the Asp91 residue of the other monomer. The MD calculations for His11Phe and His11Tyr mutants demonstrated the stability of the dimeric structures at neutral pH and the dissociation of the dimers to isolated monomers. The dissociation of the dimer caused a flexible backbone at the surface that was different from the dimeric interface at the point where the other monomer interacts to form an oligomeric structure. Further MD calculations on the tetrameric structure of neoculin suggested that the flexible backbone contributed to further dissociation of other monomers under acidic conditions. These results suggest that His11 plays a role in the formation of oligomeric structures at pH 7 and that the isolated monomer of neoculin at acidic pH is responsible for sweetness.     

Dissociation of small-intestinal sucrase - isomaltase complex into enzymatically active subunits.

1. The sucrase - isomaltase complex from rabbit small intestine dissociated into its subunits upon reaction with citraconic anhydride. They can recombine after deacylation under mild acidic conditions. 2. When citraconylated, the subunits could be separated and isolated in a catalytically active form. 3. The previously reported procedure for separation of the subunits by alkaline treatment at pH 9.6 is apparently not due to contaminating degradative enzymes (possibly still present at undetectable levels in the isolated sucrase - isomaltase complex) but to the action of alkali.     

Spectroscopic characterization of diverse amyloid fibrils in vitro by the fluorescent dye Nile red

The fluorescence of Nile red (9-diethylamino-5H-benzophenoxazine-5-one) is quenched in aqueous solutions but shows augmented fluorescence in hydrophobic environments. Nile red fluorescence was blue shifted and strongly augmented in the presence of various amyloid fibrils assayed under acidic as well as neutral pH conditions. Fibrils grown from lysozyme and insulin (at pH 1.6 and 65 °C), transthyretin (TTR) fibrils grown from the acid unfolded monomer (pH 2.0, 21 °C) or from the dissociated tetramer starting from native protein under less acidic conditions (pH 4.4, 37 °C) were detected. Nile red was also successfully employed in detecting Aβ1-42 and human prion protein (PrP90-231) amyloid fibrils grown at neutral pH. Nile red was amyloid fibril specific and did not fluoresce appreciably in the presence of the monomeric precursor proteins. Stoke's shifts of the wavelength maximum of Nile red bound to various fibrils were different (ranging from 615 nm to 638 nm) indicating sensitivity to the tertiary structure in its respective binding sites of different amyloid proteins. A polarity assay using ethanol-water mixtures and pure octanol ranging from dielectric constants between 10 and 70 showed a linear correlation of Nile red Stoke's shift and allowed assignment of amyloid fibril binding site polarity. Fluorescence resonance energy transfer between Thioflavin T (ThT) and Nile red was proven to be efficient and co-staining was employed to discriminate between conformational isoforms of Aβ1-42 amyloid fibrils grown under agitated and quiescent conditions. This paper demonstrates the complementary use of this fluorometric method for conformational typing of amyloid structures.     

CO recombination in cytochrome c peroxidase: effect of the local heme environment on CO binding explored through site-directed mutagenesis

CO recombination to the cloned cytochrome c peroxidase [CCP(MI)] and mutants of CCP(MI) prepared by site-directed mutagenesis was examined as a function of pH by flash photolysis. The mutants examined included distal Arg 48----Leu, Lys; proximal Asp 235----Asn; and His 181----Gly. At alkaline pH, ferrous CCP(MI) was converted to a hexacoordinate form by a cooperative two-proton ionization, apparent pK(a) = 8.0. This change was observed in all of the mutants, although in the His 181----Gly mutant, the conversion to the hexacoordinate form was the result of a single-proton ionization, implicating His 181 as one of the two residues deprotonated in this isomerization. The pH-dependent conversion of CO ferrous CCP(MI) from acidic to alkaline forms was also observed and was similar to that reported for cytochrome c peroxidase from bakers' yeast [Iizuka, T., Makino, R., Ishimura, Y., & Yonetani, T. (1985) J. Biol. Chem. 260, 1407-1412]. Photolysis of the acidic form of the CO complex of CCP(MI) produces a kinetic form of the ferrous enzyme (form A) which exhibits the slow rate of CO recombination (l1' approximately 10(3) M-1 s-1) characteristic of peroxidases, while photolysis of the alkaline form of the CO complex produces a second kinetic form (form B), which exhibits a much faster rate of recombination (l2' approximately 10(5) M-1 s-1). Kinetic forms analogous to forms A and B were observed in all of the mutants examined. A third kinetic form (form B*) with a bimolecular rate constant l3' approximately 10(6) M-1 s-1 was also observed in the mutants at alkaline pH. Although the pH dependence for the conversion of form A to form B with increasing pH was altered by changes in the local heme environment, the rate of CO recombination by the respective forms was not dramatically altered in the mutants. Transient spectra of the reaction of CO with ferrous CCP(MI) after photolysis show that equilibrium between penta- and hexacoordinate ferrous enzyme is rapid relative to CO recombination. The presence of the internal sixth ligand has no discernible effect on the observed rate of recombination, however. The results presented indicate that in CCP(MI) the rate of ligand binding is determined primarily by isomerization of the protein from a closed conformation at acidic pH to an open conformation at alkaline pH and that polar effects of proximal Asp 235 and distal Arg 48 are of minor significance in the rate of CO recombination in both conformations.     

Subunit structure of a potent platelet-activating glycoprotein isolated from the venom of Crotalus durissus cascavella

The potent platelet-activating factor isolated from the venom of Crotalus durissus cascavella is an acid-soluble multisubunit glycoprotein of Mr 72,000 built up of two types of subunits, alpha and beta, linked by disulphide bonds. The mean apparent Mr of the reduced complex was around 12,000 by gel filtration under denaturating conditions. The Mrs of the alpha and beta subunits, with an apparent ratio of 1/1, were 12,600 and 13,580 by SDS-PAGE respectively. The Mr 72,000 glycoprotein is thought to be an alpha 3 beta 3 complex. The urea dissociated glycoprotein (Mr 72,000) retained its platelet-stimulating activity. It is concluded that the Mr 300,000 form isolated at acidic pH under native conditions, and showing a rosette - like, ring-shaped structure in the electron microscope as well as the Mr 144,000 form isolated at physiological pH under native conditions and active on platelets were the tetrameric and dimeric states of the molecule respectively.     

The protein phosphatases involved in cellular regulation. 2. Glycogen metabolism

The nature of protein phosphatases that are active against the phosphorylated proteins of glycogen metabolism was investigated in rabbit skeletal muscle and liver. Six 32P-labelled substrates corresponding to the major phosphorylation sites on glycogen phosphorylase, phosphorylase kinase, glycogen synthase and inhibitor-1 were used in these studies. The results showed that the four protein phosphatases defined in the preceding paper, namely protein phosphatases-1, 2A, 2B and 2C [Ingebritsen, T. S. and Cohen, P. (1983) Eur. J. Biochem. 132, 255-261] were the only significant enzymes acting on these substrates. The four enzymes can be conveniently separated and identified by a combination of ion-exchange chromatography and gel filtration and by the use of specific inhibitors. Three species of protein phosphatase-2A were resolved on DEAE-cellulose, termed protein phosphatases-2Ao (0.12 M NaCl), 2A1 (0.2 M NaCl) and 2A2 (0.28 M NaCl) that had apparent molecular weights of 210000, 210000 and 150000 respectively. Protein phosphatase-2Ao was a completely inactive enzyme whose activity was only expressed after dissociation to a 34000-Mr(app) catalytic subunit by freezing and thawing in 0.2 M 2-mercaptoethanol. This treatment also dissociated protein phosphatases 2A1 and 2A2 to more active 34000-Mr(app) catalytic subunits. The catalytic subunits derived from protein phosphatases-2Ao, 2A1 and 2A2 possessed identical substrate specificities, preferentially dephosphorylated the alpha-subunit of phosphorylase kinase, were unaffected by inhibitor-1 and inhibitor-2 and were inhibited by similar concentrations of ATP. The properties of protein phosphatases-2A1 and 2A2 were very similar to those of the catalytic subunits, except that they were less sensitive to inhibition by ATP. Protein phosphatase-2B was eluted from DEAE-cellulose in the same fraction as protein phosphatase-2Ao. These activities were resolved by gel filtration, the Mr(app) of protein phosphatase-2B being 98000. Protein phosphatase-2B was completely inhibited by 100 microM trifluoperazine, which did not affect the activity of protein phosphatase-2Ao or any other protein phosphatase. Freezing and thawing in 0.2 M 2-mercaptoethanol resulted in partial inactivation of protein phosphatase-2B. Protein phosphatase-2C was eluted from DEAE-cellulose at the leading edge of the peak of protein phosphatase-2A1. These activities were completely resolved by gel filtration, since the Mr(app) of protein phosphatase-2C was 46000. Two forms of protein phosphatase-1 can be identified by chromatography on DEAE-cellulose, namely protein phosphatase-1 itself and the Mg X ATP-dependent protein phosphatase. Both these species were eluted at 0.16 M NaCl just ahead of protein phosphatases-2C and 2A1. These enzymes did not interfere with measurements of type-2 protein phosphatases, since it was possible to block their activity with inhibitor-2...     

The dissociation of the extracellular hemoglobin of Tubifex tubifex at extremes of pH and its reassociation upon return to neutrality

The dissociation of the extracellular hemoglobin of Tubifex tubifex at alkaline and acid pH, and its reassociation upon return to neutral pH, was investigated using gel filtration, ultracentrifugation, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE). Tubifex hemoglobin dissociated at pH above 8 and below 6; both dissociations appeared to be equilibrium processes. The extent of dissociation increased as the pH moved away from neutrality; although dissociation was virtually complete at pH 11, its extent at acid pH did not exceed 50–60% at pH 4. Ca(II), Mg(II), and Sr(II) cations over the range 1–100 mm decreased the extent of the dissociation only at alkaline pH. The visible absorption spectrum of the oxyhemoglobin remained unaltered in the pH range 4–9. At more extreme pH, it changed with time, altering irreversibly to that of the aquo ferri form. Gel filtration of the hemoglobin at both extremes of pH showed that it dissociated into two heme-containing fragments; one consisting of subunit 1 (M_r ~ 17,000) and the other containing subunits 2, 3, and 4 of the hemoglobin (M_r ~ 60,000). Upon return to neutral pH, the dissociated fragment reassociated to the extent of 50 to 80% to whole hemoglobin molecules. The reassociation decreased with increase in alkaline pH, and with decrease in acid pH to which the hemoglobin had been exposed; it increased in the presence of Ca(II), Sr(II), and Mg(II) only subsequent to dissociation at alkaline pH. The SDS-PAGE patterns, gel-filtration elution volumes, and α-helical contents, determined from circular dichroism at 222 nm, of the reassociated whole molecules were identical to those of the native hemoglobin.     

The role of pH in the intracellular acetylcholine receptor ligand processing

Previous studies have shown that the internalized AChRs are transported through many vesicular compartments: Golgi associated vesicles, coated vesicles, smooth vesicles, endosome-like structures and lysosomes. These compartments have an acidic pH ranging from 4.5 to 6.5. The pH differences between organelles suggests that these differences may influence the sorting and final expression of AChRs. To test this hypothesis, we measured the number of counts of 125I-alpha BTX or 125I-Mab35 dissociated from myotube membranes containing AChRs as a function of pH. Neither the 125I-alpha BTX nor 125I-Mab35 showed an enhanced dissociation in the pH range 4.0-7.0, whereas lowering the pH to 6.0 or below enhanced the dissociation of 125I-alpha 2-macroglobulin from myotubes. In other experiments using Torpedo membrane we showed that neither 125I-alpha BTX nor 125I-Mab35 appreciably dissociated from the AChR unless the pH was less than 4 or above 11. Double-label studies using a novel membrane permeable acidotropic molecule DAMP (3-(2,4 nitroanilino) 3'amino-N-methyl-dipropylamine), facilitated mapping the pH of the intracellular compartments containing internalized AChRs. This molecule accumulates inside acidic compartments in the cell and has a dinitrophenol (DNP) group recognized by DNP specific antibodies. Cells were treated with 30 micrograms DAMP for 30 min and allowed to internalize Mab35-gold (15 nm) for various periods (0-15 h). At each time point we fixed and washed the cells, and incubated with anti-DNP monoclonal antibodies followed by incubation with anti-mouse IgG and protein A colloidal gold (5 nm). Different sized gold particles allowed us to simultaneously identify the AChR compartments and estimate their pH. Sister cultures were exposed to acidotropic drugs to destroy pH gradients. Under those conditions, AChR delivery to lysosomes was blocked. Our studies show that AChRs are transported through acidic compartments ranging from pH 4.5 to 6.5 and in contrast to other ligands they do not dissociate from the intracellular membranes at low pH.     

Unstructured model for free and immobilized cell culture without pH control of Bifidobacterium animalis subsp. lactis Bb 12—Inhibitory effect of the undissociated organic acids

A model was developed to describe growth and organic acids production of Bifidobacterium animalis growing without pH control in free and immobilized cell culture. The Verlhust model was considered for growth, and to account for the inhibition observed at acidic pH, the Luedeking–Piret production model was modified by introducing an additional term involving the undissociated form of the organic acids, acetic and lactic acids, the main inhibitory species. To describe the relationship between pH and both the dissociated and the undissociated forms of organic acids, the Henderson–Hasselbach equation was considered. The model was found to satisfactory describe experimental growth and production data recorded during free and immobilized cell cultures. The part of each acid produced can be deduced from the calculated production data, since a constant lactic to acetic acid mass ratio was found, 1.29 and 1.66 during free and immobilized cell cultures. Owing to the acidic pH values recorded, 4.43 at lowest, higher amounts of undissociated acetic acid were produced, leading to a higher inhibitory effect of this acid if compared to lactic acid.     

The dissociation of the extracellular hemoglobin of Lumbricus terrestris at acid pH and its reassociation at neutral pH. A new model of its quaternary structure

Lumbricus terrestris HbO2 and HbCO dissociated below pH 5.0; a time-dependent alteration to the met form occurred at pH less than 5 and pH less than 4.5, respectively. The extent of dissociation was unaffected by alkaline earth cations but was decreased by an increase in ionic strength. HbO2 and HbCO exposed to pH 4.0-4.8 were centrifuged to obtain the undissociated pellet (P1) and dissociated supernatant (S1) fractions. S1 was reassociated at pH 7.0 by dialysis against various buffers and then centrifuged to obtain the reassociated pellet (P2) and unreassociated supernatant (S2) fractions. Reassociation was possible only if S1 was dialyzed against water prior to return to neutral pH; otherwise precipitation occurred starting at about pH 5.3. The extent of reassociation varied from about 40 to 80%, was usually higher for HbCO than HbO2, and was unaffected by an increase in ionic strength or by Ca(II). Gel filtration of P2 on Sephacryl S-300 at neutral pH gave one peak IaR, eluting at a slightly greater volume than the native Hb; S1 and S2 gave in addition, three peaks, Ib (200 kDa), II (65 kDa), and III (18 kDa). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that P2 was slightly deficient in subunit M relative to the Hb, that Ib was deficient in subunits D1 and D2 and that II and III consisted of subunits D1 + D2 + T and subunit M, respectively. Scanning transmission electron microscopy of P2 showed that it was smaller than the native hemoglobin: 25 nm in diameter and 16 nm in height, instead of 30 X 20 nm. Comparison of the results of the dissociations of Lumbricus Hb at alkaline pH (Kapp, O. H., Polidori, G., Mainwaring, M., Crewe, A. V., Vinogradov, S. N. (1984) J. Biol. Chem. 259, 628-639) with those obtained in this study suggested that the Hb quaternary structure was not multimeric and that an alternative model had to be considered. In the proposed model it is assumed that subunits D1 and D2 form a scaffolding or "bracelet," decorated with 12 complexes of M and T subunits.