The pKa values of two histidine residues in human haemoglobin, the Bohr effect, and the dipole moments of alpha-helices

Studies of abnormal and chemically modified haemoglobins indicate that in 0.1 m-NaCl about 40% of the alkaline Bohr effect of human haemoglobin is contributed by the C-terminal histidine HC3(146)β. In deoxyhaemoglobin, the imidazole of this histidine forms a salt bridge with aspartate FG1(94)β, in oxyhaemoglobin or carbonmonoxyhaemoglobin it accepts a hydrogen bond from its own NH group instead. Kilmartin et al. (1973) showed that in 0.2 m-NaCl + 0.2 m-phosphate this change of ligation lowered the pK_a of the histidine from 8.0 in Hb³ to 7.1 in HbCO, but Russu et al. (1980) claimed that in bis-Tris buffer without added NaCl its pK_a in HbCO dropped no lower than 7.85, and that in this medium the C-terminal histidine made only a negligible contribution to the alkaline Bohr effect.We have compared the histidine resonances of HbCO A with those of three abnormal haemoglobins: HbCO Cowtown (His HC3(146)β → Leu), HbCO Wood (His FG4(97)β → Leu) and HbCO Malmø (His FG4(97)β → Gln). Our results show that the resonance assigned by Russu et al. to His HC3(146)β in fact belongs to His FG4(97)β. Although in Hb the pK_a of His HC3(146)β is 8.05 ± 0.05 independent of ionic strength, in HbCO its pK_a drops sharply with diminishing ionic strength, so that in the buffer employed by Russu et al. it has a pK_a of 6.2 and makes a contribution to the alkaline Bohr effect that is 57% larger than in the phosphate buffer employed by Kilmartin et al. (1973).In HbCO A, His FG4(97)β does not contribute to the Bohr effect, but in HbCO from which His HC3(146)β has been cleaved (HbCO des-His), His FG4(97)β is in equilibrium between two conformations with different pK_a values. This equilibrium varies with ionic strength and pH, and presumably also with degree of ligation of the haem moiety.In HbCO A, His FG4(97)β has a pK_a of 7.8 compared to the pK_a value of about 6.6 characteristic of free histidines at the surface of proteins. This high pK_a is accounted for by its interaction with the negative pole at the C terminus of helices F and FG. It corresponds to a free energy change of the same order as that observed in the interaction of histidines with carboxylate ions and confirms the strongly dipolar character of α-helices, which manifests itself even when they lie on the surface of the protein.     

Structure of deoxyhaemoglobin Yakima: a high-affinity mutant form exhibiting oxy-like 1 2 subunit interactions

Crystals of deoxyhaemoglobin Yakima (Asp Gl(99)β → His) are isomorphous with those of deoxyhaemoglobin A, even though the mutation produces disturbances in both the tertiary structure of the subunits and the quaternary structure of the tetramer. Asp Gl(99)β₂ lies at the α₁β₂ subunit interface, and in deoxyhaemoglobin A forms a crucial hydrogen bond with Tyr C7(42) α₁. The histidine residue that replaces the aspartate results in the removal of this single important intersubunit bond, and it further acts as a wedge between the α₁ and β₂ subunits, so that they are pushed apart and displaced part of the way towards the oxy structure. These disturbances are accompanied by the formation of a new intersubunit hydrogen bond, which is usually only observed in the oxy quaternary structure of haemoglobin. The disturbances at the α₁β₂ contact affect the stereochemistry of the entire molecule and are transmitted to the α and β haems. The X-ray structure of deoxy Yakima therefore provides a stereochemical explanation for its abnormal function; this being an abnormally high affinity for oxygen and vastly diminished haem-haem interactions.     

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.     

Ligand binding kinetics of des arginine haemoglobin

Des arginine 141 a haemoglobin (the haemoglobin in which the C-terminal arginine of the a chain has been removed) has a high affinity for oxygen and a reduced co-operativity in its oxygen equilibrium binding. The kinetic consequences of this modification are investigated in this paper. Deoxy des Arg haemoglobin binds carbon monoxide faster than does haemoglobin A, whilst oxy des Arg haemoglobin loses oxygen more slowly. These results are correlated with the oxygen equilibrium binding properties of des Arg haemoglobin. The carbon monoxide binding kinetics have been interpreted as implying a change in the parameter c (of the allosteric model), as well as L, when this arginine is removed from haemoglobin.     

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.     

Histidine proton resonances of carbonmonoxyhaemoglobins A and Cowtown in chloride-free buffer

A re-examination of the C-2 histidine proton resonances of haemoglobins A and Cowtown (His HC3(146)β → Leu) in chloride-free Hepes buffer has shown that all the resonances present in haemoglobin A are present in haemoglobin Cowtown, so that the pK_a of His HC3(146)β cannot be determined by nuclear magnetic resonance in this buffer.     

His‐tag binding by antibody C706 mimics β‐amyloid recognition

Alzheimer's disease is a progressive neurodegenerative disease characterized by extracellular deposits of β‐amyloid (Aβ) plaques. Aggregation of the Aβ₄₂ peptide leading to plaque formation is believed to play a central role in Alzheimer's disease pathogenesis. Anti‐Aβ monoclonal antibodies can reduce amyloid plaques and could possibly be used for immunotherapy. We have developed a monoclonal antibody C706, which recognizes the human Aβ peptide. Here we report the crystal structure of the antibody Fab fragment at 1.7 Å resolution. The structure was determined in two crystal forms, P2₁ and C2. Although the Fab was crystallized in the presence of Aβ₁₆, no peptide was observed in the crystals. The antigen‐binding site is blocked by the hexahistidine tag of another Fab molecule in both crystal forms. The poly‐His peptide in an extended conformation occupies a crevice between the light and heavy chains of the variable domain. Two consecutive histidines (His4–His5) stack against tryptophan residues in the central pocket of the antigen‐binding surface. In addition, they form hydrogen bonds to the acidic residues at the bottom of the pocket. The mode of his‐tag binding by C706 resembles the Aβ recognition by antibodies PFA1 and WO2. All three antibodies recognize the same immunodominant B‐cell epitope of Aβ. By similarity, residues Phe–Arg–His of Aβ would be a major portion of the C706 epitope. Copyright © 2010 John Wiley & Sons, Ltd.     

Haemoglobin Hiroshima and the Mechanism of the Alkaline Bohr Effect

HAEMOGLOBIN Hiroshima is a variant with interesting physiological properties^1,2 discovered in a Japanese family. Its Bohr effect is halved, its oxygen affinity at physiological pH increased about three-fold and haem-haem interaction is somewhat reduced compared with normal haemoglobin. In 0.1 M NaCl solutions initially stripped of phosphate, 2,3-diphospho-glycerate (2,3-DPG) diminishes the oxygen affinity as in haemoglobin A (H. F. Bunn, unpublished results). The amino-acid substitution originally deduced for this abnormal haemoglobin was histidine 143 (H21)β ? aspartic acid¹. It was possible to conceive of a mechanism which accounted for its diminished Bohr effect³, but the normal response of its oxygen affinity to 2,3-DPG was inconsistent with the proposed role of histidine 143 in 2,3-DPG binding by haemoglobin A^4,5. An X-ray crystallographic study of deoxyhaemoglobin Hiroshima has now revealed that the replacement occurs not in position 143 but 146β. This was confirmed by chemical methods and the physiological properties of this haemoglobin are now satisfactorily accounted for. The results support the role of histidine 146β in the alkaline Bohr effect⁶.     

Arginine: Its pKa value revisited

Using complementary approaches of potentiometry and NMR spectroscopy, we have determined that the equilibrium acid dissociation constant (pK_a value) of the arginine guanidinium group is 13.8 ± 0.1. This is substantially higher than that of ∼12 often used in structure-based electrostatics calculations and cited in biochemistry textbooks. The revised intrinsic pK_a value helps explains why arginine side chains in proteins are always predominantly charged, even at pH values as great as 10. The high pK_a value also reinforces the observation that arginine side chains are invariably protonated under physiological conditions of near neutral pH. This occurs even when the guanidinium moiety is buried in a hydrophobic micro-environment, such as that inside a protein or a lipid membrane, thought to be incompatible with the presence of a charged group.     

Structure-function relationships in the low-affinity mutant haemoglobin Aalborg (Gly74 (E18)beta----Arg).

Haemoglobin Aalborg (Gly74 (E18)beta----Arg) has a reduced oxygen affinity, in both the absence and the presence of organic phosphates; it has a raised affinity for organic phosphates, and it is moderately unstable. By contrast, haemoglobin Shepherds Bush (Gly74 (E18)beta----Asp) has an increased oxygen affinity in both the absence and the presence of organic phosphates, a diminished affinity for organic phosphates and is also unstable. We have determined the crystal structure of deoxyhaemoglobin Aalborg at 2.8 A resolution and compared it to the structures of deoxy- and oxyhaemoglobin A and of deoxyhaemoglobin Shepherds Bush. The guanidinium group of Arg74(E18)beta protrudes from the haem pocket and donates hydrogen bonds to the E and F helices. The carboxylate group of Asp74(E18)beta forms a hydrogen bond only with residue EF6 and is partially buried, which may be why haemoglobin Shepherds Bush appears to be more unstable than haemoglobin Aalborg. To discover why the latter has a low oxygen affinity, we superimposed the B, G and H helices of haemoglobin A, whose conformation is known to be unaffected by ligand binding, on those of haemoglobin Aalborg. This also brought helices E and the haems into superposition, but revealed a shift of the F helix of deoxyhaemoglobin Aalborg towards the EF-corner. This shift is opposite to that which occurs on ligand binding and on transition to the quaternary oxy-structure, and is linked to an increased tilt of the proximal histidine residue away from the haem axis. Since the relative positions of helices E and F and of the haem group are thought to be the main determinants of the changes in oxygen affinity, the shift of helix F may account for the reduced oxygen affinity of haemoglobin Aalborg. The shift may be due to a combination of steric and electrostatic effects introduced by the arginine residue's side-chain. The effects of the arginine and aspartate substitutions at position E18 beta on the 2,3-diphosphoglycerate affinity are equal and opposite. They can be quantitatively accounted for by the electrostatic attraction or repulsion by the oppositely charged side-chains.     

Human and rodent amyloid-β peptides differentially bind heme: Relevance to the human susceptibility to Alzheimer’s disease

Amyloid-β (Aβ) peptides are implicated in the neurodegeneration of Alzheimer’s disease (AD). We previously investigated the mechanism of neurotoxicity of Aβ and found that human Aβ (huAβ) binds and depletes heme, forming an Aβ-heme complex with peroxidase activity. Rodent Aβ (roAβ) is identical to huAβ, except for three amino acids within the proposed heme-binding motif (Site-H). We studied and compared heme-binding between roAβ and huAβ. Unlike roAβ, huAβ binds heme tightly (K_d = 140 ± 60 nM) and forms a peroxidase. The plot of bound (huAβ-heme) vs. unbound heme fits best to a two site binding hyperbola, suggesting huAβ possesses two heme-binding sites. Consistently, a second high affinity heme-binding site was identified in the lipophilic region (site-L) of huAβ (K_d = 210 ± 80nM). The plot of (roAβ-heme) vs. unbound heme, on the other hand, was different as it fits best to a sigmoidal binding curve, indicating different binding and lower affinity of roAβ for heme (K_d = 1 μM). The effect of heme-binding to site-H on heme-binding to site-L in roAβ and huAβ is discussed. While both roAβ and huAβ form aggregates equally, rodents lack AD-like neuropathology. High huAβ/heme ratio increases the peroxidase activity. These findings suggest that depletion of regulatory heme and formation of Aβ-heme peroxidase contribute to huAβ’s neurotoxicity in the early stages of AD. Phylogenic variations in the amino acid sequence of Aβ explain tight heme-binding to huAβ and likely contribute to the increased human susceptibility to AD.     

Effect of feeding complete diets containing graded levels of Prosopis cineraria leaves on feed intake,nutrient utilization and rumen fermentation in lambs and kids

A study was conducted to assess the effect of feeding graded levels of tannin-containing Prosopis cineraria leaves in a complete feed mixture (CFM) on the performance of lambs and kids. Eighteen lambs and 18 kids of Malpura and Marwari breed, respectively of similar age (90 days) and body weight (11.0 kg), were randomly distributed in to three groups of six each. Each group was offered complete feed mixtures ad libitum in feeding troughs under group feeding system for 90 days. The concentrate component of CFM was from the commercial feed in mash form containing corn, soybean meal, wheat bran and de-oiled rice bran. Prosopis leaves were ground to pass through a 4 mm sieve before being thoroughly mixed with required quantity of concentrate mash. CFMs contained graded levels of P. cineraria leaves. In, CFM-1, ratio of P. cineraria to concentrate mixture was 25: 75 (T₁), whereas in CFM-2 and CFM-3, it was 50:50 (T₂) and 75:25 (T₃), respectively. The lambs in L₁, L₂ and L₃ and kids in K₁, K₂ and K₃, groups received CFM-1, CFM-2 and CFM-3, respectively. CP (g kg⁻¹) was 182 in CFM-1, 162 in CFM-2 and 140 in CFM-3. P. cineraria leaves contained (g kg⁻¹) CP 159, neutral detergent fibre (NDF) 567, acid detergent fibre (ADF) 360 and acid detergent lignin (ADL) 189 on dry matter (DM) basis. The extractable condensed tannin (CT; leucocyanidin equivalent), hydrolysable tannin (HT) and protein precipitation capacity (PPC) of the leaves (g kg⁻¹) were 90.7, 3.4 and 111.5, respectively. DMI in L₁ and L₂ lambs were higher than their corresponding groups in kids, whereas it was reverse in L₃ (more in kids than lambs). Significant difference in digestible crude protein (DCP) intake was recorded among the groups in both lambs and kids. In lambs, highest DCPI (g day⁻¹) was recorded in L₁ (93.3), as compared to L₂ (82.2) and L₃ (37.2), whereas in kids it was K₂ (73.4), followed by K₁ (69.2) and K₃ (43.8). Significant difference in the digestibility of DM, CP and NDF were recorded among three groups in both species. Maximum nutrient digestibility was recorded in T₁ followed by T2 and T₃ in both lambs and kids. However, digestibility of all the nutrients was more in kids than that of lambs. Maximum ammonia-N, total N and trichloro acetic acid (TCA) precipitable N was recorded in T₁ followed by T₂ and T₃ in both the species. However, there was no typical trend between the two species in rumen parameters. Blood haemoglobin (Hb) and blood urea nitrogen (BUN) values were highest in T₁ followed by T₂ and T₃ in both lambs and kids. After 90 days of intensive feeding, maximum weight gain was recorded in T₂ as compared to that of T₁ and T₃ in both lambs and kids. Although L₁ lambs performed better under high concentrate diet compared to K₁ kids, weight gain in K₂ and K₃ kids were significantly higher compared to their counter part lambs. Similar was the trend in ADG also. It was concluded that performance of lambs and kids differed with the level of CT in their diet and kids performed better on high tannin diet as compared to that of lambs.     

Arginine Metabolising Enzymes as Therapeutic Tools for Alzheimer’s Disease: Peptidyl Arginine Deiminase Catalyses Fibrillogenesis of β-amyloid Peptides

The accumulation of arginine in the cerebrospinal fluid and brains of patients suffering from acute neurodegenerative diseases like Alzheimer’s disease, point to defects in the metabolic pathways involving this amino acids. The deposits of neurofibrillary tangles and senile plaques perhaps as a consequence of fibrillogenesis of β-amyloid peptides has also been shown to be a hallmark in the aetiology of certain neurodegenerative diseases. Peptidylarginine deiminase (PAD II) is an enzyme that uses arginine as a substrate and we now show that PAD II not only binds with the peptides Aβ_1-40, Aβ_22-35, Aβ_17-28, Aβ_25-35 and Aβ_32-35 but assists in the proteolytic degradation of these peptides with the concomitant formation of insoluble fibrils. PAD was purified in 12.5% yield and 137 fold with a specific activity of 59 μmol min^?1?mg^?1 from bovine brain by chromatography on diethylaminoethyl (DEAE)-Sephacel. Characterisation of the enzyme gave a pH and temperature optima of 7.5°C and 68°C, respectively, and the enzyme lost 50% activity within 38 min at this temperature. Michaelis-Menten kinetics established a V _max and K _m of 1.57 μmol min^?1?ml^?1 and 1.35 mM, respectively, with N-benzoyl arginine ethyl ester as substrate. Kinetic analysis was used to measure the affinity (K _i) of the amyloid peptides to PAD with values between 1.4 and 4.6 μM. The formation of Aβ fibrils was rate limiting involving an initial lag time of about 24 h that was dependent on the concentration of the amyloid peptides. Turbidity measurements at 400 nm, Congo Red assay and Thioflavin-T staining fluorescence were used to establish the aggregation kinetics of PAD-induced fibril formation.     

Chemical Rescue and Inhibition Studies to Determine the Role of Arg301 in Phosphite Dehydrogenase

Phosphite dehydrogenase (PTDH) catalyzes the NAD⁺-dependent oxidation of phosphite to phosphate. This reaction requires the deprotonation of a water nucleophile for attack on phosphite. A crystal structure was recently solved that identified Arg301 as a potential base given its proximity and orientation to the substrates and a water molecule within the active site. Mutants of this residue showed its importance for efficient catalysis, with about a 100-fold loss in k _cat and substantially increased K _m,phosphite for the Ala mutant (R301A). The 2.35 Å resolution crystal structure of the R301A mutant with NAD⁺ bound shows that removal of the guanidine group renders the active site solvent exposed, suggesting the possibility of chemical rescue of activity. We show that the catalytic activity of this mutant is restored to near wild-type levels by the addition of exogenous guanidinium analogues; Brønsted analysis of the rates of chemical rescue suggests that protonation of the rescue reagent is complete in the transition state of the rate-limiting step. Kinetic isotope effects on the reaction in the presence of rescue agents show that hydride transfer remains at least partially rate-limiting, and inhibition experiments show that K _i of sulfite with R301A is ∼400-fold increased compared to the parent enzyme, similar to the increase in K _m for phosphite in this mutant. The results of our experiments indicate that Arg301 plays an important role in phosphite binding as well as catalysis, but that it is not likely to act as an active site base.     

The pH dependence of the affinity,kinetics and cooperativity of ligand binding to the root effect haemoglobin of the goldfish Carassius auratus

• 1.1. The binding of O₂ to goldfish haemoglobin showed a strong pH dependence P₅₀=5.5 mmHg; n = 2.4 at pH 8.0 and P₅₀ = 170 mmHg; n = 1.0 at pH 5.5 such that the protein is only 50% saturated in a solution of air equilibrated buffer at pH 5.5.
• 2.2. The binding of CO is cooperative at high pH (n = 2.8; L = 1000; K_R = 0.1 μM; K_T = 4 μM) and non-cooperative (n = 1) at pH 5.5.
• 3.3. The rate of O₂ dissociation is extremely fast and pH dependent; being 30 sec⁻¹ at pH 8.0 and 400 sec⁻¹ at pH 6.0 at 1°C. At 23°C the rate of this process is too fast to obtain accurate data using stopped-flow techniques.
• 4.4. Partial photolysis of the oxyhaemoglobin species leads to homogeneous recombination kinetics at pH 8.0 with an associated rate constant of 4.7 × 10⁷ M⁻¹ sec⁻¹. At pH < 7.5 the recombination process occurs in two steps. One rate is equal to that observed at pH 8.0. The slower process is favoured at low pH.
• 5.5. Photolysis of the CO haemoglobin complex indicates that, at high pH, combination of CO with deoxyhaemoglobin is cooperative, whilst recombination with Hb(CO)₃ is non-cooperative and occurs at a rate of 1.2 × 10⁶ M⁻¹ sec⁻¹.
• 6.6. At neutral pH recombination of CO with partially linganded haemoglobin occurs in a two-step process. The proportion contributed by each of these two steps in pH dependent.
• 7.7. The functioning of this Root effect haemoglobin is discussed in terms of the two state-model of cooperativity in which the αβ chain heterogeneity is minimal

     

Decay of individual Escherichia coli trp messenger RNA molecules is sequentially ordered.

Human fluoromethaemoglobin with inositol hexaphosphate (IHP) in 0.05 m-phosphate buffer was crystallized by addition of polyethylene glycol (PEG). The crystals are isomorphous with those of deoxyhaemoglobin A without IHP grown in solutions containing PEG by Ward et al. (1975). The structure was investigated by means of a difference Fourier synthesis against deoxyhaemoglobin A based on X-ray data collected within a limiting sphere of 3.5 Å^?1. The four subunits are arranged in the quaternary T structure and IHP is bound at the same site between the β chains as in deoxyhaemoglobin. In both the α and β haem regions the distance between the haem plane and the F helix is reduced in fluoromethaemoglobin relative to deoxyhaemoglobin and the iron atom is moved from the proximal towards the distal side of the plane, but the change, if any, in the distance between the iron and the N_ε of the proximal histidine cannot be clearly established. The α Fe in fluoromethaemoglobin is either in the haem plane or up to 0.8 Å on the distal side, suggesting the possibility of rupture of the bond to the histidines N_ε; it was not possible to estimate the position of the β iron. The main spectral changes associated with the reaction of fluoromethaemoglobin with IHP take place in less than 3 ms at room temperature.     

Promoting α‐secretase cleavage of beta‐amyloid with engineered proteolytic antibody fragments

Deposition of beta‐amyloid (Aβ) is considered as an important early event in the pathogenesis of Alzheimer's Disease (AD), and reduction of Aβ levels by various therapeutic approaches is actively being pursued. A potentially non‐inflammatory approach to facilitate clearance and reduce toxicity is to hydrolyze Aβ at its α‐secretase site. We have previously identified a light chain fragment, mk18, with α‐secretase‐like catalytic activity, producing the 1–16 and 17–40 amino acid fragments of Aβ40 as primary products, although hydrolysis is also observed following other lysine and arginine residues. To improve the specific activity of the recombinant antibody by affinity maturation, we constructed a single chain variable fragment (scFv) library containing a randomized CDR3 heavy chain region. A biotinylated covalently reactive analog mimicking α‐secretase site cleavage was synthesized, immobilized on streptavidin beads, and used to select yeast surface expressed scFvs with increased specificity for Aβ. After two rounds of selection against the analog, yeast cells were individually screened for proteolytic activity towards an internally quenched fluorogenic substrate that contains the α‐secretase site of Aβ. From 750 clones screened, the two clones with the highest increase in proteolytic activity compared to the parent mk18 were selected for further study. Kinetic analyses using purified soluble scFvs showed a 3‐ and 6‐fold increase in catalytic activity (k_cat/K_M) toward the synthetic Aβ substrate compared to the original scFv primarily due to an expected decrease in K_M rather than an increase in k_cat. This affinity maturation strategy can be used to select for scFvs with increased catalytic specificity for Aβ. These proteolytic scFvs have potential therapeutic applications for AD by decreasing soluble Aβ levels in vivo. © 2009 American Institute of Chemical Engineers. Biotechnol. Prog., 2009     

Determination of unfrozen water in winter cereals at subfreezing temperatures

The freezing of water in acclimated and nonacclimated cereals was studied using pulsed nuclear magnetic resonance spectroscopy. The quantity of unfreezable water per unit dry matter was not strongly dependent on the degree of cold acclimation. In contrast, the fraction of water frozen which was tolerated by nonacclimated winter cereals and by an acclimated spring wheat (Triticum aestivum L.) was less than in acclimated hardy cereals. The freezing curves had the following form:L_T = L₀ΔTm/T + KL_T and L₀ are liquid water per unit dry matter at T and 0 C, respectively. ΔTm is the melting point depression and K is the liquid water which does not freeze.     

Enhancement of a bacterial laccase thermostability through directed mutagenesis of a surface loop

Laccases (benzenediol oxygen oxidoreductases, EC 1.10.3.2) are used in many biotechnological processes, including removal of polyphenols in beverages, decolorizing and detoxifying effluents, drug analysis and bioremediation. In the present work, we have tried to increase thermal stability of laccase from Bacillus HR03 using site directed point mutations. Glu188 was substituted with 2 positive (Lys and Arg) and one hydrophobic (Ala) residues. All mutations showed improved thermal stability. Thermal activation of laccase was also increased after introducing the mutations. Remarkably, the Glu188Lys variant showed 3-fold higher thermal activation and higher T₅₀ (5 °C) with respect to the native enzyme. Furthermore steady-state k_cat and K_m values were influenced despite the distance between the mutated position and the catalytic site. In Glu188Arg mutation, the k_cat was improved 3-fold and K_m reduced by 25%. Interestingly, all three variants showed higher stability against urea as a chemical denaturant. Structural analyses of the native and mutated variants were carried out using fluorescence and far-UV circular dichroism.     

Graphic method for determination of allosteric constants

The maximum slope of the plot, appearing in the paper of Watari & Isogai (1976), was derived algebraically as a function of allosteric constants c and α_mor β_m (= cα_m), and the relation between L, c, and α_mor β_m, was also obtained, where $\text{L =}\text{T}{}_{\mathrm{o}}\text{R}{}_{\mathrm{o}}\text{, c =}\text{K}{}_{\mathrm{R}}\text{K}{}_{\mathrm{T}}\text{, α}{}_{\mathrm{m}}\text{=}\text{F}{}_{\mathrm{m}}\text{K}{}_{\mathrm{R}}\text{, β}{}_{\mathrm{m}}\text{=}\text{F}{}_{\mathrm{m}}\text{K}{}_{\mathrm{T}}\text{, R}{}_{\mathrm{o}}\text{and}\text{T}{}_{\mathrm{o}}$ are concentrations of unligated R and T states respectively, K_Rand K_T are microscopic dissociation constants, and F_m is the ligand concentration at the maximum slope of the plot. When the maximum slope is increased by one, the value becomes Hill constant, n. Nomographs which enable easier estimation of allosteric constants, L and c, were constructed from the two given values, the maximum slope of the plot, n ? 1, and α_mor β_m, in the cases where the maximum number of ligands, N, was 2 and 4. In the nomograph, log c is plotted against log L²c^N keeping the value of the maximum slope of the plot and that of α_mor β_m constant. These nomographs show that the representation is symmetrical in the cases of L²c^N > 1 and L²c^N < 1.