Interaction of chicken cystatin with inactivated papains.

Papain which was inactivated by covalent attachment of small substituents to the active-site cysteine, up to the size of a carbamoylmethyl group, bound with high affinity to chicken cystatin (Kd less than approximately 15 pM), although less tightly than did active papain (Kd approximately 60 fM). However, as the size of the substituent was increased further, the affinity decreased appreciably, generally in proportion to the size of the inactivating group. For instance the dissociation constants for papain inactivated with N-ethylmaleimide and [N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-amido-(4-guanido )butane were 0.17 and approximately 10 microM respectively. The spectroscopic changes accompanying the reaction of all but the most weakly binding (Kd greater than or equal to 2 microM) inactivated papains with cystatin were similar to those induced by the active enzyme. Interactions involving the reactive cysteine residue of papain are thus not crucial for high-affinity binding of the enzyme to cystatin, in accordance with a recently proposed model for the enzyme-inhibitor complex, based on computer docking experiments. In this model there is sufficient space around the reactive cysteine in the complex for a small inactivating group, explaining the tight binding of papains with such substituents. However, larger inactivating groups cannot be accommodated in this space and therefore must displace the inhibitor out of the tight fit with the enzyme, in agreement with the observed decrease in binding affinity with increasing size of bulkier substituents. The kinetics of binding of cystatin to inactivated papains were compatible with simple, reversible, bimolecular reactions, having association rate constants of (7-9) x 10(6) M-1 s-1 at pH 7.4, 25 degrees C, similar to what was shown previously for the binding of cystatin to active papain. The rate of association of the inhibitor with either active or inactivated papain thus appears to be primarily diffusion-controlled. The decreasing affinity of cystatin for papains inactivated with groups of increasing size was shown to be due to progressively higher dissociation rate constants, consistent with the greater impairment of fit between the binding regions of the two molecules.     

Purification and Characterization of High Molecular Mass and Low Molecular Mass Cystatin from Goat Brain

Cystatin are thiol proteinase inhibitors ubiquitously present in mammalian body and serve various important physiological functions. In the present study two cystatins were isolated from goat brain using alkaline treatment, ammonium sulphate fractionation, gel filtration and ion exchange chromatography. The high molecular mass cystatin of 70.8 kDa was named as HM-GBC (high molecular mass goat brain cystatin) and the low molecular mass cystatin of 12.72 kDa was named as LM-GBC (low molecular mass goat brain cystatin). The molecular mass determined by SDS-PAGE was found to be 70.8 and 12.88 kDa for HM-GBC and LM-GBC, respectively, however with gel filtration the masses were found to be 70.8 and 12.58 kDa. Both the cystatins were found to be stable in broad range of pH and temperature. HM-GBC was found to have 2% carbohydrate content while LM-GBC lacks any carbohydrate content. Both cystatins were found to be devoid of any sulphydryl content. Stoke's radii of 36 and 16 A, and diffusion coefficient of 6.189 x 10(-15) and 1.392 x 10(-14) cm(2)/s were calculated for HM-GBC and LM-GBC. K (i) values with papain were found to be 1.875 x 10(-8) and 3.125 x 10(-8) M for HM-GBC and LM-GBC, respectively. K (+1), K (-1) and half-life calculated along with K (i) values obtained showed that HM-GBC inhibited papain more specifically as compared to LM-GBC. The IC(50) values obtained for HM-GBC and LM-GBC also showed that HM-GBC binds more effectively to papain than LM-GBC. Ultraviolet and fluorescence spectra indicated that upon formation of papain-HM-GBC/LM-GBC complex there is significant conformational change after interaction in one or both the proteins of the complex.     

Inhibition of cysteine proteinases and dipeptidyl peptidase I by egg-white cystatin.

The interactions between egg-white cystatin and the cysteine proteinases papain, human cathepsin B and bovine dipeptidyl peptidase I were studied. Cystatin was shown to be a competitive reversible inhibitor of cathepsin B (Ki 1.7 nM, k-1 about 2.3 X 10(-3) s-1). The inhibition of dipeptidyl peptidase I was shown to be reversible (Ki(app.) 0.22 nM, k-1 about 2.2 X 10(-3) s-1). Cystatin bound papain too tightly for Ki to be determined, but an upper limit of 5 pM was estimated. The association was a second-order process, with k+1 1.0 X 10(7) M-1 X s-1. Papain was shown to form equimolar complexes with cystatin. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of complexes formed between papain or cathepsin B and an excess of cystatin showed no peptide bond cleavage after incubation for 72 h. The reaction of the active-site thiol group of papain with 5,5'-dithiobis-(2-nitrobenzoic acid) at pH 8 and 2,2'-dithiobispyridine at pH 4 was blocked by complex-formation. Dipeptidyl peptidase I and papain were found to compete for binding to cystatin, contrary to a previous report. The two major isoelectric forms of cystatin were found to have similar specific inhibitory activities for papain, and similar affinities for papain, cathepsin B and dipeptidyl peptidase I. This, together with specific oxidation of the N-terminal serine residue with periodate, showed the N-terminal amino group of cystatin 1 to be unimportant for inhibition. General citraconylation of amino groups resulted in a large decrease in the affinity of cystatin for dipeptidyl peptidase I. It is concluded that the interaction of cystatin with cysteine proteinases has many characteristics similar to those of an inhibitor such as aprotinin with serine proteinases.     

Studies on the interaction of papain with human placental cystatin by UV, fluorescence and CD spectroscopy

The interaction of activated papain with low molecular weight cystatin (Mr 12500) purified from human placenta has been studied. Analysis of inhibition of caesinolytic activity of papain by cystatin showed stoichiometry of 1:1. Kinetic studies gave an inhibition constant (K(i)) value of 5.5 x 10(-8) M and association rate constant (K(+1)) value of 3.4 x 10(4) (M(-1) s(-1)). All spectroscopic studies showed conformational changes in both papain and cystatin on formation of complex. The data suggest perturbation of environment of aromatic residues and change of their native structure and conformation thereby shedding light on the behaviour of cystatins, especially interaction of placental cystatin with thiol protease inhibitors.     

Two distinct cystatin species in rice seeds with different specificities against cysteine proteinases. Molecular cloning, expression, and biochemical studies on oryzacystatin-II.

Oryzacystatin (oryzacystatin-I) is a proteinaceous cysteine proteinase inhibitor (cystatin) in rice seeds and is the first well defined cystatin of plant origin. In this study we isolated cDNA clones for a new type of cystatin (oryzacystatin-II) in rice seeds by screening with the oryzacystatin-I cDNA probe. The newly isolated cDNA clone encodes 107 amino acid residues whose sequence is similar to that of oryzacystatin-I (approximately 55% of identity). These oryzacystatins have no disulfide bonds, and so could be classified as family-I cystatins; however, the amino acid sequences resemble those of family-II members more than family-I members. Oryzacystatin-I and -II are remarkably distinct in two respects: 1) their specificities against cysteine proteinases; and 2) the expression patterns of their mRNAs in the ripening stage of rice seeds. Oryzacystatin-I inhibits papain more effectively (Ki 3.0 x 10(-8) M) than cathepsin H (Ki 0.79 x 10(-6) M), while oryzacystatin-II inhibits cathepsin H (Ki 1.0 x 10(-8) M) better than papain (Ki 0.83 x 10(-6) M). The mRNA for oryzacystatin-I is expressed maximally at 2 weeks after flowering and is not detected in mature seeds, whereas the mRNA for oryzacystatin-II is constantly expressed throughout the maturation stages and is clearly detected in mature seeds. Western blotting analysis using antibody to oryzacystatin-II showed that, as is the case with oryzacystatin-I, oryzacystatin-II occurs in mature rice seeds. Thus, these two oryzacystatin species are believed to be involved in the regulation of proteolysis caused by different proteinases.     

Kinetics of binding of chicken cystatin to papain

The kinetics of binding of chicken cystatin to papain were studied by stopped-flow fluorometry under pseudo-first-order conditions, i.e., with an excess of inhibitor. All reactions showed first-order behavior, and the observed pseudo-first-order rate constant increased linearly with the cystatin concentration up to the highest concentration that could be studied, 35 microM. The analyses thus provided no evidence for a limiting rate resulting from a conformational change stabilizing an initial encounter complex, in contrast with previous studies of reactions between serine proteinases and their protein inhibitors. The second-order association rate constant for complex formation was 9.9 X 10(6) M-1 s-1 at 25 degrees C, pH 7.4, I = 0.15, for both forms of cystatin, 1 and 2. This value approaches that expected for a diffusion-controlled rate. The temperature dependence of the association rate constant gave an enthalpy of activation at 25 degrees C of 31.5 kJ mol-1 and an entropy of activation at 25 degrees C of -7 J K-1 mol-1, compatible with no appreciable conformational change during the reaction. The association rate constant was independent of pH between pH 6 and 8 but decreased at lower and higher pH in a manner consistent with involvement of an unprotonated acid group with a pKa of 4-4.5 and a protonated basic group with a pKa of 9-9.5 in the interaction. The association rate constant was unaffected by ionic strengths between 0.15 and 1.0 but decreased somewhat at lower ionic strengths. Incubation of the complex between cystatin 2 and papain with an excess of cystatin 1 resulted in slow displacement of cystatin 2 from the complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectroscopic studies on the interaction of bilirubin with liver cystatin

Studies on the role of endogenous metabolites such as bilirubin and their interactions with biomolecules have attracted considerable attention over the past several years. In this work, the interaction of bilirubin (BR) with purified goat liver cystatin (LC) was studied using fluorescence and ultraviolet (UV) spectroscopy. The fluorescence data proved that the fluorescence quenching of liver cystatin by BR was the result of BR–cystatin complex formation. Stern–Volmer analysis of fluorescence quenching data showed the binding constant to be 9.27 × 10⁴ M⁻¹ and the number of binding sites to be close to unity. The conformation of the BR–cystatin complex was found to change upon varying the pH of the complex. The BR–cystatin complex was found to have reduced papain inhibitory activity. Photo-illumination of BR–cystatin complex causes perturbation in the micro-environment of goat liver cystatin as indicated by red-shift. This report summarizes our research efforts to reveal the mechanism of interaction of bilirubin with liver cystatin.     

The haemocyanin of the whelk, Busycon contrarium (Conrad): aggregation states and subunit structure

1. The haemocyanin of the left-handed whelk Busycon contrarium (Conrad) exists largely as six or more multi-decameric aggregates characterized by sedimentation coefficients of approximately 105S, 132S, 155S, 170S, 185S and about 200-220S. 2. These aggregates represent di- to hepta- or octa-decameric assemblies of the basic haemocyanin decamer having a mol. wt of 4.3 x 10(6)-4.5 x 10(6). 3. The fully dissociated subunits in 8.0 M urea (pH 8.5) and at pH 11.1, 0.01 M EDTA have mol. wts of 4.78 x 10(5) and 4.62 x 10(5), close to one-tenth of the mol. wt of the basic decameric unit of most gastropod haemocyanins. 4. The pH dependence of the mol. wts (Mw), studied by light-scattering at the constant protein concentration of 0.010%, exhibit bell-shaped pH transition profiles with mol. wt values of about 16 x 10(6) in the presence of 0.01 M Mg2+, in the pH region from about pH 4.5-8.0; in the absence of stabilizing divalent ions the observed mol. wt is about 10 x 10(6) at pH 4.5-7.0. Below pH 4.5 and above 7.0-8.0 there is a sharp drop in mol. wt to about 4 x 10(5)-4.5 x 10(5). 5. The transition profiles observed with both the urea and salt series of probes investigated at concentration = 0.010% are found to produce aggregation at low reagent concentrations with mol. wt changes from about 9 x 10(6)-12 x 10(6)-14 x 10(6), followed by a decrease in mol. wt below 4.3 x 10(6)-4.5 x 10(6) of the haemocyanin decamers.(ABSTRACT TRUNCATED AT 250 WORDS)     

cDNA cloning, identification and characterization of a novel cystatin from the tentacle of Cyanea capillata

Cystatin is of interest from biochemical and evolutionary prospective, and also has been applied in biotechnology. In this paper, a novel cystatin was found by EST sequence analysis of the cDNA library of Cyanea capillata tentacle. The sequence of a full-length cDNA clone contained an open reading frame encoding a putative 18-residue signal peptide and a mature protein of 113 amino acids, which showed only 26% identities to Family 2 cystatins and had its own characteristic enzyme-binding motifs, Ser(97)-Trp(98), which had not been found in any other known cystatins. Thus, the novel cystatin cloned from jellyfish was designated as cystatin J, which may belong to a new family of cystatin, called Family 4. The mature cystatin J was produced in Escherichia coli as a thioredoxin (Trx) fusion protein using the pET expression system and purified by affinity and cation exchange chromatography. The recombinant cystatin J of approximately M(r) = 12,800 displayed an obvious inhibition of papain (K(i) value below 0.5 nM), in competition with substrate. Thus, the recombinant cystatin J was a functional cystatin in spite of relatively lower sequence similarity with other cystatins. Activity of the novel cystatin was stable at pH 4-11 at 4 degrees C, but unstable at neutral pH at >50 degrees C.     

Light-scattering and scanning transmission electron microscopic investigation of the hemocyanin of the bivalve, Yoldia limatula (Say)

1. The hemocyanin of the bivalve, Yoldia limatula (Say) was found by light-scattering to have a mol. wt of 8.0 +/- 0.6 x 10(6). Mass measurements by scanning transmission electron microscopy (STEM) gave a particle mass of 8.25 +/- 0.42 x 10(6) for the native particle and 4.09 +/- 0.20 x 10(6) for the half-molecule. 2. The hemocyanin subunits fully dissociated in 8.0 M urea and 6.0 M GdmCl at pH 8.0, and at pH 11.0, 0.01 M EDTA have mol. wts of 4.38 x 10(5), 4.22 x 10(5) and 4.71 x 10(5), close to one-twentieth of the parent molecular weight of Y. limatula hemocyanin and most gastropod hemocyanins. 3. Analyses of the urea dissociation transitions studied at pH 8.0, 1 x 10(-2) M Mg2+, 1 x 10(-2) M Ca2+ and pH 8.0, 3 x 10(-3) M Ca2+ suggest few hydrophobic amino acid groups, of the order of 10 to 15 at the contact areas of each half-molecule or decamer. 4. The further dissociation of the decamers to dimers and the dimers to monomers indicates the presence of a larger number of amino acid groups of ca 35-40/dimer and 100-120/monomer. 5. This suggests hydrophobic stabilization of the dimer to dimer and monomer to monomer contacts within the decamers, as observed with other molluscan hemocyanins.     

Preparation of analogues of ATP, ADP and AMP suitable for binding to matrices and the enzymic interconversion of ATP and ADP in solid phase.

Alkylation of ATP with iodoacetic acid at pH 6.5 yielded 1-carboxymethyl-ATP which, after alkaline rearrangement, gave N-6-carboxymethyl-ATP. Condensation of this analogue with 1,6-diaminohexane in the presence of a water-soluble carbodiimide generated N-6-[(6-aminohexyl)carbamoylmethyl]-ATP in an overall yield of 40% based on the parent nucleotide ATP. The coenzymic activities of both N-6-adenine-substituted derivatives of ATP were tested with three kinases. Both derivatives showed coenzymic function against hexokinase with the "long" derivative having highest activity (95%) relative to unsubstituted ATP. Their activities towards the other two kinases tested was negligible except with the "long" analogue against glycerokinase (20%). The latter ATP analogue, when bound to Sepharose through its terminal amino group, could be dephosphorylated to the corresponding ADP analogue with soluble hexokinase yielding glucose 6-phosphate in an enzymic "solidphase" fashion. The Sepharose-bound ADP formed could subsequently be phosphorylated back to ATP using soluble acetate kinase. Sepharose-ATP preparations were also used in preliminary affinity chromatography studies using citrate synthase. Alkylation of ADP following the above procedure yielded the corresponding ADP analogue, N-6-[(6-aminohexyl)carbamoylmethyl]-ADP in an overall yield of 40%. Alkylation of AMP yielded the corresponding N-6-[(6-aminohexyl)carbamoylmethyl]-AMP in an overall yield of 45%.     

Interaction of the cysteine proteinase inhibitor chicken cystatin with papain

The two forms of chicken cystatin, with different isoelectric points, that have been described previously were indistinguishable in analyses of amino- and carboxy-terminal residues, amino acid composition, and peptide maps. The two forms thus are highly similar and most likely differ only in an amide group or in a small charged substituent. The binding of either cystatin form to highly purified, active papain was accompanied by the same pronounced changes in near-ultraviolet circular dichroism, ultraviolet absorption, and fluorescence emission. These changes were compatible with perturbations of the environment of aromatic residues in one or both proteins of the complex, arising from local interactions or from a conformational change. Modification of the single tryptophan residue of cystatin, at position 104, with N-bromosuccinimide resulted in considerably smaller spectroscopic changes on binding of the inhibitor to papain, indicating that the environment of this residue is affected by the binding. Analogous modification of Trp-69 and Trp-177 of papain markedly affected the fluorescence changes observed on binding of cystatin to the enzyme, similarly suggesting that these two residues of papain are involved in the interaction. The fluorescence increase of papain at alkaline pH, arising from Trp-177 and due to deprotonization of the adjacent His-159, was abolished on binding of cystatin to the enzyme, further supporting the proposal that this region of papain participates in the interaction with the inhibitor. A stoichiometry of binding of either cystatin form to papain of 1:1 and a lower limit for the binding constant of 10(9) M-1 were determined by titrations monitored by either the ultraviolet absorption or fluorescence changes induced by the interaction.     

Kinetic study of the slow cyanide binding to Glycera dibranchiata monomer hemoglobin components III and IV

Compared to other monomeric heme proteins and the heme peroxidases, the Glycera dibranchiata monomer hemoglobin components III and IV exhibit very slow cyanide binding kinetics. This is agreement with the previously reported behavior of component II. Similar to component II, components III and IV have been studied under pseudo-first-order conditions at pH 6.0, 7.0, 8.0, and 9.0 by using a 100-250-fold excess of potassium cyanide at each pH. At 20 degrees C with micromolar protein concentrations, kobs for component III varies between 7.08 x 10(-5) s-1 at pH 6.0 and 100-fold cyanide excess and 1.06 x 10(-2) s-1 at pH 9.0 and 250-fold cyanide excess. For component IV, the values are 2.03 x 10(-4) s-1 for 100-fold cyanide excess at pH 6.0 and 4.13 x 10(-2) s-1 for 250-fold cyanide excess at pH 9.0. In comparison to other heme proteins, our analysis shows that the bimolecular rate constant (klapp) is small. For example, at pH 7.0, it is 3.02 x 10(-1) M-1 s-1 for component III and 1.82 M-1 s-1 for component IV, compared to 400 M-1 s-1 for sperm whale metmyoglobin, 692 M-1 s-1 for soybean metleghemoglobin a, 111 M-1 s-1 for guinea pig methemoglobin, and 1.1 x 10(5) M-1 s-1 for cytochrome c peroxidase. Our results also show that the dissociation rates (k-lapp) are extremely slow and no larger than 10(-6) s-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of a cystatin from the leaves of methyl jasmonate treated tomato plants

A multidomain cystatin was purified from the leaves of mature and seedling tomato plants (Lycopersicon esculentum, cv Bonnie Best) that had been sprayed with methyl jasmonate. For seedlings, cystatin purification was accomplished using EDTA washing, KCI extraction, 70 degrees C heat treatment, ammonium sulfate fractionation and gel filtration chromatography. For mature plants, DEAE chromatography was also needed to separate a protease, hydrolysis products of cystatin and serine proteinase inhibitors from the intact cystatin. Purified tomato cystatin has a molecular weight (Mr) of 88 kDa, eight papain binding domains, is a non-competitive inhibitor of papain with K1 of 1.4 nM and is not a glycoprotein. Tryptic peptides (Mr 26, 13 kDa) and most chymotryptic peptides (Mr 33, 13 kDa) of tomato cystatin retain inhibitor activity. Amino acid analysis revealed no Cys; Asx, Glx, Gly, Ser accounted for almost half the residues and there was some homology with potato multicystatin. Activity is stable at pH 4-11 at 4 degrees C, but unstable at neutral pH at > 60 degrees C (Ea = 92.5 kJ/mole). Extracts of mature plants treated with methyl jasmonate contain lower Mr cystatins that appear to result from the action of an endogenous 26 kDa protease on the 88 kDa inhibitor.     

Enhanced anti-rotavirus action of human cystatin C by site-specific glycosylation in yeast

The cDNA encoding human cystatin C (HCC) was subjected to site-specific substitution of alanine for serine at the position 37, to obtain the Asn(35)-Lys(36)-Ser(37) sequence that is a signal for asparagine-linked (N-linked) glycosylation of protein in eukaryotes, and was transformed into Pichia pastoris X33. As a result, 1.2 mg/L oligomannosyl HCC with a carbohydrate chain of Man(10)GlcNAc(2) was produced by the Pichia transformant. The oligomannosyl HCC was more stable at the low ionic strength condition of 50 mM potassium phosphate buffer, pH 7.0, than the wild-type. In addition, the oligomannosylation substantially improved the molecular stability of cystatin against an aspartic proteinase, cathepsin D, in which the susceptibility decreased to less than 50% of nonglycosylated one. The anti-rotavirus activity of HCC was substantially enhanced by the site-directed glycosylation using the yeast expression system. A MA-104 cell line was used as a host cell for human rotavirus type-2 Wa strain in this study, to which both the wild-type and oligomannosyl HCCs did not show cytotoxicity at a concentration of 100 mug/mL. More than 80% viability of the host cell infected with 1.0 x 10(5) PFU/mL of rotavirus was conserved under the condition coexisting with 75 mug/mL of the oligomannosyl HCC, which was 15.2% higher than that of wild-type HCC. Thus, the in vitro anti-rotavirus assay indicated that the supplement of a proper amount of the oligomannosyl HCC could be used as an anti-rotavirus agent.     

Characterization of pH-induced transitions of beta-lactoglobulin: ultrasonic, densimetric, and spectroscopic studies.

Depending on solution conditions, beta-lactoglobulin can exist in one of its six pH-dependent structural states. We have characterized the acid and basic-induced conformational transitions between these structural states over the pH range of pH 1 to pH 13. To this end, we have employed high-precision ultrasonic and densimetric measurements coupled with fluorescence and CD spectroscopic data. Our combined spectroscopic and volumetric results have revealed five pH-induced transitions of beta-lactoglobulin between pH 1 and pH 13. The first transition starts at pH 2 and is not completed even at pH 1, our lowest experimental pH. This transition is followed by the dimer-to-monomer transition of beta-lactoglobulin between pH 2.5 and pH 4. The dimer-to-monomer transition is accompanied by decreases in volume, v degrees (-0.008(+/-0.003) cm3 x g(-1)), and adiabatic compressibility, k degrees (S) (-(0.7(+/-0.4))x10(-6) cm3 x g(-1) x bar(-1)). We interpret the observed changes in volume and compressibility associated with the dimer-to-monomer transition of beta-lactoglobulin, in conjunction with X-ray crystallographic data, as suggesting a 7 % increase in protein hydration, with the hydration changes being localized in the area of contact between the two monomeric subunits. The so-called N-to-Q transition of beta-lactoglobulin occurs between pH 4.5 and pH 6 and is accompanied by increases in volume, v degrees (0.004(+/-0.003) cm3 x g(-1)), and compressibility, k degrees (S) ((0.7(+/-0.4))x10(-6) cm3 x g(-1) x bar(-1)). The Tanford transition of beta-lactoglobulin is centered at pH 7.5 and is accompanied by a decrease in volume, v degrees (-0.006(+/-0.003) cm3 x g(-1)), and an increase in compressibility, k degrees (S) ((1.5(+/-0.5))x10(-6) cm3 x g(-1) x bar(-1)). Based on these volumetric results, we propose that the Tanford transition is accompanied by a 5 to 10 % increase in the protein hydration and a loosening of the interior packing of beta-lactoglobulin as reflected in a 12 % increase in its intrinsic compressibility. Finally, above pH 9, the protein undergoes irreversible base-induced unfolding which is accompanied by decreases in v degrees (-0.014(+/-0.003) cm3 x g(-1)) and k degrees (S) (-(7.0(+/-0.5))x10(-6) cm3 x g(-1) x bar(-1)). Combining these results with our CD spectroscopic data, we propose that, in the base-induced unfolded state of beta-lactoglobulin, only 80 % of the surface area of the fully unfolded conformation is exposed to the solvent. Thus, in so far as solvent exposure is concerned, the base-induced unfolded states of beta-lactoglobulin retains some order, with 20 % of its amino acid residues remaining solvent inaccessible.     

The interaction of inositol pentaphosphate with the hemoglobins of highland and lowland geese.

We have studied the binding of inositol pentaphosphate (IPP) to the hemoglobins from two species of goose living at low and high altitudes, using the proton absorption method. Measurements were done at 25 and 37 degrees C in a pH range between 6.0 and 8.8. The bird hemoglobins show a high affinity and a binding stoichiometry of 1 IPP molecule/hemoglobin tetramer both in the ligated and unligated state, indicating the same binding site for IPP in oxy- and deoxyhemoglobin. The results indicate that the interaction of IPP with both geese hemoglobins is very similar. For the deoxyhemoglobins of both species the IPP-binding constant shows a strong pH dependence extending over a wide pH range (i.e. +/- 2 x 10(6) M at pH 8.8 and +/- 6 x 10(10) M at pH 6.0). The binding constant of IPP for the oxyhemoglobins shows a much weaker pH dependence (i.e. +/- 4 x 10(4) M at pH 8.8 and +/- 3 x 10(6) M at pH 6.0), indicating that the interaction of IPP with the goose hemoglobin is strongly dependent on the state of ligation of the protein. The IPP binding constants for the oxy- and deoxyhemoglobins are found to be in good agreement with the IPP-induced change in oxygen affinity of both hemoglobins as estimated from oxygen binding curves.     

Carp ovarian cystatin binds and agglutinates spermatozoa via electrostatic interaction

A sperm-agglutinating factor was purified from ovulated carp eggs and the conditioned medium (CM) of cortical-reacted eggs. It was identified to be the carp ovarian cystatin. Three cystatin isoforms were found. The cystatin isolated from the CM had a higher sperm-agglutinating activity than that isolated from eggs, although the cystatins have identical N-terminal amino acid sequences, masses, and positive charges. Differences in sperm-agglutinating activity between the cystatins of the CM and eggs may be caused by the different conformations because they differed in circular dichroism spectrum and tryptic map. Cystatin was discharged from cortical granules to the perivitelline space after fertilization and is abundant in the perivitelline fluid (PVF) of early stage embryos. Cystatin rapidly agglutinated spermatozoa via an electrostatic interaction. Other basic proteins also agglutinated carp spermatozoa. Their activities were inhibited by salt and high pH. Cystatin bound to the entire surface of carp spermatozoa. The PVF of early embryos agglutinated carp spermatozoa. The activity was related to the cystatin content and influenced by ionic strength and pH. Therefore, cystatin is the major sperm-agglutinating factor of PVF. Owing to the rapid action of cystatin on spermatozoa agglutination and the presence of a high concentration of cystatin in PVF, cystatin is considered important for preventing polyspermy in carp eggs.     

Radical scavenging properties of genistein

The reactivity of genistein toward reactive radical species has been investigated by means of pulse radiolysis. The values of rate constants, respectively 2.3 x 10(10) M(-1)s(-1) and 1.3 x 10(10) M(-1)s(-1) for the reaction with hydroxyl radical at pH 8.3 and 3.0, are close to diffusion limit indicating that genistein is a potent hydroxyl radical scavenger. The reactivity of genistein towards one-electron oxidants has also been investigated. The rate constants k = 4.6 x 10(9) M(-1)s(-1) (pH 8.3) and 6.7 x 10(8) M(-1)s(-1) (pH 7.6) have been determined for the reaction of genistein with *N3 and Br2*- radicals, respectively. For both oxidants the rate constants at pH 3 does not exceed 10(8) M(-1)s(-1). The differences in reactivity of genistein towards the oxidants at different acidity of the solution have been assumed to arise from the acid-base equilibria of genistein. The dissociation constants for genistein (pKa: 7.2, 10.0, and 13.1) have been evaluated spectroscopically. The influence of acid-base equilibria on bond dissociation energy and ionization potential for genistein has also been investigated by means of DFT calculations. It has been concluded on the basis of these calculations that monoanionic form of genistein existing at physiological pH is more powerful radical scavenger than the neutral molecule.     

Kinetic studies on thermal denaturation of C-phycocyanin

The kinetics of thermal denaturation of a biliprotein, C-phycocyanin (C-PC) isolated from Spirulina platensis were studied at different pH values, ranging from 4.0 to 8.0. The denaturation of C-PC follows the first order kinetics and rate constant at pH 5.0 and temperature 55 degrees C is found to be 4.37 x 10(-5) s(-1), which increases to 5.46 x 10(-1) s(-1) at pH 7.0. The denaturation rate is much higher at 65 degrees C and pH 7.0 (7.96 x 10(-4)), as compared to at pH 5.0 (1.46 x 10(-4)). The thermal stability of C-PC is more at pH 5.0, as compared to other pH values. The observed differences in entropy values at pH 5.0, as compared to other pH values indicate a considerably close fit structure of the protein at pH 5.0, which increases the stability of native structure, even at higher temperature (65 degrees C).