Molar Urea Research Articles

Protein stability: functional dependence of denaturational Gibbs energy on urea concentration.

Determination of protein stability (DeltaGD0) from the conformational transition curve induced by a chemical denaturant is problematic; for different values of DeltaGD0, the value of the Gibbs energy change on denaturation (DeltaGD) in the absence of the denaturant are obtained when different extrapolation methods are used to analyze the same set of (DeltaGD, denaturant concentration) data [Pace, C. N. (1986) Methods Enzymol. 131, 266-280]. We propose a practical solution to this problem and use it to test the dependence of DeltaGD of lysozyme, ribonuclease-A, and cytochrome-c on [urea], the molar urea concentration. This method employs (i) measurements of the urea-induced denaturation in the presence of different guanidine hydrochloride (GdnHCl) concentrations which by themselves disrupt the native state of the protein at the same temperature and pH at which denaturations by urea and GdnHCl have been measured; (ii) estimation of DeltaGDcor, the value of DeltaGD corrected for the effect of GdnHCl on the urea-induced denaturation using the relation (DeltaGDcor = DeltaGD + mg [GdnHCl] = DeltaGD0 - mu [urea], where mg and mu are the dependencies of DeltaGD on [GdnHCl] and [urea], respectively) whose parameters are all determined from experimental denaturation data; and (iii) mapping of DeltaGDcor onto the DeltaGD versus [urea] plot obtained in the absence of GdnHCl. Our results convincingly show that (i) [urea] dependence of DeltaGD of each protein is linear over the full concentration range; (ii) the effect of urea and GdnHCl on protein denaturation is additive; and (iii) KCl affects the urea-induced denaturation if the native protein contains charge-charge interaction and/or anion binding site, in a manner which is consistent with the crystal structure data.

Reliability of low-avidity IgG and of IgA in the diagnosis of primary infection by rubella virus with adaptation of a commercial test

The detection of IgA and low-avidity IgG and antibodies in serum is a potentially useful marker of recent infection by a microorganism. We studied the reliability of IgG avidity and presence of IgA for the diagnosis of recent acute infection by rubella virus. Low-avidity IgG (Avy-EIA test) was determined with a modified commercial test using 8 molar urea (indirect ELISA, DiaSorin, Italy) and IgA was determined with a homemade indirect ELISA test. Twenty-five patients with recent primary infection by rubella virus (group I) and 50 healthy subjects (group II) were studied. In group I low-avidity IgG varied between 100 and 0% (67.3+/-21.8%); IgA was present in 24 patients (96%). In group II low-avidity IgG varied from 50.4 to 0% (19.8+/-16.9%). IgA was present in 2 subjects (4%). The sensitivity of the Avi-EIA and the IgA test was 92 and 96%, respectively; specificity was 100 and 96%, respectively. We conclude that both low-avidity IgG and IgA tests are helpful and reliable for the diagnosis of recent primary infection.

Reliability of low‐avidity IgG and of IgA in the diagnosis of primary infection by rubella virus with adaptation of a commercial test

The detection of IgA and low-avidity IgG and antibodies in serum is a potentially useful marker of recent infection by a microorganism. We studied the reliability of IgG avidity and presence of IgA for the diagnosis of recent acute infection by rubella virus. Low-avidity IgG (Avy-EIA test) was determined with a modified commercial test using 8 molar urea (indirect ELISA, DiaSorin, Italy) and IgA was determined with a homemade indirect ELISA test. Twenty-five patients with recent primary infection by rubella virus (group I) and 50 healthy subjects (group II) were studied. In group I low-avidity IgG varied between 100 and 0% (67.3 ± 21.8%); IgA was present in 24 patients (96%). In group II low-avidity IgG varied from 50.4 to 0% (19.8 ± 16.9%). IgA was present in 2 subjects (4%). The sensitivity of the Avi-EIA and the IgA test was 92 and 96%, respectively; specificity was 100 and 96%, respectively. We conclude that both low-avidity IgG and IgA tests are helpful and reliable for the diagnosis of recent primary infection. J. Clin. Lab. Anal. 13:1–4, 1999. © 1999 Wiley-Liss, Inc.

Epididymal Sperm Transport in Normal and Recent Spinal Cord Injured Sprague Dawley Rats

Causes of poor semen quality following spinal cord injury (SCI) are not known. One possible reason, based upon studies that reported improved semen quality in SCI men after several induced ejaculations, is delayed epididymal sperm transport. Our study was designed to establish baseline epididymal sperm transport values in the Sprague Dawley rat and evaluate effects of SCI on this process. Spermatozoa protamine was labeled with tritiated arginine, and the rats were sacrificed various times after injection. Each epididymis was divided into six equal sections from proximal to distal. Sperm tails were dissolved with 8 molar (M) urea in the presence of 2mM dithiothreitol (DTT); sperm heads were collected by centrifugation (3,000 rpms, 10 min.). The radioactivity in sperm heads from each section was counted and expressed as counts per million sperm heads. To account for different rates of labeled arginine incorporation, the percentage of counts per million sperm heads in each section was calculated relative to the total number of counts in all six sections. Our results showed there was an orderly progression of sperm through the epididymis. It took 8 days for labeled sperm to enter the epididymis and 28 days to peak in the caudal (tail) section in non-SCI rats. Stasis was present 10 days after T-9 SCI in rats compared with transport in sham controls. This was evidenced by a significant increase in the percentage of labeled sperm in proximal sections of the epididymis (sections 1, 2, and 4) in T-9 transected animals (p < 0.01). If similar stasis occurs in SCI men, it could obviously contribute to poor semen quality. However, it remains to be determined how long this stasis persists after SCI in rats. (J Spinal Corel Med 1999;22:102–106)

Mechanism of hydration of urea and guanidium ion: A model study of denaturation of proteins

Abstract

Change in conductivity in non-cross-linked polyacrylamide capillary electrophoresis Effects of aging polyacrylamide and buffer composition

Upon application of an electric field to a polymer-filled capillary, there is a rapid expulsion of excess ions associated with the polymerization mixture. This expulsion is complete in about 10–15 min. A much slower transport of ions occurs at one end of the capillary; the transport arises because of the difference in transference number between the buffer reservoir and the polymer filled capillary. This ionic transport leads to a change in the conductivity of the polymer at one end of the capillary. We have monitored the conductivity profile in 5% T non-cross-linked polyacrylamide capillaries at 300 V/cm for different ages of polyacrylamide. The profile was fit with a double-exponential, corresponding to the rapid expulsion of reaction ions and the slow transport of ions due to differences in transference number. The fit indicates that there are more ions in polyacrylamide as it is aging, and that the depletion of ionic concentrations is less important in aged polyacrylamide than in fresh polyacrylamide. The effect of the buffer composition was studied. By changing from 1 × TBE buffer reservoirs to a mixture of 30 m M KCl and 1 × TB buffer, the depletion of ionic concentration at the injection end disappears and is replaced by an increase of ionic concentration at the ground end. By changing 1 × TBE buffer reservoirs to 1 × TBE, 5% T non-cross-linked polyacrylamide and seven molar urea buffer reservoirs the depletion of ionic concentration at the injection end disappears.

Kinetics of NOx reduction by urea solution in a pilot scale reactor.

The effects of urea molar ratio (1.0–2.0), reaction temperature and additives on NO reduction in flue gas have been determined in a pilot-scale flow reactor. A kinetic model is proposed for NO reduction rate with -NH and -CN in selective reducing agents (urea, cyanuric acid, ammonia). Two primary reactions can be expressed kinetically in the DeNOx process as:N.S. (nitrogenous species) + Ox (oxidants) → NO + … (kf; rate constant for NO formation)N.S. + NO → N2 + … (kr; rate constant for NO reduction)in which the rate constants of kf and kr in the model are determined from the experimental data in the temperature range 1173 K–1373 K as:kf = 4.01 × 1011 exp(–267/RT)kr = 7.24 × 1011 exp(–230/RT)The proposed model can predict the lowered activation energies caused by the addition of different species and molar ratio to NO.

Dielectric spectroscopy analysis on the interfacial composition of non-ionic micellar solutions

By iterative use of a suitable mixture equation for the dielectric permittivity of heterogeneous systems we investigate C 12E 6 solutions as a function of the monomer concentrations and of the temperature at four different solvent compositions: 0, 2, 4 and 6 molar urea in water. With this methodology we obtain the permittivity of the micellar head group crown termination as a function of the hydrated volume of the monomer head group, composed in our case by 6 PEO (polyethylene oxide) units and by the interdigitated solvent molecules. Then the intercept between the dielectric behaviour as a function of the PEO molar concentration of the micellar crown region and of a solution of PEO-water-urea gives an estimate of the actual composition within the interface, at a fixed temperature and solvent composition. Our results suggest a temperature dependence of the degree of hydration at the micellar interface in pure water solution. With mixed water-urea solutions, cosolvent adsorbtion at the micellar interface is observed.

The solubilities of five cyclic dipeptides in water and in aqueous urea at 298.15 K: a quantitative model for the denaturation of proteins in aqueous urea solutions

The solubilities of cyclo( l-alanylglycine), cyclo( l-alanyl- l-alanine), cyclo(glycyl- l-leucine), cyclo( l-valyl- l-valine) and cyclo(glycyl- l-phenylalanine) were determined in water and in aqueous urea solutions up to concentrations of 9 molar urea at 298.15 K. The solubilities of all cyclic dipeptides increase with increasing urea concentration. A simple equilibrium model, taking into account the activity of urea and that of water, fits the solubility data yielding apparent equilibrium constants describing the interactions occurring between urea and the peptide groups plus the alkyl groups that are next to these peptide groups. The apparent equilibrium constants were converted to Gibbs energy parameters for each amino acid residu which were then used to make a quantitative estimate of the contribution of urea to the denaturation of proteins.

Unusually stable beta-sheet formation in an ionic self-complementary oligopeptide.

A 16-residue amphiphilic oligopeptide (EAK16) with every other residue alanine and also containing glutamic acid and lysine (Ac-NH-AEAEAKAKAEAEAKAK-CONH2) is able to form an unusually stable beta-sheet structure. The beta-sheet structure is stable at very low concentrations in water and at high temperatures. Various pH changes at 1.5, 3, 7, and 11 had little effect on the stability of the beta-sheet structure. The beta-sheet structure was not altered significantly even in the presence of 0.1% SDS, 7 molar guanidine hydrochloride, or 8 molar urea. One of the structural characteristics of the EAK16 is its ionic self-complementarity in that ionic bonds and hydrogen bonds between Glu and Lys can form readily between two oligopeptide beta-sheet structures. This structural feature is probably one of the factors that promotes its extreme stability. This is the first example of such an extended ionic self-complementarity in a protein structure. EAK16 and its related peptides may have applications as useful biomaterials. It also offers a good model for studying the mechanism of beta-sheet formation. Because the oligopeptide can self-assemble to form a membranous structure, it may have relevance to origin of life research.

NMR determination of residual structure in a urea-denatured protein, the 434-repressor.

A nuclear magnetic resonance (NMR) structure determination is reported for the polypeptide chain of a globular protein in strongly denaturing solution. Nuclear Overhauser effect (NOE) measurements with a 7 molar urea solution of the amino-terminal 63-residue domain of the 434-repressor and distance geometry calculations showed that the polypeptide segment 54 to 59 forms a hydrophobic cluster containing the side chains of Val54, Val56, Trp58, and Leu59. This residual structure in the urea-unfolded protein is related to the corresponding region of the native, folded protein by simple rearrangements of the residues 58 to 60. Based on these observations a model for the early phase of refolding of the 434-repressor(1-63) is proposed.

Microheterogeneity in Purified Broad Bean Polyphenol Oxidase

Polyphenoloxidase was purified from chloroplasts of broad bean leaves (Vicia faba L.) to apparent homogeneity. The enzyme was composed of two proteins with an apparent mass of 65 and 68 kilodaltons after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated enzyme contained covalently attached carbohydrates and bound concanavalin A, Phaseolus vulgaris erythroagglutinin, and Ricinus communis agglutinin lectins. Under native isoelectric focusing, several charged isoforms were present in the pH range of 4 to 6. Many, if not all, of the isoforms separated by isoelectric focusing were glycosylated and bound concanavalin A. All these isoforms shared a 65 kilodalton protein in common, and some of the isoforms were associated with both a 65 and 68 kilodalton protein. Isoforms separated by isoelectric focusing in the presence of 9 molar urea followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a similar pattern of proteins within a slightly higher pH range from 5 to 6.5.

Identification of Low Molecular Mass GTP-Binding Proteins in Membranes of the Halotolerant Alga Dunaliella salina

A family of specific guanine nucleotide-binding proteins in Dunaliella salina was studied. Polypeptides of different subcellular fractions were separated by electrophoresis and transferred to nitrocellulose or Immobilon membranes. Incubation of the transfer blots with [(35)S]GTPgammaS or [alpha-(32)P]GTP showed no evidence for GTP-binding proteins in the chloroplast and cytosol fractions. However, two GTP-binding proteins with molecular masses of 28 and 30 kilodaltons were present in the plasma membrane and microsomal fractions. An additional 29 kilodalton GTP-binding protein was detected in the plasma membrane. The mitochondrial fraction contained significant amounts of only the 28 kilodalton GTP-binding protein. Binding of [(32)P]GTP to the protein blots was completely prevented by 10 micromolar GTP or guanosine 5'-O-(2-thiodiphosphate) (added in 3 x 10(4)-fold excess), whereas ATP or CTP had no effect on the binding. The 28 kilodalton GTP-binding protein was recognized by polyclonal antibodies to the ras-related YPT1 protein of yeast but not by the anti-ras Y13-259 monoclonal antibody. GTP-binding proteins present in the microsomal fraction could not be solubilized by incubation of microsomes with 1 molar NaCl or 0.2 molar Na(2)CO(3), but some GTP-binding activity was solubilized when microsomes were treated with 6 molar urea. These results indicate that D. salina GTP-binding proteins are tightly associated with the membranes. The covalent attachment of fatty acids to these proteins was also investigated. Electrophoresis followed by fluorography of delipidated microsomal proteins extracted from [(3)H]myristic acid-labeled cells showed an intense labeling of a 28 kilodalton protein. We conclude that D. salina contains proteins resembling the ras-related proteins found in animal cells and higher plants.

Expression of Constitutive and Tissue-Specific Acyl Carrier Protein Isoforms in Arabidopsis

We have characterized the occurrence and expression of multiple acyl carrier protein (ACP) isoforms in Arabidopsis thaliana (L.) Heynh ecotype Columbia. Immunoblot analysis of ACPs from Arabidopsis tissues separated by native polyacrylamide gel electrophoresis and 1 molar urea polyacrylamide gel electrophoresis revealed a complex pattern of multiple ACP isoforms. All tissues examined (leaves, roots, and seeds) expressed at least three forms of ACP. The immunoblot identifications of ACP bands were confirmed by acylation of ACP extracts with Escherichia coli acyl-ACP synthetase. A full-length cDNA clone has been isolated that has 70% identity with a previously characterized Arabidopsis genomic ACP clone (ACP-1) (MA Post-Beittenmiller, A Hlousek-Radojcić, JB Ohlrogge [1989] Nucleic Acids Res 17: 1777). Based on RNA blot analysis, the cDNA clone represents an ACP that is expressed in leaves, seeds, and roots. In order to identify the protein products of each known ACP gene, their mature coding sequences have been expressed in E. coli. Using polymerase chain reactions, exons II and III of the genomic ACP-1 clone and the mature coding sequences of the ACP-2 cDNA clone were subcloned into E. coli expression vectors. Site-directed mutagenesis was used to convert the amino acid sequence of the ACP-2 cDNA clone to that of the A2 clone of Lamppa and Jacks ([1991] Plant Mol Biol 16: 469-474), ACP-3. The three E. coli-expressed proteins have different mobilities on polyacrylamide gel electrophoresis gels and each comigrates with a different Arabidopsis ACP isoform expressed in leaves, seeds, and roots. Thus, all of the three cloned ACPs appear to be constitutively expressed Arabidopsis ACPs. In addition to these three ACP isoforms, protein blots indicate that seed, leaf, and root each express one or more tissue-specific isoforms.

Isoelectric Focusing of Plant Plasma Membrane Proteins

Detergent-solubilized plasma membrane proteins from pea (Pisum sativum L.) stem tissue were separated by isoelectric focusing (IEF) using a Bio-Rad Rotofor cell, with the goal of identifying protein(s) involved in beta-1,3-glucan synthase (GS-II) activity. Ordinary IEF procedures result in membrane protein precipitation. Inclusion of 10% glycerol mitigates this problem in digitonin-solubilized preparations, but not in those solubilized in 3-[(-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Loss of GS-II activity during IEF is minimized by improved cooling of the Rotofor cell. GS-II focuses at pH 5.1. Antiserum against a 55 kilodalton (kD) polypeptide that was recognized from other evidence as involved in GS-II activity, detects this polypeptide in exact correspondence with the GS-II activity peak. A presumptive P-type ATPase, detected using an antibody against corn root plasma membrane 97 kD ATPase, focuses at pH 5.3. In this digitonin/glycerol medium, most of the membrane proteins focus within the relatively narrow pH range of 4.5 to 6, compared to pH 5.5 to 8.5 for IEF in the presence of 9 molar urea, 2% Nonidet P-40 (NP-40), and 5% mercaptoethanol, a medium that inactivates GS-II. This latter medium increases the apparent isoelectric point (pl) values of the abovementioned 55 and 97 kD polypeptides to 5.8 and 7.3, respectively. In the digitonin/glycerol medium, membrane polypeptides apparently focus at pH values lower than their true pls, because of adhering negatively charged phospholipids, which can be at least partially removed by the detergent NP-40 in the presence of urea. These results provide independent evidence that the 55 kD polypeptide is associated with the GS-II activity and indicate that inclusion of urea and a strong nonionic detergent such as NP-40 is necessary if membrane proteins are to be focused at pH values near their true pls.

Purification and Partial Characterization of Two Soluble NAD(P)H Dehydrogenases from Arum maculatum Mitochondria

Two enzyme systems carrying out the oxidation of NAD(P)H in the presence of various electron acceptors have been isolated and partially characterized from the supernatant of frozen-thawed mitochondria from Arum maculatum spadices. The two systems contain flavoproteins and differ by their ability to oxidize NADH or NADPH, optimum pH and pI values, sensitivity to Ca(2+) and EGTA, denaturation by 4 molar urea, molecular mass, and number of subunits. These properties, together with methodological considerations, are compatible with the location of these enzyme activities on the outer surface of the inner mitochondrial membrane, and support the hypothesis of the existence of two separate dehydrogenases responsible for the mitochondrial oxidation of cytosolic NADH and NADPH.

The ternary system water + 1-propanol + urea AT 298.15K. Activity coefficients, partial molal volumes and kirkwood-buff integrals.

The activity coefficiens, γi, and the partial molal volumes, V i, of the three components of the ternary system water(1)+1-propanol(2)+urea(3) have been measured at 298.15 K in the entire domain of full miscibility. These data have been used to calculate the six Kirkwood-Buff (K-B) integrals, G ij, of the ternary mixture. γ 1 and γ 2 are found very little dependent on urea mole fraction x 3. indicating the scarce influence of urea on the interaction free energy between water and propanol. V 1 and V 2 too are little dependent on urea concentration; V 2 shows a characteristic minimum in dilute alcohol solution even at high x 3 values. The increase of V 3 with x 3, a characteristic of hydrophilic compounds in water, is found even at high x 2. The observed increase of the three homologous G ii's and of G 12 and the decrease of G 23 and G 12 with addition of urea indicate that this compound brings about an enhanced homocoordination of the three components, as well as an increase of the urea-water affinity, and suggest that the observed increase of G 22 with urea addition may be due to the abduction of water molecules from the solvation sphere of propanol.

Digesta kinetics, ruminal fermentation characteristics and serum metabolites of pregnant and lactating ewes fed chopped alfalfa hay.

Eight ruminally cannulated ewes (four control and four bred; average BW = 85.7 kg), limit-fed alfalfa hay (1.86% BW), were used in two experiments to determine the effects of pregnancy and lactation on digestive function and serum metabolites. Seven-day sampling periods were used starting on d 102, 118 and 132 of gestation (Exp. 1) and d 14 and 32 of lactation (Exp. 2). Particulate (6.8 vs 4.9%/h; PPR) and fluid passage rates (13.9 vs 10.9%/h) were greater (P less than .05) and gastrointestinal mean retention time (28.9 vs 35.7 h) and fluid turnover time (FTT, 7.5 vs 9.5 h) were lesser (P less than .05) in pregnant than in nonpregnant ewes, respectively. Isobutyrate concentration was lower (P less than .05) in pregnant (1.7 mol/100 mol) than in nonpregnant (1.9 mol/100 mol) ewes. No differences (P greater than .10) were noted for any other ruminal fermentation measures between pregnant and nonpregnant ewes. In Exp. 2, no differences (P greater than .10) were noted in digesta kinetics or ruminal fermentation measures except for isobutyrate and isovalerate molar proportions and serum urea N (SUN) concentration. Isobutyrate, isovalerate and SUN concentrations (21.8 vs 26.1 mg/dl) were lower (P less than .05) in lactating ewes than in nonlactating ewes. Gastrointestinal fill (5.7 vs 7.7 g/kg BW) and FTT (9.3 vs 12.7 h) were lesser and DM digestion (66.7 vs 57.4%) was greater (P less than .05) in lactating than in nonlactating ewes. Data suggest that, during pregnancy, passage rate increases occur without affecting DM digestion and that, during lactation, PPR is not affected when ewes are limit-fed.

Reduction of nitrogen oxides by injection of urea in the freeboard of a pilot scale fluidized bed combustor

The ‘thermal deNO x’ process using urea has been investigated in a 1 MW fluidized bed combustor. NO x reductions of up to 76% were obtainable by using this method. The experimental results show that urea is at least as active as NH 3, which is commonly used in this application, but which is far more toxic and corrosive. Emission levels of 200 mg m −3 for NO x could be achieved by injecting the urea at a height of 2 m above the distribution plate in a molar ratio urea: NO x = 1.5. The SO 2 emission value also appeared to be reduced when the urea was injected at a urea: NO x molar ratio > 4.

Purification and identification of the fusicoccin binding protein from oat root plasma membrane.

Fusicoccin (FC), a fungal phytotoxin, stimulates the H(+) -ATPase located in the plasma membrane (PM) of higher plants. The first event in the reaction chain leading to enhanced H(+) -efflux seems to be the binding of FC to a FC-binding protein (FCBP) in the PM. We solubilized 90% of the FCBP from oat (Avena sativa L. cv Victory) root PM in an active form with 1% octyl-glucoside. The FCBP was stabilized by the presence of protease inhibitors. The FCBP was purified by affinity chromatography using FC-linked adipic acid dihydrazide agarose (FC-AADA). Upon elution with 8 molar urea, two major protein bands on sodium dodecyl sulfate-polyaerylamide gel electrophoresis with molecular weights of 29,700 and 31,000 were obtained. Successive chromatography on BBAB Bio-Gel A, hexyl agarose, and FC-AADA resulted in the same two bands when the FC-AADA was eluted with sodium dodecyl sulfate. A direct correlation was made between 3H-FC-binding activity and the presence of the two protein bands. The stoichiometry of the 29,700 and 31,000 molecular weight bands was 1:2. This suggests that the FCBP occurs in the native form as a heterotrimer with an apparent molecular weight of approximately 92,000.