Concentrated Guanidine Research Articles

Abstract 5701: Innovative approach for isolation of circulating cell-free DNA from liquid biopsies

Abstract We developed a new approach for isolation of circulating cell-free DNA (cfDNA) from liquid biopsies (plasma or serum). Our technology is based on using the proprietary bi-functional substance X (SubX®) that binds DNA under physiological conditions (e.g. directly in biological liquids), followed by adsorption of [DNA- SubX®] complex on a solid phase matrix. Since SubX®) captures DNA via phosphate groups it allows for elimination of bias related to both AT/CG content and DNA fragments length, thus improving extraction efficacy and accuracy of downstream applications. All currently available commercial kits are [silica-chaotropic salt]-based and exhibit bias for either short or long DNA fragments, as well as for the GC content. In addition, such systems require at least 4- fold dilution of starting volume of bio liquid with concentrated (~5.0 M) guanidine thiocyanate (GTC) to keep resulted GTC concentration at about least 3.0 M. Thus, already low concentrations of cfDNA are significantly reduced and may negatively influence extraction efficacy of low abundance DNA fragments. Since our method does not require chaotropic salt for DNA capture, no dilution of starting material (and thus cfDNA) takes place. DNA-binding matrix SubX® allows either scaling the procedure to either large volumes of starting material or extra low volumes and adaptation of the protocol for 96/384 well format. Separation of cfDNA from the bulk of proteins occurs in a single vortex-spin step without employing chaotropic agents thus speeding-up isolation procedure and reducing the costs. The whole procedure can be completed within an hour and cfDNA is eluted in small volume of TE buffer. Average DNA yield from 0.2 ml of double centrifuged blood plasma from normal donors, cancer and multiple sclerosis patients was 7.5±4.2 ng (n=96). Citation Format: Andrei Malykh, Vladimir Evtushenko. Innovative approach for isolation of circulating cell-free DNA from liquid biopsies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5701. doi:10.1158/1538-7445.AM2017-5701

Improved yield of high molecular weight DNA coincides with increased microbial diversity access from iron oxide cemented sub-surface clay environments.

Despite over three decades of progress, extraction of high molecular weight (HMW) DNA from high clay soils or iron oxide cemented clay has remained challenging. HMW DNA is desirable for next generation sequencing as it yields the most comprehensive coverage. Several DNA extraction procedures were compared from samples that exhibit strong nucleic acid adsorption. pH manipulation or use of alternative ion solutions offered no improvement in nucleic acid recovery. Lysis by liquid N2 grinding in concentrated guanidine followed by concentrated sodium phosphate extraction supported HMW DNA recovery from clays high in iron oxides. DNA recovered using 1 M sodium phosphate buffer (PB) as a competitive desorptive wash was 15.22±2.33 µg DNA/g clay, with most DNA consisting of >20 Kb fragments, compared to 2.46±0.25 µg DNA/g clay with the Powerlyzer system (MoBio). Increasing PB concentration in the lysis reagent coincided with increasing DNA fragment length during initial extraction. Rarefaction plots of 16S rRNA (V1–V3 region) pyrosequencing from A-horizon and clay soils showed an ∼80% and ∼400% larger accessed diversity compared to the Powerlyzer soil DNA system, respectively. The observed diversity from the Firmicutes showed the strongest increase with >3-fold more operational taxonomic units (OTU) recovered.

Slow Unfolding Pathway of Hyperthermophilic Tk-RNase H2 Examined by Pulse Proteolysis Using the Stable Protease Tk-Subtilisin

The unfolding speed of some hyperthermophilic proteins is significantly slower than those of their mesostable homologues. Ribonuclease H2 from the hyperthermophilic archaeon Thermococcus kodakarensis (Tk-RNase H2) is stabilized by its remarkably slow unfolding rate. In this work, we examined the slow unfolding pathway of Tk-RNase H2 by pulse proteolysis using a superstable subtilisin-like serine protease from T. kodakarensis (Tk-subtilisin). Tk-subtilisin has enzymatic activity in highly concentrated guanidine hydrochloride (GdnHCl), in which Tk-RNase H2 unfolds slowly. The native state of Tk-RNase H2 was completely resistant to Tk-subtilisin, whereas the unfolded state (induced by 4 M GdnHCl) was degraded by Tk-subtilisin. Degradation products of Tk-RNase H2 created from pulse proteolysis during its unfolding were detected by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We identified the cleavage sites in Tk-RNase H2 by N-terminal sequencing and mass spectrometry and constructed mimics of the unfolding intermediate of Tk-RNase H2 by protein engineering. The mimics were biophysically characterized. We found that the native state of Tk-RNase H2 (N-state) changed to the I(A)-state that was digested by Tk-subtilisin in the early stage of unfolding. In the slow unfolding pathway, the I(A)-state shifted to two intermediate forms, I(B)-state and I(C)-state. The I(B)-state was digested by Tk-subtilisin in the C-terminal region, but the I(C)-state was a Tk-subtilisin resistant form. These states gradually unfolded through the I(D)-state, in which the N-terminal region was digested. The results indicate that pulse proteolysis, by a superstable protease, was a suitable strategy and an effective tool for analyzing intermediate structures of proteins with slow unfolding properties. We also showed that the N-terminal region contributes to the slow unfolding of Tk-RNase H2, and the C-terminal region is important for folding and stability.

Nonnative Aggregation of an IgG1 Antibody in Acidic Conditions: Part 1. Unfolding, Colloidal Interactions, and Formation of High-Molecular-Weight Aggregates

Monomeric and aggregated states of an IgG1 antibody were characterized under acidic conditions as a function of solution pH (3.5-5.5). A combination of intrinsic/extrinsic fluorescence (FL), circular dichroism, calorimetry, chromatography, capillary electrophoresis, and laser light scattering were used to characterize unfolding, refolding, native colloidal interactions, aggregate structure and morphology, and aggregate dissociation. Lower pH led to larger net repulsive colloidal interactions, decreased thermal stability of Fc and Fab regions, and increased solubility of thermally accelerated aggregates. Unfolding of the Fab domains, and possibly the CH3 domain, was inferred as a key step in the formation of aggregation-prone monomers. High-molecular-weight soluble aggregates displayed nonnative secondary structure, had a semi-rigid chain morphology, and bound thioflavin T (ThT), consistent with at least a portion of the monomer forming amyloid-like structures. Soluble aggregates also formed during monomer refolding under conditions moving from high to low denaturant concentrations. Both thermally and chemically induced aggregates showed similar ThT binding and secondary structural changes, and were noncovalent based on dissociation in concentrated guanidine hydrochloride solutions. Changes in intrinsic FL during chemical versus thermal unfolding suggest a greater degree of structural change during chemical unfolding, although aggregation proceeded through partially unfolded monomers in both cases.

A Multivalent Assay to Detect Glycosaminoglycan, Protein, Collagen, RNA, and DNA Content in Milligram Samples of Cartilage or Hydrogel-Based Repair Cartilage

The biochemical measure of success in assisted cartilage repair is normally judged by repair tissue cell density, mRNA and protein expression, and accumulation of extracellular matrix molecules. Existing methods to solubilize cartilage matrix proteoglycans and cellular DNA for quantification, such as papain digestion, often destroy one or more species of the above-named parameters, in order to render others measurable. We have therefore developed a methodology to measure specific levels of mRNA, protein, DNA, glycosaminoglycan, and collagen content on single pulverized 10-mg samples of cartilage, or tissue-engineered cartilage, using successive extractions in concentrated guanidine hydrochloride (GuCl) and guanidine thiocyanate (GITC) solutions. Conditions were developed to solubilize most cellular proteins, DNA, proteoglycans, and some matrix proteins with an initial GuCl extraction step. A subsequent extraction with GITC was essential to solubilize the majority of the cellular RNA. Guanidine-insoluble material was rendered soluble by papain digestion, to enable quantification of collagen, residual glycosaminoglycan, and residual unextracted DNA in individual samples. In general, total collagen, GAG, and DNA content measured in multivalent-extracted samples was similar to that obtained with samples digested directly with papain. Moreover, we were able to reliably detect, in these same multivalent extracts, expressed mRNA as well as specific cellular and extracellular matrix proteins. This multivalent assay could be applied to a variety of cells cultured in biopolymers and to tissues from which biochemical components may be otherwise difficult to extract.

Activity of enzyme immobilized on silanized Co-Cr-Mo.

The surface of an orthopedic biomaterial was modified by the covalent immobilization of biomolecules. Derivatization of Co-Cr-Mo samples with organic and aqueous solutions of gamma-aminopropyltriethoxysilane (APS) resulted in a concentration-dependent number of reactive NH2 groups on the surface available for coupling to protein. The enzyme trypsin was used as a model biomolecule to investigate the effect of immobilization on proteolytic activity. Trypsin was coupled to the silanized samples by formation of Schiff's base linkages via glutaraldehyde. The nature of the interaction between trypsin and biomaterial was then probed by treatment with concentrated guanidine hydrochloride (GuHCl) and urea. Residual activity (following treatment with chaotropic agents) of trypsin immobilized on silanized Co-Cr-Mo was dependent both on the nature of the silane solution and on the type of chaotropic agent. Organic silanization with APS required a minimum density of approximately 49 NH2 per nm2 of nominal surface area (> 0.021 M APS) for residual activity of immobilized trypsin. For aqueous silanization, approximately 5.4 NH2/nm2 (0.51 M APS) resulted in maximal residual trypsin activity. Treatment with GuHCl removed more trypsin activity from Co-Cr-Mo samples silanized with organic solutions of APS than did treatment with urea. On the contrary, with aqueous silanization the samples possessed greater residual activity following treatment with GuHCl than following urea. Compared to simple adsorption with protein onto Co-Cr-Mo, both methods of silanization with APS resulted in superior residual immobilized enzyme activity.

Domain organization of Bacillus thuringiensis CryIIIA delta-endotoxin studied by denaturation in guanidine hydrochloride solutions and limited proteolysis.

Denaturation of Bacillus thuringiensis CryIIIA delta-endotoxin--an insecticidal protein, active against Coleoptera larvae--in concentrated guanidine hydrochloride solutions was pursued by fluorescence and circular dichroism spectroscopy and limited proteolysis. It was found that the protein consists of two fragments that differ by their stability to denaturation by guanidine hydrochloride at pH 3. The less stable fragment corresponds to the N-terminal alpha-helical domain limited by Leu-279; the more stable one starts with Ile-280, contains about 330 amino acid residues, and corresponds to the molecule C-terminal moiety that consist of its two beta-structural domains forming a superdomain.

Kinetic mechanism of cytochrome c folding: involvement of the heme and its ligands.

The covalently attached heme and its axial ligands not only are essential for the structure and function of cytochrome c but they also play an important role in the folding process. Under typical denaturing conditions (concentrated guanidine hydrochloride or urea near pH 7), one of the axial ligands, His 18, remains bound to the oxidized heme iron, but the second ligand, Met 80, is replaced by a non-native histidine ligand (His 26 or His 33 in horse cytochrome c). Using quenched-flow and NMR methods, hydrogen exchange rates were measured for several individual amide protons in guanidine-denatured horse cytochrome c. The observation of a single highly protected (140-fold) backbone amide, that of His 18, suggests the presence of a persistent H-bond consistent with heme ligation of the His 18 side chain in the unfolded state. Heme absorbance changes induced by rapid acidification of oxidized cytochrome c in 4.5 M guanidine hydrochloride from pH 7.8 to 4.6 or below exhibit two kinetic phases with rates of 110 and 25 s-1, attributed to the dissociation of non-native histidine ligands from the heme in the unfolded state. The kinetics of folding from guanidine-denatured cytochrome c under a variety of initial and final conditions was investigated by stopped-flow methods, using tryptophan fluorescence as a conformational probe and Soret absorbance as a probe for the ligation state of the heme. A fast kinetic phase (80 s-1) accompanied by a major decrease in fluorescence and a minor absorbance change coincides with the formation of a partially folded intermediate with interacting chain termini detected in earlier pulsed NH exchange measurements [Roder, H., Elöve, G. A., & Englander, S. W. (1988) Nature 335, 700]. At neutral pH, an intermediate kinetic phase (1.8 s-1) accounts for 78% of the absorbance change and 47% of the fluorescence change. In contrast, the folding kinetics at pH 5 is dominated by the fast phase, and the amplitude of the intermediate phase is reduced to approximately 10%. The pH-dependent amplitude changes show titration behavior with an apparent pK of approximately 5.7, consistent with the protonation of a single histidine residue. The intermediate phase can also be suppressed by the addition of 20 mM imidazole. Since both of these conditions interfere with histidine ligation, the intermediate kinetic phase is attributed to the presence of a non-native histidine ligand (His 26 or His 33) that can become trapped in a partially folded intermediate.(ABSTRACT TRUNCATED AT 400 WORDS)

Quantitative measurement of calretinin and β-actin mRNAIN rat brain micropunches without prior isolation of RNA

A microdissection technique for quantitation of neurochemicals in discrete brain nuclei has been applied to quantitative measurement of mRNA. The method permits quantitation of low abundance mRNA from submilligram amounts of tissue (10–500 μ protein). Discrete nuclei and other regions of the brain are solubilized in concentrated guanidine thiocyanate solution, mRNA is directly hybridized with riboprobes, and detected with a ribonuclease protection assay. This method eliminates the necessity for RNA isolation from solid tissue. No assumptions regarding RNA recovery are necessary since tissue specimens are solubilized, hybridized and treated with ribonuclease in a single tube. We have determined the mRNA levels of calretinin, a predominantly neuron-specific calcium binding protein in microdissected nuclei and other regions of rat brain. For interassay comparison, measurement of sample protein and β-actin mRNA permits normalization and quantitation in terms of these internal controls. The quantity of calretinin mRNA ranged from 281 ± 35 fg/μg protein in the thalamic paraventricular nucleus to 2.3 ± 0.5 fg/μg protein for the cerebral cortex. The calretinin/β-actin ratios ranged from 79.9 ± 9.3% to 1.3 ± 0.1%, respectively. The combination of microdissection techniques with a lysate RNase protection assay: (1) establishes this technique as quantitative for detection of high and low abundance mRNAs from microdissected brain specimens; (2) bypasses the inefficiencies and uncertainties associated with isolating RNA; and (3) enables large numbers of determinations from discrete brain nuclei to be analyzed in 2 to 3 days.

Physical and immunochemical studies of the globular domain of type IV collagen. Cryptic properties of the Goodpasture antigen.

The globular domain of type IV collagen from bovine glomerular basement membrane was isolated under nondenaturing conditions. It was shown to exist in a hexameric form comprising monomeric and dimeric subunits, with the Goodpasture antigen residing in monomer M2 and dimer D2 as previously described (Butkowski, R. J., Wieslander, J., Wisdom, B. J., Barr, J. F., Noelken, M. E., and Hudson, B. G. (1985) J. Biol. Chem. 260, 3739-3747). The epitope, however, is sequestered inside the hexamer, but becomes exposed and binds with the Goodpasture antibody upon dissociation of the hexamer into its subunits after treatment with concentrated guanidine HC1 or dilute acetic acid (pH less than 3.0). The process is completely reversible even from the denatured state. Circular dichroism studies show that the conformation of each subunit is unusually resistant to change in 6 M guanidine HC1 at 25 degrees C. This suggests that exposure of the epitope by dissociation requires minimal or no unfolding of subunits. The results provide additional evidence for localization of the Goodpasture antigen to the globular domain of type IV collagen. Moreover, these studies extend the conclusion (Weber, H., Engel, J., Wiedemann, H., Glanville, R., and Timpl, R. (1984) Eur. J. Biochem. 139, 401-410) about a tumor basement membrane, to an authentic physiological membrane, that the globular domain is a major cross-linking site in the type IV collagen matrix.

Renaturation of the allosteric phosphofructokinase from Escherichia coli.

The allosteric phosphofructokinase from Escherichia coli has been renatured after complete unfolding in concentrated guanidine hydrochloride. The enzyme regains both its catalytic and regulatory abilities quantitatively. The kinetics of reactivation are biphasic and are consistent with a two-step mechanism in which a monomolecular reaction precedes a bimolecular one. The presence of ATP during reactivation increases the rate at which phosphofructokinase is renatured; the second order rate constant of the bimolecular step increases from about 10(4) M-1 S-1 in the absence of ATP to about 2 X 10(5) M-1 S-1 in the presence of 1 mM ATP. The other ligands of the enzyme have no effect on reactivation. It is tentatively proposed that a folded monomer is the intermediate species which already possesses a functional ATP-binding site and that the rate-limiting association step is the formation of dimeric species. This interpretation is compatible with the known three-dimensional structure of another bacterial phosphofructokinase, that from Bacillus stearothermophilus.

Unfolding pathway of myoglobin. Evidence for a multistate process.

The free energy of unfolding of horse myoglobin has been calculated from the denaturation pattern induced by guanidine hydrochloride as well as by acid. The delta GH2O, i.e., the value in the absence of denaturant obtained by using the two-state transition model, was found to be 25% lower than that determined from the acid denaturation pattern, i.e., 12.0 kcal/mol, although the extent of protein denaturation produced by acid was much lower. The amount of helical structure surviving the acid-induced conformational change was estimated to be 50% of that present in the native protein, and it could be destroyed only after exposure of myoglobin samples kept at pH 3.0 to concentrated guanidine. From the guanidine denaturation pattern at acidic pH, a further variation of free energy of unfolding of 5.5 kcal/mol could be calculated, thus indicating that the overall free energy of unfolding determined from the two consecutive processes corresponds to 17.5 kcal/mol. The discrepancy between the two sets of data, i.e., guanidine unfolding at neutral pH and acid unfolding followed by addition of denaturant, has been considered to depend on the general assumption that the guanidine unfolding of myoglobin is a two-state process in the transition region. According to the recent experimental evidence showing the occurrence of at least two molecular events during the guanidine unfolding of apomyoglobin [Colonna, G., Balestrieri, C., Bismuto, E., Servillo, L., & Irace, G. (1982) Biochemistry 21, 212-215], the guanidine denaturation pattern of myoglobin was analyzed in terms of two independent steps.(ABSTRACT TRUNCATED AT 250 WORDS)

Stability of acetylcholinesterase in guanidine hydrochloride solution.

The irreversible unfolding of acetylcholinesterase (acetylcholine hydrolyase, EC 3.1.1.7) by guanidine hydrochloride was studied by difference spectral, circular dichroic, and enzyme activity measurements. At pH 7.0 and in 1.1 M denaturant solution, a conformational state in which enzyme is completely inactive was detected. It is identical to the native enzyme as far as sedimentation coefficient and molecular weight are concerned, but differs from the native molecule by a slight loss in secondary structure and by a small perturbation of aromatic residues. Acetylcholinesterase in concentrated guanidine hydrochloride solution containing beta-mercaptoethanol dissociates and exists as a random coil of molecular weight 68 000.

Cell-free translation of messenger RNAs from adult and fetal human muscle. Characterization of neosynthesized glycogen phosphorylase, phosphofructokinase and glucose phosphate isomerase.

Using a procedure of ethanol precipitation in concentrated guanidine . HCl solutions followed by chloroform/isoamylic alcohol extraction and washing in 3 M sodium acetate, we isolated high-molecular-weight cellular RNA from human fetal and adult skeletal muscle. About 500 micrograms RNA were obtained/g of fetal muscle and 50 micrograms RNA/g of adult muscle. Both RNA preparations were efficiently translated in a cell-free reticulocyte lysate system and directed synthesis of various polypeptides, one of them of Mr 200,000 probably corresponding to myosin heavy chains. Dodecylsulphate/polyacrylamide gel electrophoretic pattern of polypeptides neosynthesized using either fetal or adult RNA exhibited several differences. Three neosynthesized cytosolic muscle enzymes were purified from the translation mixtures using a micro-method of immunoaffinity chromatography; specificity of the neosynthesized polypeptides purified according to this procedure was checked by immunological competition with the corresponding unlabeled pure muscle enzymes. Successful cell-free translation of RNA from adult skeletal muscle and purification of neosynthesized human enzymes are reported for the first time. These methods, indispensable for further studies on human adult muscle gene expression, could also shed light on the mechanism of some inherited molecular diseases and tumoral or dystrophic processes.

Purification and characterization of Band 3, the major intrinsic membrane protein of the bovine erythrocyte membrane.

Band 3 from bovine erythrocyte membranes was isolated in a state of high purity by the following steps in the presence of a nonionic detergent, nonaethyleneglycol n-dodecyl ether (C12E9): (1) selective removal of Band 2.6 from ghosts by solubilization with 2% C12E9 (2) extraction of Band 3-rich fraction with 4% C12E9 from 2% C12E9-treated membrane residues, and (3) purification of Band 3 by aminoethyl-conjugated Sepharose 4B column chromatography. Human Band 3 was also purified in good yield by aminoethyl-conjugated Sepharose 4B column chromatography of erythrocyte membrane proteins solubilized with 1% C12E9 and treated with 2,3-dimethymaleic anhydride. There were no significant differences in CD spectra in C12E9, amino acid compositions, and migration mobilities in sodium dodecyl sulfate-gel electrophoresis between bovine and human Band 3. Calculations of average hydrophobicity and discriminant function demonstrated that bovine Band 3 could be categorized as a typical integral membrane protein. Bovine Band 3 showed a tendency to form a dimer and higher aggregates in 0.1% C12E9; these were resistant to dissociation into monomers in sodium dodecyl sulfate solution and, further, the protein retained residual secondary structure in highly concentrated guanidine hydrochloride solution, indicating the possible presence of an extended sequence of hydrophobic amino acid residues.

Alteration of human intrinsic factor during affinity chromatography purification using concentrated guanidine.

Alteration of human intrinsic factor during affinity chromatography purification using concentrated guanidine.

CIRCULAR DICHROISM OF HUMAN PITUITARY LUTEINIZING HORMONE AND ITS GLYCOPEPTIDES

The circular dichroic (CS) spectrum of the glycoprotein hormone, human pituitary luteinizing hormone (hLH), has been determined between 195-320 nm and resolved into gaussian constituents. Below 230 nm the CD spectrum is characterized by a negative extremum at 207 nm with a shoulder at 217 nm. Resolution into gaussian constituents of the 200-230 nm CD spectrum resulted in two resolved negative bands, one at 206 nm and the other at 215 nm. The latter band is assigned to beta-structure which is estimated to be about 25%. The 206 nm resolved band is assigned to the N-acetylated carbohydrate groups (e.g. N-acetyl glucosamine, galactosamine, and neuraminic acid). This is based partly on the evidence that the CD spectrum of the hLH glycopeptide fraction (prepared by a pronase digestion of s-carboxymethylated hLH) exhibited a negative extremum at 207.5 nm, which is close to the resolved 206 nm band in hLH. Above 230 nm the CD spectrum is characterized by a negative extremum at about 275 nm. Most of the ellipticity in this region is attributed to the disulfides in hLH. Both strong acid (0.1 N hcl) and concentrated guanidine hydrochloride (4 M) affect the ellipticity in the vicinity of 275 nm, but only the latter (as well as concentrated urea) has a major effect on the CD spectrum below 230 nm indicating extensive conformational changes. There is, however, some loss of beta-structure in 0.1 N hcl. Thus, it appears that the conformation of the hLH subunits in these subunit-dissociating agents is rather different. There was no dramatic change in the magnitude of the 207 nm extremum of native hLH between 10-50C.

Studies on phytohemagglutinins XVI. Subunit structure of the pea isophytohemagglutinins

The existence of subunits in the molecules of the two pea isophytohemagglutinins was demonstrated by sodium dodecylsulfate polyacrylamide gel electrophoresis, gel filtration and ultracentrifugation in concentrated guanidine · HCl solutions. Molecules of both pea isophytohemagglutinins (mol. wt 55 000) were found to be composed of two heavy (mol. wt 18 000) and two light (mol. wt 10 500) subunits. Subunits are bound together in the intact isophytohemagglutinin molecules apparently by non-covalent bonds. As far as molecular weight and N- and C-terminal amino acids are concerned the heavy and the light subunits of both isophytohemagglutinins are identical. The light subunit contains valine as the N-terminal amino acid and serine as the C-terminal. The C-terminal sequence Ser-Val-Leu …… has been found. Threonine is the N-terminal amino acid of the heavy subunit polypeptide chain. Unlike the native isophytohemagglutinins both the subunits are devoid of manganese and exhibit no hemagglutinating activity.

Behavior of Glutenin in Different Concentrations of Guanidine Hydrochloride

In order to determine the limiting dispersion glutenin in concentrated guanidine hydrochloride, the behavior of glutenin was observed in different concentrations of guanidine hydrochloride. Increase of intrinsic viscosity and decrease of turbidity were observed with increasing concentrations of guanidine hydrochloride up to 6 m and a limiting state was attained above 6 m. The molecular weight determination also gave a limiting value above 6 m. The values obtained were 850,000 for one preparation and 640,000 for another. While the turbidity and viscosity in 6.5 m guanidine hydrochloride were constant in the pH region from 4 to 5.5, they increased slightly around pH 6 and decreased toward pH 7. The viscosity of glutenin around pH 7 was lower than that around pH 4, but the same molecular weight was obtained at pH 4.0 and 6.9.

Studies on the Structure and Function of Muscle Aldolase: V. MOLECULAR CHARACTERIZATION OF THE ENZYME FROM SHARK (MUSTELUS CANIS)

Abstract Sedimentation equilibrium experiments conducted under neutral conditions (0.05 m KPO4, pH 7.0) show that native shark muscle aldolase has a mass of approximately 160,000 g per mole. Upon exposure to an acidic environment (0.1 m glycine, pH 2.35) or to a solution of concentrated guanidine hydrochloride (6 m), the mass drops to about 40,000 g per mole, indicating the tetrameric nature of the enzyme's quaternary structure. Fingerprint analyses after tryptic digestion suggest that each of the aldolase subunits is similar in chemical composition. This hypothesis finds further substantiation in end group analyses which show that the carboxyl- and amino-terminal residues of each chain are tyrosine and proline, respectively. The loss of aldolase activity toward fructose 1,6-diphosphate in the presence of carboxy-peptidase A correlates with the liberation of the enzyme's carboxyl-terminal tyrosines. Despite the many striking similarities found between shark and rabbit muscle aldolase, there are large differences in the amino acid compositions of these two homologous proteins.