Guanidine Hydrochloride Solutions Research Articles

Oligonucleotide Probes That Hybridize with rRNA as a Tool To Study Frankia Strains in Root Nodules

Oligonucleotide probes that hybridize with specific sequences in variable regions of the 16S rRNA of the nitrogen-fixing actinomycete Frankia were used for the identification of Frankia strains in nodules. Frankia cells were released from plant tissue by grinding glutaraldehyde-fixed root nodules in guanidine hydrochloride solution. rRNA was obtained after sonication, precipitation with ethanol, and purification by phenolchloroform extraction. Degradation of rRNA, evident in Northern blots, did not affect hybridization with the oligonucleotides. Nodules of about 1 mg (fresh weight) provided sufficient rRNA for reliable detection of the Frankia strain. The utility of this rRNA extraction method was tested in a competition experiment between two effective Frankia strains on cloned Alnus glutinosa plants.

Isolation and analysis of ribonucleic acids from skeletal tissues

We report a method for the isolation of total cellular RNA from mineralized or cartilaginous tissues. The procedure accommodates the large amount of hydroxyapatite and high buoyant density proteoglycans present in skeletal tissue samples, as well as the low cell density characteristic of these tissues. The procedure can be reliably used for processing a large number of small (100–800 mg) tissue samples. Tissues are homogenized in guanidine hydrochloride solution, then centrifuged at low speed, and filtered to remove the nonsolubilized extracellular matrix proteins. Subsequent high speed density gradient centrifugation produces a high yield of RNA (0.2–0.6 μg RNA/mg tissue) which is precipitated in a low pH sodium acetate solution. RNA extracted by this method has been analyzed for the expression of various genes by Northern blotting. In addition to mRNAs of bone- and cartilage-specific proteins, messenger RNA for growth factors, proto-oncogenes, and heat shock proteins can be detected.

Isolation of domain-sized fragments of bovine serum albumin by limited peptic digestion and their secondary structural changes in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate

Three fragments of bovine serum albumin (BSA) by limited peptic hydrolysis were isolated and their positions in the parent molecule were identified on the basis of the analyses of plural amino acid residues at N- and C-terminals of each fragment. The sequences of the three fragments correspond to residue Nos. 1–306, 193–368, and 383–511. The molecular weights of the three fragments were determined to be 34,900, 20,100, and 14,700 from their sequences. As is well known, BSA consists of three domains, each comprising three subdomains (disulfide double loops) and containing about 190 amino acid residues with a molecular weight of approximately 22,000. The present three fragments approximately corresponded to regions representing 1.5, 1, and 0.5 domains of BSA based on their positions in the parent molecule and their molecular weights. The relative proportions of a-helix, β-structure, and disordered structure in conformations of the fragments were determined by the curve-fitting method of circular dichroism (CD) spectra. The a-helical portions occupied 50–60% in these fragments. Most of the helices in the intact BSA molecule appeared to remain after digestion into the fragments. The helical proportions of the fragments decreased stepwise in SDS solutions in the same way as intact BSA. This suggests that the decrease in helical content of BSA might represent the sum of the reductions in helical content of the three domains by SDS binding. The helices in the fragments resisted structural changes by dilute urea and guanidine in a manner similar to that of BSA.

Secondary structural changes in the intact and the disulfide bridges cleaved beta-lactoglobulin A and B in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate.

The relative proportions of alpha-helix, beta-sheet, and unordered form in beta-lactoglobulin A and B were examined in solutions of urea, guanidine, and sodium dodecyl sulfate (SDS). In the curve-fitting method of circular dichroism (CD) spectra, the reference spectra of the corresponding structures determined by Chen et al. (1974) were modified essentially according to the secondary structure of beta-lactoglobulin B predicted by Creamer et al. (1983), i.e., that the protein has 17% alpha-helix and 41% beta-sheet. The two variants showed no appreciable difference in structural changes. The reduction of disulfide bridges in the proteins increased beta-sheet up to 48% but did not affect the alpha-helical proportion. The alpha-helical proportions of nonreduced beta-lactoglobulin A and B were not affected below 2 M guanidine or below 3 M urea, but those of the reduced proteins began to decrease in much lower concentrations of these denaturants. By contrast, the alpha-helical proportions of the nonreduced and reduced proteins increased to 40-44% in SDS. The beta-sheet proportions of both nonreduced and reduced proteins which remained unaffected even in 6 M guanidine and 9 M urea, decreased to 24-25% in SDS.

Tenascin/hexabrachion in human skin: biochemical identification and localization by light and electron microscopy.

Tenascin/hexabrachion is a large glycoprotein of the extracellular matrix. Previous reports have demonstrated that tenascin is associated with epithelial-mesenchymal interfaces during embryogenesis and is prominent in the matrix of many tumors. However, the distribution of tenascin is more restricted in adult tissues. We have found tenascin to be present in normal human skin in a distribution distinct from other matrix proteins. Immunohistochemical studies showed staining of the papillary dermis immediately beneath the basal lamina. Examination of skin that had been split within the lamina lucida of the basement membrane suggested a localization of tenascin beneath the lamina lucida. In addition, there was finely localized staining within the walls of blood vessels and in the smooth muscle bundles of the arrectori pilorem. Very prominent staining was seen around the cuboidal cells that formed the basal layer of sweat gland ducts. The sweat glands themselves did not stain. The distribution of tenascin in the papillary dermis was studied at high resolution by immunoelectron microscopy. Staining was concentrated in small amorphous patches scattered amongst the collagen fibers beneath the basal lamina. These patches were not associated with cell structures, collagen, or elastic fibers. Tenascin could be partially extracted from the papillary dermis by urea, guanidine hydrochloride, or high pH solution. The extracted protein showed a 320-kD subunit similar to that purified from fibroblast or glioma cell cultures. We have developed a sensitive ELISA assay that can quantitate tenascin at concentrations as low as 5 ng/ml. Tests on extracts of the papillary dermis showed tenascin constituted about 0.02-0.05% of the protein extracted.

Features of collagen matrix reconstructed with proteodermatan sulfate from pigskin insoluble collagen.

Pigskin insoluble collagen was step-wise extracted with 0.5 m and 4 m guanidine hydrochloride (GuHCl) solution. Two proteoglycan preparations were obtained by DEAE-Toyopearl chromatography of the extracts, PG-I from 0.5 m GuHCl extract and PG-II from 4 m GuHCl extract. The proteoglycan preparations both contained at least two components having different mobilities on SDS-PAGE (Mr, about 60 × 103 and 90 × 103), and were identified as proteodermatan sulfate (PDS) by two-dimensional electrophoresis on a cellulose acetate membrane. The two proteodermatan sulfates (PG-I and PG-II) accelerated the rate of the reconstruction of collagen matrix from neutral collagen solution, while the accelerating effect of PG-I was greater than that of PG-II. Electron microscopic observation showed that collagen matrices reconstructed with the PDSs consisted of thick fibrils with the banding pattern of native type and thin fibrils with an indistinct banding pattern. Collagen matrix reconstructed with addition of PDSs showed low...

Secondary structural changes of non-reduced and reduced ribonuclease A in solutions of urea, guanidine hydrochloride and sodium dodecyl sulfate

The secondary structures of ribonuclease A (RNAase A) before and after reduction of the disulfide bridges and blockage of the thiol groups with iodoacetamide were examined in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate (SDS). The relative proportions of α-helix, β-structure, and disordered structure were estimated by the curve-fitting method of circular dichroism (Chen, Y.H., Yang, J.T. and Chau, K.H. (1974) Biochemistry 13, 3350–3359). The native RNAase A, with the disulfide bridges intact, contained 19% helix and 38% β-structure. Reduction of its disulfide bridges led to a decrease in the proportion of these structures to 9% for the α-helix and 17% for the β-structure. The non-reduced RNAase A resisted unfolding in low concentrations of urea and guanidine hydrochloride. The β-structure which remained after reduction appeared to be stable even in solutions of 6 M guanidine and 9 M urea. A considerable amount of the β-structure in both the non-reduced and the reduced RNAase A remained unaffected by high concentrations of SDS.

Separation of lipid-free egg yolk proteins by high-pressure liquid chromatography using solvents containing formic acid

A method for obtaining total protein patterns from lipid-containing systems, in particular egg yolk, is described. After dispersion of the yolk in 8 m guanidine hydrochloride solution, lipid is removed by extraction with chloroform-methanol and petrol. The protein solution is applied to a high-pressure liquid chromatograph and eluted with a gradient of formic acid, isopropanol, and acetonitrile. In measurements on a known yolk protein, duck apovitellenin I, the method did not cause irreversible formylation of N-terminal or other residues. The method was used (1) to compare protein patterns of whole yolk from hen and quail eggs; (2) to isolate and partially sequence quail apovitellenin I; and (3) to compare protein patterns of “white yolk” and “yellow yolk”.

Secondary structure changes of disulfide bridge-cleaved bovine serum albumin in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate

Secondary structure of bovine serum albumin, disulfide bridges of which were reduced and blocked by iodoacetamide, was examined in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate. The profile of structural changes in the reduced albumin was compared with that in the unreduced protein with the intact disulfide bridges. The relative proportions of α-helix, β-structure, and random coil were estimated by simulating a mixed circular dichroism (CD) spectrum of reference spectra of the corresponding structures to the experimentally obtained spectrum of the protein. The helical proportion was 25% for the reduced albumina dn the proportion increased up to 50% in a dodecyl sulfate solution. On the other hand, the helical proportion is 66% for the unreduced protein and it decreases down to 50% by the addition of the identical surfactant [K. Takeda et al., J. Colloid Interface Sci. 117, 120 (1987)]. The helical proportions of both the reduced and the unreduced proteins decreased in the presence of urea and guanidine. the unreduced albumin resisted the unfolding of the helix below 4 M urea and a 1.5 M guanidine. In contrast, the helical proportion in the reduced protein began to decrease even in lower concentrations of the two denaturants.

The major antigen of elastin-associated microfibrils is a 31-kDa glycoprotein.

The major antigen derived from elastic fiber microfibrils was identified as a Mr = 31,000 glycoprotein, using immunoblotting and immunohistochemical techniques with antisera raised to "reductive guanidine extracts" of fetal bovine nuchal ligament, and to subfractions of these. A second, elastic fiber-derived, but unidentified, antigen of large molecular size (Mr greater than 200,000) was present in these extracts. Antisera raised to the purified 31-kDa glycoprotein were shown, by immunoelectron microscopy, to localize specifically to the elastin-associated microfibrils. Thus, the macro-molecule was called "microfibril-associated glycoprotein" or MAGP. MAGP is an acidic glycoprotein with a distinctive amino acid composition, being exceptionally rich in glutamic acid, rich in cystine, and low in glycine. MAGP was extractable from tissue homogenates using NaCl, urea, or guanidine hydrochloride solutions, only if a strong reducing agent was present. Thus, disulfide bonding is important for the strong association of MAGP with elastic fibers. Immunoblotting with anti-MAGP antiserum identified two additional reactive species, of Mr = 60,000 and Mr approximately 300,000, in tissue extracts. As only the 31-kDa species was detected in fibroblast culture medium, these additional species were probably aggregates, rather than precursors. MAGP did not react with antilysyl oxidase antiserum on immunoblots or by enzyme-linked immunosorbent assay. MAGP is the first macromolecule to have been established to be a constituent of elastin-associated microfibrils in both developing and mature elastic tissues.

Spin-label study of the conformational states of polypeptide models of histones

The conformational states of the regular polypeptides (Gly-Lys-Gly)n, (Ala-Orn-Gly)n, and (Ala-Orn-Ala)n have been studied by the spin-label method. Their behavior in solutions of guanidine hydrochloride and urea and in solutions of salts of bivalent metals does not contradict the presence of an extended levohelical conformation in their polypeptide chains. In CaCl2 (5 N) solutions the polypeptides exhibit aggregation properties. A study of the behavior of the poly peptide at these temperatures has shown that with a rise in temperature there is a monotonic change in the structures of the polypeptide chains that is characteristic for a conformation of the polyproline-II type. Differences have been observed in the behavior of glycine-and alanine-containing polypeptides in the presence of sodium dodecyl sulfate with a change in the temperature.

Partial specific volume and interaction of glycinin with solvent components in urea and guanidine hydrochloride

The interaction of glycinin with urea/guanidine hydrochloride was investigated by determining the apparent partial specific volume of the protein in these solvents. The apparent partial specific volume was determined under both isomolal and isopotential conditions. The preferential interaction parameter with solvent components had values of 0.10 and 0.11 g urea and guanidine hydrochloride respectively per g of protein. In both the cases the interaction was not preferential with water. The total binding of urea/guanidine hydrochloride to glycinin was calculated. Excellent correlation between the calculated value and the total number of peptide bonds and aromatic amino acids residues of protein was observed. The changes in volume upon transferring glycinin from dilute salt solution to urea and guanidine hydrochloride solution were also calculated.

Isolation, fractionation, and preliminary characterization of a novel class of sulfated glycans from the tunic of Styela plicata (Chordata Tunicata).

The sulfated glycans in the tunic of Styela plicata differ from the glycosaminoglycans of animal tissues and also from the sulfated polysaccharides isolated from marine algae. The ascidian glycans occur primarily as three fractions that differ markedly in molecular weight and chemical composition. The high molecular weight fraction encompasses a broad range of molecular weights but is chemically homogeneous and contains an unusual amount of galactose. The 20,000 molecular weight polysaccharide is rich in galactose and glucose while the 8,000 molecular weight fraction is rich in amino sugars and contains the neutral hexoses galactose, glucose, and mannose. All fractions contain large amounts of sulfate esters. The ascidians polysaccharides can be extracted from the tissue by proteolytic enzyme or by guanidine hydrochloride solutions. The high molecular weight fraction is preferentially extracted by papain while guanidine hydrochloride removes mainly the low molecular weight polysaccharides. We speculate that these sulfated glycans are essential for maintaining the structural integrity of the tunic, in analogy with the glycosaminoglycans of vertebrate connective tissues.

Association-dissociation of glycinin in urea, guanidine hydrochloride and sodium dodecyl sulphate solutions

The effect of urea, guanidine hydrochloride and sodium dodecyl sulphate on glycinin, the high molecular weight protein fraction from soybean has been investigated by analytical ultracentrifugation. Urea and guanidine hydrochloride dissociate the protein to a ‘2S’ protein through the intermediary 7S and 4S proteins. Howeαer, in sodium dodecyl sulphate the protein directly dissociates to a 2S protein. Analysis of the data by calculation of per cent fraction and S20,w value indicates that dissociation and denaturation of glycinin occur simultaneously in the presence of the aboαe reagents but to different extents.

Purification and some properties of prothoracicotropic hormone in the silkworm, Bombyx mori

Prothoracicotropic hormone was purified from the heads of Bombyx mori so that the nature of the hormone could be elucidated. The hormone extracted from acetone powder with 2% NaCl at pH 6.8 was partially purified by heat treatment, followed successively by ammonium sulphate precipitation, acetone fractionation and picric acid precipitation. Further purification was carried out by successive column chromatography on DEAE-cellulose, CM-Sephadex C-50, hydroxylapatite and Bio-Gel P-60. Finally, the hormone was isolated by preparative polyacrylamide gel disc-electrophoresis. These purification steps effected a 1700-fold increase in specific activity with a 4% yield of hormone preparation. From 1.5 × 10 6 heads, 5.3 mg of the hormone with a specific activity of 100,000 units per mg was isolated. The hormone labelled with 125I appeared as a single band with an apparent molecular weight of 18,000 on SDS-polyacrylamide gel electrophoresis. No loss of hormonal activity occurred in 6 M urea and 6 M guanidine hydrochloride solution. The isolated hormone was capable of inducing adult development of a debrained, dormant Bombyx pupa at a dose of 10ng, and was evidently different from the prothoracicotropic hormone (mol. wt 4400) obtained from the adult heads of Bombyx mori by Nagasawa et al. (1979) [ Devl. Growth Differ. 21, 29–38], which induced adult development of a brainless Samia pupa alone.

Effect of temperature on the conformation of soybean glycinin in 8 M urea or 6 M guanidine hydrochloride solution

The effect of temp. on soybean glycinin in 8M urea or 6M guanidine hydrochloride solution was studied by circular dichroism and optical rotatory dispersion measurements. At room temp. the protein assumes an unordered conformation in the presence of the denaturant. With increase in temp., mean residue ellipticity in the range 250-210 nm assumes more negative values, suggesting formation of ordered conformations at higher temperatures.

Brain-derived fibroblast growth factor: A study of its inactivation

Brain fibroblast growth factor has been identified as a component of myelin basic protein. Its activity is destroyed when treated with a variety of solvents including dilute acid, organic solvents and solutions of guanidine hydrochloride. These conditions do not alter the encephalitogenic properties of myelin basic protein.

Histone H1: Ultracentrifugation studies of the effects of ionic strength and denaturants on the solution conformation

The conformation of histone H1 has been examined under native and denaturing conditions in the absence of DNA or chromatin. Sedimentation coefficients were determined for Histone H1 in 0.1 m KCl and in 6 m guanidine hydrochloride solutions at pH 7.4. The influence of ionic strength on the conformation of histone H1 has been determined by measurement of the sedimentation coefficient in tetramethylammonium chloride solutions of up to 2.5 m and extrapolated to infinite ionic strength. Results from these experiments suggest that the native conformation of histone H1 is very asymmetric in shape. The molecule is best described as a prolate ellipsoid with axes of 312 Å (2a) and 16 Å (2b) in low ionic strength media and also as a prolate ellipsoid with axes of 202 Å (2a) and 20 Å (2b) at high ionic strength or when associated with polyanions, e.g., DNA. Denaturation of histone H1 by guanidine hydrochloride was found to be completely reversible. In 6 m guanidine hydrochloride, the H1 molecule collapses to a sphere but the original extended conformation of the protein is readily restored on dialysis. This suggests rigid conformational requirements for the H1 molecule as incorporated into chromatin. The shape and dimensions for the H1 molecule at high ionic strength are not sufficiently conclusive to locate H1 in the chromatin structure. It is proposed, however, that viable models for chromatin architecture must be consistent with the histone H1 solution dimensions obtained here.

The Spectrofluorometric Determination of 9,10-Phenanthrenequinone

Abstract 9,10-Phenanthrenequinone (9,10-PQ) reacts with guanidine in an alkaline medium to form 2-amino-1H-phenanthro[9,10-d]imidazole. The reaction can be used for the fluorometric determination of 9,10-PQ, since the reaction product is fluorescent in an acidic solution. A dimethyl sulfoxide-methanol mixture is suitable as the solvent. A sample solution was allowed to stand for 15 min at room temperature after the additions of guanidine hydrochloride and sodium methoxide solutions, followed by the addition of hydrochloric acid. After the solution had been allowed to stand for 20 min, the fluorescence intensity of the solution was measured at 403 nm with excitation at 363 nm. The fluorescence intensity was stable for at least 8 h. The determination limit of 9,10-PQ was 50 ng.

Thermodynamic characterization of hog kidney D-amino acid oxidase apoenzyme in concentrated guanidine hydrochloride solution. Preferential interaction with the solvent components and the molecular weight of the monomeric unit.

This paper describes the physical characterization of the monomeric unit of hog kidney D-amino acid oxidase apoenzyme in 6 M guanidine hydrochloride (GuHCl) solution by means of differential refractometry, densimetry, light scattering, equilibrium sedimentation, and high-speed gel filtration chromatography. In 6 M GuHCl solution, the oxidase interacts preferentially with GuHCl: the values of the preferential interaction parameter are 0.11 +/- 0.03 (S.D.) g/g of protein by densimetry and 0.14 +/- 0.04 g/g of protein by refractometry. The volume change, delta V, of the oxidase on transfer from the native to the denatured state is -350 ml/mol. The molecular weight of the monomeric apoenzyme is 39,600 +/- 1,700 by light scattering and 38,000 +/- 1,200 by high-speed equilibrium sedimentation. The values of the molecular weight estimated by the empirical methods, i.e., sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and high-speed gel filtration chromatography in 6 M GuHCl, agree well with those obtained by the thermodynamic methods mentioned above. These results confirm definitely that the complex of the apoenzyme with SDS normally behaves in the same manner as those of standard proteins in SDS-gel electrophoresis. This is also supported in this study by the analysis of the electrophoretic data at several gel concentrations by Ferguson plots. The molecular weight of quasi-D-amino acid oxidase apoenzyme was also examined by the empirical methods.