Difference of molecular association in two types of curdlan gel

The molecular association in a curdlan gel formed by neutralizing an alkaline solution of curdlan with carbon dioxide was compared with those in gels obtained by heating aqueous suspensions of curdlan at various temperatures.

The neutralized and 60°C-set preparations were soluble in 0·01 sodium hydroxide, whereas preparations set at above 90°C were soluble only in concentrations of sodium hydroxide above 1 . The absorption of Aniline blue or Congo red to the preparations decreased with an increase in the temperature of heat treatment and the adsorption to a gel heated at 120°C for 4 h was about 30% of that for the unheated neutralized gel. Seventy-three per cent of the heated preparation was resistant to treatment with 32% sulfuric acid at 32°C for 30 days, whereas none of the neutralized gel was resistant. An electron micrograph of the resistant part of the curdlan showed that it had a pseudocrystalline form. X-ray studies showed a much higher crystalline structure in the resistant part than in the preparation without heat treatment. The X-ray patterns were almost the same for preparations treated with 32% sulfuric acid or (1 → 3)-β-glucanase.     

Curdlan gels as protein drug delivery vehicles

The use of curdlan, a natural -1,3-glucan, in protein drug delivery vehicles was studied by carrying out in vitro release studies with curdlan gels containing bovine serum albumin (BSA) as a model protein. Addition of urea (8 M) decreased the gel formation temperature to 37°C. Curdlan was hydroxyethylated in order to form gels under mild conditions such as physiological temperature and pH. In gels formed in 8 M urea solution, urea was almost released after 2 h while BSA was completely released after 45–100 h. The total time for complete release of BSA increased with curdlan concentration within gels. The strength of hydroxyethylated curdlan gels (385.7 dyne cm^–2) was weaker than that of curdlan gels formed in 8 M urea solution (6277 dyne cm^–2).     

Gel-Electrophoretic Separation, Detection, and Characterization of Plant and Bacterial UDP-Glucose Glucosyltransferases

We have developed procedures for detection and characterization of UDP-glucose: glucosyltransferases following electrophoretic separation in nondenaturing polyacrylamide gels. Using digitonin-solubilized membrane protein preparations from a variety of plants and two cellulose-producing bacteria, activity can be demonstrated for several UDP-glucose:β-glucan synthases with an in situ assay following gel electrophoresis. These enzymes can be characterized within the gels with respect to effector requirements and products produced, and several advantages of this assay over solution assays are demonstrated. For example, the clear dependence of plant UDP-glucose:(1→3)-β-glucan synthase on both Ca²⁺ and a β-linked glucoside is shown; bacterial cellulose synthases show direct stimulation within the gel by guanyl oligonucleotide, and the Acetobacter xylinum enzyme appears more stable in the gel assay than in solution assay.     

Mitogenic activity of particulate yeast β-(1 → 3)-d-glucan and its water-soluble derivatives

Particulate β-d-glucan was isolated from baker's yeast using autolysis and delipidization of the cells, followed by alkaline and acid treatment. The residual water-insoluble glucan termed cerevan has a β-(1→ 3)-linked backbone with β-(1 → 6)-linked short side chains. In order to achieve water solubility of the glucan, various derivatives were prepared (car☐ymethyl-, car☐yethyl-, hydroxyethyl-, sulfoethyl-), and the β-glucan was oxidized to glucuronoglucan. Their solubility, degree of substitution (DS), and molecular weight distribution (M_w) were compared. The immunomodulatory activity of these preparations was investigated in mitogenic and co-mitogenic tests on rat thymocytes. Cerevan showed higher stimulation indices compared with the known immunomodulator zymosan. Of the water-soluble derivatives, sulfoethylglucan was found to be the most active. Of the car☐ymethyl derivatives of various DS, the preparation with DS=0.75 exhibited the highest activity. Water-soluble car☐ymethyl preparations with DS > 1.0 and low-molecular-weight glucuronoglucan were inactive.     

Rapid isoelectric focusing in a vertical polyacrylamide minigel system

A rapid method is described for the resolution of proteins employing isoelectric focusing in a vertical polyacrylamide minigel system. Isoelectric focusing can be performed in only 3 h, utilizing low voltage, under either native conditions or denaturing conditions in the presence of 8 M urea. The procedure permits the application of larger sample volumes containing more protein than other isoelectric focusing procedures, and provides the additional advantages of slab gels over tube gels for analytical purposes. The procedure is also well adapted for use in two-dimensional electrophoretic techniques, making it possible to complete a two-dimensional gel in 1 day.     

Additional components of bovine heart cytochrome c oxidase demonstrated by high-resolution polyacrylamide-gel electrophoresis in the presence of chloral hydrate.

We have shown that aq. 100% (w/v) chloral hydrate (2,2,2-trichloroethane-1,1-diol) dissociates bovine heart cytochrome c oxidase. We have developed new procedures of polyacrylamide-gel electrophoresis in the presence of chloral hydrate that permit variation in the pH of the separation, and, by using these procedures, we have observed 15 components in preparations of the enzyme. This number contrasts with the eight bands that were seen on electrophoresis in the presence of SDS (sodium dodecyl sulphate) and urea. We have isolated material from these eight bands and have characterized each by electrophoresis in the presence of chloral hydrate. Twelve of the fifteen components that were seen by electrophoresis in chloral hydrate were identified as constituents of the eight bands seen by electrophoresis in the presence of SDS and urea. Two-dimensional electrophoretic separations confirmed these identifications ans showed that the other three components which were resolved as discrete bands by electrophoresis in the presence of chloral hydrate appeared to be diffusely present in the electrophoretic separations performed in the presence of SDS and urea, which suggested anomalous behaviour in that detergent. Trypsin treatment of cytochrome c oxidase caused total loss, as observed by electrophoretic separations in the presence of chloral hydrate, of a number of components. The trypsin-sensitive components included all of those that behaved anomalously in the presence of SDS and urea. Chloral hydrate is a potent non-ionic dissociating agent for cytochrome c oxidase and its use in polyacrylamide-gel electrophoresis, with variation in the pH of the gel, permits charge-dependent separations that should have general application in the analysis of membrane proteins.     

Specific dimerization of the light chains of human immunoglobulin

1. The light chains of human immunoglobulin were allowed to dimerize in vitro on removal of the dispersing agents acetic acid or urea. 2. On electrophoresis in polyacrylamide gel at pH8.8 the dimers yielded up to nine regularly spaced bands. This approximates to the number of electrophoretic components known to occur among the monomers. 3. Single electrophoretic components of the dimers were isolated from the gel, dissociated into monomers, and subjected as such to electrophoresis in urea-containing gels. Each gave two adjacent bands. 4. Similarly, after all the light chains as monomers had been subjected to electrophoresis in urea-containing gels, single electrophoretic components were isolated and allowed to dimerize. When examined now as dimers in the absence of urea, each component gave two adjacent bands. 5. These findings are explicable on the following basis. (a) The dimerization of the light chains is specific, at least inasmuch as it occurs between monomers of the same electrophoretic mobilities. (b) With the buffer constant, different light chains undergo different changes in net charge on being transferred from urea-containing to urea-free solution; in this way two different chains of the same initial charge can acquire a charge difference of 1. 6. Experiments with Bence-Jones proteins and other homogeneous light chains gave results substantiating the conclusions (a) and (b).     

Substrate-containing gel electrophoresis: sensitive detection of amylolytic, nucleolytic, and proteolytic enzymes

Electrophoresis of hydrolytic enzymes under nondenaturing conditions on acrylamide gels containing the appropriate high-molecular-weight substrates entrapped on the gel has been explored as a general method for sensitive enzyme resolution and detection. Under electrophoresis conditions of optimal enzyme activity, the enzymes may bind tightly to the fixed substrate and can only migrate in the electrophoretic field as the substrate is hydrolyzed. When the gels after electrophoresis in this “binding mode” are stained with substrate-detecting reagents, clear tracks of enzyme migration are observed, and the length of each track is a function of the amount of enzyme present in that track. Multiple forms of a given enzyme activity have not been and are not likely to be observed under these conditions. Under electrophoresis conditions of minimal (or suboptimal) enzyme activity, the enzymes do not bind to the fixed substrate and their mobility in the electrophoretic field does not appear to be significantly affected by the presence of substrate. After electrophoresis in this “nonbinding mode” the gels are incubated under conditions of optimal enzyme activity to allow substrate hydrolysis to take place before they are stained with substrate-detecting reagents, and active enzymes are detected as clear bands. Multiple forms of a given activity which were resolved during electrophoresis in the nonbinding mode are reflected by the presence of individual bands. The substrate-containing gel electrophoresis technique does not appear to be amenable to precise quantification of enzymes. By comparing the length of the clear tracks or the degree of staining of the activity bands for a range of enzyme concentrations, however, it is possible to establish the smallest amount of enzyme that can unequivocally be detected under a given set of conditions; from such studies we estimate that the sensitivity of detection with the substrate-containing gel electrophoresis technique can be orders of magnitude better than that obtained with other methods. The levels of detection observed in the work presented here were about 50 pg for α-amylase run on starch-containing gels, 1 pg to 1 ng for nucleases run on DNA- or RNA-containing gels, and 100 pg to 10 ng for 11 different pure and crude protease preparations run on gels containing heat-denatured bovine serum albumin.     

Relationships between conformation of β-lactoglobulin in solution and gel states as revealed by attenuated total reflection Fourier transform infrared spectroscopy

Attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR) has been used to compare the structure of β-lactoglobulin, the major component of whey proteins, in solution and in its functional gel state. To induce variation in the conformation of β-lactoglobulin under a set of gelling conditions, the effect of heating temperature, pH, and high pressure homogenization on the conformation sensitive amide I band in the infrared spectra of both solutions and gels has been investigated. The results showed that gelification process has a pronounced effect upon β-lactoglobulin secondary structure, leading to the formation of intermolecular hydrogen-bonding β-sheet structure as evidenced by the appearance of a strong band at 1614 cm⁻¹ at the expense of other regular structures. These results confirm that this structure may be essential for the formation of a gel network as it was previously shown for other globular proteins. However, this study reveals, for the first time, that there is a close relationship between conformation of β-lactoglobulin in solution and its capacity to form a gel. Indeed, it is shown that conditions which promote predominance of intermolecular β-sheet in solution such as pH 4, prevent the formation of gel in conditions used by increasing thermal stability of β-lactoglobulin. On the basis of these findings, it is suggested that by controlling the extent of intermolecular β-structure of the protein in solution, it is possible to modify the ability of protein to form a gel and as a consequence to control the properties of gels.     

Visualization by chilling of protein bands in polyacrylamide gels containing 8 M urea: preparation and quantitation of foot-and-mouth disease virus capsid proteins

Protein bands become visible in polyacrylamide gels containing 8 m urea after chilling the gels in air for 5 to 10 min at ?70°C. Urea appears to crystallize preferentially as opaque bands in regions of the gel where protein reduces the amount of free water available as solvent for the urea molecules. Thus detected, the gel sections containing protein bands from foot-and-mouth disease virus can be immediately cut out, and their proteins obtained by electrophoretic elution or extraction procedures. Analysis of the proteins for purity and concentration is then carried out by electrophoresing measured aliquots on analytical gels, staining with Coomassie brilliant blue, scanning the gels for absorbance at 600 nm, and converting peak areas to micrograms of protein using Folin phenol standard curves determined for each purified capsid protein. The most basic capsid protein and its in virion proteolytic-cleavage products stain metachromatically.     

Rheology and fracture of mixed ι- and κ-carrageenan gels: Two-step gelation

It is shown that under certain circumstances, on cooling mixed ι- and κ-carrageenan solutions, the two forms gel separately at different temperatures, with the ι form gelling first. This ‘two-step gelation’ was only observed when both sodium and potassium ions were present, with a sodium/potassium mole ratio of between 1 and 100. For such mixed gels, a κ fraction as low as 2·5% of the total carrageenan has significant effects on their rheology, both at low deformation and fracture. In these systems, the κ form, gelling in the presence of an existing ι gel, produces measurable rheological effects at much lower concentrations than if it were alone. This behaviour can be used as a sensitive test of the ‘rheological purity’ of samples of ι-carrageenan.     

Visualization by chilling of protein bands in polyacrylamide gels containing 8 urea: Preparation and quantitation of foot-and-mouth disease virus capsid proteins

Protein bands become visible in polyacrylamide gels containing 8 urea after chilling the gels in air for 5 to 10 min at −70°C. Urea appears to crystallize preferentially as opaque bands in regions of the gel where protein reduces the amount of free water available as solvent for the urea molecules. Thus detected, the gel sections containing protein bands from foot-and-mouth disease virus can be immediately cut out, and their proteins obtained by electrophoretic elution or extraction procedures. Analysis of the proteins for purity and concentration is then carried out by electrophoresing measured aliquots on analytical gels, staining with Coomassie brilliant blue, scanning the gels for absorbance at 600 nm, and converting peak areas to micrograms of protein using Folin phenol standard curves determined for each purified capsid protein. The most basic capsid protein and its in virion proteolytic-cleavage products stain metachromatically.     

Nematode chromosomal proteins--I. Isolation of chromatin and preliminary characterization of the chromosomal proteins of Caenorhabditis elegans.

1. A procedure is described which gives clean chromatin preparations from the free-living nematode Caenorhabditis elegans. It involves homogenization using glass beads, collection of the precipitate from a low speed centrifugation, removal of cell membranes with Triton X-100, several washes with 0.14 M NaCl, sucrose density gradient centrifugation, a cycle of extraction and reprecipitation using dilute Tris buffer and 0.14 M NaCl respectively, and final extraction of the purified deoxyribonucleoprotein in 10 mM Tris-HCl (pH 8). 2. Acidic urea gel electrophoresis of the histones from C. elegans yielded 4 main groups which were preliminary identified as H1, H2a (+ H3?), H2b, H4 and moved on the gels in that order of increasing mobility. the coincidence of histone H3 with H2a was putative, but its presence was firmly suggested by the generation of a dimeric form in oxidizing conditions. 3. By SDS-Tris-glycine gel electrophoresis of the non-histone chromosomal proteins of C. elegans, about 18 proteins were distinguished with molecular weights ranging from 15,000 to 100,000 daltons.     

Secreted adenylate cyclase of Bordetella pertussis: calmodulin requirements and partial purification of two forms.

The extracellular adenylate cyclase of Bordetella pertussis was partially purified and found to contain high- and low-molecular-weight species. The high-molecular-weight form had a variable molecular weight with a peak at about 700,000. The smaller species had a molecular weight of 60 to 70,000 as determined by gel filtration. The low-molecular-weight form could be derived from the high-molecular-weight species. The high-molecular-weight complex purified from the cellular supernatant was highly stimulated by calmodulin, while the low-molecular-weight enzyme was much less stimulated. Active enzyme could be recovered from sodium dodecyl sulfate (SDS) gels at positions corresponding to molecular weights of about 50,000 and 65,000. Active low-molecular-weight enzyme recovered from SDS gels migrated with a molecular weight of about 50,000, which coincides with a coomassie blue-stained band. However, when both high- and low-molecular weight preparations were analyzed in 8 M urea isoelectrofocusing gels, the enzyme activity recovered did not comigrate with stained protein bands. The enzyme recovered from denaturing isoelectrofocusing or SDS gels was activated by calmodulin, indicating a direct interaction of calmodulin and enzyme. The high-molecular-weight form of the enzyme showed increasing activity with calmodulin concentrations ranging from 0.1 to 500 nM, while the low-molecular-weight form was fully activated by calmodulin at 20 nM. Adenylate cyclase on the surface of living cells was activated by calmodulin in a manner which resembled that found for the high-molecular-weight form.     

Small nuclear RNAs from Drosophila KC-H cells; characterization and comparison with mammalian RNAs

Small molecular weight nuclear RNAs were extracted from cultured Drosophila KC-H cells and characterized by their electrophoretic mobilities in 5–15% gradient acrylamide gels or in 10% acrylamide-7 M urea gels. Comparison between the electrophoretic profiles of these SnRNAs with those from human and mouse cells revealed striking similarities and allowed for assignation of band nomenclatures as established for mammalian cells. Comparison of mobilities in the two gel systems also permitted correspondence between the different nomenclatures established by various groups for this class of RNAs, as well as an approximate estimate of their molecular sizes.     

Gel electrophoresis in studies of protein conformation and folding

Electrophoresis through polyacrylamide gels is a useful method for distinguishing conformational states of proteins and analyzing the thermodynamic and kinetic properties of transitions between conformations. Although the relationship between protein conformation and electrophoretic mobility is quite complex, relative mobilities provide qualitative estimates of compactness. Conformational states which interconvert slowly on the time scale of the electrophoretic separation can often be resolved, and the rates of interconversion can be estimated. If the transitions are more rapid, then the electrophoretic mobility represents the equilibrium distribution of conformations. Protein unfolding transitions induced by urea are readily studied using slab gels containing a gradient of urea concentration perpendicular to the direction of electrophoresis. Protein applied across the top of such a gel migrates in the presence of continuously varying urea concentrations, and a profile of the unfolding transition is generated directly. Transitions induced by other agents could be studied using analogous gradient gels. Electrophoretic methods are especially suited for studying small quantities of protein, and complex mixtures, since the different components can be separated during the electrophoresis.     

Experimental and theoretical studies of DNA separations by capillary electrophoresis in entangled polymer solutions.

Studies of electrophoretic separations of DNA restriction fragments by capillary electrophoresis in solutions of (hydroxyethyl) cellulose (HEC) were performed. Rheological studies were used to confirm that the entanglement threshold (phi*) for the HEC solutions used is approximately 0.004 g/mL, in good agreement with theoretical predictions. A mesh size an order of magnitude smaller than that found in agarose gels (on a per weight basis) was calculated using polymer-entanglement theory and was confirmed by electrophoretic measurements. Electrophoretic migration was shown to follow the Ogston regime under most conditions. An approach for obtaining smaller mesh sizes is presented.     

Nematode chromosomal proteins--III. Some structural properties of the histones of Caenorhabditis elegans

The histones of Caenorhabditis elegans (Nematoda) have been identified by correlating criteria of electrophoresis and amino acid composition with the five main histones from calf thymus. C. elegans H1(1) consists of at least two subtypes with approximate molecular weights of 20,000 and 18,500 daltons as resolved by SDS polyacrylamide gel electrophoresis. They are some 10% smaller than the two subtypes of calf histone H1. The differences are also corrobated by the amino acid composition of the nematode and calf H1 complements. Nematode H2A resembles calf H2A in chromatographic and electrophoretic properties and in the amino acid composition, although it lacks histidine, which seems to be replaced by lysine. Like calf H2A, it is dimorphic as shown by Triton/acid/urea polyacrylamide gel electrophoresis. The H2B complement from C. elegans consists of two proteins with a molecular weight of approximately 12,500. They can be separated by ion-exchange chromatography, but they are very analogous to each other and to calf H2B in amino acid composition. Each form is also resolved into two more subtypes by Triton/acid/urea polyacrylamide gel electrophoresis. Nematode H3 resembles calf thymus H3 in its electrophoretic behaviour; three subfractions can be distinguished in Triton/acid/urea gels. C. elegans H4 is very similar to calf H4 in its chromatographic, electrophoretic and solubility properties, but differs significantly in composition. The meaning of this difference is discussed with regard to the generally observed stringent conservation of H4 sequences between distantly related species.     

Cooperative biotin binding by streptavidin. Electrophoretic behavior and subunit association of streptavidin in the presence of 6 M urea

We describe the cooperativity in the biotin binding of streptavidin. We have developed an electrophoretic method which can separate streptavidin molecules with bound biotin from those without biotin. In 6 M urea, the electrophoretic mobility of streptavidin in polyacrylamide gels becomes significantly faster upon biotin binding. When streptavidin was titrated with biotin, only two major bands were observed on the gel, consisting of streptavidin molecules without bound biotin and those saturated with biotin. The change in mobility is due partly to the negative charge of the bound biotin, but it must reflect conformational changes of the protein molecule associated with biotin binding. Gel filtration chromatography showed that the streptavidin molecule dissociates into two subunit dimers in the presence of 6 M urea. These results suggest that the biotin binding by the streptavidin subunit dimer is cooperative and that some communication must exist between the two subunits.     

The polymorphism of the vitamin D-binding protein (Gc); Isoelectric focusing in 3 M urea as additional method for identification of genetic variants

Since the last report numerous new DBP (Gc) variants have been observed; at present a total of 84 different mutants can be distinguished. Several of them have similar electrophoretic mobilities and/or isoelectric points of conventional isoelectric focusing (IEF). IEF in polyacrylamide gels in the presence of 3 M urea is a convenient and efficient method for the detection of hidden variation.