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PubMed1997

Analysis of interacting biopolymer systems by analytical ultracentrifugation.

Many of the functions of biological macromolecules are based on specific interactions. Extended concentration dependent studies of sedimentation coefficients or molecular masses of biopolymers are highly useful for describing the different kinds of association phenomena. These studies allow one to determine the partial concentrations of monomers and associates or reactants and complexes in self-associating systems or heterologous associations, respectively. Furthermore, in combination with corresponding measurements of biological activity these data allow one to estimate the individual activity parameters of components involved in equilibrium processes. The study of self-association and heterologous association using analytical ultracentrifugation, some recent developments therein, and its application to different examples are outlined here.

Biopolymers↗
PubMed1997

A trimeric, alpha-helical, coiled coil peptide: association stoichiometry and interaction strength by analytical ultracentrifugation.

Alpha-helical coiled coils are proving to be almost ideal systems for the modelling of peptide and protein self-association processes. Stable oligomeric systems, in which the stoichiometry is well defined, can be produced by the careful selection of the appropriate amino acid sequence, although the principles behind this are still not fully understood. Here we report on a 35 residue peptide, FZ, synthesized by the solid phase method, which was originally designed to form a dimer, but which, in fact, associates to the trimeric state. A detailed characterization of the associative properties of the peptide has been performed by circular dichroism spectroscopy and, in particular, by sedimentation equilibrium in the analytical ultracentrifuge. The presence of the trimeric state, which is stable even at low peptide concentrations, has been confirmed by various independent methods of analysis for molar mass. The effects of both temperature and of guanidinium chloride on the peptide have been investigated and both found to be peptide-concentration dependent. The unfolding induced by the denaturant cannot be adequately described by a simple, two state monomer-trimer equilibrium.

Amino Acid Sequence↗
PubMed1997

A case study and use of sedimentation equilibrium analytical ultracentrifugation as a tool for biopharmaceutical development.

Analytical ultracentrifugation (AUC) has reemerged as a powerful technique for protein characterisation. We report the pivotal role sedimentation equilibrium AUC has played in the development of macrophage inflammatory protein-1 alpha (MIP-1 alpha) as a protein therapeutic. MIP-1 alpha has potential clinical applications in cancer but its clinical use is limited, since it associates to form large insoluble aggregates in physiological buffers. Using AUC as a screening technique, we have produced a biologically active variant of MIP-1 alpha, BB-10010, which has a reduced tendency to aggregate in physiological buffers. The aggregation of protein based pharmaceuticals is routinely monitored by size exclusion chromatography (SEC). Comparison of the data acquired by SEC and AUC, demonstrates that owing to the complexity of BB-10010, AUC analysis is required in addition to SEC to provide a rigorous characterisation of molecular association. This work has been extended to include the use of AUC as an analytical tool to monitor the quality of BB-10010 during formulation and stability studies.

Biopharmaceutics↗
PubMed1997

Characterisation of the low affinity interaction between rat cell adhesion molecules CD2 and CD48 by analytical ultracentrifugation.

CD2 is a cell adhesion molecule found on the plasma membrane of T-lymphocytes. Its counter-receptor in rat is the structurally related CD48. This interaction is believed to contribute to the adhesion of T-cells to other cells such as cytotoxic targets and antigen presenting cells. Cell-cell adhesion involves the formation of multiple cell adhesion molecule complexes at the cell surface and if cell-cell de-adhesion is to occur, these complexes need to be disrupted. The affinities of cell adhesion molecule interactions are suggested to be relatively weak to allow this de-adhesion of cell-cell interactions. The CD2/CD48 interaction has been studied using recombinant extracellular proteins and the affinity of the interaction of soluble recombinant rat CD2-CD48 has been determined (at 37 degrees C) using surface plasmon resonance (and shown to be weak), with the dissociation constant Kd = 60-90 microM. The values determined by surface plasmon resonance results could be affected by the immobilisation of the ligand on the chip and any self-association on the chip. We used three different analytical ultracentrifuge procedures which each allowed the interaction to be studied in free solution without the need for an immobilisation medium. Both sedimentation equilibrium (using direct analysis of the concentration distribution and also modelling of molecular weight versus concentration data) and sedimentation velocity at 5 degrees C yielded dissociation constants in the range of 20-110 microM, supporting the surface plasmon resonance findings showing that binding between these cell adhesion molecules is relatively weak. These studies also ruled out the presence of any significant self-association of the reactants which could lead to systematic error in the surface plasmon resonance results.

Animals↗
PubMed1998

Ultracentrifugation studies on the transmembrane domain of the human erythrocyte anion transporter band 3 in the detergent C12E8.

The dilute solution behaviour of the transmembrane domain (TMD) of the human erythrocyte anion exchanger Band 3 was studied by analytical ultracentrifugation. Sedimentation velocity and equilibrium studies of the TMD solubilized with the detergent C12E8 demonstrate that the protein is a stable dimer in the concentration range 0.1 to 1 mg/ml. There is no evidence of a dissociation at low concentrations or of an association at higher concentrations. Hydrodynamic calculations applying a prolate ellipsoid of revolution and assuming a hydration of w = 0.35 result in an asymmetrical particle with an axial ratio (a/b) of approximately 3.5.

Algorithms↗
PubMed1983

The resolution of bimodal peaks in the analytical ultracentrifuge in the presence and absence of ligands.

The patterns for peak resolution in the analytical ultracentrifuge characteristic of systems associating by different mechanisms are analyzed. These are (i) that in which reequilibration is infinitely slow; (ii) that in which reequilibration is rapid, i.e., a Gilbert-type system; and (iii) that in which reequilibration is rapid, but the self-association is mediated by a ligand. Criteria for distinguishing the three are described and illustrated by results on tubulin self-association under various conditions.

Buffers↗
PubMed1983

An automated method for determination of the molecular weight of macromolecules via sedimentation equilibrium in a preparative ultracentrifuge.

An automated method for quantitating the concentration gradient of a macromolecule brought to sedimentation equilibrium in a preparative ultracentrifuge is described. Between 30 and 80 microliter of macromolecular solution are spun in a combination centrifuge tube and optical cell fabricated from quartz. Immediately following the conclusion of centrifugation the tube is optically scanned along its length using a spectrophotometer sample cell modified for this purpose. Successive scans of the same tube containing first solution and then solvent permit the differential absorbance due to macromolecule to be measured precisely as a function of position within the centrifuge tube, and hence the radial position within the rotor. Molecular weights calculated from data so obtained from 32 centrifugations of eight proteins ranging in Mr from 12K to 340K using either swinging-bucket or fixed-angle rotors agree with previously published values with a standard deviation of 5%.

Autoanalysis↗
PubMed1983

Molecular weight of human high-molecular-weight kininogen light chain by equilibrium sedimentation in an air-driven ultracentrifuge.

High-molecular-weight kininogen, a nonenzymatic glycoprotein of the intrinsic blood coagulation system, is proteolytically cleaved by kallikrein as an early event in the activation of this system. The light chain of cleaved kininogen retains the ability to form specific noncovalent complexes with prekallikrein and factor XI, other members of this system. We have determined the molecular weight of human kininogen light chain by equilibrium sedimentation in buffers of differing density, using an air-driven benchtop ultracentrifuge. The resulting molecular weight (30,500 +/- 800 g/mol) and partial specific volume (0.660 +/- 0.008 ml/g) are consistent with the idea that a sizeable fraction of the carbohydrate of high-molecular-weight kininogen is associated with the light chain. This level of precision is relatively easy to attain. The procedures are detailed, along with expressions for error propagation, to permit ready application of the technique.

Air↗
PubMed1989

A method for replacing intravesicular contents of Golgi vesicles using an air-driven ultracentrifuge.

Golgi membrane vesicles can be easily and very rapidly (within 10 min.) loaded with solutions of desired composition by centrifugation of the vesicles at high g force in an air-driven ultracentrifuge and subsequent resuspension of the vesicle pellet. This centrifugal/mechanical loading procedure does not destroy the integrity of these vesicles, as demonstrated by the ability of loaded vesicles to (i) retain their contents, (ii) maintain a K+ gradient when loaded with K+ ions, and (iii) exchange internal UMP for external [3H]UMP when loaded with UMP. When radiolabeled solutes are loaded into vesicles, the displaced internal volume can be measured using a rapid filtration assay. This simple and rapid technique of replacing the intravesicular contents of Golgi membrane vesicles should prove useful in studying transport across this membrane and may have a variety of other applications, such as intravesicular volume measurements, macromolecule and drug delivery protocols, and the study of membrane fusion events.

Animals↗
PubMed1989

Easily assembled digital data acquisition system for the analytical ultracentrifuge.

A system for the acquisition of digital data from the analytical ultracentrifuge which uses a commercially available data acquisition board, a standard IBM compatible personal computer (PC), and an interface circuit has been developed. The system uses the signal from the standard Beckman scanner. Preliminary analysis and data reduction are performed at the PC within minutes of data acquisition using simple commercially available software, and final data fitting is performed with a mainframe computer. Procedures are described which allow approach to equilibrium to be followed and attainment of equilibrium to be demonstrated. Data density of approximately 200 points per millimeter column height (approximately 500 points per 100 microliters of sample) allows the use of short columns and hence short run times. Only 2 min are required to collect a complete scan, which is recorded in a format suitable for direct analysis by standard spreadsheet software. This allows multiple sequential scans to be quickly recorded at equilibrium and averaged to reduce noise prior to analysis. The combination of characteristics allows molecular weight determinations to be performed relatively quickly with only a few micrograms of protein. The system is inexpensive and easy to assemble given the centrifuge and a PC.

Computer Systems↗
PubMed1983

Protein-protein interactions: analysis of the interaction of concanavalin A with serum glycoproteins by sedimentation equilibrium using an air-driven ultracentrifuge.

An air-driven ultracentrifuge has been used for the quantitative analysis of the interaction of concanavalin A with a number of serum glycoproteins. Concanavalin A significantly affected the sedimentation equilibrium behavior of 125I-labeled human alpha 1-acid glycoprotein, rat alpha 1-acid glycoprotein, and rat transferrin containing two N-acetylneuraminic acid residues per molecule (Tf2). The weight-average molecular weight of the labeled component increased and there was a corresponding decrease in the concentration of the labeled component at the meniscus. In contrast, concanavalin A did not significantly alter the sedimentation equilibrium behavior of the 125I-labeled rat transferrin containing three N-acetyl neuraminic acid residues per molecule. Sedimentation equilibrium results for interacting mixtures of concanavalin A and labeled glycoprotein and at various concentrations of the competitive inhibitor alpha-methyl mannoside were analyzed in terms of a model. Values of 5 X 10(4), 2 X 10(5), and 6 X 10(5) M-1 were obtained for the equilibrium constants for the interaction of concanavalin A with Tf2, human alpha 1-acid glycoprotein, and rat alpha 1-acid glycoprotein, respectively.

Chemical Phenomena↗
PubMed1985

Ultracentrifugation of concentrated biopolymer solutions and effect of ascorbate.

Highly concentrated solutions of bovine methemoglobin, human transferrin, proteoglycan-protein complex from bovine cartilage, and human hyaluronate were run to equilibrium in the preparative ultracentrifuge. The mass fraction, wo, of water in the compact part of the sediment ("the pellet") was studied as a function of the centrifugal force, ionic strength and pH. For the proteins, wo was close to 0.4 and varied only slightly on variation of the ionic strength and the force. For the proteoglycan-protein complex, wo was close to unity at low force but decreased sharply with increasing force, a finding which agrees well with the specific physical properties of the cartilage matrix. For hyaluronate, wo exceeded 0.8 even at the highest forces, but decreased sharply with increasing ionic strength. There appeared to be a relationship between wo and the carbohydrate mass fraction, wc, of the dry polymer material, wo increasing linearly with wc at 440,000g. A series of biologically or pharmacologically interesting substances was tested for a possible effect on wo of hyaluronate, and it was found that the addition of ascorbate (0.2 g/liter) caused a reduction of 0.040 in wo, independent of the force at which this parameter was measured. Simultaneously, the sedimentation coefficient was doubled. These findings suggest that, on reacting with ascorbate, the hyaluronate molecule changes from a random coil to a more compact molecule.

Animals↗
PubMed1989

Composition and structure of lipopolysaccharide-human plasma low density lipoprotein complex. Analytical ultracentrifugation, 31P-NMR, ESR and fluorescence spectroscopy studies.

Complexes of Salmonella typhimurium lipopolysaccharide toxin (LPS) with low density lipoproteins (LDL) prepared in vitro have been analyzed. LPS-LDL complexes were found to comprise approx. 0.24 mg LPS/mg LDL protein. The major protein of complexes was apolipoprotein apoB-100 (greater than or equal to 90-95%). Incorporation of LPS molecules into LDL was accompanied by small changes in lipid composition, i.e. the phosphatidylcholine content was diminished by approx. 11% and the free fatty acid concentration was raised 2-fold. Analytical ultracentrifugation showed that insertion of LPS into LDL results in the increase of a portion of particles with higher density (lower flotation coefficient) compared to initial LDL. As was evidenced by ESR, in LPS-LDL complexes, the phospholipid hydrocarbon chains are more ordered than in LDL. 31P-NMR spectra indicated that in LPS-LDL complexes the mobility of phospholipid polar headgroups is restricted in comparison with LDL. Application of the shift reagent (Pr3+) revealed that phospholipid molecules form a monolayer structure on the surface of complexes. Upon binding of LPS to LDL, a maximum of the apoB intrinsic fluorescence was slightly red-shifted (1-2 nm) which may testify that the localization of apoB remains nearly unchanged. For LPS-LDL complexes, the accessibility of apoB fluorophores to quenchers (I-, Cs+, acrylamide) did not dramatically differ from that of LDL. It is concluded that rather large amounts of LPS (about 9-10 molecules) can accommodate in one LDL particle without severely perturbing its original composition and structure. Moreover, in the LPS-LDL complexes, oligosaccharide chains of LPS screen notably neither phospholipid polar headgroups nor, what is very important, apoB. LPS-LDL complexes are suggested to be able in vivo to bind to cellular apoB/E receptors, possible LPS receptors and scavenger-receptors of macrophages (monocytes).

Electron Spin Resonance Spectroscopy↗
PubMed1984

Characterization with zonal ultracentrifugation of low-density lipoproteins in type V hyperlipoproteinemia.

Low-density lipoproteins (density = 1.019-1.063 g/ml) were isolated in 10 subjects with type V hyperlipoproteinemia by ultracentrifugation in a zonal rotor under rate flotation conditions. Plasma LDL concentrations in these patients were extremely reduced, as well as being heterogeneous, and two different subclasses consisting of LDL2 (density = 1.019-1.045 g/ml) and LDL3 (density = 1.045-1.063 g/ml) were observed. LDL2 and LDL3 have similar electrophoretic mobilities in beta position in agarose gel, and their diameters, calculated from gel filtration studies, were inversely proportional to their densities. LDL2 and LDL3 have a mean hydrated density of 1.034 and 1.054 g/ml, respectively. In comparison with normal LDL2, the LDL2 and LDL3 of hypertriglyceridemic subjects are particularly rich in triacylglycerols and poor in cholesteryl esters and free cholesterol, while they have an increasing amount of proteins. The protein moiety is composed almost exclusively of apolipoprotein B-100 in IDL, LDL2 and LDL3 ; in addition, IDL also contain apolipoprotein C peptides. This characterization of LDL heterogeneity in type V hyperlipoproteinemia should be considered in interpreting kinetic data in human normal and pathological lipid metabolism and in evaluating the atherogenic risk of hypertriglyceridemia.

Adult↗
PubMed1978

Ultracentrifugation studies of yeast valyl-tRNA synthetase and of its interaction with tRNAVal.

Yeast valyl-tRNA synthetase and its complexes with yeast tRNAVal were investigated by means of analytical ultracentrifugation. A molecular weight of 125 700 +/- 1500 and a sedimentation coefficient (SO 20, w) of 6.3 +/- 0.3 were found for the native enzyme. When the enzyme (3--60 muM) was mixed with its cognate tRNA, several types of complex were observed, depending on the relative amounts of the two macromolecules. In the presence of equimolecular amounts of tRNA and enzyme, a complex formed by the association of one of each molecule was observed with a sedimentation coefficient of about 7.3 S. However, for tRNA/enzyme stoichiometries lower than one, beside the 1 : 1 complex, a complex of higher molecular weight was observed, with a sedimentation coefficient of about 10.0 S which fits with the association of two valyl-tRNA synthetase molecules with one tRNA molecule. This 2 : 1 complex was predominant from tRNA/enzyme stoichiometries lower than 0.3. It dissociated into the 1 : 1 complex upon addition of monovalent salts or MgCl2, suggesting the electrostatic nature of the interaction in this association. All these association and dissociation phenomena were detected over a large range of pH (6.0--7.5) and in various buffers.

Amino Acyl-tRNA Synthetases↗
PubMed1977

Partial dissociation and renaturation of embryonic chick delta-crystallin. Characterization by ultracentrifugation and circular dichroism.

1. delta-Crystallin from 15-day-old embryonic chick lenses was characterized by circular dichrosim (CD) spectroscopy. Examination by CD spectroscopy in the far ultraviolet (190-250 nm) demonstrated that the secondary structure of delta-crystallin has at least 75% alpha-helix; the delta-crystallin subunits dissociated in sodium dodecyl sulfate retain their alpha-helical content. This appreciable alpha-helical content of delta-crystallin contrasts with the absence of alpha-helix in other lens crystallins. 2. As judged by CD spectroscopy in the near ultraviolet (250-320 nm) the tertiary structure of embryonic delta-crystallin is not readily disrupted by environmental changes, such as NaCl, KSCN or the non-ionic detergent Emulphogene BC 720, and is stable to temperature fluctuation between 2 and 56 degrees C. 3. Experiments were directed towards deaggregation and renaturation of the four subunits of embryonic delta-crystallin by treatment with urea or guanidine hydrochloride. The native tertiary structure of delta-crystallin was lost above 4 M urea or 2 M guanidine hydrochloride, as judged by CD spectroscopy in the near ultraviolet. Ultracentrifugation at sedimentation equilibrium showed that in 4 M urea delta-crystallin dissociates into dimeric subunits, while in 2 M guanidine hydrochloride delta-crystallin exists as a mixture of dimeric and tetrameric subunits. Dialysis of delta-crystallin from 4 M urea resulted in reaggregation of the subunits into tetramers, about 50% of which showed native tertiary structure. Dialysis from 2 M guanidine hydrochloride also resulted in tetramer formation, and about 35% was recovered with native conformation. Removal of denaturant by dialysis produced no native teritary structure after treatment with 8 M urea, but about 15% native conformation after treatment with 6 M guanidine hydrochloride.

Animals↗
PubMed1981

Photometric measurement of lipoprotein-cholesterol after agarose electrophoresis: comparison with single-spin ultracentrifugal analysis.

A method is presented for the evaluation of lipoprotein-cholesterol after fractionation of 20 microliter of serum in agarose electrophoresis in a discontinuous buffer system. The prestained lipoprotein bands are separated in less tha 1 h, cut out and extracted with butanol. The cholesterol in the extract is measured by a simple colorimetric method. The recoveries of lipoprotein cholesterol were 95.3% +/- 13.0 (S.D.), and the average coefficient of variation was 8% for alphalipoprotein, 6.4% for betalipoprotein and 8.0% for pre-betalipoproteins. Lipoprotein cholesterol concentrations, determined by the proposed method correlated well with those analyzed following ultracentrifugation. The method can handle up to 4 serum samples run in duplicate per day and only requires equipment available in conventional clinical laboratories, and stable reagents.

Adult↗