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The solution properties of two different glycoforms of IgG1 (IgG1Cri and IgG1Wid) are compared using primarily sedimentation equilibrium analysis with two complementary analysis routines: SEDFIT-MSTAR and MULTISIG. IgGCri bears diantennary complex-type glycans on its Fc domain that are fully core fucosylated and partially sialylated, whilst on IgGWid, they are non-fucosylated, partially galactosylated and non-sialylated. IgGWid is also Fab glycosylated. Despite these differences, SEDFIT-MSTAR analysis shows similar weight average molar masses Mw of ~ (150 ± 5) kDa for IgGCri and ~ (154 ± 5) kDa for IgGWid and both glycoforms show evidence of the presence of a small fraction of dimer confirmed by MULTISIG analysis and also by sedimentation coefficient distributions from supportive sedimentation velocity measurements. The closeness of the sedimentation equilibrium behaviour and sedimentation coefficient distributions with a main peak sedimentation coefficient of ~ 6.4S for both glycoforms at different concentrations suggest that the different glycosylation profiles do not significantly impact on molar mass (molecular weight) nor conformation in solution.

The Oseberg ship is one of the most important archaeological testimonies of the Vikings. After excavation in 1904, the wooden gravegoods were conserved using alum salts. This resulted in extreme degradation of a number of the objects a hundred years later through acid depolymerisation of cellulose and lignin. The fragile condition of the artefacts requires a reconsolidation which has to be done avoiding water as solvent. We synthesized tert-butyldimethylsilyl (TBDMS) chitosan which is soluble in a 50:50 solution of ethyl acetate and toluene. Measurement of its molecular weight, to anticipate its penetration, provided a challenge as the density difference of the polymer and solvent was too small to provide adequate solute redistribution under a centrifugal field, so a two-stage process was implemented (i) determination of the weight-average molar mass of the aqueous soluble activated precursor, chitosan mesylate, Mw,mc using sedimentation equilibrium with the SEDFIT-MSTAR algorithm, and determination of the degree of polymerisation DP; (ii) measurement of the average degree of substitution DSTBDMS of the TBDMS group on each chitosan monosaccharide monomer unit using NMR, to augment the Mw,mc value to give the molar mass of the TBDMS-chitosan. For the preparation, we find Mw = 9.8 kg·mol−1, which is within the acceptable limit for penetration and consolidation of degraded wood. Future work will test this on archaeological wood from different sources.

Multiple myeloma (MM) patients have increased risk of severe coronavirus disease 2019 (COVID-19) when infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Monoclonal gammopathy of undetermined significance (MGUS), the precursor of MM has been associated with immune dysfunction which may lead to severe COVID-19. No systematic data have been published on COVID-19 in individuals with MGUS. We conducted a large population-based cohort study evaluating the risk of SARS-CoV-2 infection and severe COVID-19 among individuals with MGUS. We included 75,422 Icelanders born before 1976, who had been screened for MGUS in the Iceland Screens Treats or Prevents Multiple Myeloma study (iStopMM). Data on SARS-CoV-2 testing and COVID-19 severity were acquired from the Icelandic COVID-19 Study Group. Using a test-negative study design, we included 32,047 iStopMM participants who had been tested for SARS-CoV-2, of whom 1754 had MGUS. Among these participants, 1100 participants, tested positive, 65 of whom had MGUS. Severe COVID-19 developed in 230 participants, including 16 with MGUS. MGUS was not associated with SARS-CoV-2 infection (Odds ratio (OR): 1.05; 95% confidence interval (CI): 0.81–1.36; p = 0.72) or severe COVID-19 (OR: 0.99; 95%CI: 0.52–1.91; p = 0.99). These findings indicate that MGUS does not affect the susceptibility to SARS-CoV-2 or the severity of COVID-19.

Vancomycin, a branched tricyclic glycosylated peptide antibiotic, is a last-line defence against serious infections caused by staphylococci, enterococci and other Gram-positive bacteria. Orally-administered vancomycin is the drug of choice to treat pseudomembranous enterocolitis in the gastrointestinal tract. However, the risk of vancomycin-resistant enterococcal infection or colonization is significantly associated with oral vancomycin. Using the powerful matrix-free assay of co-sedimentation analytical ultracentrifugation, reinforced by dynamic light scattering and environmental scanning electron microscopy, and with porcine mucin as the model mucin system, this is the first study to demonstrate strong interactions between vancomycin and gastric and intestinal mucins, resulting in very large aggregates and depletion of macromolecular mucin and occurring at concentrations relevant to oral dosing. In the case of another mucin which has a much lower degree of glycosylation (~60%) – bovine submaxillary mucin - a weaker but still demonstrable interaction is observed. Our demonstration - for the first time - of complexation/depletion interactions for model mucin systems with vancomycin provides the basis for further study on the implications of complexation on glycopeptide transit in humans, antibiotic bioavailability for target inhibition, in situ generation of resistance and future development strategies for absorption of the antibiotic across the mucus barrier., The authors are grateful for a grant from the Independent Diabetes Trust (G.G.A. and S.E.H.) and the Engineering and Physical Sciences Research Council, grant number EP/L015633/1 (I.F., S.E.H., G.G.A., V.D.).

The analytical ultracentrifuge (AUC) invented by T. Svedberg has now become an extremely versatile and diverse tool in Biochemistry and Polymer Science for the characterisation of the sizes, shapes and interactions of particles ranging in size from a few nanometres to tens of microns, or in molecular weight, M (molar mass) terms from a few hundred daltons to hundreds of megadaltons. We illustrate this diversity by reviewing recent work on (1) small lignin-like isoeugenols of M ~ 0.4–0.9 kDa for archaeological wood conservation, (2) protein-like association of a functional amino-cellulose M = 3.25 kDa, (3) a small glycopeptide antibiotic (M ~ 1.5 kDa) and its association with a protein involved in antibiotic resistance (M ~ 47 kDa), (4) tetanus toxoid protein TTP (M ~ 150 kDa) and (5) the incorporation of TTP into two huge glycoconjugates considered in glycovaccine development with molecular weight species in a broad distribution appearing to reach 100 MDa. In illustrating the diversity, we will highlight developments in hydrodynamic analysis which have made the AUC such an exciting and important instrument, and point to a potential future development for extending its capability to highly concentrated systems.

The major challenges in biophysical characterization of human protein-carbohydrate interactions are obtaining monodispersed preparations of human proteins that are often post-translationally modified and lack of detection of carbohydrates by traditional detection systems. Light scattering (dynamic and static) techniques offer detection of biomolecules and their complexes based on their size and shape, and do not rely on chromophore groups (such as aromatic amino acid sidechains). In this study, we utilized dynamic light scattering, analytical ultracentrifugation and small-angle X-ray scattering techniques to investigate the solution properties of a complex resulting from the interaction between a 15 kDa heparin preparation and miniagrin, a miniaturized version of agrin. Results from dynamic light scattering, sedimentation equilibrium, and sedimentation velocity experiments signify the formation of a monodisperse complex with 1:1 stoichiometry, and low-resolution structures derived from the small-angle X-ray scattering measurements implicate an extended conformation for a side-by-side miniagrin‒heparin complex.

The purpose of this special issue is to honour Professor Donald J. Winzor’s long career as a researcher and scientific mentor, and to celebrate the milestone of his 80th birthday. Throughout his career, Don has been renowned for his development of clever approximations to difficult quantitative relations governing a range of biophysical measurements. The theme of this special issue, ‘Quantitative and analytical relations in biochemistry’, was chosen to reflect this aspect of Don’s scientific approach.

We report the novel application of Analytical Ultracentrifugation (AUCF) to characterise the polymeric proanthocyanidin fraction of hops. Extraction of hop samples with 70% acetone (aq) followed by a C-18 Solid Phase Extraction yielded polyphenolic fractions for AUCF analysis. Sedimentation velocity experiments demonstrated the presence of discrete molecular weight bands of proanthocyanidins, as opposed to a continuous distribution of molecular weights. There were 4 such bands for Saaz hop (0.15, 1.1, 2.7 and 4.4S) and 3 bands for Magnum (0.15, 1.6 and 3.0S). The method resulted in a reproducible size (sedimentation coefficient) distribution for replicate runs of the same extract and for extracts prepared from different samples of the same hop variety. Sedimentation equilibrium experiments were then used to fit molecular weight distributions using the new SEDFIT-MSTAR method for the same samples. Thus we report for the first time polymeric proanthocyanidins in hops with molecular weights of up to 100 kDa in Saaz hop (or up to 56 kDa in Magnum). This represents the first application of AUCF to characterise complex fractions of polyphenolics extracted from botanical sources and the methodology developed should find wider application in the study of this diverse and bioactive class of compounds.

Polysaccharides, the most abundant biopolymers, are required for a host of activities in lower organisms, animals, and plants. Their solution characterization is challenging due to their complex shape, heterogeneity, and size. Here, recently developed data analysis approaches were applied for traditional sedimentation equilibrium and velocity methods in order to investigate the molar mass distribution(s) of a subtype of polysaccharide, namely, mannans from four Candida spp. The molecular weight distributions of these mannans were studied using two recently developed equilibrium approaches: SEDFIT-MSTAR and MULTISIG, resulting in corroboratory distribution profiles. Additionally, sedimentation velocity data for all four mannans, analyzed using ls-g*(s) and Extended Fujita approaches, suggest that two of the fungal mannans (FM-1 and FM-3) have a unimodal distribution of molecular species whereas two others (FM-2 and FM-4) displayed bi-modal and broad distributions, respectively: this demonstrates considerable molecular heterogeneity in these polysaccharides, consistent with previous observations of mannans and polysaccharides in general. These methods not only have applications for the characterization of mannans but for other biopolymers such as polysaccharides, DNA, and proteins (including intrinsically disordered proteins).

Mucins are the primary macromolecular component of mucus--nature's natural lubricant--although they are poorly characterised heterogeneous substances. Recent advances in hydrodynamic methodology now offer the opportunity for gaining a better understanding of their solution properties. In this study a combination of such methods was used to provide increased understanding of a preparation of porcine intestinal mucin (PIM), MUC2 mucin, in terms of both heterogeneity and quantification of conformational flexibility. The new sedimentation equilibrium algorithm SEDFIT-MSTAR is applied to yield a weight average (over the whole distribution) molar mass of 7.1 × 10(6) g mol(-1), in complete agreement with size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS), which yielded a value of 7.2 × 10(6) g mol(-1). Sedimentation velocity profiles show mucin to be very polydisperse, with a broad molar mass distribution obtained using the Extended Fujita algorithm, consistent with the elution profiles from SEC-MALS. On-line differential pressure viscometry coupled to the SEC-MALS was used to obtain the intrinsic viscosity [η] as a function of molar mass. These data combined with sedimentation coefficient data into the global conformation algorithm HYDFIT show that PIM has a flexible linear structure, with persistence length L p ~10 nm and mass per unit length, M L ~2380 g mol(-1) nm(-1), consistent with a Wales-van Holde ratio of ~1.2 obtained from the concentration dependence of the sedimentation coefficient.

Single, double and triple doses of the synthetic insulins glargine and degludec currently used in patient therapy are characterised using macromolecular hydrodynamic techniques (dynamic light scattering and analytical ultracentrifugation) in an attempt to provide the basis for improved personalised insulin profiling in patients with diabetes. Using dynamic light scattering and sedimentation velocity in the analytical ultracentrifuge glargine was shown to be primarily dimeric under solvent conditions used in current formulations whereas degludec behaved as a dihexamer with evidence of further association of the hexamers (“multi-hexamerisation”). Further analysis by sedimentation equilibrium showed that degludec exhibited reversible interaction between mono- and-di-hexamer forms. Unlike glargine, degludec showed strong thermodynamic non-ideality, but this was suppressed by the addition of salt. With such large injectable doses of synthetic insulins remaining in the physiological system for extended periods of time, in some case 24–40 hours, double and triple dose insulins may impact adversely on personalised insulin profiling in patients with diabetes.

MultiSig is a newly developed mode of analysis of sedimentation equilibrium (SE) experiments in the analytical ultracentrifuge, having the capability of taking advantage of the remarkable precision (~0.1 % of signal) of the principal optical (fringe) system employed, thus supplanting existing methods of analysis through reducing the ‘noise’ level of certain important parameter estimates by up to orders of magnitude. Long-known limitations of the SE method, arising from lack of knowledge of the true fringe number in fringe optics and from the use of unstable numerical algorithms such as numerical differentiation, have been transcended. An approach to data analysis, akin to ‘spatial filtering’, has been developed, and shown by both simulation and practical application to be a powerful aid to the precision with which near-monodisperse systems can be analysed, potentially yielding information on protein-solvent interaction. For oligo- and poly-disperse systems the information returned includes precise average mass distributions over both cell radial and concentration ranges and mass-frequency histograms at fixed radial positions. The application of MultiSig analysis to various complex heterogenous systems and potentially multiply-interacting carbohydrate oligomers is described.

The last three decades has seen some important advances in our ability to represent the conformation of proteins in solution on the basis of hydrodynamic measurements. Advances in theoretical modeling capabilities have been matched by commensurate advances in the precision of hydrodynamic measurements. We consider the advances in whole-body (simple ellipsoid-based) modeling—still useful for providing an overall idea of molecular shape, particularly for those systems where only a limited amount of data is available—and outline the ELLIPS suite of algorithms which facilitates the use of this approach. We then focus on bead modeling strategies, particularly the surface or shell–bead approaches and the HYDRO suite of algorithms. We demonstrate how these are providing great insights into complex issues such as the conformation of immunoglobulins and other multi-domain complexes.

Alcohol and nicotine are the most commonly abused drugs, and they are often taken together. We have developed a procedure in which rats self-administer nicotine intravenously and alcohol orally during the same operant session.Male Wistar rats were trained to self-administer alcohol (12%, w/v; 0.19 ml/alcohol delivery) or implanted with jugular catheters and trained to self-administer nicotine (30 microg/kg i.v./infusion) by pressing a lever or were trained to self-administer both drugs, some with alcohol first, and others with nicotine first. The effects of extinction of responding for either or both drugs in animals trained to coadminister alcohol and nicotine and the effects of alcohol and nicotine primes on reinstatement were also determined.Animals readily coadministered alcohol and nicotine concurrently. Access to alcohol reduced nicotine self-administration significantly. When responding for alcohol was extinguished with nicotine still available, extinction of alcohol seeking was slowed significantly. In rats trained to coadminister nicotine and alcohol, priming with nicotine or alcohol reinstated extinguished responding for both drugs. Reinstatement of extinguished nicotine or alcohol seeking by, respectively, nicotine or alcohol priming was unaffected by continued access to the other drug.These results show that rats will self-administer relevant amounts of intravenous nicotine and oral alcohol concurrently. They also provide further support for the important relationship between nicotine and alcohol.

WOS:000272868600005 PubMed: 19669133 A study of the heterogeneity and conformation in solution [in 70% (v/v) aq. ethanol] of gliadin proteins from wheat was undertaken based upon sedimentation velocity in the analytical ultracentrifuge, analysis of the distribution coefficients and ellipsoidal axial ratios assuming quasi-rigid particles, allowing for a range of plausible time-averaged hydration values. All classical fractions (alpha, gamma, omega(slow), omega(fast)) show three clearly resolved components. Based on the weight-average sedimentation coefficient for each fraction and a weight-average molecular weight from sedimentation equilibrium and/or cDNA sequence analysis, all the proteins are extended molecules with axial ratios ranging from similar to 10 to 30 with alpha appearing the most extended and gamma the least.

We present a scheme, based on existing and newly developed computational tools, for the determination of the overall conformation of biological macromolecules composed by domains or subunits, using from such structural determination easily available solution properties. In a multi-scale approach, atomic-level structures are used to provide simple shapes for the subunits, which are put together in a coarse grained model, with a few parameters that determine the overall shape of the macromolecule. Computer programs, like those in the HYDRO suite that evaluate the properties of either atomic or coarse-grained models. In this paper we present a new scheme for a global fit of multiple properties, implemented in a new computer program, HYDROFIT, which interfaces with the programs of the HYDRO suite to find an optimum, best-fitting structure in a robust but simple way. The determination of the overall structure of the native antibody IgG3, bearing a long hinge, and that of the hingeless mutant m15 is presented to test and confirm the validity of this simple, systematic and efficient scheme.

We previously found that systemic injections of the 5-HT uptake blocker fluoxetine attenuate intermittent footshock stress-induced reinstatement of alcohol seeking in rats, while inhibition of 5-HT neurons in the median raphe induces reinstatement of alcohol seeking. In this study, we further explored the role of 5-HT in footshock stress-induced reinstatement of alcohol seeking by determining the effects of the 5-HT releaser and reuptake blocker dexfenfluramine, and the 5-HT receptor antagonists ondansetron and tropisetron, which decrease alcohol self-administration and anxiety-like responses in rats, on this reinstatement. Different groups of male Wistar rats were trained to self-administer alcohol (12% v/v) for 28–31 days (1 h/day, 0.19 ml per alcohol delivery) and then their lever responding for alcohol was extinguished over 9–10 days. Subsequently, the effect of systemic injections of vehicle or dexfenfluramine (0.25 or 0.5 mg/kg, i.p), ondansetron (0.001, 0.01, or 0.1 mg/kg, i.p), or tropisetron (0.001, 0.01, and 0.1 mg/kg, i.p) on reinstatement induced by 10 min of intermittent footshock (0.8 mA) was determined. Systemic injections of dexfenfluramine, ondansetron or tropisetron attenuated footshock-induced reinstatement of alcohol seeking. Injections of dexfenfluramine, ondansetron, or tropisetron had no effect on extinguished lever responding in the absence of footshock. The present results provide additional support for the hypothesis that brain 5-HT systems are involved in stress-induced reinstatement of alcohol seeking. The neuronal mechanisms that potentially mediate the unexpected observation that both stimulation of 5-HT release and blockade of 5-HT3 receptors attenuate footshock-induced reinstatement are discussed.

Attempts have been made to correlate estimates of molecular weight for a group of cationic polysaccharides known as chitosans between the highly popular technique of size-exclusion chromatography coupled to multi-angle laser light scattering, "SEC-MALLS", and the less convenient but more established technique of sedimentation equilibrium in the analytical ultracentrifuge. Four pharmaceutical grade chitosans of various molecular weights and degrees of acetylation (4-30%) were chosen. Better correlation than previous was achieved, although some batch variability was observed. Despite the broad spectrum in degree of acetylation, a log s degrees(20,w) versus log Mw scaling plot appeared to fit a straight line with power-law exponent b=0.25 +/- 0.04, i.e. between the limits of rod (0.15) and coil (0.4-0.5), although this may be the average of a lower b value at low Mw and higher b at high Mw. With regard to viscosity, a logeta versus logMw scaling plot appeared to also fit a straight line with power-law exponent a=0.96 +/- 0.10, again between the coil (0.5-0.7) and rod (1.8) limits.

A modified crystallohydrodynamic approach introduced in 2001 is applied to two human IgG4 constructs from mouse IgG1. The constructs were point mutants of the chimeric antibody molecule cB72.3(gamma4): cB72.3(gamma4A), devoid of inter-chain disulfide bridging, and cB72.3(gamma4P), which has full inter-chain bridging. As before, the known crystallographic structures for the Fab and Fc domains were combined with the measured translational frictional ratios to obtain an estimate for the apparent time-averaged hydration of the domains and hence for that of the intact molecule. The original approach was modified with the hydrated dimensions of the domains being applied, rather than the anhydrous crystallographic dimensions, for assessing the inter-domain orientations using the algorithms HYDROSUB and SOLPRO. Both chimeric IgG4 molecules were found to have open, rather than compact, structures, in agreement with the previous study on wild-type human IgG4. The insertion of a frictionless connector between the domains was necessary, however, for representing the cB72.3(gamma4A) chimera. It therefore appears that the inter-chain disulfide bonds act as physical constraints in the cB72.3(gamma4P) chimera, forcing the antibody domains together and producing a less elongated structure than that of cB72.3(gamma4A). The open structures produced for the two IgG4 chimeras showed similarity to those structures identified for murine IgG1 and IgG2a molecules through X-ray crystallography.

Water-soluble N-vinylpyrrolidone-crotonic acid-2-hydroxyethyl methacrylate terpolymers, potential polymeric carriers for biologically active substances, were prepared. The composition of the terpolymers was determined, their chemical structure was confirmed, and their hydrodynamic characteristics and structural features in aqueous solutions were studied.

Unusual protein-like, partially reversible associative behaviour has recently been observed in solutions of the water soluble carbohydrates known as 6-deoxy-6-(ω-aminoalkyl)aminocelluloses, which produce controllable self-assembling films for enzyme immobilisation and other biotechnological applications. Now, for the first time, we have found a fully reversible self-association (tetramerisation) within this family of polysaccharides. Remarkably these carbohydrate tetramers are then seen to associate further in a regular way into supra-molecular complexes. Fully reversible oligomerisation has been hitherto completely unknown for carbohydrates and instead resembles in some respects the assembly of polypeptides and proteins like haemoglobin and its sickle cell mutation. Our traditional perceptions as to what might be considered “protein-like” and what might be considered as “carbohydrate-like” behaviour may need to be rendered more flexible, at least as far as interaction phenomena are concerned.

The paper presents preparation and characterization of starch-modified Fe3O4 nanoparticles (NPs) in aqueous dispersion after impregnation into healthy and damaged types of cartilage. We show that starch-modified dispersion has a narrower size distribution than a non‐stabilized one. The average hydrodynamic radius of magnetite NPs in a dispersion used for impregnation into cartilage is (48 ± 1) nm with the width of the distribution from 5 to 200 nm. We investigate stability of aqueous magnetite NPs dispersions during storage and with increase in temperature (up to 70 °C). We find that polydisperse magnetite NPs can penetrate into cartilage and the size and concentration of impregnated particles depend on the organization of the tissue structure. The results confirm the possibility of application of magnetite NPs in diagnostics and laser treatment of degenerative cartilage deceases.

Rationale: Intermittent footshock stress reliably reinstates extinguished alcohol-taking behavior in drug-free rats, but the neurochemical events involved in this effect are not known. Objective: We studied here whether two main modulators of stress responses, corticotropin-releasing factor (CRF) and corticosterone, are involved in reinstatement of alcohol seeking induced by the intermittent footshock stressor. Methods: Rats were given alcohol in a two-bottle choice procedure (water versus alcohol) for 30 days and were then trained for 60 min per day to press a lever for alcohol (12% w/v) for 24–30 days in operant conditioning chambers. After stable drug-taking behavior was obtained, lever pressing for alcohol was extinguished by terminating drug delivery for 5–8 days. Reinstatement of alcohol seeking was then determined after exposure to intermittent footshock (0.8 mA; 10 min) in different groups of rats that were pretreated with CRF receptor antagonists or underwent adrenalectomy (ADX) to remove endogenous corticosterone from the body. Results: The CRF receptor antagonists, d-phe-CRF (0.3 or 1.0 µg; ICV) and CP-154,526 (15, 30 or 45 mg/kg; IP) attenuated footshock-induced reinstatement of alcohol seeking in a dose dependent manner. In contrast, the removal of circulating corticosterone by ADX had no effect on footshock stress-induced reinstatement of alcohol-taking behavior. In addition, the prevention of the footshock-induced rise in corticosterone while maintaining basal levels of the hormone by providing adrenalectomized rats with corticosterone pellets (50 mg/kg per day), had no effect on stress-induced reinstatement. Conclusions: These data suggest that CRF contributes to stress-induced relapse to alcohol seeking via its actions on extra-hypothalamic sites. The present data, and previous data with heroin- and cocaine-trained rats, point to a general role of CRF in relapse to drugs induced by stressors.

Serotonergic agents in general and the 5-HT1A agonist 8-OH DPAT in particular, reduce alcohol intake in rats and primates but the mechanism of this effect is not known. Previous studies have shown a correlation between alcohol consumption and the propensity to consume sweet substances. Indeed, certain biochemical events accompanying glucose utilization have been proposed as satiety signals in the control of feeding. Since 8-OH DPAT produces hyperglycemia, we tested the hypothesis that its effect on alcohol intake may be partly mediated through an increase blood glucose. Male Wistar rats were trained to drink a bout of 6% (w/v) alcohol using the limited access procedure which offers a daily 40-min access to alcohol and water. On consecutive test trial days separated by intervening non-drug days, the amount of alcohol consumed (1 g/kg on intervening days) was measured following the administration of 8-OH DPAT (150 micrograms/kg 10 min prior to drinking) alone or in combination with the prior (20 min) injection of idazoxan (2 mg/kg), an alpha-2 adrenoceptor antagonist with hypoglycemic properties. Idazoxan attenuated the hyperglycemic effect of 8-OH DPAT and completely reversed 8-OH DPAT's inhibitory effect on alcohol intake. Idazoxan alone produced a mild hypoglycemia and stimulated alcohol intake. These results support a role for glucoregulatory processes in serotonergically-mediated changes in alcohol consumption.

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.

This paper describes a series of four programmes for the PC based on ellipsoidal representations of macromolecular shape in solution using Universal shape functions, ELLIPS1 is based on simple ellipsoid of revolution models (where two of the three axes of the ellipsoid are fixed equal to each other). If the user types in a value for a shape function from sedimentation or other types of hydrodynamic measurement, it will return a value for the axial ratio of the ellipsoid. ELLIPS2 is based on the more general triaxial ellipsoid with the removal of the restriction of two equal axes. The user enters the three semi-axial dimensions of the molecule or the equivalent two axial ratios and ELLIPS2 returns the value of all the hydrodynamic shape functions. It also works of course for ellipsoids of revolution. ELLIPS3 and ELLIPS4 do the reverse of ELLIPS2, that is they both provide a method for the unique evaluation of the triaxial dimensions or axial ratios of a macromolecule (and without having to guess a value for the so-called "hydration") after entering at least three pieces of hydrodynamic information: ELLIPS3 requires EITHER the intrinsic viscosity with the second virial coefficient (from sedimentation equilibrium, light scattering of osmometry) and the radius of gyration (from light or x-ray scattering) OR the intrinsic viscosity with the concentration dependence term for the sedimentation coefficient and the (harmonic mean) rotational relaxation time from fluorescence depolarisation measurements. ELLIPS4 evaluates the tri-axial shape of a macromolecule from electro-optic decay based Universal shape functions using another Universal shape function as a constraint in the extraction of the decay constants.

The distribution of molecular weights for polymeric colonic mucus glycoprotein or ``mucin'' isolated and solubilised in the presence of protease inhibitors from pig colons is shown to be considerably greater than its ``subunit'' (thiol reduction product) and papain digested forms using the technique of size-exclusion chromatography coupled to multi-angle laser light scattering, and confirmed by sedimentation equilibrium measurements. The conformation of this mucin is probed by examining the molecular weight – intrinsic viscosity relationship in terms of the Mark-Houwink-Kuhn-Sakurada analysis for its polymeric (or ``whole''), reduced and papain-digested forms: an exponent ``a'' of (1.1±0.1) is obtained indicating a linear random coil conformation consistent with other mucins. Size-exclusion chromatography coupled to multi-angle laser light scattering is shown to provide a relatively simple complementary technique to sedimentation equilibrium for the molecular weight distribution analysis of polydisperse materials.

This paper describes a program available for PC's for the evaluation of molecular weights from sedimentation equilibrium. This program, in its two forms – MSTARA for absorption optical records and MSTARI for interference optical records – requires no prior assumption of the nature of the system (ideal, non-ideal, monodisperse, polydisperse, self-associating etc.) and takes into consideration the whole solute distribution (i.e. from solution meniscus to cell base) in the ultracentrifuge cell rather than just a selected data-set. MSTARA or MSTARI are therefore recommended as a first analysis programme of sedimentation equilibrium data coming off an absorption or interference based analytical ultracentrifuge. These programmes are therefore particularly well suited if heterogeneity (polydispersity or interaction phenomena) or non-ideality is suspected. Their use is demonstrated for a series of data-set types (ideal, non-ideal, polydisperse and self-associating). Although MSTARA and MSTARI are model independent, they provide the basis for more detailed analysis of interactions, polydisperse distributions or non-ideality via easy export of ASCII datafiles to model dependent routines.

The solution behaviour of electron transferring flavoprotein (ETF) from Methylophilus methylotrophus was investigated at low temperature (4 °C) by analytical ultracentrifugation. The concentration dependence of the apparent weight average molecular weight, Mw,app, established the existence of the protein in heterodimeric state (M = 63,700 Da), but also signified the possible dissociation of the heterodimer at lower concentrations into its constituent subunits (M = 28,900 Da and 33,700 Da, together with FAD and AMP cofactors of collective M = 1120 Da). This similarity in subunit size allows approximate quantification of the dissociation in terms of expressions for a monomer-dimer equilibrium. The dissociative behaviour was confirmed by determination of the point average molecular weight, Mw,app(r), as a function of the ETF concentration, c(r), throughout the sedimentation equilibrium distributions obtained with loading concentrations of 0.4 and 0.7 mg/ml. By means of the recently formulated ``psi'' procedure for direct analysis of solute self-association a value of (1.5 ± 0.1) µM has been obtained for the dissociation constant Kd. Sedimentation velocity experiments yielded an estimate of the heterodimer sedimentation coefficient, s020,w, of (4.5 ± 0.2) S which for M = 63,700 Da suggests a globular structure.

X-ray crystallographic studies have previously shown that glutamate dehydrogenase from Clostridium symbiosum is a homohexamer. Mutation of the active-site aspartate-165 to histidine causes an alteration in the structural properties of the enzyme. The mutant enzyme, D165H exists predominantly as a single species of lower molecular mass than the wild-type enzyme as indicated by gel filtration and sedimentation velocity analysis. The latter technique gives an S20,w value for D165H of (6.07 +/- 0.01)S which compares with (11.08 +/- 0.01)S for the wild-type, indicative of alteration of the homohexameric quaternary structure of the native enzyme to a dimeric form, a result confirmed by sedimentation equilibrium experiments. Further support for this is provided by chemical modification by Ellman's reagent of cysteine-144 in the mutant, a residue which is buried at the dimer-dimer interface in the wild-type enzyme and is normally inaccessible to modification. The results suggest a possible structural route for communication between the active sites and subunit interfaces which may be important for relaying signals between subunits in allosteric regulation of the enzyme.

Single-valued hydrodynamic coefficients of a rigid particle can be calculated from existing theories and computer programs for either bead models or ellipsoids. Starting from these coefficients, we review the procedures for the calculation of complex solution properties depending on rotational diffusion, such as the decays of electric birefringence and fluorescence anisotropy. We also describe the calculation of the scattering from factor of bead models. The hydrodynamic coefficients and solution properties can be combined to give universal, shape-dependent functions, which were initially intended for ellipsoidal particles, and are extended here for the most general case. We have implemented all three developments in a new computer program. SOLPRO, for calculation of SOLution PROperties, which can be linked to existing software for bead models or ellipsoids.

The SH2 domain from Fyn tyrosine kinase, corresponding to residues 155-270 of the human enzyme, was expressed as a GST-fusion protein in a pGEX-E. coli system. After thrombin cleavage and removal of GST, the protein was studied by heteronuclear NMR. Two different phosphotyrosyl-peptides were synthesized and added to the SH2 domain. One peptide corresponded to the regulatory C-terminal tail region of Fyn. Sequence-specific assignment of NMR spectra was achieved using a combination of 1H-15N-correlated 2D HSQC, 15N-edited 3D TOCSY-HMQC, and 15N-edited 3D NOESY-HMQC spectra. By analysis of the alpha-proton chemical shifts and NOE intensities, the positions of secondary structural elements were determined and found to correspond closely to that seen in the crystal structure of the, homologous, Src-SH2 domain. To investigate the internal dynamics of the protein backbone, T1 and T2 relaxation parameters were measured on the free protein, as well as on both peptide complexes. Analytical ultracentrifugation and dynamic light scattering were employed to measure the effect of concentration and peptide-binding on self-association. The results suggest that, at NMR-sample concentrations, the free protein is present in at least dimeric form. Phosphopeptide binding and lower concentration significantly, but not completely, shift the equilibrium towards monomers. The possible role of this protein association in the regulation of the Src-family tyrosine kinases is discussed.

It has previously been suggested that the DNA binding domain (residues 1 to 147) of the yeast transcriptional activator GAL4 exists in solution in dimeric form, with the region responsible for dimerisation somewhere between residues 74 and 147. In this study limited proteolysis and carboxy-terminal deletions of the DNA binding domain (residues 1 to 147) of the yeast transcriptional activator GAL4 followed by subsequent characterization by equilibrium sedimentation in the analytical ultracentrifuge have been used to define more precisely the regions required for DNA binding and protein self-association. Sedimentation equilibrium analyses confirmed that the 'hydrophobic region' of the protein (residues 54-97, which contains a larger proportion of alpha-helix), is essential for dimerisation, with an apparent dissociation constant K(D,app), of approximately 50 microM for the 1-94 residue peptide and approximately 20 microM for the 1-147 residue peptide. Our studies do not rule out the possible formation of small amounts of additional higher order complexes.

Earlier studies using x-ray crystallography have shown that trimethylamine dehydrogenase (TMADH) from methylotropic bacteria exists as homodimers in the crystalline state. In this present hydrodynamic study we show that this is true also in dilute solution conditions and investigate the degree of swelling or relaxation of the protein in solution. Analytical ultracentrifugation was used to determine the molar mass and to investigate whether the homodimeric nature of this molecule in crystal form — as visualized by x-ray crystallography — is reproduced in dilute solution at temperatures between 4 and 40°C. The globular solution structure determined at 4 and 40°C is in good agreement with crystallographic data although trimethylamine dehydrogenase was found to be either more asymmetric in solution — or highly hydrated —, a phenomenon found to increase with temperature. In agreement with the crystallographic structure, the enzyme sediments as a homodimer with a molar mass of (163,000±5,000) g/mol. The concentration dependence of the sedimentation coefficient in the range of 0–1 mg/ml, indicates that no association or dissociation occurs. These findings are additionally supported by sedimentation equilibrium data in the concentration range of 0 to 1.8 mg/ml. Finally, from the sedimentation coefficient distribution at various temperatures, it was concluded that the enzyme is conformationally flexible and assumes an even more expanded structure at higher temperatures which is in good agreement with the hydrodynamic calculations performed.

Weight restriction has frequently been used to induce consumption of pharmacologically significant amounts of alcohol by rats. When previously weight-restricted rats are fed ad lib., however, their alcohol consumption is substantially reduced. This could occur because weight restriction per se causes increased alcohol consumption, or because the stimulus conditions (in this case, largely interoceptive) that were originally associated with drinking have changed, resulting in a stimulus generalization decrement. The present experiment was designed to discriminate between these two possibilities. Two groups of animals, one at free-feeding weight (FFW) and one at 80% FFW were initially trained to drink alcohol in a limited access paradigm. Each group was then divided into two, such that one half remained at its original weight and the other was gradually shifted to the reverse feeding condition in a double cross-over design. When alcohol consumption was again stable the experimental groups were returned to their original weights. If the weight restriction hypothesis were true, animals should drink more when weight-reduced and less when at FFW. According to the stimulus generalization decrement hypothesis, drinking should decrease with any shift from their initial weight. The direction of the results was such as to support the weight restriction hypothesis, but the magnitude of the changes was greater than would be expected on the basis of the respective control groups, and the effects were often transient. In these ways the results more closely resembled positive and negative contrast effects that have traditionally been described following shifts in amount of reinforcement. Possible mechanisms and the importance of an animal's history as a determinant of alcohol consumption are discussed.