Automatic lipid extraction and thin-layer chromatography application with a prgrammed flow system

An eight-channel programmed flow system for automatic lipid extraction and TLC application is described. Each channel has a container for lipid extraction connected by Acidflex tubing through an AutoAnalyzer pump to a TLC applicator needle. Extraction containers are prepared from disposable Oxford sampler pipet tips by inserting a small cotton filter into their lower, narrower end, which is connected to the pump tubing. The applicator needles are supported vertically in a manifold, and their tips rest on a TLC plate placed on a hot plate. Serum is added to isopropanol in each extraction container, and proteins are completely precipitated in 2 min and retained in the extraction chambers by the cotton filters; lipid extracts are then transferred on to the heated TLC plate by intermittent pumping at a rate allowing for continuous evaporation of isopropanol under streams of warmed air or nitrogen. The lipids accumulate on the plate in eight small spots, one for each channel. Solvent is proportionally added into the extraction chambers from a common reservoir through Acidflex tubing in a second AutoAnalyzer pump. During the extraction procedure, both pump motors are automatically operated by a programmed timer with a solid-state switch. Of several different solvents tested, isopropanol is the fastest for protein precipitation and lipid extraction and does not extract substances from the Acidflex tubing which interfere with chromatographic separation.     

Automated protein crystallization and a new crystal form of a subtilisin:eglin complex

A procedure is described for automating labour-intensive steps of the 'hanging drop' protein crystallization method. An automatic sample changer is employed to fill the wells in a multi-well plate so that concentration gradients in various components are obtained. The sample changer is also used for preparing droplets on a second multi-well plate. Subsequently, this second plate is manually turned around and placed on top of the first multi-well plate such that a large number of chambers with different conditions is obtained simultaneously. During initial trials a new crystal form of a subtilisin:eglin complex was obtained. The crystals have space group P2(1), contain two enzyme inhibitor complexes per asymmetric unit and diffract beyond 2.2 A.     

Multiple heavy-atom reagents for macromolecular X-ray structure determination: Application to the nucleosome core particle

The X-ray structure of the nucleosome core particle was solved at 7 Å resolution using the method of multiple isomorphous replacement based on two isomorphous derivatives, each containing a different multiple heavy-atom compound. The preparation of these heavy-atom compounds and their application to this macromolecular structure determination are described. The first of these reagents, TAMM (tetrakis(acetoxymercuri)methane), was solubilized by the addition of an excess of glycylglycine and, when added to crystals of the nucleosome core particle, produced a derivative with a single major site. Despite the large mass of 206,000 daltons per asymmetric unit, the position of the TAMM molecule was found in these crystals using the difference Patterson technique. This compound was sufficiently electron-dense to produce a unique solution, whereas the mono-mercurial, methylmercury nitrate had been inadequate. The second reagent, PIP (di-μ-iodobis(ethylenediamine)diplatinum(II) nitrate), is freely soluble in aqueous solution and, on addition to the crystals, labelled the histone proteins at several sites. The locations of the PIP groups were determined from difference Fourier and Patterson maps. The X-ray structure and solution characterization of this compound are reported. These multiple heavy-atom compounds appear to be generally applicable to X-ray structure determination, and are particularly useful in conjunction with crystals having asymmetric units of large volume but lacking non-crystallographic symmetry elements.     

Robotic nanolitre protein crystallisation at the MRC Laboratory of Molecular Biology

We have set up high-throughput robotic systems to screen and optimise crystallisation conditions of biological macromolecules with the aim to make difficult structural biology projects easier. The initial screening involves two robots. A Tecan Genesis liquid handler is used to transfer commercially available crystallisation reagents from 15 ml test tubes into the reservoirs of 96-well crystallisation plates. This step is fully automated and includes a carousel for intermediate plate storage, a Beckman plate sealer and a robotic arm, which transfers plates in between steps. For adding the sample, we use a second robot, a 17-tip Cartesian Technologies PixSys 4200 SynQuad liquid handler, which uses a syringe/solenoid valve combination to dispense small quantities of liquid (typically 100 nl) without touching the surface of the plate. Sixteen of the tips are used to transfer the reservoir solution to the crystallisation wells, while the 17th tip is used to dispense the protein. The screening of our standard set of 1440 conditions takes about 3 h and requires 300 microl of protein solution. Once crystallisation conditions have been found, they are optimised using a second Tecan Genesis liquid handler, which is programmed to pipette gradients from four different corner solutions into a wide range of crystallisation plate formats. For 96-well plates, the Cartesian robot can be used to add the sample. The methods described are now used almost exclusively for obtaining diffraction quality crystals in our laboratory with a throughput of several thousand plates per year. Our set-up has been copied in many institutions worldwide.     

Two-dimensional parallel array technology as a new approach to automated combinatorial solid-phase organic synthesis

An automated, 96-well parallel array synthesizer for solid-phase organic synthesis has been designed and constructed. The instrument employs a unique reagent array delivery format, in which each reagent utilized has a dedicated plumbing system. An inert atmosphere is maintained during all phases of a synthesis, and temperature can be controlled via a thermal transfer plate which holds the injection molded reaction block. The reaction plate assembly slides in the X-axis direction, while eight nozzle blocks holding the reagent lines slide in the Y-axis direction, allowing for the extremely rapid delivery of any of 64 reagents to 96 wells. In addition, there are six banks of fixed nozzle blocks, which deliver the same reagent or solvent to eight wells at once, for a total of 72 possible reagents. The instrument is controlled by software which allows the straightforward programming of the synthesis of a larger number of compounds. This is accomplished by supplying a general synthetic procedure in the form of a command file, which calls upon certain reagents to be added to specific wells via lookup in a sequence file. The bottle position, flow rate, and concentration of each reagent is stored in a separate reagent table file. To demonstrate the utility of the parallel array synthesizer, a small combinatorial library of hydroxamic acids was prepared in high throughput mode for biological screening. Approximately 1300 compounds were prepared on a 10 μmole scale (3-5 mg) in a few weeks. The resulting crude compounds were generally >80% pure, and were utilized directly for high throughput screening in antibacterial assays. Several active wells were found, and the activity was verified by solution-phase synthesis of analytically pure material, indicating that the system described herein is an efficient means for the parallel synthesis of compounds for lead discovery. Copyright 1998 John Wiley & Sons, Inc.     

Chemiluminescence ELISA for the detection of antibodies to bovine leukaemia virus antigens

A chemiluminescence enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to bovine leukaemia virus antigens (BLV) has been developed. The possibility of using an enhanced chemiluminescence reaction for the determination of adsorbed immunoperoxidase conjugates was studied in this work. The intensity of chemiluminescence depends on both the concentration of reagents and experimental conditions used. The efficiency of the assay is determined by the formation of an immobilized antigen monolayer. A relationship between the quantity of the protein added and adsorbed has been shown. The optimal time and temperature for the antigen–antibody incubation steps have been estimated for each system (3h at 37°C was chosen as a standard incubation time). A linear dependence of the chemiluminescence intensity and optical density on the concentration of antibodies to the BLV antigens was observed. The detection limit of antibodies in the chemiluminescence ELISA is 2–3 times lower than that in the spectrophotometric one. The results obtained indicate the possibility of using both methods.     

Crystallization and preliminary X-ray crystallographic analysis of Mirabilis antiviral protein.

Mirabilis antiviral protein is a single-chain ribosome-inactivating protein purified from the tuberous root of Mirabilis jalapa L. We obtained several forms of crystals of the protein by the hanging drop vapor diffusion method, but most of these crystals were not suitable for X-ray crystallography. After refining the growth conditions, crystals of crystallographic quality were grown in 20-microliters droplets of an equi-volume mixture of 1.5% (w/v) protein solution and a reservoir solution containing 49 to 50% (w/v) ammonium sulfate and 50 mM-ammonium citrate (pH 5.4) at room temperature. Addition of 2 mM-adenine sulfate reduced twinning and "crystal shower". The resulting trigonal crystals diffract beyond 2.5 A resolution using a rotating anode X-ray generator. The space group was determined to be P3(1)21 or P3(2)21 (a = b = 103.9.A, c = 134.6 A, alpha = beta = 90 degrees, gamma = 120 degrees) based on their precession photography of h0l and hk0 zones. There seems to be three monomers in an asymmetric unit for VM = 2.51 A3/Da.     

MERCURY INHIBITION ON PRIMARY PRODUCTIVITY USING LARGE VOLUME PLASTIC CHAMBERS IN SITU1

Approximately 6000 l of lake water was suspended in large, clear plastic chambers in Lake Powell, Arizona. The chambers were monitored during 24 h incubation periods in the spring and summer 1974 and 1975. Physico-chemical differences between the natural lake system and within the experimental chambers were negligible. Experimental mercury concentrations ranging from < 0.02 to 1.25 ppm were added to the chambers to determine the effect of elevated Hg concentrations on in situ primary production. Mercury concentrations were monitored in the water column of each chamber during each 24 h incubation period to determine absolute concentrations following reduction through adsorption, absorption and vaporization. At least a 40% reduction in photosynthetic activity occurred at Hg concentrations as low as 0.06 ppm. A toxic Hg threshold concentration of 0.06 ppm was demonstrated for the summer phytoplankton assemblage, but a distinct threshold concentration was absent for the spring diatom assemblage. Differences in spring and summer phytoplankton populations may suggest subtle differences in Hg sensitivity between phytoplankton assemblages in combination with temperature acting on total community metabolism.     

Repeated seeding technique for growing large single crystals of proteins

A seeding method has been developed for growing large single crystals of globular proteins once small, preliminary specimens have been obtained. A small, carefully washed, crystal is used to seed a protein solution. After growth has stopped, the crystal is removed and inserted into a fresh protein solution, which allows it to grow further. This process can be repeated until the crystal has reached the desired dimensions. In several instances isomorphous heavy-atom derivatives could be obtained by including heavy-atom reagents in the seeded protein solution. This seeding technique is shown to work reproducibly with several proteins and under different conditions, suggesting that it might be generally applicable.     

Relationship of crystallization to the nature of polyhedron protein,with reference to a nuclear-polyhedrosis virus of the silkworm, Bombyx mori

When the silkworm nuclear polyhedrosis inclusion bodies were exposed to a drop of weak alkaline solution, the protein part of the bodies dissolved, and subsequently recrystallized out with the gradual evaporation of water from the preparation under ordinary room condition. The crystals assumed a variety of crystalline forms. These crystalline forms could be demonstrated by a simulation of folding paper strips along the edge or diagonal lines of rhombi which are to be formed on the strips serially by passing parallel lines with an inclination of 60° against the strip edge. Polyhedron protein was very homogeneous and simple in nature, the sedimentation constant being around 6.26 Svedberg units (S). These protein particles were easily associated to form large complexes which gave 13.6, 19.7, 29.7, and 38.0 S in neutral solutions. Associated polyhedron protein complexes were separable into various sizes by ultracentrifuge and disc electrophoresis.     

Effect of Coccolith Polysaccharides Isolated from the Coccolithophorid, <Emphasis Type="Italic">Emiliania huxleyi</Emphasis>, on Calcite Crystal Formation in In Vitro CaCO<Subscript>3</Subscript> Crystallization

Marine coccolithophorids (Haptophyceae) produce calcified scales “coccoliths” which are composed of CaCO₃ and coccolith polysaccharides (CP) in the coccolith vesicles. CP was previously reported to be composed of uronic acids and sulfated residues, etc. attached to the polymannose main chain. Although anionic polymers are generally known to play key roles in biomineralization process, there is no experimental data how CP contributes to calcite crystal formation in the coccolithophorids. CP used was isolated from the most abundant coccolithophorid, Emiliania huxleyi. CaCO₃ crystallization experiment was performed on agar template layered onto a plastic plate that was dipped in the CaCO₃ crystallization solution. The typical rhombohedral calcite crystals were formed in the absence of CP. CaCO₃ crystals formed on the naked plastic plate were obviously changed to stick-like shapes when CP was present in the solution. EBSD analysis proved that the crystal is calcite of which c-axis was elongated. CP in the solution stimulated the formation of tabular crystals with flat edge in the agarose gel. SEM and FIB-TEM observations showed that the calcite crystals were formed in the gel. The formation of crystals without flat edge was stimulated when CP was preliminarily added in the gel. These observations suggest that CP has two functions: namely, one is to elongate the calcite crystal along c-axis and another is to induce tabular calcite crystal formation in the agarose gel. Thus, CP may function for the formation of highly elaborate species-specific structures of coccoliths in coccolithophorids.     

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein''s phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.     

Lysozyme crystal growth, as observed by small angle X-ray scattering, proceeds without crystallization intermediates

A combination of small angle X-ray scattering and gel techniques was used to follow the kinetics of protein crystal growth as a function of time. Hen egg white lysozyme, at different protein concentrations, was used as a model system. A new sample holder was designed, in which supersaturation is induced in the presence of salt by decreasing the temperature. It had been shown previously that a decrease in temperature and/or an increase in crystallizing agent induces an increase in the attractive interactions present in the lysozyme solutions, the lysozyme remaining monomeric. In the present paper we show that similar behaviour is observed in NaCl when agarose gels are used. During crystal growth, special attention was paid to determine whether oligomers were formed as the protein in solution was incorporated in the newly formed crystals. From these first series of experiments, we did not find any indication of oligomer formation between monomer in solution and crystal. The results obtained are in agreement with the hypothesis that lysozyme crystals in NaCl grow by addition of monomeric particles. Received: 28 July 1997 / Revised version: 4 December 1997 / Accepted: 5 December 1997     

Factors affecting struvite (MgNH4PO4.6H2O) crystallization in feline urine

Factors affecting struvite, a magnesium-ammonium-phosphate complex (MgNH(4)PO(4).6H(2)O), in feline urine were evaluated. Incubation of just "urine mineral (UM)" solution, in which mineral concentrations are compatible with those in feline urine, for 4 h at 37 degrees C did not induce the formation of crystals. Similarly, incubation of urine alone did not produce crystals. However, struvite crystals were formed by the addition of urine to UM solution. Mg, NH(3) and P were all required for urine-induced struvite crystallization. The lower molecular weight (LMW) fraction of urine was essential for struvite crystal formation, and the higher molecular weight (HMW) fraction enhanced formation of LMW-induced struvite crystals. The effects of urine proteins further fractionated by column chromatography were examined. A protein at >250 kDa and cauxin, a major urine protein recently identified as a regulator of felinine production, potentiated struvite crystal formation induced by the LMW fraction. In contrast, Tamm-Horsfall glycoprotein, a urine protein thought to promote struvite crystallization, did not have this activity. The present study reveals a novel mechanism of feline struvite crystallization.     

Metal ion dependence of a heat-modifiable protein from the outer membrane of Escherichia coli upon sodium dodecyl sulfate-gel electrophoresis.

One heat-modifiable protein of Escherichia coli outer membrane does not completely change to the high-temperature form in the presence of magnesium ion in sodium dodecyl sulfate solution. When the metal ion complexing reagents ethylenediaminetetraacetic acid, phosphate ion, hydroxyl ion, or the competitive cations Zn2+ or Ca2+ are added to the sodium dodecyl sulfate-solubilized sample of outer membrane, and then the sample is heated to 100 degrees C and recooled to room temperature, the protein is almost completely converted to the high-temperature form. In control samples, or if sodium chloride, magnesium chloride, or manganous chloride are added to these samples and treated the same way, a large amount of the low-temperature form of the protein is preserved. beta-Mercaptoethanol additions gave the same results as the metal ion complexing reagents and may owe its activity in these solutions to metal-binding activity and not to its role as a reducing reagent. We concluded that magnesium ion may be involved with stabilization of the low-temperature form of the protein either by directly binding the magnesium or by mediating interaction with other components of the membrane.     

Use of layer silicate for protein crystallization: effects of Micromica and chlorite powders in hanging drops

Two kinds of layer silicate powder, Micromica and chlorite, were used to aid protein crystallization by the addition to hanging drops. Using appropriate crystallization buffers, Micromica powder facilitated crystal growth speed for most proteins tested in this study. Furthermore, the addition of Micromica powder to hanging drops allowed the successful crystallization of lysozyme, catalase, concanavalin A, and trypsin even at low protein concentrations and under buffer conditions that otherwise would not generate protein crystals. Except for threonine synthase and apoferritin, the presence of chlorite delayed crystallization but induced the formation of large crystals. X-ray analysis of thaumatin crystals generated by our novel procedure gave better quality data than did that of crystals obtained by a conventional hanging drop method. Our results suggest that the speed of crystal growth and the quality of the corresponding X-ray data may be inversely related, at least for the formation of thaumatin crystals. The effect of Micromica and chlorite powders and the application of layer silicate powder for protein crystallization are discussed.     

Elimination of Fat and Protein Prior to the Selective Staining of Bone

The technic of staining skeletal systems previously described is often unsatisfactory for fetal specimens of Aves, because of the large amount of fat and protein. The writer avoids this by introducing two preliminary steps: (1) The specimen is placed in equal parts of glycerin, 95% alcohol and distilled water, and 10% aqueous pepsin (with a drop of 6N HC1 added) injected into the yoik sac, with 2-3 hours incubation at 40^oC. (2) While in 5% aqueous KOH (with a few drops of 2% H₂O₂), the fat areas are injected with cellosolve; and the specimen is left in this solution until skeletal elements become clearly visible. Staining in alizarin red S then follows.     

High throughput metabolic stability screen for lead optimization in drug discovery

A high throughput approach for the determination of in vitro metabolic stability and metabolic profiles of drug candidates has been developed. This approach comprises the combination of a Biomek FX liquid handling system with 96-channel pipetting capability and a custom-designed 96-well format on-line incubator with efficient thermal conductivity. This combination facilitates automated reagent preparation, sample incubation, and sample purification for microsome stability studies. The overall process is both fast and accurate and meets the challenges of high throughput screening for drug discovery. A custom designed, user-friendly computer program has been incorporated for large-scale data processing and report generation. Several applications are discussed that implement this strategy for rapid selection of compounds in early drug discovery.     

The effect of adenosine triphosphate,magnesium chloride and phospholipids on crystal formation in the demineralized shell-repair membrane of the snail,Helix pomatia L.

Summary The effect of adenosine triphosphate (ATP), magnesium chloride (MgCl₂) and phospholipids on the calcium-binding activity and crystal formation within the decalcified shell-repair membrane of the snail, Helix pomatia, was studied in vitro. The application of ATP produced a characteristic dual effect on calcification: (1) It strongly inhibited the formation of inorganic calcium carbonate (CaCO₃) crystals. (2) It stimulated the development of organic crystalline bodies and induced deposition of amorphous calcium carbonate. The demineralized shell-repair membranes became white and rigid after incubation for 7 days in the medium containing 1.0mM ATP. The inhibitory effect of Mg²⁺ on CaCO₃ crystal formation was diminished by reduction of the concentration of MgCl₂ in the incubation solution. Thus, after incubation for only 24h, 1.0mM MgCl₂ promoted the formation of birefringent CaCO₃ crystals within the repair membranes. The principal effect of phospholipids on the demineralized shell-repair membrane was stimulatory, but after application of phospholipids to the medium, the formation of crystals proceeded slowly. The very large, composite crystals that were formed within the repair membranes showed strong birefringence. In all cases the development of the crystals and the organic crystalline bodies occurred in close vicinity to the amoebocytes. The role of ATP, MgCl₂ and phospholipids in the recalcification of shell-repair membrane is discussed.The author wishes to thank Mrs. E. Hellmén for valuable technical assistance