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Proteins implement most biological functions at the molecular level. As one might expect based on this fact, it appears that the altered functional states associated with toxic effects involve changes in the abundance or structure of proteins. Although numerous specific assays exist to measure changes in the abundance of individual proteins, practical limitations have prevented widespread use of multiple protein assays for the global characterization of toxicity. Recent developments in protein analytical technology are rapidly changing this picture. Two-dimensional gel electrophoresis, a technique capable of resolving and quantitating hundreds of proteins simultaneously, is becoming an automated, high-throughput tool. In parallel, techniques have been developed that allow the resulting deluge of protein measurements to be organized into a prototype Molecular Effects Database™ describing xenobiotic effects in rodent liver. This database can detect, classify, and characterize a broad range of liver toxicity mechanisms. It currently contains approximately 10 million protein measurements, including data on the liver effects of 43 compounds, with a further 50 compounds to be added in 1995. Observed effects range from very broad (sex steroids alter levels of 45% of all liver proteins) to very specific (e.g., hepatic hydroxymethyl glutaryl coenzyme A reductase inhibitors). Companion 2-dimensional databases describing rodent brain and kidney have been initiated, as have linkages to the genomic sequence databases. Assimilation of this approach into research and regulatory toxicology poses an interesting challenge—one that is likely to lead to a radically more sophisticated understanding of toxicity and its biological basis.

The ISO-DALT system of two-dimensional electrophoresis allows high-resolution separations of proteins and protein subunits. However, the conventional isoelectric focusing employed in this system does not give satisfactory resolution of the more basic proteins, such as histones. The BASO-DALT system was designed to obtain improved resolution of these basic proteins in the first dimension. In this system, phosphatidyl choline is used as the solubilization agent, and allows resolution of many low molecular weight basic proteins that were not seem with more conventional detergents. Using the BASO-DALT system, Novikoff hepatoma chromosomal proteins have been analyzed, and the five histones identified.

Most human serum proteins consistently appear as more than one spot when analyzed by high resolution two-dimensional electrophoresis. We have characterized the “micro-patterns” corresponding to several of the principal proteins. Three minor forms of transferrin are observed (one larger than the predominant form and probably a precursor) and each shows carbohydrate heterogeneity similar to the main form. The haptoglobin β-chain exhibits stepwise heterogeneity in both pI (sialic acid variation) and SDS molecular weight (neutral sugar structure attachment), and is shown to give rise to a smaller, likewise heterogeneous product. The two frequent alleles of α2HS glycoprotein are shown to differ predominantly in sialic acid content rather than polypeptide charge, suggesting a sequence difference at a carbohydrate attachment site. All of the expected sources of microheterogeneity (sialic acid, proteolysis, allelism) are observed, plus an SDS molecular weight microheterogeneity probably due to variation in attachment of large neutral sugar structures.

Two-dimensional electrophoresis of proteins and protein subunits, employing isoelectric focusing in the first dimension and electrophoresis in the presence of sodium dodecyl sulfate in the second, yields the highest resolutions currently available. In this paper separations in the second dimension are considered (the so called DALT system). Methods for multiple-parallel casting of gradient gels in slab gel holders are described. The problem of electrical isolation of the ends of the slabs together with continuous cooling of both surfaces of the slab gel holder along their entire length has been achieved by running the gels in a horizontal direction in a three-compartment tank with the holders inserted in insulating septa. In the system described, 10 slabs are run simultaneously. This, however, is not the upper limit of the number of slabs which can be conveniently run in parallel.

The preparation and use of rat heart whole homogenate as a standard for the sodium dodecyl sulfate (SDS) electrophoresis dimension of two-dimensional electrophoresis is described. By including the rat heart homogenate in the agarose overlay used to hold an isofocusing gel (first dimension) in contact with a slab gel (second dimension), 80 horizontal lines can be superimposed on a two-dimensional electrophoresis pattern. Such an internal standard is useful as a reference marker for the intercomparison of many gels and also, when calibrated, can be used to determine the approximate molecular weight of proteins.

The ISO-DALT two-dimensional electrophoretic system (1,2), based on the method of O'Farrell (3), is capable of performing large numbers of analysis on complex mixtures of proteins. However, both separations employed are carried out under dissociating or denaturing conditions and no enzyme activities are readily observable in the analyzed proteins. In order to identify the spots corresponding to particular enzymes, it is therefore necessary to employ some nondestructive resolving technique first and as a second step to perform both enzyme and two-dimensional electrophoretic analyses on the fractions generated. By correlating enzyme activity with intensity of various spots on the two-dimensional gels throughout the series of initial fractions, identifications, can be made. This approach, unlike the more direct immunoprecipitation methods (4), requires the running of large numbers of enzyme analyses and two-dimensional gels and some convenient initial resolving procedure. Convenient and rapid techniques for the analyses (5,6) and gels (1,2) have been described previously in this series and elsewhere. This paper deals with the use of selective denaturation in a temperature gradient as an initial resolving procedure and describes a simple thermal gradient device for generating such a gradient.

Human erythrocyte lysate proteins were resolved into over 250 discrete spots by two-dimensional electrophoresis using isoelectric focusing in the first dimension and electrophoresis in the presence of sodium dodecyl sulfate, (SDS) in the second. The overwhelming excess of hemoglobin has made such analyses difficult in the past. However, with the ISO-DALT two-dimensional electrophoresis system, large numbers of red cell proteins can be mapped in the presence of hemoglobin. When hemoglobin and several other major proteins are removed by adsorption to DEAE-cellulose, additional minor components are seen, giving a total of over 275. With the use of purified preparations, the map positions of five cell enzymes or their subunits were determined: pyruvate kinase, catalase, glucose-6-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase, and carbonic anhydrase. The mapping techniques described complement and extend those traditionally used to find human red cell protein variants.

Principles and applications of an automatic cyclic affinity chromatography system, especially as applied to separation of proteins by immunosorption, are described. Use is made of columns of either immobilized antigens or antibodies to separate from a mixture, in a repetitive fashion, a desired protein or proteins by immunoadsorption followed by elution or to take from a mixture all proteins but the desired ones by allowing these latter to go through unadsorbed. The amplification provided by the cyclic use of the system and the biological amplification of hyperimmunization in achieving useful yields of desired proteins is discussed. Experience in the use of eluting solutions which do not cause damage to either the fixed or eluted proteins is presented. Representative separations done with the system are described, illustrating the different modes of use.

1. 1. Treatment of dilute rat thymus breis with concentrated NaCl solutions produced viscous solutions that were permanently disrupted by shearing. Attempts to measure the viscosity of such extracts in ordinary capillary viscometers were unsuccessful; however, reproducible measurements of apparent relative viscosity (± 2–3 per cent) were obtained by timing the first passage of the extracts through a modified Cannon-Fenske viscometer. 2. 2. The viscosity of extracts prepared in 1 M NaCl reached a maximum and constant value only after 10–12 hours of extraction at room temperature (25–27 °C). 3. 3. The maximum apparent relative viscosity of the extracts varied logarithmically with homogenate concentration. 4. 4. The viscosity of the extracts was markedly affected by pH. From pH 6.4 to pH 8.5, the viscosity was relatively constant; below pH 6.4, it fell rapidly, and, at lower pH's, was the same as for the solvent. Above pH 8.5, the specific viscosity increased rapidly to a maximum of about pH 10 and then dropped to zero at pH 11.5. 5. 5. The viscosity of the extracts was low in 0.05 to 0.4 M NaCl concentrations but increased rapidly in concentrations of 0.6 to 0.9 M. The viscosity of the extracts continued to rise slowly as the NaCl concentration was raised to 2 M but then decreased when the concentration was raised above 2 M. 6. 6. Viscosity decreased when the extracts or the breis from which the extracts were prepared were frozen. 7. 7. The results indicate that at least two types of protein-DNA bonds exist. The first, which cross-links DNA-protein strands in the nuclear gelwork, is easily broken by 1 M NaCl. The second, which binds the DNA molecules end to end to form long strands, is not broken in the initial extract and accounts for the high viscosity. The strands are believed to be the fundamental units of chromosome structure.

The distribution of catalase, rhodanese, acid phosphatase, nucleoside phosphorylase, esterase, adenosine deaminase, glutathione reductase, and β-glucuronidase in the free-electrophoresis pattern of the soluble phase of rat liver at pH 7.5 has been determined.

The taper in the K-II core was designed to aid in maintaining resolution during dynamic unloading. Static unloading has proved to be superior in practice however, thereby eliminating the requirement for a taper. Since, as shown here, superior recovery is obtained with a nontapered core, the latter design has been adopted for the K-III titanium continuous-sample-flow-with-banding rotor (17).

A number of artefacts which prevent ideal sedimentation have been found during the centrifugation of rat liver breis. These include hydrodynamic effects which cause local areas containing a number of large particles to move as a unit dragging along associated smaller particles, turnover effects at density boundaries, convection currents due to heat and mechanical agitation, and wall effects. Both breis and suspensions of red cells have been used in these studies. A separation approaching the ideal is obtained by the use of more dilute breis, careful layering over a continuous density gradient sufficiently steep to prevent the anomalies mentioned, and a rigid adherence to a gradual acceleration and deceleration schedule. Gradients of known characteristics are produced by a mechanical gradient engine. A stroboscopic illumination system has been devised for studying the behavior of layered systems in a refrigerated centrifuge having an observation port. A speed-versus-time recording system allows integration of the centrifuging done. Speeds are accurately measured to 0.02 per cent. New sector-shaped centrifuge tubes which minimize wall effects and a sliding device for recovering all levels of the gradient from the tube after centrifugation are described. Breis may now be quantitatively fractionated in one or two steps.

1. 1. One molar NaCl extracts of rat thymus are highly sensitive to X rays, and an exposure of 10 r causes a detectable loss in viscosity. An aftereffect was observed, and the viscosity of irradiated samples continued to decrease for 4–5 hours after irradiation. Dose curves were sigmoid. The effects of X rays on the extracts were largely indirect. Freezing, the addition of chemical protective agents, and increasing the concentration of the gell all had a dosereducing effect. There seemed to be a small contribution from direct effects not exceeding a few per cent of the total. 2. 2. Except for cyanide, all of the compounds tested that are known to protect animals from the lethal effects of irradiation also protected in the extracts. However, several compounds that had no protective action on animals gave large dose-reduction factors in the extracts. No good correlation exists between chemical agents protecting animals and those giving dosereducing effects in the extracts. 3. 3. The high sensitivity of the extracts to X-irradiation appeared to be a direct consequence of the high dilutions employed. Direct irradiation of the homogenate and excised tissue required, respectively, 100 and 700 times the X-ray dose necessary to produce the same effect in the extracts. The higher doses probably reflect protection from indirect effects by the high concentration of DNP in the cell nucleus and by protective substances such as proteins. The fact that one can demonstrate an effect by direct irradiation of the tissue is of interest, since no effects on DNA have been detected after irradiation in vivo .

A cuvet-rotor, designated G-IIB, has been developed that contains small-bore syphons attached to each of the fifteen cuvets. Drainage of reactants from the transfer disc was found to be approximately 90% complete in 1 second and 99% complete after 2.5 seconds during acceleration to 1000 rpm. Mixing of protein-biuret reagent solution and 30% sucrose occurred in 1 second when a vacuum was used to pull air back through the syphons and cuvets. Drainage of the cuvets through the syphons was almost quantitative (>99%) when air pressure and one cycle from 400 rpm to 1000 rpm and back to 400 rpm was used. The interval between successive sets of analyses may therefore be 1 minute or less. A method of heat cycling to make the Telflon cuvet-spacer conform to the glass windows is presented.

Information on the condensation of chromatin by organic substances is of interest in relation to attempts to formulate the properties of the chromosomal condensant active during cell division. The relation of nuclear volume to concentration has therefore been studied. n -Propylamine, an example of an aliphatic monoamine, produced swelling in low concentrations (0.001 to 0.01 M ) but little volume change in the range 0.025 to 0.36 M , closely paralleling results obtained with NaCl and KC1. Diamines (diaminopropane, -butane, -pentane, -hexane, -heptane, and -decane) produced shrinkage and granulation and nucleolar condensation in ≈ 0.025 M solutions and less shrinkage as the concentration of the diamines was increased. Condensation was readily reversed by either distilled water or the isolation medium. The effects of diamines resemble those of MgCl 2 and CaCl 2 . Spermidine and spermine, which possess three and four positive charges, respectively, produce condensation at very low (0.001 M ) concentrations, with marked swelling at approximately 0.2 M . Condensation was not reversed by distilled water and was slowly reversed by the phosphate-containing isolation medium. Chromosome-like structures were observed in a number of solutions but accurate counts were not possible. In a comparison of Amphiuma erythrocyte nuclei and rat liver nuclei, relatively few granules were seen in the former (2n = 24) whereas many were seen in liver nuclei (4n = 84), suggesting that the structures observed may be chromosomal in origin. Available evidence suggests that the prophase condensing agent contains more than two charges if condensation is caused only by charge effects.

Summary Performance of the K-II continuous-sample-flow-with-banding zonalcentrifuge rotor was evaluated using sucrose gradients and polystyrene latex beads having a mean diameter of 0.091 μ and a banding density of 1.049 gm/cm3 in sucrose at 20°C. Narrower particle zones were recovered with static unloading as compared with dynamic unloading. With the former, the average band width at half-height was 64 ml in the total volume of 3600 cm3, corresponding, to a width of 0.24 mm in the rotor. Multiple reorientations of the gradient produced only a small loss of resolution. Measurements of gradient decay and sucrose loss during continuous flow indicate that gradients suitable for influenza virus recovery may be retained in the rotor for 10 hr, while 200 liters of fluid passes through the rotor. The particle capture efficiency of the rotor is slightly less than predicted by the Berman theory.

A simple gradient device is described which allows convex gradients to be formed for use in zonal centrifuge rotors. The configuration chosen produces very rapid mixing of the very viscous sucrose solutions. By varying the total volume of the mixer, the shape of the gradient may be altered within rather wide limits. With large volume mixers, gradients may be obtained which are very nearly linear with radius when placed in the sector-shaped compartments of the rotor. With smaller volume mixers, gradients that are convex with respect to radius when in position in the rotor may be generated which are more useful where high-capacity high-resolution separations are required.

1. Two months after injection of rat bone marrow into lethally X-irradiated mice (950 r-RBM mice), 100% of the circulating RBC were serologically of the rat type, indicating that the surface molecular configuration of RBC from these experimental mice are of the rat type. 2. The hemoglobin was found to be also very much like the rat type in its ease in crystallization, its alkali denaturation property, its electrophoretic property, and its tendency to form a paracrystalline state at low temperature. 3. These cells possessed dual osmotic properties; the relative hemoglobin concentration released when cells were lysed in water was more comparable to the rat type, but its temperature dependency was more comparable to the mouse type.

An inexpensive yet versatile device for driving the mechanical stage of a microscope is described. The device provides for continuous variable-speed control and positively monitors the distance traversed at short and known increments of stage travel. This device was attached to the horizontal stage of a microscope used as the optical component of a microdensitometer in order to facilitate the accurate and precise determination of migration distances when scanning developed electrophoresis patterns.

A simple system, based on a series of vessels arranged so that the top of each is at the midlevel of its predecessor, and all draining by gravity into a common line, has been shown to be capable of producing very complex, predictable gradients. By simply changing the levels, so that the top of each chamber is below the level of its predecessor, a step elution system may be obtained.

The gel released from isolated rat liver nuclei in response to heparin treatment has been found to stain with methylene blue, azure A, and methyl green when the dyes were added to the salt-sucrose nuclear isolation medium. Azure A and methylene blue caused rapid nuclear shrinkage to as little as 1 4 the original nuclear volume. Subsequent treatment with heparin caused the nuclei to fade rapidly and swell to approximately 5 4 of the original volume. With methylene blue stained nuclei heparin caused the extrusion of deeply stained, slightly birefringent rods through apertures on the nuclear surface. Methyl green also caused nuclear shrinkage, but to a lesser degree. Studies with the Feulgen reaction demonstrated structural damage in isolated rat liver nuclei as a result of heparin action. The viscous material released by heparin was shown to be Feulgen positive by resort to hydrolysis without prior fixation, since after customary fixatives the presence of a Feulgen positive reaction outside the nucleus could not be clearly demonstrated. The possibility is suggested that the Feulgen reaction following the customary fixatives depends in part on the manner in which the DNA is bound. The nuclei of leucocytes with visually intact cell membranes included in the nuclear preparations failed to show structural damage due to heparin and it is suggested that either the cell membrane provides some protection against heparin action or that damaged cells are more susceptible to this action. Observations made provide additional basis for the conclusion that heparin replaces DNA in the nucleo-histone of the nucleus, resulting in the structural damage observed, and releasing DNA in the form of a soluble viscous protein containing complex.

1. The effects of morganic ions, electrolyte concentration, and pH on the appearance and volume of the isolated rat liver nucleus have been studied. Nuclei were isolated by differential centrifugation in a buffered salt-sucrose mixture at pH 7.1. Nuclear volumes were determined photographically. 2. In solutions of NaCl, of KCl, and in potassium phosphate buffers the nuclear volume decreased markedly with an increase in concentration from 0.001 M to 0.05 M but remained essentially constant with further increase in concentration to 1.0 M. The effects of CaCl2 and MgCl2 differed from those of NaCl and KCl in that a smaller volume was obtained in concentrations less than 0.15 M, and in the case of CaCl2 an increase in volume was obtained in more concentrated solutions. The volume changes are considered to be due primarily to ionic effects on the nuclear colloids rather than to osmotic behavior. 3. Treatment of nuclei with DNAase prevented the characteristic volume changes resulting from ion effects, suggesting the importance of DNA in nuclear volume changes. 4. The optical changes in isolated nuclei in various concentrations of KCl, NaCl, CaCl2, MgCl2, and in potassium phosphate buffers as observed under phase contrast illumination are described. CaCl2 gave the most marked nuclear changes from the conditions in the uninjured cell and caused shrinkage and granulation in 0.001 M concentration. The effects of CaCl2 were also manifested in 0.88 M sucrose, in mixtures with monovalent salts, and in serum. Changes in nuclear volume and optical appearance which occurred in salt solutions and in 0.1 N HCl were readily reversible. 5. Nuclear volume remained constant between pH 8.91 and 5.12 and decreased in more acid solutions. 6. Sucrose had no appreciable osmotic effect, and in hyperosmotic solution. (0.88 M) nuclei showed swelling and rupture comparable to that in distilled water. 7. The results are considered in relation to the requirements of nuclear isolation media. 8. Rat liver nuclei isolated in a buffered salt-sucrose medium by differential centrifugation exhibited a pattern of size distribution similar to that of fixed nuclei but were of considerably larger volume. The ratio of the volumes of the peak frequencies of the two chief size groups was 1:1.9.

The zonal ultracentrifuge was used to separate the subcellular components of rat liver brei into soluble phase, microsomal, mitochondrial, membranous fragments, and nuclear fractions during a single centrifugation. The centrifuge was run at 10,000 to 30,000 RPM for 15 to 240 minutes, and the rotor contained a 1200 ml sucrose gradient, varying linearly with radius from 17 to 55 per cent sucrose with a "cushion" of 66 per cent sucrose at the rotor edge. The distribution of the mitochondria was determined using cytochrome oxidase as the marker enzyme. An automated assay system for cytochrome oxidase was developed utilizing reduced cytochrome c as substrate, modules of the Technicon Autoanalyzer, and the Beckman DB Spectrophotometer. All of the cytochrome oxidase activity was restricted to a single peak in the gradient, and no activity could be detected in the zones occupied by the microsomes and nuclei. The mitochondrial fraction was isolated from rat liver brei in 0.25 M sucrose by differential centrifugation, and then run in the zonal ultracentrifuge.This fraction behaved in the zonal ultracentrifuge in the same way as mitochondria separated directly from intact brei. Observations of the isolated fractions in the phase contrast microscope indicated that a wide variety of granules was present in the mitochondrial zone in addition to the true mitochondria. Under the conditions employed, the mitochondria were sedimented essentially to their isopycnic position in the gradient at approximately 43.8 per cent sucrose, density 1.20 gm/cc.

Zonal separation of particles in density gradients contained in hollow or tubeless rotors has been explored with the aim of developing a preparative counterpart of the analytical ultracentrifuge. A rotor with a capacity of 1625 milliliters has been tested up to 22,500 revolutions per minute, with sucrose density gradients used. Excellent separation of subcellular particles has been achieved as well as partial separation of the albumin and globulin peaks of serum.

Complex manifolds are required to arrange and control flow of liquids during the loading and unloading of zonal centrifuges, in vaccine production using the continuous-sample-flow-with-banding K-type centrifuge, in rapid recycling affinity chromatography, in continuous flow analyzers, and in a variety of devices and systems using density gradients. In many instances permanent metal manifolds or valves are to be avoided because they are expensive to fabricate, are difficult to clean, and may accumulate sediments and microorganisms which are not observed because they are opaque. Glass manifolds are fragile and are not easily made by the uninitiated. Connections between plastic tubing and glass may separate under pressure, especially when detergents are present. The advantages of using plastic tubing, and hemostats in place of valves to control complex flow patterns, have been previously stressed. A simple solution to the problems of manifolds of metal or glass is to use various sizes of Tygon tubing cemented together with cyclohexanone containing approximately 5 to 10% dissolved Tygon. Very small tubing (less than 1/8-in. i.d.) may be clogged by the cement and should be cleared of liquid cement with low-pressure air. Very large pieces of Tygon tubing may need to be held in place with clampsmore » or jigs until the cement has set. These Tygon manifolds have been found very resistant to tearing and easily cleaned and replaced.« less

Liquid density gradients and experimental sample bands have been successfully recovered from a hollow-bowl centrifuge rotor in which a static gradient is reoriented into a radial configuration during acceleration and is subsequently recovered in its original orientation at rest. Analysis of fluid movements in the rotor indicate that very little shearing occurs during gradient reorientation. The reorienting gradient rotor principle makes feasible the construction of high-speed, large-volume rotors suitable for banding nucleic acids and proteins in salt gradients, and for the separation of macromolecules on the basis of their sedimentation rate through density gradients.

A method is designed for the rapid fractionation of large volumes of suspensions of particles varying in size and density by gradient differential centrifugation. A centrifuge distribution head is used that permits the rapid construction of continuous liquid density gradients since the gradients are stabilized by centrifugal force as they are formed. The application of the method to the fractionation of rat liver breis is described.

A variety of structures, many of which resemble components of liver cells, have been shown to form in clear ultracentrifuged extracts of rat liver. The structures appear after dialysis against isotonic sucrose, pH 7.5 phosphate buffers, or after standing in the cold. When the pH is lowered to 5 or 6, fibrils are readily formed. It is suggested that these structures may form in rat liver breis. The possibility is discussed that cell components exist in equilibrium with their constituent molecules in solution and that mechanisms similar in part to those involved in structure formation in solution may also obtain in the living cell.

Summary The K-II continuous-sample-flow-with-banding-9 zonal centrifuge hasbeen developed for large-scale virus isolation. The cylindrical aluminum rotor (capacity 3600 ml) contains a 3 liter gradient and has a 700 ml stream volume. Fluid line seals are located on opposite ends of the rotor, eliminating the possibility of cross-leakage. An air turbine drive is used to accelerate the rotor to 35,000 rpm (83,440g, maximum). The development of safe armor and control systems is described.

Loss of viability, most often expressed as a failure of irradiated cells to divide indefinitely, has been the major criterion in evaluating radiation damage at the cellular level. In terms of the cell cycle, the ultimate aim is to find radiation-sensitive phases in the cell cycle, especially if they are intimately associated with cell division (1-8). A variety of approaches have been devised to single out the response of those individual cells that were irradiated at a given stage in the cell cycle. Cells have been labeled with radioactive precursors of nucleic acids, irradiated, and followed for several generations (1-3). Puck and Steffen (9) have developed a system of mathematical analysis based on the kinetics of cellular proliferation that follows the introduction of a metabolic or radiological block of cell division. This approach permits the localization of radiation lesions in the cell cycle of cells whose pattern of division fits the mathematically derived formulations. Terasima and Tolmach (10) have developed a technique for isolating mitotic HeLa cells from exponentially growing cultures. These cells then divide synchronously for a limited number of generations. They can then be used to study radiation sensitivity throughout the cell cycle. Such a method, essentially representing a technique for synchronization of cell division, has been subsequently modified so that large populations of HeLa cells can be isolated and employed in biochemical studies (11). The technique has also been extended to other cell types (12, 13). Other methods of synchronization of mammalian cells have included the use of nucleic acid precursors or analogs (14, 15) and tem