Dependence upon temperature of corrected sedimentation coefficients measured in a Beckman analytical ultracentrifuge

The corrected sedimentation coefficient (s_{20, w}) has been examined as a function of temperature for bovine liver catalase and for a small linear duplex DNA in a Beckman Model E analytical ultracentrifuge. These measurements were carried out using both the standard Beckman rotor temperature indicator and control system and a modified version of this system which was designed to reduced thermal gradients in the rotor [Hearst, J. E., and Gray, H. B., Jr. (1968) Anal. Biochem.24, 70–79)]. The temperature dependence of s_{20, w} for both the protein and the DNA were identical between the two systems. The slight increase of s_{20, w} with increasing temperature observed for the DNA is in agreement with that predicted on the basis of the temperature dependence of the Kuhn statistical length for DNA. Intrinsic viscosity measurements as a function of temperature were also carried out for the DNA in a low-shear viscometer. These data indicate a slight decrease of intrinsic viscosity with increasing temperature. Previous investigations, which have suggested that the actual temperature of the ultracentrifuge cell is substantially higher [Incardona, N. L., Notarius, H., and Flanegan, J. B. (1971) Anal. Biochem.40, 267–280] or lower [Rowe, A. J., and Khan, G. M. (1972) Anal. Biochem.45, 488–497] than that indicated by the temperature monitoring system of the Model E centrifuge at temperatures below ambient, are not supported by the present studies. Discrepancies between the actual temperature of the sedimenting sample and the indicated temperature are thus shown in this work not to be an inherent problem of the design of the Beckman instrument.     

Evaluation of diffusion coefficients by means of an approximate steady-state condition in sedimentation velocity distributions

This investigation examined the feasibility of manipulating the rotor speed in sedimentation velocity experiments to spontaneously generate an approximate steady-state condition where the extent of diffusional spreading is matched exactly by the boundary sharpening arising from negative s–c dependence. Simulated sedimentation velocity distributions based on the sedimentation characteristics for a purified mucin preparation were used to illustrate a simple procedure for determining the diffusion coefficient from such steady-state distributions in situations where the concentration dependence of the sedimentation coefficient, s = s⁰/(1 + Kc), was quantified in terms of the limiting sedimentation coefficient as c → 0 (s⁰) and the concentration coefficient (K). Those simulations established that spontaneous generation of the approximate steady state could well be a feature of sedimentation velocity distributions for many unstructured polymer systems because the requirement that Kc_oω²s⁰/D be between 46 and 183 cm⁻² is not unduly restrictive. Although spontaneous generation of the approximate steady state is also a theoretical prediction for structured macromolecular solutes exhibiting linear concentration dependence of the sedimentation coefficient, s = s⁰(1 − kc), the required value of k is far too large for any practical advantage to be taken of this approach with globular proteins.     

Solubility of hemoglobin S by sedminetation, equilibrium, and antisickling compounds.

The effect of the compounds guanidine, arginine, lysine, and aspartic acid and the salt arginine aspartate on the solubility of deoxyhemoglobin S (Hb S) was studied by sedimentation equilibrium at 20–22 °C. Guanidine and arginine were found to be most effective, whereas aspartic acid and lysine had only a small effect. The effectiveness of these compounds in solubilizing Hb S is relatively pH independent. It is unlikely that the small effect of lysine and aspartic acid on the solubility of Hb S can account for the antisickling properties of lysine and aspartic acid previously reported (Sophianopoulos, A. J., et al. (1974) Clin. Biochem.7, 112–118). The effect of guanidine and arginine is large enough to account for a large part of such antisickling properties (Sophianopoulos et al. (1974). The nonideality of concentrated hemoglobin solutions (up to 0.3 g cm^?3) has been studied in detail. By using the liganded as well as the unliganded forms of both Hb S and Hb A, it was found that the magnitude of the virial (nonideality) coefficients can change with varying solution conditions. A comparison of pure Hb S with hemolysates is made using viscosity and sedimentation velocity.     

Inactivation of luciferase from the Luminous marine bacterium Beneckea harveyi by proteases: evidence for a protease labile region and properties of the protein following inactivation

We have previously reported (T. O. Baldwin, J. W. Hastings, and P. L. Riley, 1978, J. Biol. Chem., 253, 5551–5554) that the proteolytic inactivation of the luciferase from the luminous marine bacterium Beneckea harveyi is due to hydrolysis of one or a small number of peptide bonds within the α subunit, and that following proteolytic inactivation, the molecular weight of the protein measured by sedimentation equilibrium is unaltered. We have continued these investigations and have made the following observations: (1) The proteaselabile region of the α subunit is about 20–25 residues in length, and located approximately 100–125 residues from one of the termini of the subunit; it possesses five to six trypsin-sensitive sites and two chymotrypsin-sensitive sites. (2) No binding could be measured between either the substrate, reduced flavin mononucleotide, or the product, oxidized flavin mononucleotide, and the luciferase inactivated with trypsin. (3) The results of sedimentation velocity measurements (Holzman, T. F., and Baldwin, T. O., 1980, Proc. Nat. Acad. Sci. USA, in press), reaction of the protein thiols with 5,5′-dithiobis(2-nitrobenzoic acid), and ultraviolet circular dichroism measurements suggest that following proteolytic inactivation, the luciferase has a slightly more expanded structure. (4) Analysis of a mutant luciferase, AK-6, having an α-subunit lesion that results in a red-shifted bioluminescence emission spectrum and a greatly reduced affinity for reduced flavin mononucleotide shows that alterations in the active center can result in a dramatically altered sensitivity to proteases, both in the rate of inactivation and in the sites of peptide bond hydrolysis.     

Diffusion of the Reaction Boundary of Rapidly Interacting Macromolecules in Sedimentation Velocity

Sedimentation velocity analytical ultracentrifugation combines relatively high hydrodynamic resolution of macromolecular species with the ability to study macromolecular interactions, which has great potential for studying dynamically assembled multiprotein complexes. Complicated sedimentation boundary shapes appear in multicomponent mixtures when the timescale of the chemical reaction is short relative to the timescale of sedimentation. Although the Lamm partial differential equation rigorously predicts the evolution of concentration profiles for given reaction schemes and parameter sets, this approach is often not directly applicable to data analysis due to experimental and sample imperfections, and/or due to unknown reaction pathways. Recently, we have introduced the effective particle theory, which explains quantitatively and in a simple physical picture the sedimentation boundary patterns arising in the sedimentation of rapidly interacting systems. However, it does not address the diffusional spread of the reaction boundary from the cosedimentation of interacting macromolecules, which also has been of long-standing interest in the theory of sedimentation velocity analytical ultracentrifugation. Here, effective particle theory is exploited to approximate the concentration gradients during the sedimentation process, and to predict the overall, gradient-average diffusion coefficient of the reaction boundary. The analysis of the heterogeneity of the sedimentation and diffusion coefficients across the reaction boundary shows that both are relatively uniform. These results support the application of diffusion-deconvoluting sedimentation coefficient distributions c(s) to the analysis of rapidly interacting systems, and provide a framework for the quantitative interpretation of the diffusional broadening and the apparent molar mass values of the effective sedimenting particle in dynamically associating systems.     

Low and high temperature asymmetric forms of pig kidney and rabbit muscle phosphofructokinases and reversible, temperature-dependent transitions.

Previous viscometric studies from this laboratory (Johnson, C. S., Vogtmann, L., and Deal, W. C., Jr. (1976) Biochem. Biophys. Res. Commun.73, 391–395) have shown that at 3.5 ° C, pig kidney phosphofructokinase (PFK) is markedly asymmetric and rabbit muscle PFK is moderately asymmetric. The present viscometric and ultracentrifugal studies show that both enzymes are also asymmetric at near-physiological temperatures, that both exist in high-temperature and low-temperature forms, and that the high-temperature forms of both are less asymmetric and more dissociated than the low-temperature forms. The risults also show that the transitions from low- to high-temperature forms are reversible if the exposure to 35 °C is short enough that no irreversible chemical modification occurs. For pig kidney PFK, intrinsic viscosity values of 34.0, 25.6, and 13.8 ml/g were obtained at 3.5, 20 and 35 °C, respectively, whereas rabbit muscle PFK yielded values of 6.9, 6.2, and 5.2 ml/g at the corresponding temperatures. These data clearly show a decrease in asymmetry with increase in temperature. However, both enzymes are still asymmetric at the higher temperature, inasmuch as most globular macromolecules have intrinsic viscosity values in the range of 3 to 4 ml/g, regardless of molekular weight. Studies from 1 to 45 ° C at a fixed protein concentration (4.8 mg/ml) showed that pig kidney PFK has reduced viscosity values of 51.0 ml/g (low-temperature form) and 20.4 ml/g (high-temperature form) in plateau regions of the viscosity graph at the temperature extremes; the mid-point of the transition between the two forms is at about 22–24 °C. Rabbit muscle PFK at 4.2 mg/ml reproducibly gave corresponding reduced viscosity values of 6.9 and 4.8 ml/g for the low- and high-temperature forms, respectively; the transition mid-point between the two forms is at about 16 °C. The first reported sedimentation velocity studies of rabbit muscle PFK at near-physiological temperature (35 °C) show that with near-physiological protein concentration (1.25 mg/ml), the enzyme is in a much more dissociated form, s_20,w(weight average) = 14. 5 S; s_20,w(peak leading edge) = 17 S, than that previously reported at lower temperatures, s_20,w(fastest peak) = 23–30 S. Similarly, the first sedimentation studies on the pig kidney enzyme indicate a lower sedimentation coefficient at 35 ° C (s^0.39%_20,w = 48 S) than at 3.5 ° C(65 S).     

Studies on Transformations of Hemophilus influenzae : II. The molecular weight of transforming DNA by sedimentation and diffusion measurements

The sedimentation and diffusion coefficients have been determined for Hemophilus influenzae transforming activity and DNA using P³²-labeled DNA. The methods employed the Spinco fixed boundary separation cell for measurements of the sedimentation coefficient and the Northrop-Anson diffusion cell to determine the diffusion coefficient. There was a very close correlation between the amount of DNA and transforming activity sedimented or diffused. The sedimentation coefficient (s_20°), for both biological activity and DNA was 27 and the diffusion coefficient (D_20°) 1 x 10^-8 cm²/sec. The molecular weight calculated from these coefficients gave a value of 16 million. There was no difference in the sedimentation coefficients for the two unlinked markers, streptomycin and erythromycin resistance, and the diffusion coefficients for single markers or the linked markers, streptomycin and cathomycin, were the same.     

Molecular characteristics of bovine factor X activated in concentrated sodium citrate solution

Preparations of the zymogen form of bovine factor X were incubated in 25% $\text{w}\text{v}$ sodium citrate at room temperature. The rate of activation of factor X was dependent on the extent of contamination with factor VII, prothrombin, and thrombin. The activated factor X was isolated by DEAE-cellulose chromatography. Analysis of the final product by sedimentation velocity centrifugation coupled with measurements of the rate of boundary spreading, high-speed sedimentation equilibrium, and gel filtration chromatography provided evidence for a single molecular species undergoing reversible association-dissociation with a monomeric molecular weight of 48,000. In the absence of mercaptoethanol a single band was seen by disc electrophoresis and by SDS-acrylamide electrophoresis but after disulfide reduction two components of molecular weights 30,000 and 17,000 were visible. The protein contained large amounts of acidic amino acids but no carbohydrate. The N-terminal amino acids were alanine and isoleucine and 1 mole C-terminal arginine per mole protein was found. These characteristics are very similar to those of factor X activated with Russell's viper venom.When a BaSO₄ eluate of bovine plasma rich in prothrombin was allowed to stand in 25% sodium citrate both thrombin and activated factor X were generated. Chromatography of the isolated activated factor X on Sephadex G-200 as well as disc electrophoresis showed that it behaved identically with the enzyme obtained from purified zymogen and was clearly distinguishable from autoprothrombin c, a glycoprotein possessing qualitatively similar biological activity (Seegers, W. H., Cole, E. R., Harmison, C. R., and Marciniak, E. (1963) Can. J. Biochem. Physiol.41, 1047).     

AIAP: A Quality Control and Integrative Analysis Package to Improve ATAC-seq Data Analysis

Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) is a technique widely used to investigate genome-wide chromatin accessibility. The recently published Omni-ATAC-seq protocol substantially improves the signal/noise ratio and reduces the input cell number. High-quality data are critical to ensure accurate analysis. Several tools have been developed for assessing sequencing quality and insertion size distribution for ATAC-seq data; however, key quality control (QC) metrics have not yet been established to accurately determine the quality of ATAC-seq data. Here, we optimized the analysis strategy for ATAC-seq and defined a series of QC metrics for ATAC-seq data, including reads under peak ratio (RUPr), background (BG), promoter enrichment (ProEn), subsampling enrichment (SubEn), and other measurements. We incorporated these QC tests into our recently developed ATAC-seq Integrative Analysis Package (AIAP) to provide a complete ATAC-seq analysis system, including quality assurance, improved peak calling, and downstream differential analysis. We demonstrated a significant improvement of sensitivity (20%–60%) in both peak calling and differential analysis by processing paired-end ATAC-seq datasets using AIAP. AIAP is compiled into Docker/Singularity, and it can be executed by one command line to generate a comprehensive QC report. We used ENCODE ATAC-seq data to benchmark and generate QC recommendations, and developed qATACViewer for the user-friendly interaction with the QC report. The software, source code, and documentation of AIAP are freely available at https://github.com/Zhang-lab/ATAC-seq_QC_analysis.     

The molecular properties of the copper enzyme galactose oxidase

Galactose oxidase (EC 1.1.3.9) has been purified 140-fold by DEAE- and CM-cellulose chromatography from cultures of Polyporus circinatus. The enzyme has a molecular weight of 68,000 ± 3,000 as determined by sedimentation equilibrium, sodium dodecyl sulfate-acrylamide gel electrophoresis, Sephadex G-150 chromatography, and osmometry. Galactose oxidase is a single-chain protein which does not self-associate. Charge isozymes of the enzyme are detected by ion-exchange chromatography and gel electrophoresis. The amino acid composition determined herein is significantly different from that previously reported (Kelly-Falcoz, F., Greenberg, H., And Horecker, B. L. (1965) J. Biol. Chem.240, 2966–2970). The enzyme contains 1% by weight of neutral carbohydrate.Galactose oxidase contains 1 g-atom of copper per 70,000 g of protein. The metal does not contribute to the electrophoretic or isozymic properties of the protein. However, the sedimentation coefficients of the holo- and apoenzymes, 4.76S and 4.83S, respectively, do suggest that small differences in protein conformation accompany the removal of the copper from the holoenzyme.Attempted sulfhydryl group titration of galactose oxidase shows that the holoenzyme is resistant to denaturation. However, in β-mercaptoethanol-guanidine HCl 5 half-cystine residues are titrated in the apoenzyme. On a dry-weight basis, the E_1cm^1% value for galactose oxidase at 280 nm is 15.4. Galactose oxidase has an isoelectric point above pH 10 which is a probable source of some of its anomalous behavior in physical measurements and enzyme-activity determinations.     

Difference sedimentation velocity adapted to low molecular weight proteins

The difference sedimentation velocity technique reported by Kirschner and Schachman (1971, Biochemistry10, 1900–1919) has been modified to eliminate the need for a supernatant region. The method is now applicable to the measurement of small changes in sedimentation coefficient for low molecular weight proteins and other small macromolecules. Procedural changes necessary to overcome the absence of a supernatant region until late in the run have been devised and tested. A modified double-sector centerpiece was used to match the radial positions of the two menisci. The integration of the moment of the concentration difference was carried out from the meniscus to the plateau region, rather than over the peak only. The interference baseline was measured on photographs at the start of each run and after remixing. Some instability of baseline height was noted. The calculation method adjusted the baseline height to correspond with the concentration difference in the plateau region arising from unequal radial dilution. Tests of the method have been made using D₂O to retard the sedimentation of lysozyme. The interference results at low D₂O concentration (small values of Δs) are in agreement with schlieren results at high D₂O concentrations. Changes of 0.005 S have been detected.     

Determination os sedimentation coefficient distributions for cartilage proteoglycans

Sedimentation coefficient distributions of widely polydisperse proteoglycan preparations were made using a previously developed transport sedimentation methodology. Boundary stability was improved by centrifuging samples in a preformed CsCl density gradient (0.016 g/cm⁴). The results were compared with the distributions obtained with an interferometric analytical centrifugation method. When these two techniques were applied to analyze A1 and A1–D1 proteoglycan preparations, results were in substantial agreement with respect to the mean sedimentation coefficients of the peaks, average S value, sedimentation coefficient distribution, skewness, proportion of monomer and aggregates, and linearity of the plot ln(s) versus C extrapolations to zero concentration. The lower solute concentration compatible with the transport (velocity gradient) method makes this technique particularly suitable for studying the details of proteoglycan distribution of molecular sizes, especially for aggregates.     

Pig liver fatty acid synthetase: purification and physicochemical properties.

This paper reports the first detailed study of the physicochemical properties of a fatty acid synthetase multienzyme complex from a mammalian liver. Fatty acid synthetase from pig liver was purified by a procedure including the following main steps: (i) preparation of a clarified supernatant solution (50,000 g), (ii) ammonium sulfate fractionation, (iii) DEAE-cellulose chromatography to separate 11 S catalase from the 13 S fatty acid synthetase, (iv) a preparative sucrose density gradient step to remove a 7 S impurity, and (v) a calcium phosphate gel step to remove an unusual yellow 16 S heme protein to yield a colorless preparation. The purified fatty acid synthetase was colorless and showed a single symmetrical peak in sucrose density gradient and conventional sedimentation velocity experiments. Fatty acid synthetase was very stable at 4 °C in the presence of 1 mm dithiothreitol and 25% sucrose. Extrapolation to zero protein concentration yielded values of S^o_20,w = 13.3 S and D^o_20,w = 2.60 × 10^?7cm²/s for the sedimentation and diffusion coefficients of the enzyme. Frictional coefficient values of 1.55 and 1.56 × 10^?7 cm, respectively, were calculated from the values for the sedimentation and diffusion coefficients. Based on these frictional coefficient values, the Stokes radius of the enzyme was calculated to be 82.4 Å. Sedimentation and diffusion coefficient data yielded a molecular weight value of $\text{M}{}_{\mathrm{<mtext>w</mtext>}}\text{(}\text{s}\text{D}\text{) = 478,000}$ and sedimentation equilibrium data yielded a value of M_w = 476,000. Preliminary intrinsic viscosity measurements at 20 °C gave a value of 7.3 ml/g, indicating that the enzyme is somewhat asymmetric. This is supported by the value of 1.58 calculated for the frictional ratio and by the fact that the values for the sedimentation and diffusion coefficients are both slightly lower than expected for a globular protein of molecular weight 478,000. The enzyme possesses about 90 SH groups per molecule, assuming a molecular weight of 478,000. The ultraviolet absorption spectrum of the enzyme shows a maximum at 280 nm and an unusual shoulder at 290 nm. The fluorescence spectrum of the enzyme is dominated by tryptophan fluorescence and, over the excitation range of 260–300 nm, there is a single emission maximum at 344 nm.     

Methods of cell disintegration and the sedimentation patterns of ribosomes inEscherichia coli

A number of methods for the disintegration ofEscherichia coli cells was followed to investigate their suitability for the isolation of ribosomes. The decrease of viability and the conditions for fine disruption of the cells for all methods were established. The sedimentation coefficients of ribosomes and their sizedistribution in the cell-free extracts in dependence upon the method employed and the concentration of Mg⁺⁺ ions were determined by analytical ultracentrifugation. The sedimentation coefficients and the sizedistribution of ribosomes did not differ in most methods and the results obtained were basically in agreement with the data given by other authors. The method of grinding with alumina seemed to be too vigorous and the results obtained were less reproducible. Ribosomes from extracts prepared by osmotical shocking of lysozyme spheroplasts showed ultracentrifugation patterns differing from those found with other methods: In extracts with low (0.00025m) and high (0.01m) concentration of Mg⁺⁺ almost the whole range of ribosomes was found whereas in the buffer with 0.001m Mg⁺⁺ only 30 and 50s ribosomes were present. alumina Particles with a sedimentation constant higher than 100s (polysomes) were not found in any of our extracts. We attempted to discuss our results and compare them with the data given in other reports. The method of osmotical lysis of spheroplast seems to give results most closely related to thein vivo situation in the cell and enables us to study the functional relationship of ribosomes to other cell components namely the cytoplasmic membrane.     

A method for measuring sedimentation and diffusion of macromolecules in capillary tubes by total intensity and quasi-elastic light-scattering techniques.

Light scattered from a macromolecular solution in a capillary tube is used to determine both the sedimentation and translational diffusion coefficients. The capillary tube is spun in a preparative centrifuge, removed, and placed in a light-scattering photometer equipped with a scanning mechanism. The intensity distribution of scattered light along the tube represents the concentration profile in the tube and provides the measure of boundary migration. The sedimentation coefficient is determined from this measure and the applied centrifugal field. The diffusion coefficient is obtained from a time-autocorrelation analysis of fluctuations in intensity of light scattered from any fixed point of the profile. These coefficients were obtained for two monodisperse systems, R17 bacteriophage and 28s ribosomal rat liver RNA. The molecular weights obtained from ratios of these coefficients are in good agreement with literature values. In the sedimentation analysis, deviations from linearity between boundary displacement and applied field were found to be less than 1%. This precision confirms that the boundary is stable for the capillary geometry even in the absence of a preformed density gradient. The sedimentation coefficients of identical samples were also measured with the Spinco Model E analytical ultracentrifuge; results of the two methods agree to within 4%. As a consequence of the capillary tube geometry and light-scattering detection, sedimentation coefficients can be obtained from sample volumes of less than 100 μl. This detection techniques is thus far demonstrated to be at least an order of magnitude more sensitive than Schlieren optics, thereby useful when uv absorption is not applicable. For diffusion measurements there are also several inherent advantages. The diffusion coefficient is obtained from the identical sample, and scanning provides the capability to measure D from various parts of the sedimentation profiles and thereby directly explore concentration dependence, homogeneity, and integrity of the sample. The capillary tube with a layer of silicone oil over the sample and centrifugation provides an effective method to cleanse the solution and trap all dust.     

Statistical evaluation of ways to analyze nonideal systems by sedimentation equilibrim

Three equations describing sedimentation equilibrium are examined and tested for their ability to analyze data. The testing procedure using simulated data is similar to that described previously (Holladay, L. A., and Sophianopoulos, A. J. (1972) J. Biol. Chem.247, 427–439) and used with another equation. The equations examined here are found to be of much less statistical reliability and of a more restricted range of application than the previously examined equation. The equation described previously, (Holladay, L. A., and Sophianopoulos, A. J. (1972) J. Biol. Chem.247, 427–439) is also used here to examine the conditions necessary to detect isodesmic systems of more than four components. The self-association of lysozyme reported previously (Sophianopoulos, A. J., and Van Holde, K. E. (1964) J. Biol. Chem.239, 2516–2524) is reexamined at pH 8.2, 0.15 ionic strength, and 13°C. The tentative conclusion is that the system is mainly a monomer-dimer, with a small, uncertain amount of tetramer possibly present. Under the above conditions the second virial coefficient, B, is estimated to lie in the range 0–4.4 × 10^?6 mole·dl·g^?2, the dimerization constant. K₂₁, lies in the range 2.3–2.7 × 10^?3m, and the tetramerdimer constant, K₄₂, is in the range 1.5–15 × 10^?3m.     

Sedimentation coefficients of self-associating species: I. Basic theory

Under the same solution conditions, the apparent weight average sedimentation coefficient, s_wa, and some quantities obtained from it can be combined with the equilibrium constant or constants, K_i, and the monomer concentration, c_I, obtained from sedimentation equilibrium, light scattering or osmotic pressure experiments on the same self-associating solute, so that the individual sedimentation coefficients, s_i, of the self-associating species, and also the hydrodynamic concentration dependence parameter,g or $\text{g}$, can be evaluated. Using two different models for the hydrodynamic concentration parameter, four different methods are presented for the evaluation of the s_i's. Methods for evaluating g or $\text{g}$, once the s_i's are known, are presented. A method for obtaining the number average sedimentation coefficient, s_N, and its application to self-associations is presented. Methods are shown for the evaluation of Z average properties, x_zc, as well as number average properties,x_Nc, of a self-associating solute from its weight average properties, x_wc.     

Neutral sucrose sedimentation of very large DNA from Bacillus subtilis. I. Effect of random double-strand breaks and centrifuge speed on sedimentation

A method is presented for preparing very large DNA from Bacillus subtilis protoplasts. When the DNA is characterized by sedimentation in neutral sucrose gradients, a fast-sedimenting component is found whose sedimentation coefficient varies with centrifuge speed. By use of [³H]thymine label for the DNA and a ¹⁴C-labeled amino acid, it is shown that less than 5% cellular material other than DNA is associated with this component. Irradiation of this DNA in solution with gamma rays forms a slower component, called the “main peak”, whose sedimentation coefficient also depends on centrifuge speed. More irradiation breaks down this main peak into even slower-sedimenting DNA; it is shown that for low doses, double-strand breaks are formed in both the B. subtilis DNA and in bacteriophage T2 DNA at the same rate linear in dose, 0.018 double-strand breaks per kilorad per mass equal to that of T2 DNA.The speed dependence of the DNA sedimenting at the main peak is compared with an approximate theory of the speed dependence of the sedimentation coefficient of linear DNA by B. H. Zimm (unpublished calculations). The comparison suggests that for sufficiently high centripedal acceleration, DNA molecules larger than a critical mass will sediment at much the same velocity. The theory, and data on the break-up of the DNA with gamma rays, are used to estimate that the DNA extracted is at least 13 times the mass of T2 DNA, and possibly larger.In the Appendix, data from the literature are put together with data taken during this work to make plausible the assumption that the usual theory for the sedimentation of DNA molecules, experimentally tested in salt solutions, may also be applied to sucrose solutions. If, in neutral sucrose gradients, the distance sedimented is proportional to a power α of the mass, the best value of α = 0.38.