Molecular weights from approach-to-sedimentation equilibrium data using nonlinear regression analysis

Data obtained from early times during the transient period of sedimentation equilibrium experiments are analyzed using an approximate solution to the Lamm equation to estimate s/D. The Cr versus r data obtained at several times during approach-to-equilibrium are analyzed using a nonlinear least squares algorithm and Fujita's approximate solution. This procedure was tested using D-Ser13-somatostatin, ribonuclease, and ovalbumin. The results obtained demonstrate that for monodisperse samples s/D may be rapidly and reliably estimated using this method.     

Boundary analysis in sedimentation transport experiments: a procedure for obtaining sedimentation coefficient distributions using the time derivative of the concentration profile.

A procedure is described for computing sedimentation coefficient distributions from the time derivative of the sedimentation velocity concentration profile. Use of the time derivative, (delta c/delta t)r, instead of the radial derivative, (delta c/delta r)t, is desirable because it is independent of time-invariant contributions to the optical baseline. Slowly varying baseline changes also are significantly reduced. An apparent sedimentation coefficient distribution (i.e., uncorrected for the effects of diffusion), g*(s), can be calculated from (delta c/delta t)r as [formula: see text] where s is the sedimentation coefficient, omega is the angular velocity of the rotor, c0 is the initial concentration, r is the radius, rm is the radius of the meniscus, and t is time. An iterative procedure is presented for computing g*(s)t by taking into account the contribution to (delta c/delta t)r from the plateau region to give (delta c/delta t)corr. Values of g*(s)t obtained this way are identical to those of g*(s) calculated from the radial derivative to within the roundoff error of the computations. Use of (delta c/delta t)r, instead of (delta c/delta r)t, results in a significant increase (greater than 10-fold) in the signal-to-noise ratio of data obtained from both the uv photoelectric scanner and Rayleigh optical systems of the analytical ultracentrifuge. The use of (delta c/delta t)r to compute apparent sedimentation coefficient distributions for purposes of boundary analysis is exemplified with an antigen-antibody system.     

Non-ideality by sedimentation velocity of halophilic malate dehydrogenase in complex solvents

We have investigated the potential of sedimentation velocity analytical ultracentrifugation for the measurement of the second virial coefficients of proteins, with the goal of developing a method that allows efficient screening of different solvent conditions. This may be useful for the study of protein crystallization. Macromolecular concentration distributions were modeled using the Lamm equation with the approximation of linear concentration dependencies of the diffusion constant, D = D(o) (1 + k(D)c), and the reciprocal sedimentation coefficient s = s(o)/(1 + k(s)c). We have studied model distributions for their information content with respect to the particle and its non-ideal behavior, developed a strategy for their analysis by direct boundary modeling, and applied it to data from sedimentation velocity experiments on halophilic malate dehydrogenase in complex aqueous solvents containing sodium chloride and 2-methyl-2,4-pentanediol, including conditions near phase separation. Using global modeling for three sets of data obtained at three different protein concentrations, very good estimates for k(s) and s degrees and also for D degrees and the buoyant molar mass were obtained. It was also possible to obtain good estimates for k(D) and the second virial coefficients. Modeling of sedimentation velocity profiles with the non-ideal Lamm equation appears as a good technique to investigate weak inter-particle interactions in complex solvents and also to extrapolate the ideal behavior of the particle.     

A method for directly fitting the time derivative of sedimentation velocity data and an alternative algorithm for calculating sedimentation coefficient distribution functions

The time-derivative method for deriving the sedimentation coefficient distribution, g(s*), from sedimentation velocity data that was developed by Walter Stafford has many advantages and is now widely used. By fitting Gaussian functions to the g(s*) distribution both sedimentation and diffusion coefficients (and therefore molecular masses) for individual species can be obtained. However, some of the approximations used in these procedures limit the accuracy of the results. An alternative approach is proposed in which the dc/dt data are fitted rather than g(s*). This new approach gives improved accuracy, extends the range to sedimentation coefficients below 1 S, and enhances resolution of multiple species. For both approaches the peaks from individual species are broadened when the data cover too wide a time span, and this effect is explored and quantified. An alternative algorithm for calculating ?(s*) from the dc/dt curves is presented and discussed. Rather than first averaging the dc/dt data for individual scan pairs and then calculating ?(s*) from that average, the ?(s*) distributions are calculated for every scan pair and then subsequently averaged. This alternative procedure yields smaller error bars for g(s*) and somewhat greater accuracy for fitted hydrodynamic properties when the time span becomes large.     

Influence of pulsatility on the development of intracardiac jets: an in vitro laser Doppler study.

So far, it has been hypothesized that numerical data obtained in steady flow conditions apply to pulsatile flows. In order to study the modifications of the velocity fields due to pulsatility, jets were produced by 8 orifices (with a diameter "D" of 4.4 to 11.3 mm) included in a chamber of 50 mm. The velocity was measured using laser Doppler anemometry with a pulsatile flow ("pf") and compared to the values obtained in steady ("sf"): at maximum velocity, the longitudinal velocity profile is qualitatively similar to this observed in steady flow: it is made of a plateau followed by an hyperbolic velocity decay in the turbulent area. The length of the core ("Lpf") is strongly related to "D" (Lpf = 3.72 D + 5.49, r = .99) and the velocity decay depends on the ratio between the distance "x" from the orifice and "D" (V/Vo = 2.83D/x + 3.46, r = .85, where V is the velocity at "x" and Vo the initial velocity). During the acceleration and the deceleration, the laminar core is disturbed by turbulences. The comparison of "pf" data with "sf" data demonstrated similar diameters at the origin of the jets (Dpf = 0.96 Dsf + .12, r = .99), but significant (p less than .0001) differences both for "L" and "V/Vo": Lpf = .91Lsf + 6.58, r = .97, V/Vopf = .63 V/Vosf + .34, r = .76. Thus, pulsatility modifies velocity fields and the results obtained in steady flow conditions do not apply to pulsatile jets.     

Estimation of muscle fiber conduction velocity from two-dimensional surface EMG recordings in dynamic tasks

The aims of this study are (1) to demonstrate that multi-channel surface electromyographic (EMG) signals can be detected with negligible artifacts during fast dynamic movements with an adhesive two-dimensional (2D) grid of 64 electrodes and (2) to propose a new method for the estimation of muscle fiber conduction velocity from short epochs of 2D EMG recordings during dynamic tasks. Surface EMG signals were collected from the biceps brachii muscle of four subjects with a grid of 13 × 5 electrodes during horizontal elbow flexion/extension movements (range 120–170°) at the maximum speed, repeated cyclically for 2 min. Action potentials propagating between the innervation zone and tendon regions could be detected during the dynamic task. A maximum likelihood method for conduction velocity estimation from the 2D grid using short time intervals was developed and applied to the experimental signals. The accuracy of conduction velocity estimation, assessed from the standard deviation of the residual of the regression line with respect to time, decreased from (range) 0.20–0.33 m/s using one column to 0.02–0.15 m/s when combining five columns of the electrode grid. This novel method for estimation of muscle fiber conduction velocity from 2D EMG recordings provides an estimate which is global in space and local in time, thus representative of the entire muscle yet able to track fast changes over the execution of a task, as is required for assessing muscle properties during fast movements.     

Activated ERK2 is a monomer in vitro with or without divalent cations and when complexed to the cytoplasmic scaffold PEA-15

The extracellular signal-regulated protein kinase, ERK2, fully activated by phosphorylation and without a His(6) tag, shows little tendency to dimerize with or without either calcium or magnesium ions when analyzed by light scattering or analytical ultracentrifugation. Light scattering shows that ~90% of ERK2 is monomeric. Sedimentation equilibrium data (obtained at 4.8-11.2 μM ERK2) with or without magnesium (10 mM) are well described by an ideal one-component model with a fitted molar mass of 40180 ± 240 Da (without Mg(2+) ions) or 41290 ± 330 Da (with Mg(2+) ions). These values, close to the sequence-derived mass of 41711 Da, indicate that no significant dimerization of ERK2 occurs in solution. Analysis of sedimentation velocity data for a 15 μM solution of ERK2 with an enhanced van Holde-Weischet method determined the sedimentation coefficient (s) to be ~3.22 S for activated ERK2 with or without 10 mM MgCl(2). The frictional coefficient ratio (f/f(0)) of 1.28 calculated from the sedimentation velocity and equilibrium data is close to that expected for an ~42 kDa globular protein. The translational diffusion coefficient of ~8.3 × 10(-7) cm(2) s(-1) calculated from the experimentally determined molar mass and sedimentation coefficient agrees with the value determined by dynamic light scattering in the absence and presence of calcium or magnesium ions and a value determined by NMR spectrometry. ERK2 has been proposed to homodimerize and bind only to cytoplasmic but not nuclear proteins [Casar, B., et al. (2008) Mol. Cell 31, 708-721]. Our light scattering data show, however, that ERK2 forms a strong 1:1 complex of ~57 kDa with the cytoplasmic scaffold protein PEA-15. Thus, ERK2 binds PEA-15 as a monomer. Our data provide strong evidence that ERK2 is monomeric under physiological conditions. Analysis of the same ERK2 construct with the nonphysiological His(6) tag shows substantial dimerization under the same ionic conditions.     

Oligomeric structure of mammalian purine nucleoside phosphorylase in solution determined by analytical ultracentrifugation

The influence of phosphate, ionic strength, temperature and enzyme concentration on the oligomeric structure of calf spleen purine nucleoside phosphorylase (PNP) in solution was studied by analytical ultracentrifugation methods. Sedimentation equilibrium analysis used to directly determine the enzyme molecular mass revealed a trimeric molecule with Mr = (90.6 +/- 2.1) kDa, regardless the conditions investigated: protein concentration in the range 0.02-1.0 mg/ml, presence of up to 100 mM phosphate and up to 200 mM NaCl, temperature in the range 4-25 degrees C. The sedimentation coefficient (6.04 +/- 0.02) S, together with the diffusion coefficient (6.15 +/- 0.11) 10(-7) cm2/s, both values obtained from the classic sedimentation velocity method at 1.0 mg/ml PNP concentration in 20 mM Hepes, pH 7.0, yielded a molecular mass of (90.2 +/- 1.6) kDa as expected for the trimeric enzyme molecule. Moreover, as shown by active enzyme sedimentation, calf spleen PNP remained trimeric even at low protein concentrations (1 microg/ml). Hence in solution, similar like in the crystalline state, calf spleen PNP is a homotrimer and previous suggestions for dissociation of this enzyme into more active monomers, upon dilution of the enzyme or addition of phosphate, are incorrect.     

Molecular mass determination by sedimentation velocity experiments and direct fitting of the concentration profiles.

A new method for the direct molecular mass determination from sedimentation velocity experiments is presented. It is based on a nonlinear least squares fitting procedure of the concentration profiles and simultaneous estimation of the sedimentation and diffusion coefficients using approximate solutions of the Lamm equation. A computer program, LAMM, was written by using five different model functions derived by Fujita (1962, 1975) to describe the sedimentation of macromolecules during centrifugation. To compare the usefulness of these equations for the analysis of hydrodynamic results, the approach was tested on data sets of Claverie simulations as well as experimental curves of some proteins. A modification for one of the model functions is suggested, leading to more reliable sedimentation and diffusion coefficients estimated by the fitting procedure. The method seems useful for the rapid molecular mass determination of proteins larger than 10 kDa. One of the equations of the Archibald type is also suitable for compounds of low molecular mass, probably less than 10 kDa, because this model function requires neither the plateau region nor a meniscus free of solute.     

Determination of sedimentation coefficients for small peptides.

Direct fitting of sedimentation velocity data with numerical solutions of the Lamm equations has been exploited to obtain sedimentation coefficients for single solutes under conditions where solvent and solution plateaus are either not available or are transient. The calculated evolution was initialized with the first experimental scan and nonlinear regression was employed to obtain best-fit values for the sedimentation and diffusion coefficients. General properties of the Lamm equations as data analysis tools were examined. This method was applied to study a set of small peptides containing amphipathic heptad repeats with the general structure Ac-YS-(AKEAAKE)nGAR-NH2, n = 2, 3, or 4. Sedimentation velocity analysis indicated single sedimenting species with sedimentation coefficients (s(20,w) values) of 0.37, 0.45, and 0.52 S, respectively, in good agreement with sedimentation coefficients predicted by hydrodynamic theory. The described approach can be applied to synthetic boundary and conventional loading experiments, and can be extended to analyze sedimentation data for both large and small macromolecules in order to define shape, heterogeneity, and state of association.     

The purification and properties of isocitrate lyase from Chlorella

1. Isocitrate lyase (threo-d(s)-isocitrate glyoxylate-lyase, EC 4.1.3.1) has been purified from acetate-adapted cells of Chlorella pyrenoidosa. 2. The final preparation was homogeneous by the criteria of sedimentation, diffusion and polyacrylamide-gel electrophoresis. 3. The sedimentation coefficient (S(20,w)) was 9.04x10(-13)sec. and the diffusion coefficient (D(20,w)) 4.62x10(-7)cm.(2)/sec.; from these values the molecular weight of the enzyme was calculated to be 170000 and its Stokes radius to be 4.63x10(-7)cm. 4. The elution of the enzyme from Sephadex G-100 was studied and estimates of molecular weight and Stokes radius were obtained from the elution data. 5. The turnover number of the enzyme was 5950moles of glyoxylate formed/min./mole of enzyme at 30 degrees . 6. With threo-d(s)(+)-isocitrate as substrate, the K(m) of the enzyme was 0.023mm.     

Pro-(carboxypeptidases A) from whole pig pancreas. Their mass, size, shape and solvation.

Sedimentation analysis and light-scattering measurements were made with the two forms of pig pancreas pro-(carboxypeptidase A), in order to determine some of their physical properties. The following values were found (the first value applies to the binary complex and the second one to the monomer). The A 1%/280.1 cm values were 19.9 +/- 0.3 and 16.3 +/- 0.3. The partial specific volumes v -0 were 0.707 +/- 0.016 cm3/g and 0.714 +/- 0.015 cm3/g. The sedimentation coefficients S 0/20,w were 4.90 +/- 0.15S and 3.75 +/- 0.15 S. The diffusion coefficients D 0/20,w were (5.8 +/- 0.1) X 10(-7) cm2/s and (6.95 +/- 0.15) X 10(-7) cm2/s. From these data the following values were calculated. Relative molecular masses Mr were 71 000 +/- 4000 and 46 000 +/- 3000. The frictional ratios f/fmin. were 1.37 +/- 0.06 and 1.31 +/- 0.07; assuming a value for the solvation of the molecules (delta = 0.5 g/g) the asymmetry values range from 3 to 5 for the binary complex and from 2 to 4 for the monomer. The Mr values found in the present work coincide with those found by means of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate [Martínez, Avilés, SanSegundo & Cuchillo (1981) Biochem. J. 197, 141-147]. Therefore the low values obtained by those authors when using gel-filtration chromatography must be the result of the interaction of the zymogens with the gel matrix, as the asymmetry is too small to justify the large discrepancies found.     

Analysis of high-affinity assembly for AMPA receptor amino-terminal domains

Analytical ultracentrifugation (AUC) and steady-state fluorescence anisotropy were used to measure the equilibrium dissociation constant (Kd) for formation of dimers by the amino-terminal domains (ATDs) of the GluA2 and GluA3 subtypes of AMPA receptor. Previous reports on GluA2 dimerization differed in their estimate of the monomer-dimer Kd by a 2,400-fold range, with no consensus on whether the ATD forms tetramers in solution. We find by sedimentation velocity (SV) analysis performed using absorbance detection a narrow range of monomer-dimer Kd values for GluA2, from 5 to 11 nM for six independent experiments, with no detectable formation of tetramers and no effect of glycosylation or the polypeptide linker connecting the ATD and ligand-binding domains; for GluA3, the monomer-dimer Kd was 5.6 μM, again with no detectable tetramer formation. For sedimentation equilibrium (SE) experiments, a wide range of Kd values was obtained for GluA2, from 13 to 284 nM, whereas for GluA3, the Kd of 3.1 μM was less than twofold different from the SV value. Analysis of cell contents after the ～1-week centrifuge run by silver-stained gels revealed low molecular weight GluA2 breakdown products. Simulated data for SE runs demonstrate that the apparent Kd for GluA2 varies with the extent of proteolysis, leading to artificially high Kd values. SV experiments with fluorescence detection for GluA2 labeled with 5,6-carboxyfluorescein, and fluorescence anisotropy measurements for GluA2 labeled with DyLight405, yielded Kd values of 5 and 11 nM, consistent with those from SV with absorbance detection. However, the sedimentation coefficients measured by AUC using absorbance and fluorescence systems were strikingly different, and for the latter are not consistent with hydrodynamic protein models. Thus, for unknown reasons, the concentration dependence of sedimentation coefficients obtained with fluorescence detection SV may be unreliable, limiting the usefulness of this technique for quantitative analysis.     

Spatio-temporal parameters of gait measured by an ambulatory system using miniature gyroscopes

In this study we describe an ambulatory system for estimation of spatio-temporal parameters during long periods of walking. This original method based on wavelet analysis is proposed to compute the values of temporal gait parameters from the angular velocity of lower limbs. Based on a mechanical model, the medio-lateral rotation of the lower limbs during stance and swing, the stride length and velocity are estimated by integration of the angular velocity. Measurement's accuracy was assessed using as a criterion standard the information provided by foot pressure sensors. To assess the accuracy of the method on a broad range of performance for each gait parameter, we gathered data from young and elderly subjects. No significant error was observed for toe-off detection, while a slight systematic delay (10 ms on average) existed between heelstrike obtained from gyroscopes and footswitch. There was no significant difference between actual spatial parameters (stride length and velocity) and their estimated values. Errors for velocity and stride length estimations were 0.06 m/s and 0.07 m, respectively. This system is light, portable, inexpensive and does not provoke any discomfort to subjects. It can be carried for long periods of time, thus providing new longitudinal information such as stride-to-stride variability of gait. Several clinical applications can be proposed such as outcome evaluation after total knee or hip replacement, external prosthesis adjustment for amputees, monitoring of rehabilitation progress, gait analysis in neurological diseases, and fall risk estimation in elderly.     

Progesterone and prostanoid production by bovine binucleate trophoblastic cells

A procedure for preparing highly enriched suspensions of bovine binucleate trophoblastic cells was developed and data showing that these cells produce progesterone, prostacyclin (PGI2), and prostaglandin E2 (PGE2) were obtained. Approximately 200 X 10(6) enzymatically dissociated cells from bovine cotyledons were applied to the surface of a density gradient of 2% to 4% Ficoll-400 using the Wescor CELSEP sedimentation chamber. After 90-120 min of sedimentation at unit gravity, fractions containing binucleate trophoblastic cells were obtained and washed in HEPES-buffered Medium 199. Preparations of 90% to 100% binucleate trophoblastic cells were obtained routinely; viability was 50% to 80%. After incubation at 37 degrees C, concentrations (ng/10(5) cells) of progesterone were greater in those fractions containing binucleate cells than in those containing primarily smaller, mononucleate cells. Total progesterone secreted (mean +/- SEM) after 4 h by 1 X 10(5), 2 X 10(5), 4 X 10(5), 8 X 10(5), and 1.6 X 10(6) binucleate cells was 0.27 +/- 0.03, 1.01 +/- 0.09, 4.02 +/- 0.37, 10.31 +/- 0.92, and 20.96 +/- 2.23 ng, respectively (r = 0.997). Addition of 10% fetal bovine serum (FBS) or normal anestrous cow serum increased (P less than 0.05) production of progesterone by binucleate trophoblastic cells. Luteinizing hormone, follicle-stimulating hormone, prolactin, thyrotropin, and 8-bromo-adenosine 3',5'-cyclic monophosphate had no effect. Binucleate trophoblastic cells also produced PGI2 in relation to number of cells incubated (r = 0.996). Time courses for production of PGI2, PGE2, and progesterone were similar. Aspirin inhibited production of PGI2 and PGE2 by about 50% at a dose of 100 microM; FBS stimulated production of both prostanoids.     

Re-examining the oligomerization state of macrophage migration inhibitory factor (MIF) in solution

The state of oligomerization of macrophage migration inhibitory factor (MIF, also known as glycosylation inhibiting factor, GIF) in solution has been variously reported as monomer, dimer, trimer, or mixtures of all three. Several crystal structures show MIF to be a trimer. Sedimentation velocity shows a recombinant human MIF sample is quite homogeneous, with 98% as a species with s(20,w)=3.07 S and D(20,w)=8.29 x 10(-7) cm(2)/s. Using the partial specific volume calculated from the amino acid composition these values imply a mass of 33.56 kDa, well above that of dimer, but also 9% below the trimer mass of 37.035 kDa. Sedimentation equilibrium data at loading concentrations from 0.01 to 1 mg/ml show unequivocally that the self-association is extremely tight. However, the apparent mass is 33.53 kDa [95% confidence 33.25-33.82], again 9% below that expected for 100% trimer. To examine the possibility this protein has an unusual partial specific volume, sedimentation equilibrium was also done in H(2)O/D(2)O mixtures, giving 0.765+/-0.017 ml/g rather than the calculated 0.735 ml/g. With this revised partial specific volume, the equilibrium and velocity data each give M=37.9+/-2.8 kDa, fully consistent with a strongly-associated trimeric quaternary structure.     

Some examples of the use of computer-produced contour plots in the fitting of enzyme rate equations to reaction-velocity measurements

The use of computer-based isotach plots, relating reaction velocity to simultaneous variation of two substrates or effectors of an enzyme, in producing estimates of the parameters of enzyme rate equations was investigated. The computer program (;SYMAP') incorporates an interpolation algorithm, and the superiority of this over visual estimation in producing interpolated velocity values for the estimation of parameter values by conventional double-reciprocal plots is described. The usefulness of the SYMAP program in monitoring the process of fitting data obtained by simultaneous changes in two experimental variables is also described. It is shown that if the residual errors are weighted by a procedure described elsewhere (Ottaway, 1971b, 1973), the percentage error of the computed velocity is distributed evenly over a plot which contains a 100-fold variation in the concentration of one substrate and a 500-fold variation in the concentration of Mg(2+), and in which the velocity of the reaction (that catalysed by NAD kinase) varies over a 60-fold range. The two-dimensional percentage error plot was used to assess the limits within which an incomplete inhibition equation is valid, and to detect a discrepancy in an expected good fit, caused by an impurity in one of the substrates.     

Studies on polydisperse systems using an air-driven ultracentrifuge: application to phosphatidylcholine vesicles.

A high-speed air-driven ultracentrifuge (Airfuge) has been used to determine the molecular weight and effective specific volume of phosphatidylcholine vesicles. The method used to determine the effective specific volume involved varying the solution density until zero sedimentation of the vesicles occurred. The value obtained for the effective specific volume of 0.9885 ml/g agrees well with previously reported values. The determination of the molecular weight of the vesicles is based on a method in which the fraction of vesicles remaining in an upper fraction of the solution column is compared with the values obtained using standard proteins. The values obtained for the molecular weight of the vesicles range from 1.7 X 10(6) to 2.3 X 10(6) and are in good agreement with results obtained using the analytical ultracentrifuge and with previously reported results. Possible effects due to the polydispersity of the solute are assessed using theoretical calculations and the possibility of using the Airfuge for the study of other polydisperse systems is discussed.     

Conformation parameters of linear macromolecules from velocity sedimentation and other hydrodynamic methods

Linear macromolecules constitute a broad class of synthetic and natural polymers which are highly useful in various technologies and represent the key molecular systems in living nature. The study of the molecular characteristics of these polymers represents an important problem in fundamental and applied science. The methods of molecular hydrodynamics have been and remain an important way of studying the molar mass, molar mass distribution, size and conformation of linear polymers. This paper discusses the approaches to the problems of hydrodynamic methods, in particular analytical velocity ultracentrifugation, in the study of various types of linear macromolecule. The velocity sedimentation data were processed with three different methods: Sedanal and Sedfit software, and the classical approach of evaluating the rate at which the sedimentation boundary moves. The Sedfit program also allows an evaluation of the frictional ratio values, i.e., the coefficient of translational diffusion. It will be discussed for which systems the estimation of the frictional ratio obtained by Sedfit is adequate and for which it is not. The applications of other hydrodynamic methods (intrinsic viscosity, translational diffusion) are also discussed with a view to obtaining the conformational characteristics of linear macromolecules.     

DNA supercoiled domains and radiosensitivity of subpopulations of human peripheral blood lymphocytes

Enriched human B- and T-lymphocyte subpopulations were isolated by means of a Percoll step gradient centrifugation procedure. 60Co gamma-irradiation dose-response curves for these subpopulations were obtained by applying a modified nucleoid sedimentation technique, which was also employed for the determination of the superhelical content by means of ethidium bromide intercalation. Although a similarity in the average superhelical density of B- and T1-lymphocytes was shown, B-lymphocytes exhibited a more pronounced reduction in sedimentation ratio, suggesting a higher radiosusceptibility than the T1-lymphocytes. By applying the single hit kinetics of the target theory to the dose-response curves, an estimation of the supercoil domain sizes was made: B- cells, 5.5 X 10(9), 1.78 X 10(9) and 7.78 X 10(8) D; T-cells, 4.55 X 10(9), 1.75 X 10(9) and 7.67 X 10(8) D. The differences in radiosensitivity of lymphocyte subpopulations can not, therefore, be entirely ascribed to differences in DNA superstructure.