Spectroscopic studies have identified a number of proteins that appear to retain significant residual structure under even strongly denaturing conditions. Intrinsic viscosity, hydrodynamic radii, and small-angle x-ray scattering studies, in contrast, indicate that the dimensions of most chemically denatured proteins scale with polypeptide length by means of the power-law relationship expected for random-coil behavior. Here we further explore this discrepancy by expanding the length range of characterized denatured-state radii of gyration (RG) and by reexamining proteins that reportedly do not fit the expected dimensional scaling. We find that only 2 of 28 crosslink-free, prosthetic-group-free, chemically denatured polypeptides deviate significantly from a power-law relationship with polymer length. The RG of the remaining 26 polypeptides, which range from 16 to 549 residues, are well fitted (r2 = 0.988) by a power-law relationship with a best-fit exponent, 0.598 ± 0.028, coinciding closely with the 0.588 predicted for an excluded volume random coil. Therefore, it appears that the mean dimensions of the large majority of chemically denatured proteins are effectively indistinguishable from the mean dimensions of a random-coil ensemble

An objective measure of the length scale of turbulent overturning events, the Thorpe scale, Lr, is compared to the Ozmidov scale Lo = (e/N3) 1/2, where N is the buoyancy frequency and e is the kinetic energy dissipation rate. Far from the surface in wind-forced mixing layers and in the seasonal thermocline, Lo and Lr are of the same order, but near the surface of a mixing layer, Lo is significantly larger than Lr. The change in the ratio Lo/Lr is attributed to a decrease in the gradient Richardson number in the highly energetic zone near the surface. Another length scale, LB = (DCx/N) •/2, where Cxis the Cox number and D is the molecular diffusivity of temperature, is the same order as Lr near the surface of a mixing layer as well as in the layer interior and in the seasonal thermocline. It is shown, by using the turbulent kinetic energy budget, that LB/Lr is only weakly dependent on the gradient Richardson number as long as the ratio of eddy viscosity to eddy diffusivity is constant. The temperature variance dissipation rate is compared to the product of the buoyancy frequency and the existing temperature variance. Temperature fluctuations are defined as the temperature difference between the observed temperature profile and the Thorpe profile (the temperature profile which would result if an overturning patch gravitationally collapsed without dissipation). It is shown that the major balance in the temperature variance equation is between the rate at which variance is produced and the rate at which it is dissipated and that the rate of change of temperature variance can be an important modification to this balance only if the variance decays in a time much smaller than a buoyancy period. a signature which might be expected for a large overturning eddy: sharp upper and lower boundaries with intense mixing inside. While common in surface layers strongly forced by the wind, these large features are not always found (Figures 3 and 4). When wind forcing is absent, they are not found at all in surface layers. Other features often found in windforced surface layers are smaller, some having an 'eddylike' shape similar to the larger disturbances, some a random mix of small-scale fluctuations without sharp boundaries (Figure 3). The Thorpe scale Lr is defined as the root mean square Thorpe displacement, LT = {d'2) 1/2 where ( ) signifies an appropriate averaging process. While the displacement dn' is not necessarily the distance sample Pn actually traveled (eddies are not one-dimensional), The Thorpe scale is proportional to the mean eddy size as long as the mean horizontal density gradient is much smaller than the vertical gradient. It has been suggested that the maximum displacement be used rather than the Thorpe scale for the Ozmidov scale comparison (C. Gibson, private communication, 1981), and all that follows could be cast in these terms. We have chosen to use the Thorpe scale rather than the maximum displacement because we only sample verti-9601 9602 DILLON: VERTICAL OVERTURNS DEPTH DENSITY THORPE DISPLACEMENTS TH...

we have identified that the human IgG2 subclass exists as an ensemble of distinct isoforms, designated IgG2-A, -B, and -A/B, which differ by the disulfide connectivity at the hinge region. In this report, we studied the structural and functional properties of the IgG2 disulfide isoforms and compared them to IgG1. Human monoclonal IgG1 and IgG2 antibodies were designed with identical antigen binding regions, specific to interleukin-1 cell surface receptor type 1. In vitro biological activity measurements showed an increased activity of the IgG1 relative to the IgG2 in blocking interleukin-1␤ ligand from binding to the receptor, suggesting that some of the IgG2 isoforms had lower activity. Under reduction-oxidation conditions, the IgG2 disulfide isoforms converted to IgG2-A when 1 M guanidine was used, whereas IgG2-B was enriched in the absence of guanidine. The relative potency of the antibodies in cell-based assays was: IgG1 > IgG2-A > IgG2 Ͼ Ͼ IgG2-B. This difference correlated with an increased hydrodynamic radius of IgG2-A relative to IgG2-B, as shown by biophysical characterization. The enrichment of disulfide isoforms and activity studies were extended to additional IgG2 monoclonal antibodies with various antigen targets. All IgG2 antibodies displayed the same disulfide conversion, but only a subset showed activity differences between IgG2-A and IgG2-B. Additionally, the distribution of isoforms was influenced by the light chain type, with IgG2 composed mostly of IgG2-A. Based on crystal structure analysis, we propose that IgG2 disulfide exchange is caused by the close proximity of several cysteine residues at the hinge and the reactivity of tandem cysteines within the hinge. Furthermore, the IgG2 isoforms were shown to interconvert in whole blood or a "bloodlike" environment, thereby suggesting that the in vivo activity of human IgG2 may be dependent on the distribution of isoforms.

In this work, we present studies of the covalent structure of human IgG2 molecules. Detailed analysis showed that recombinant human IgG2 monoclonal antibody could be partially resolved into structurally distinct forms caused by multiple disulfide bond structures. In addition to the presently accepted structure for the human IgG2 subclass, we also found major structures that differ from those documented in the current literature. These novel structural isoforms are defined by the light chain constant domain (C L ) and the heavy chain C H 1 domain covalently linked via disulfide bonds to the hinge region of the molecule. Our results demonstrate the presence of three main types of structures within the human IgG2 subclass, and we have named these structures IgG2-A, -B, and -A/B. IgG2-A is the known classic structure for the IgG2 subclass defined by structurally independent Fab domains and hinge region. IgG2-B is a structure defined by a symmetrical arrangement of a (C H 1-C Lhinge) 2 complex with both Fab regions covalently linked to the hinge. IgG2-A/B represents an intermediate form, defined by an asymmetrical arrangement involving one Fab arm covalently linked to the hinge through disulfide bonds. The newly discovered structural isoforms are present in native human IgG2 antibodies isolated from myeloma plasma and from normal serum. Furthermore, the isoforms are present in native human IgG2 with either or light chains, although the ratios differ between the light chain classes. These findings indicate that disulfide structural heterogeneity is a naturally occurring feature of antibodies belonging to the human IgG2 subclass.

The status of the N-terminus of proteins is important for amino acid sequencing by Edman degradation, protein identification by shotgun and top-down techniques, and to uncover biological functions, which may be associated with modifications. In this study, we investigated the pyroglutamic acid formation from N-terminal glutamic acid residues in recombinant monoclonal antibodies. Almost half the antibodies reported in the literature contain a glutamic acid residue at the N-terminus of the light or the heavy chain. Our reversed-phase high-performance liquid chromatography-mass spectrometry method could separate the pyroglutamic acid-containing light chains from the native light chains of reduced and alkylated recombinant monoclonal antibodies. Tryptic peptide mapping and tandem mass spectrometry of the reduced and alkylated proteins was used for the identification of the pyroglutamic acid. We identified the formation of pyroglutamic acid from N-terminal glutamic acid in the heavy chains and light chains of several antibodies, indicating that this nonenzymatic reaction does occur very commonly and can be detected after a few weeks of incubation at 37 and 45 degrees C. The rate of this reaction was measured in several aqueous buffers with different pH values, showing minimal formation of pyroglutamic acid at pH 6.2 and increased formation of pyroglutamic acid at pH 4 and pH 8. The half-life of the N-terminal glutamic acid was approximately 9 months in a pH 4.1 buffer at 45 degrees C. To our knowledge, we showed for the first time that glutamic acid residues located at the N-terminus of proteins undergo pyroglutamic acid formation in vitro.

No abstract

Observations of vertical temperature microstructure at ocean station P during the mixed layer experiment (Mile) indicate that the shape of the high-frequency temperature gradient spectrum depends on the relative strengths of turbulence and stratification. For low Cox number ((dT/dz) 2) /(dT/dz) • the linear range of the Batchelor spectrum is not well approximated by observed spectra, while for high Cox number a remarkably close correspondence to the Batchelor spectrum is found. Dissipation rates calculated by the temperature gradient spectrum cutoff wave number method show a dramatic contrast in turbulence between low and high wind speed periods separated by only 3 hours, showing that the response of the mixed layer and transition zone to wind forcing is rapid. Some indication is found that the thermocline may also respond rapidly to surface forcing. E 1.0 Spectrum of heated laminar jet ß ß ß ß _ ß ß Uncorrected Spectrum o Corrected Spectrum, i,af •' ,bf4.cf 6

Microstructure profiles of temperature, conductivity, and velocity shear during the Arctic Internal Wave Experiment (AIWEX) in March-April 1985 in the Beaufort Sea are used to investigate the thermodynamic processes in a diffusive thermohaline staircase. The staircase occurs between depths of about 320 and 430 m, above the core of the relatively warm, salty Atlantic water, where the mean temperature and salinity are increasing with depth. Individual isothermal layers can be tracked for at least several hours, suggesting a horizontal length scale of several hundred meters or more, assuming a typical relative velocity of 0.01 m s-x at this time. Over the depth range 320-430 m the mean (average over several steps) density ratio (R,)=/•(S:)/ot(T:) varies between 4 and 6, while the typical temperature difference between layers decreases from 0.012 ø to 0.004øC. The mean thickness of the layers also varies, from 1 m at 320 m depth to 2 m at 430 m. The relationship proposed by Kelley (1984), relating layer height to (N'•), (Rp), and molecular properties of the fluid, overestimates the mean layer thickness by about a factor of 2. The variability of staircase characteristics suggests that oceanic staircases may rarely, if ever, be steady state, but in general be slowly evolving from previous perturbations. Heat fluxes estimated from laboratory-based flux laws, involving Rp and AT, are in the range 0.02 < F n < 0.1 W m -'•, which is in agreement with the molecular heat fluxes through the maximum interfacial temperature gradients. There are no interfaces where the kinetic energy dissipation rate (averaged over 0.5 m) exceeds the lower limit for diapycnal mixing, 24.5vN 2.