Hydrodynamic properties of human cervical-mucus glycoproteins in 6M-guanidinium chloride.

Cervical mucins and fragments thereof were studied by sedimentation-velocity, rotatory viscometry and laser light-scattering performed as photon-correlation spectroscopy as well as low-angle total-intensity measurements. The Mr of the whole mucins is 10 X 10(6)-15 X 10(6), whereas fragments obtained after reduction of disulphide bonds ('subunits') have Mr 2.1 X 10(6)-2.9 X 10(6), depending on the method used. Subsequent trypsin digestion of subunits afforded glycopeptides with Mr approx. 0.4 X 10(6). The high frictional ratio for the whole mucins is interpreted as a large degree of expansion. The Stokes radius calculated from the diffusion coefficient is approx. 110nm for the whole mucins, which is in agreement with that estimated from the radius of gyration (130nm) by using the concept of the equivalent hydrodynamic sphere. The ratio of the concentration-dependence parameter for the reciprocal sedimentation coefficient (Ks) to the intrinsic viscosity ( [eta] ) for the whole mucins is 1.42, suggesting that the individual macromolecule occupies a spheroidal domain in solution. The relationship between [eta] and Mr for whole mucins, subunits and T-domains suggests that they are linear flexible macromolecules behaving as somewhat 'stiff' random coils. This conclusion is supported by the relationships between the sedimentation coefficients, the diffusion coefficients and the Mr. The hydrodynamic behaviour of the mucins is thus close to that expected for coiling macromolecules entrapping a lot of solvent, which is consistent with the postulated polymeric structure.     

Assembly of amyloid protofibrils via critical oligomers--a novel pathway of amyloid formation

The amyloid formation of phosphoglycerate kinase (PGK) was investigated by static and dynamic light-scattering. The time-course of the scattering intensity and the hydrodynamic radius scale with initial monomer concentration in a linear fashion over a range of about 50 in concentration. This sets limits on theories for aggregation kinetics that can be used, and points towards irreversible, cascade type models. In addition, circular dichroism (CD) was used to monitor the transition between a predominantly alpha-helical spectrum to a beta-sheet enriched one. The time-course of the CD also proves to scale linearly with initial monomer concentration. Electron microscopy shows that small oligomers as well as protofibrils are present during aggregation. The found coupling between growth of intermediates and acquisition of beta-sheet structure is interpreted in terms of a generalized diffusion-collision model, where stabilization of beta-strands takes place by intermolecular interactions.     

Thermally induced aggregation of human transferrin receptor studied by light-scattering techniques.

The thermal stability of transferrin receptor isolated from human placenta in detergent-free solution has been investigated by static light-scattering and photon correlation spectroscopy. In detergent-free solution at 293.2 K, human transferrin receptor (hTfR) forms stable associates with a hydrodynamic radius of 16 nm. With increasing temperature the particles get more compact, above 340 K a phase transition takes, place and spontaneous aggregation of the receptor occurs. Under these conditions large clusters are formed that lead to fractal aggregates, coexisting with dendritic crystalline structures. The experimental findings are compatible with a model, which involves a reaction limited cluster-cluster aggregation mechanism in conjunction with a nucleation process. The molar enthalpy change associated with the phase transition was determined to be (1860 +/- 150) kJ/mol(-1) at a transition temperature of (341.3 +/- 0.2) K.     

Regulation and processing of a secreted protein that mediates sensing of cell density in Dictyostelium.

During Dictyostelium development, the expression of some genes is dependent on cell density. This effect is mediated by soluble factors referred to as conditioned medium factors (CMFs) which the developing cells secrete at very low rates and simultaneously sense. There are at least two classes of CMFs: one is an 80 x 10(3) Mr glycoprotein and the other is a heterogeneous group of molecules, with relative molecular masses between 6.5 x 10(3) and 0.65 x 10(3). Interestingly, the two classes of molecules do not need to be combined for activity. We find that the 80 x 10(3) Mr CMF but not the small CMF is sequestered in vegetative cells. The 80 x 10(3) Mr CMF is then secreted by cells during early development, while the small CMF appears only during late development. Like the 80 x 10(3) Mr CMF, the small CMFs are trypsin-sensitive and contain N- and O-linked glycosylation. The breakdown products of a fraction containing 80 x 10(3) Mr CMF cochromatographed from a Sephadex G-50 column and a reverse-phase HPLC column with small CMFs. The specific activity of CMF increases roughtly 100-fold upon breakdown. The results suggest that, during differentiation, the slowly diffusing 80 x 10(3) Mr CMF is first produced from a precursor pool already present in vegetative cells, allowing differentiation of only those cells in the immediate vicinity of the aggregation center. The breakdown of 80 x 10(3) Mr CMF to a faster-diffusing, higher specific activity form then might enable cells farther from the aggregation center to differentiate.     

Hydrodynamic diameters of murine mammary, Rous sarcoma, and feline leukemia RNA tumor viruses: studies by laser beat frequency light-scattering spectroscopy and electron microscopy.

We have studied purified preparations of murine mammary tumor virus (MuMTV), Rous sarcoma virus (RSV; Prague strain), and feline leukemia virus (FeLV) by laser beat frequency light-scattering spectroscopy, ultra-centrifugation, and electron microscopy. The laser beat frequency light-scattering spectroscopy measurements yield the light-scattering intensity, weighted diffusion coefficients. The corresponding average hydrodynamic diameters, as calculated from the diffusion coefficients by the Stokes-Einstein equation for MuMTV, RSV, and FeLV, respectively, are: 144 +/- 6 nm, 147 +/- 7 nm, and 168 +/- 6 nm. Portions of the purified RSV and MuMTV preparations, from which light-scattering samples were obtained, and portions of the actual FeLV light-scattering samples were examined by negatively stained, catalase crystal-calibrated electron microscopy. The light-scattering intensity weighted averages of the electron micrograph size distributions were calculated by weighing each size by its theoretical relative scattering intensity, as obtained from published tables computed according to the Mie scattering theory. These averages and the experimentally observed hydrodynamic diameters agreed to within +/- 5%, which is the combined experimental error in the electron microscopic and light-scattering techniques. We conclude that the size distributions of singlet particles observed in the electron micrographs are statistically true representations of the sedimentation-purified solution size distributions. The sedimentation coefficients (S20, w) for MuMTV, RSV, and FeLV, respectively, are: 595 +/- 29S, 689 +/- 35S, and 880 +/- 44S. Virus partial specific volumes were taken as the reciprocals of the buoyant densities, determined in sucrose density gradients. The Svedberg equation was used to calculate particle weights from the measured diffusion and sedimentation coefficients. The particle weights for MuMTV, RSV, and FeLV, respectively, are: (3.17 +/- 0.32) x 10(8), (4.17 +/- 0.42) x 10(8), and (5.50 +/- 0.55) x 10(8) daltons.     

Purification of octyl beta-D-glucopyranoside and re-estimation of its micellar size

The commercial non-ionic detergent octyl beta-D-glucopyranoside is often contaminated by significant amounts of UV absorbing and/or ionic compounds that can associate with membrane proteins. Such impurities can be monitored by several techniques (i.e., spectrophotometry, size exclusion chromatography, and pH, conductivity, and surface tension measurements) and can be removed using mixed-bed ion exchange chromatography. High performance size exclusion chromatography, dynamic light scattering, and ultracentrifugation have been used to re-estimate the size of micelles of octyl beta-D-glucopyranoside since previously published data varied over a wide range. Aggregation numbers were 27 to 100 for micellar molecular weights 8000 to 29,000. Direct physical methods that do not perturbate the sample indicated a large size for the micelles (hydrodynamic radius 23 +/- 3 A; Mr 22,000 +/- 3000; aggregation number 75 +/- 10 for a 34 mM aqueous solution). In contrast the chromatographic micellar size appeared to be smaller (hydrodynamic radius 15 +/- 1 A; Mr 8000 +/- 1000; aggregation number 27). This underestimation may be the result of adsorption and/or alteration of the micelles.     

Hydrodynamic properties of the purified glutamate-binding protein subunit of the N-methyl-D-aspartate receptor.

The hydrodynamic properties of the previously purified glutamate-binding protein from rat synaptic membranes were determined in order to estimate the molecular size of the protein in its native state. This protein is apparently a subunit of a multisubunit complex that forms the N-methyl-D-aspartate subtype of glutamate receptor and has a molecular size of approximately 70 kDa based on electrophoretic migration under denaturing conditions. On the basis of results obtained from H2O/D2O sucrose density gradient sedimentation and gel filtration chromatography of the purified glutamate-binding protein we calculated the partial specific volume of the protein-detergent complex to be 0.766 cc3/g, the Stokes radius of the complex as 4.9 nm, the Mc of the complex as 203,000 +/- 22,000 and the Mr of the protein as 182,000 +/- 19,000. These results are indicative of stable self-association of the glutamate-binding protein and are in agreement with recent studies indicating that more than one molecule of glutamate may be required to activate the N-methyl-D-aspartate receptor-associated ion channel.     

The macromolecular structure of human cervical-mucus glycoproteins. Studies on fragments obtained after reduction of disulphide bridges and after subsequent trypsin digestion.

Human cervical-mucus glycoproteins (mucins) were extracted with 6 M-guanidinium chloride in the presence of proteinase inhibitors and purified by isopycnic density-gradient centrifugation. The whole mucins (Mr approx. 10 X 10(6] were degraded into 'subunits' (Mr approx. 2 X 10(6] by reduction of disulphide bonds. Trypsin digestion of the 'subunits' produced glycopeptides with Mr approx. 380000, which appear to be rod-like with a length of approx. 105 nm. The relationship between the radius of gyration and the Mr value obtained by light-scattering for whole mucins, 'subunits' and 'domains' suggest that cervical-mucus glycoproteins are linear flexible macromolecules composed of, on the average, four or five 'domains'/subunit and four subunits/whole mucin macromolecule. The shape-dependent particle scattering function for the whole mucins and the 'subunits' are in accordance with that of a linear flexible chain. No evidence for a branched or a star-like structure was found. A tentative model for cervical mucins is proposed.     

Purification of glycoproteins IIb and III from human platelet plasma membranes and characterization of a calcium-dependent glycoprotein IIb-III complex

Human platelet membrane glycoproteins IIb and III are two major integral membrane components that have been identified as sites mediating thrombin-induced aggregation. For purposes of our study, glycoproteins IIb and III were solubilized by extracting platelet plasma membranes with a buffer containing 0.1% Triton X-100 and were separated by gel filtration chromatography on Sephacryl S-300, employing Triton X-100-containing column buffers with or without urea or guanidine hydrochloride. The physical properties of the purified glycoproteins were: for glycoprotein IIb, Rs = 61 A, s20.w = 4.7, f/f0 = 1.7, Mr = 125,000 (hydrodynamic values), Mr = 136,000 (sodium dodecyl sulfate gels); for glycoprotein III, Rs = 67 A, s20,w = 3.2 f/f0 = 2.1, Mr = 93,000 (hydrodynamic values), Mr = 95,000 (sodium dodecyl sulfate gels). Although the amino acid compositions of the two glycoproteins were similar, antibodies raised against glycoprotein IIb did not crossreact with glycoprotein III. If divalent cations were not chelated in the Triton extract, glycoproteins IIb and III coeluted during gel filtration chromatography (apparent Stokes radius of 71 A) and co-sedimented on sucrose gradients (apparent s20.w of 8.6), from which Mr = 265,000 was calculated. Glycoproteins IIb and III were coprecipitated by an antibody monospecific for glycoprotein IIb. The two glycoproteins dissociated into monomers when EDTA was added to Triton lysates. Readdition of Ca2+ caused them to reassociate into a complex with physical properties similar to those of the complex in the original Triton lysate. The data show that glycoproteins IIb and III are a heterodimer complex, that complex formation depends upon the presence of Ca2+, and that chelation of Ca2+ causes dissociation into monomeric glycoproteins.     

Molecular and catalytic properties of angiotensin converting enzyme-I from bovine seminal plasma

Angiotensin converting enzyme [EC 3.4.15.1] was shown to exist in two distinct forms in bovine seminal plasma. The higher molecular weight form of the enzyme (angiotensin convering enzyme I) was purified to homogeneity by Sephadex G-200 gel filtration, and DEAE-Sepharose, blue Sepharose, and concanavalin A-Sepharose column chromatography. Final recovery of the enzyme was 9.0. The molecular weight of the enzyme was estimated to be 8 x 10(5) by the gel filtration method. A value of 4.6 x 10(5) was obtained for the reduced and denatured enzyme by dodecylsulfate polyacrylamide gel electrophoresis. The Stokes' radius, diffusion coefficient, and intrinsic viscosity of the purified enzyme were determined to be 95 A, 2.3 x 10(-7) cm2/s, and 6.76 ml/g. The enzyme had a specific activity of 105.12 mumol/min/mg protein for hippurylhistidylleucine. The Km value for hippurylhistidylleucine was found to be 20 mM. Studies with EDTA suggest that metal ions which are tightly bound are required for its activity. The enzyme was inhibited by some heavy metal ions but did not required sulfhydryl groups for its activity. Trypsin treatment of the urea-denatured enzyme produced a catalytically active fragment with an Mr of 30,000. Chemical hydrolysis of the native enzyme did not produce any active fragment.     

Effect of α-crystallin on thermal aggregation of glycogen phosphorylase <Emphasis Type="Italic">b</Emphasis> from rabbit skeletal muscle

Thermal aggregation of rabbit skeletal muscle glycogen phosphorylase b (Phb) has been investigated using dynamic light scattering under conditions of a constant rate of temperature increase (1 K/min). The linear behavior of the dependence of the hydrodynamic radius on temperature for Phb aggregation is consistent with the idea that thermal aggregation of proteins proceeds in the kinetic regime wherein the rate of aggregation is limited by diffusion of the interacting particles (the regime of "diffusion-limited cluster-cluster aggregation"). In the presence of alpha-crystallin, a protein exhibiting chaperone-like activity, the dependence of the hydrodynamic radius on temperature follows the exponential law; this suggests that the aggregation process proceeds in the kinetic regime where the sticking probability for colliding particles becomes lower than unity (the regime of "reaction-limited cluster-cluster aggregation"). Based on analysis of the ratio between the light scattering intensity and the hydrodynamic radius of Phb aggregates, it has been concluded that the addition of alpha-crystallin results in formation of smaller size starting aggregates. The data on differential scanning calorimetry indicate that alpha-crystallin interacts with the intermediates of the unfolding process of the Phb molecule. The proposed scheme of thermal denaturation and aggregation of Phb includes the stage of reversible dissociation of dimers of Phb into monomers, the stage of the formation of the starting aggregates from the denatured monomers of Phb, and the stage of the sticking of the starting aggregates and higher order aggregates. Dissociation of Phb dimer into monomers at elevated temperatures has been confirmed by analytical ultracentrifugation.     

Structural and enzymological characterization of the homogeneous deoxyribonucleic acid polymerase from Mycoplasma orale.

We have purified the DNA polymerase from Mycoplasma orale to homogeneity. The protein structure of the enzyme was declined by sodium dodecyl sulfate gel electrophoresis, which revealed a single band of 116 000 daltons that was coincident with the polymerase activity profile in the final step of DNA--cellulose chromatography, and by two-dimensional gel analysis, which demonstrated a single protein species at pI = 6.8 that was congruent with enzyme activity and contained the same 116 000 polypeptide. although severe enzyme aggregation occurs during nondenaturing gel electrophoresis, a monomer species can be resolved with a Mr of 140 000 by the Ferguson plot analysis. Gel filtration and velocity gradient centrifugation yield a Stokes radius of 4.8 nm and a sedimentation coefficient of 5.6 S, respectively, from which Mr values of 106 000--128 000 can be computed. The different size values suggest that the polymerase molecule is asymmetric. The purified enzyme has a specific activity of approximately 6 x 10(5) units/mg of protein and in completely devoid of exodeoxyribonuclease and endodeoxyribonuclease activities, at exclusion limits of 10(-4)--10(-6%) of the polymerase activity. The mechanism of polymerization is moderately processive, with an average of 14 +/- 4 nucleotides incorporated per binding event, and the "effective template length" on activated DNA is approximately 40 nucleotides.     

Interactions involved in ovomucin gel-forming properties: a rheological-biochemical approach

Different kinds of interactions involved in the properties of ovomucin gel formation from hen egg white were studied by combining physical and biochemical methods. A decrease in viscosity of the ovomucin gel was observed when it was subjected to chymotrypsin or sonication treatment. The viscosity decrease correlated with a change from non-Newtonian to Newtonian properties of the ovomucin gel. By treatment of the gel with either 5 M guanidinium HCl, 6 M urea, or 5% sodium dodecyl sulfate a change from non-Newtonian to Newtonian properties was also obtained. Although high ionic strength or sialic acid liberation from the ovomucin gel by neuraminidase treatment provoked a decrease in viscosity, it was not followed by a change in non-Newtonian properties. The results obtained suggest that different noncovalent interactions might be involved in gel formation. Electrostatic interactions (partially destroyed by sialic acid removal or 2 M NaCl) and hydrophobic interactions might be responsible for protein-mucin and mucin-mucin interactions. Other bonds susceptible to chymotrypsin treatment and sonication would be involved in the interaction between mucin subunits.     

Size heterogeneity of human cervical mucus glycoproteins. Studies performed with rate-zonal centrifugation and laser light-scattering.

Mucus glycoproteins (mucins) from cervical pregnancy mucus were fractionated by using rate-zonal centrifugation in a gradient of guanidinium chloride. The distribution of the macromolecules, as assessed by using sialic acid determination, suggested the presence of three populations of different size. Individual fractions were subjected to laser light-scattering performed as total-intensity measurements as well as photon correlation spectroscopy. The results showed that points of inflexion were present in the distribution of both Mr and DT (translational diffusion coefficient) and that the three populations have Mr values of approx. 24 X 10(6), 16 X 10(6) and 6 X 10(6) respectively. The weight-average Mr for the whole distribution, as calculated from the values obtained for the individual fractions, was 13.6 X 10(6), which is in good agreement with that found for the unfractionated material (11.1 X 10(6]. Plots of log RG (radius of gyration) and log (1/DT) versus log Mr are in keeping with the macromolecules being linear flexible chains.     

Muscarinic cholinergic receptor of rat brain. Factors influencing migration in electrophoresis and gel filtration in sodium dodecyl sulphate

The muscarinic cholinergic receptor present in synaptosomal membranes of rat brain was covalently labelled with the alkylating muscarinic antagonist, tritiated propylbenzilylcholine mustard. The labelled receptor was then solubilized in sodium deoxycholate and sodium dodecyl sulphate, and its migration in polyacrylamide gel electrophoresis and gel filtration in the presence of sodium dodecyl sulphate analysed. Provided both proteolysis and inter-chain disulphide bond formation were vigorously prevented, the receptor from rat forebrain (cerebral cortex plus caudate putamen) migrated, in sodium dodecyl sulphate/polyacrylamide gel electrophoresis, as a broad band of apparent Mr 66000-76000. Two dominantly labelled polypeptides, of apparent Mr 68000 and 73000, could be distinguished as the major components of this band. These multiple species seen in electrophoresis may reflect a structural diversity related to the different binding properties, and modes of action, of this receptor. In electrophoresis using discontinuous buffer systems the labelled receptor readily formed intermolecular disulphide bonds and so aggregated. In particular, if solubilized membranes were reduced with 2-mercaptoethanol, and reformation of disulphide bonds during electrophoresis not prevented, then formation of a dimeric species (apparent Mr 119000-128000) occurred. This probably explains previous reports in the literature of larger-Mr species seen in electrophoresis. During gel filtration, the receptor formed intra-chain disulphide bonds which produced conformational heterogeneity, leading to polydisperse migration. In addition, extensive proteolytic degradation of the receptor occurred due to a protease migrating slightly ahead of the receptor. Both effects were eliminated by alkylation of the solubilized membranes with iodoacetamide before gel filtration. Alkylated receptor migrated on Sephacryl S-300 in 0.5% sodium dodecyl sulphate with an equivalent Stokes' radius of 6.1 nm. This is identical to that of reduced ovalbumin, a molecule with an apparent Mr in gel electrophoresis of 43000. On a different gel matrix, TSK HW 55(S), the receptor migrated with a somewhat larger Stokes' radius, eluting just behind reduced bovine serum albumin (Stokes' radius 8.5 nm; apparent Mr in electrophoresis 67000). Thus the receptor appears to adsorb to the Sephacryl matrix, although even on the TSK gel the receptor eluted as a somewhat smaller protein than expected from its behaviour in gel electrophoresis. Solubilized, alkylated receptor, partly purified by gel filtration so that it was not degraded by endogenous proteases, was not cleaved by mild hydroxylamine treatment.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stages of bacteriophage lambda head morphogenesis: physical analysis of particles in solution

Intermediates in the morphogenesis of bacteriophage lambda are characterized in solution by classical light-scattering, using a modified version of the Zimm plot procedure, by quasi-elastic light-scattering and analytical ultracentrifugation. Partial specific volumes are determined simultaneously with molecular weights by a variant of the conventional combination of sedimentation and diffusion constants. Our measurements were performed within a short time and allowed the characterisation of metastable intermediates.Comparison of hydration of DNA-containing and empty heads shows that dehydration plays a minor role in the stabilisation of the DNA within the heads. The molecular weight of the scaffolding protein is 4 × 10⁶, about twice the value estimated so far. Enlargement of preheads (21% and 13% increase in dry and hydrodynamic radius, respectively) leaves the molecular weight unchanged, whereas the volume of hydration water increases from 70% to 90% of the total hydrodynamic volume. Addition of protein pD to the enlarged preheads leads to a further increase in the radius, indicating that pD is attached to the outside of the protein shell.In order to determine simultaneously the molecular weight and the partial specific volume of large and sometimes labile structures, such as a virus, the conventional sedimentation-diffusion method is modified by measuring sedimentation and diffusion coefficients in buffers containing different amounts of ²H₂O. If diffusion coefficients are determined by quasi-elastic light-scattering, experiments can be performed in a few hours. In addition, the method allows a check on the sample for changes in the frictional coefficient due, for instance, to DNA abortively ejected from a virus preparation. This method is described in the Appendix.     

Light-scattering studies on deoxyribonucleic acid flexibility. The solution properties of a small circular deoxyribonucleic acid molecule

Previous investigations on the persistence length of DNA in solution have revealed large discrepancies between hydrodynamic results and those from light-scattering techniques which have potentially a greater resolving power. The information obtained from experiments on a small circular DNA molecule has resolved these discrepancies. The non-superhelical circular double-stranded DNA molecule from bacteriophage [unk]X174-infected cells is small enough to permit accurate light-scattering extrapolations, and its solutions have negligible anisotropy. The persistence length obtained from experimental investigations on this molecule is comparable with that obtained by hydrodynamic techniques, even with variation of the excluded-volume factor.     

Conformation of xanthan dissolved in aqueous urea and sodium chloride solutions

Quasielastic light-scattering and other physical-chemical techniques have been used to compare the conformation and intermolecular interactions of xanthan in water, aqueous sodium chloride, and urea solutions. The results showed that xanthan dissolved in 4m urea has a disordered conformation after the solution has been maintained for 3 h at 95° and then cooled to room temperature. This conformation is similar to that previously observed only in solutions having low ionic strength at higher temperatures, following disruption of the ordered, low-temperature form. “Anomalous” behavior is seen for xanthan as a function of ionic strength, in that the hydrodynamic radius increases with increase in ionic strength, whereas a decrease is typical for polyelectrolytes. These observations suggest that aggregation of rod-like chains, similar to that seen for other stiff-chain polymers, occurs for xanthan in salt solutions, where the charged groups of the polyelectrolyte are screened by the salt ions. This aggregation may explain some of the high values reported in the literature for the molecular weight.     

Kinetic studies on the nitrite reductase of Wolinella succinogenes.

Calibration relationships were derived for cartilage proteoglycan subunit (PGS) that relate the inverse z-average hydrodynamic radius (Rs) and the weight-average Mr (Mw) to the partition coefficient (Kav.) for PGS when chromatographed on a Sepharose CL-2B column. PGS isolated from chick limb-bud chondrocyte cell cultures was fractionated chromatographically into eight pools, for which Mw and Rs were determined by total-intensity and dynamic light-scattering measurements. These data were found to be related to Kav. through the following empirical equations: log Mw = -(1.65 +/- 0.27)Kav. +(6.58 +/- 0.08); log Rs = -(0.69 +/- 0.04)Kav. +(2.75 +/- 0.01). Application of these relationships to the chromatographic data led to Mw = 1.48 X 10(6) and Rs = 38.7 nm (387 A) for the unfractionated specimens compared with values of Mw = 1.46 X 10(6) and Rs = 38.2 nm (382 A) determined by light-scattering. Our results were found to be consistent with previously proposed phenomenological models for the gel-filtration mechanism. Application of these calibration relationships to Kav. for several unfractionated specimens led to predicted values of Mw and Rs that are accurate to within 10%.