Expression in Escherichia coli and Simple Purification of Human Fhit Protein

The fragile histidine triad (Fhit) protein is a homodimeric protein with diadenosine 5′,5-P¹,P³-triphosphate (Ap₃A) asymmetrical hydrolase activity. We have cloned the human cDNA Fhit in the pPROEX-1 vector and expressed with high yield in Escherichia coli with the sequence Met-Gly-His₆-Asp-Tyr-Asp-Ile-Pro-Thr-Thr followed by a rTEV protease cleavage site, denoted as “H6TV,” fused to the N-terminus of Fhit. Expression of H6TV–Fhit in BL21(DE3) cells for 3 h at 37°C produced 30 mg of H6TV–Fhit from 1 L of cell culture (4 g of cells). The H6TV–Fhit protein was purified to homogeneity in a single step, with a yield of 80%, using nickel-nitrilotriacetate resin and imidazole buffer as eluting agent. Incubation of H6TV–Fhit with rTEV protease at 4°C for 24 h resulted in complete cleavage of the H6TV peptide. There were no unspecific cleavage products. The purified Fhit protein could be stored for 3 weeks at 4°C without loss of activity. The pure protein was stable at −20°C for at least 18 months when stored in buffer containing 25% glycerol. Purified Fhit was highly active, with a K_m value for Ap₃A of 0.9 μM and a k_cat(monomer) value of 7.2 ± 1.6 s⁻¹ (n = 5). The catalytic properties of unconjugated Fhit protein and the H6TV–Fhit fusion protein were essentially identical. This indicates that the 24-amino-acid peptide containing the six histidines fused to the N-terminus of Fhit does not interfere in forming the active homodimers or in the binding of Ap₃A.     

Fluorometric determination of N-terminal prolyl dipeptides, proline and hydroxyproline in human serum by pre-column high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride

A highly sensitive HPLC method for the determination of prolyl dipeptides, Pro and Hyp in serum was developed. After deproteinization of serum and pretreatment with o-phthalaldehyde, the analytes were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70°C for 10 min. The fluorescent derivatives of prolyl dipeptides, Pro and Hyp, were separated on tandem reversed-phase columns by a gradient elution at 55°C and detected by fluorescence measured at 318 nm (excitation) and 392 nm (emission). The detection limits for prolyl dipeptides were 2–5 fmol/injection (S/N=3). Pro–Hyp, Pro–Gly and Pro–Pro were identified as serum prolyl dipeptides. The within-day and between-day relative standard deviations were 1.5–7.9 and 2.4–10.8%, respectively. The recoveries were in the range of 90.8–97.3%. The concentrations of Pro–Hyp, Pro–Gly, Pro–Pro, Pro and Hyp in normal human serum (n=10) were 0.64±0.35, 0.078±0.047, 0.022±0.016, 177.0±43.0 and 11.1±3.5 μM, respectively. The concentrations of Pro–Hyp and Pro–Pro in serum of a patient with bone metastases of prostatic cancer were about three times and 50 times, respectively, higher than those in normal human serum.     

Fine structure of mononucleosomes derived by bright- and dark-field electron microscopy

Images of intact and disrupted mononucleosomes, obtained by both bright-field (BF) and dark-field (DF) microscopy, were compared and analysed to determine the degree of continuity of morphological structures from the two visualization modes. DF images were about 10–13 % larger than BF images. Unless fixed with glutaraldehyde or stained with uranyl acetate, mononucleosomes may be partly unfolded. The 10.3 ± 1.1 nm diameter glutamate dehydrogenase hexamer, aggregates of histones H2a, H2b, H3 and H4 and naked DNA were used to estimate the contribution of protein and DNA to the images of mononucleosomes. The major contribution to the electron-dense regions of mononucleosomes viewed by the DF process is from histones and not DNA alone. Image analogs and autocorrelation methods were used to solve the three-dimensional organization of the nucleosome images in relation to the eight histones and the DNA. The structure of the mononucleosome which is most consistent with the data is that of a supercoiled nucleoprotein of 1.5 gyrs or greater with 8 roughly evenly spaced globular histone domains, each about 4.2 ± 0.7 nm in diameter, arranged as a pseudo-penta or hexamer as viewed along the central axis of symmetry. The structure is in essential agreement with models specified by Finch & Klug [12], and Trifonov [50].     

Characterization of the size distribution of unilamellar vesicles by gel filtration, quasi-elastic light scattering and electron microscopy

The size and size distribution of unilamellar phospholipid vesicles present in unsonicated phosphatidic acid and mixed phosphatidic acid/phosphatidylcholine dispersions were determined by gel filtration, quasi-elastic light scattering and freeze-fracture electron microscopy. The vesiculation in these dispersions was induced by a transient increase in pH as described previously (Hauser, H. and Gains, N. (1982) Proc. Natl. Acad. Sci. USA 79, 1683–1687). The resulting phospholipid dispersions are heterogeneous consisting of small unilamellar vesicles (average radius r < 50 nm) and large unilamellar vesicles (average r ranging from about 50 to 500 nm). The smallest vesicles with r = 11 ± 2 nm are observed with dispersions of pure phosphatidic acid, the population of these vesicles amounting to about 80% of the total lipid. With increasing phosphatidylcholine content the radius of the small unilamellar vesicles increases and at the same time the population of small unilamellar vesicles decreases. The average radius of small unilamellar vesicles present in phosphatidic acid/phosphatidylcholine dispersions (mole ratio, 1:1) is 17.5 ± 2 nm, the population of these vesicles amounting to about 70% of the total lipid. By a combination of gel filtration, quasi-elastic light scattering and freeze-fracture electron microscopy it was possible to characterize the large unilamellar vesicles. This population is heterogeneous with its mean radius also increasing with increasing phosphatidylcholine content. After separating the large unilamellar vesicles from small unilamellar vesicles on Sepharose 4B it can be shown by quasi-elastic light scattering that in pure phosphatidic acid dispersions 80–90% of the large unilamellar vesicle population consist of vesicles with a mean radius of 170 nm. In mixed phosphatidic acid/phosphatidylcholine dispersions this radius increases to about 265 nm as the phosphatidylcholine content is raised to 90 mol%.     

Highly sensitive determination of N-terminal prolyl dipeptides, proline and hydroxyproline in urine by high-performance liquid chromatography using a new fluorescent labelling reagent, 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride

A highly sensitive pre-column HPLC method for simultaneous determination of prolyl dipeptides, Pro and Hyp in urine was developed. The analytes were labelled with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70°C for 20 min. The derivatives separated on tandem reversed-phase columns by a gradient elution and were monitored with fluorescence detection at 318 nm (excitation) and 392 nm (emission). The detection limits for prolyl dipeptides, Pro and Hyp were 1–5 fmol/injection (S/N=3). Urine samples were treated with o-phthalaldehyde, followed by purification on a Bond Elut C₁₈ column before conducting the labelling reaction. Pro–Hyp, Pro–Gly and Pro–Pro were identified as prolyl dipeptides in urine. The within-day and between-day relative standard deviations were 1.5–4.8 and 1.7–5.8%, respectively. The concentrations of Pro–Hyp, Pro–Gly, Pro–Pro, Pro and Hyp in normal human urine were 97.6±28.2, 2.74±1.48, 2.08±1.13, 6.71±3.34 and 2.30±1.59 nmol/mg creatinine, respectively.     

Architecture of the cell wall of a green alga,Oocystis apiculata

Summary The cell wall of a green alga,Oocystis apiculata, was visualized by electron microscopy after preparation of samples by rapid-freezing and deep-etching techniques. The extracellular spaces clearly showed a random network of dense fibrils of approximately 6.4 nm in diameter. The cell wall was composed of three distinct layers: an outer layer with a smooth appearance and many protuberances on its outermost surface; a middle layer with criss-crossed cellulose microfibrils of approximately 15–17 nm in diameter; and an inner layer with many pores between anastomosing fibers of 8–10 nm in diameter. Both the outer and the inner layer seemed to be composed of amorphous material. Cross-bridges of approximately 4.2 nm in diameter were visualized between adjacent microfibrils by the same techniques. The cross-bridges were easily distinguished from cellulose microfibrils by differences in their dimensions.     

Three-Dimensional Structure of the Porcine Gastric H,K-ATPase from Negatively Stained Crystals

A low-resolution three-dimensional model of membrane-bound H,K-ATPase from pig gastric mucosa has been reconstructed by electron microscopy and image processing of two-dimensional crystals in negative stain. The crystal formation is induced by magnesium and vanadate, which stabilize the E₂conformation of the enzyme. The unit cell, with a size ofa=b= 123 Å, γ = 90°, has tetragonal p4 symmetry. There are four separate αβ protomers within each unit cell. The high-contrast region is limited to the cytoplasmic part of the protein. The total volume of the observed asymmetric protein domain corresponds to a molecular mass of 80–90 kDa. It consists mainly of a large pear-shaped domain measuring 60 × 45 Ų, with a height of 50 Å as measured perpendicular to the membrane plane. A small stalk segment, 20 Å in length, forms a connection to the transmembrane region.     

Antiestrogen binding sites in rat liver nuclei

Isolated, intact rat liver nuclei have high-affiity (K_d=10⁻⁹ M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [³H]tamoxifen binding capacity is thermolabile, being most stable at 4°C and rapidly lost at 37°C. More [³H]tamoxifen, however, is specifically bound at incubation temperatures of 25°C and 37°C than at 4°C although prewarming nuclei has no effect, suggesting exchange of [³H]tamoxifen for an unidentified endogenous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (K_d=10⁻⁹ M) [³H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78±0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000×g, 30 min) contains high-capacity (955±405 (S.D.) fmol/mg protein), low-affinity (K_d=10.9±4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000×g, 60 min) contains low-capacity (46±15 (S.D.) fmol/mg protein), high-affinity (K_d=0.61± 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%–3.2%, and nuclear sites less than 0.5% of total sites.     

Fast liquid chromatographic determination of urinary trans,trans-muconic acid

trans,trans-Muconic acid (1,3-butadiene-1,4-dicarboxylic acid, MA), a minor urinary metabolite of benzene exposure, was determined, after clean-up by solid-phase anion-exchange chromatography, by reversed-phase HPLC on a C₁₈ column (5 × 0.46 cm I.D., 3 μm particle size), using formic acid-tetrahydrofuran-water (14:17:969) as mobile phase and UV detection at 263 nm. The recovery of MA from spiked urine was > 95% in the 50–500 μg/l range; the quantification limit was 6 μg/l; day-to-day precision, at 300 μg/l, C.V. = 9.2%; the run time was less than 10 min. Urinary MA excretion was measured in two spot urine samples of 131 benzene environmentally exposed subjects: midday values obtained in non-smokers (mean±S.D.=77±54 μg/l, N = 82) were statistically different from those of smoerks (169±85 μg/l, N = 30) (P<0.0001); each group showed a statistically significant increase between MA excretion in midday over morning samples. Moreover, in subjects grouped according to tobacco-smoke exposure level, median values of MA were positively associated with and increased with daily smoking habits.     

Quantitative gap junction alterations in mammalian heart cells quickly frozen or chemically fixed after electrical uncoupling

Summary The gap junction morphology was quantified in freeze-fracture replicas prepared from rat auricles that had been either quickly frozen at 6 K or chemically fixed by glutaraldehyde, in a state of normal cell-to-cell conduction or in a state of electrical uncoupling. The general appearance of the gap junctions was similar after both preparative procedures. A quantitative analysis of three gap junctional dimensions provided the following measurements in the quickly frozen conducting auricles (mean±sd): (a) P-face particles' diameter 8.27±0.74 nm (n =5709), (b) P-face particles' center-to-center distance 10.78±2.12 nm (n=4800), and (c) E-face pits' distance 9.99±2.19 nm (n=1600). Corresponding values obtained from chemically fixed tissues were decreased by about 3% for the particle's diameter and about 5% for the particles' and pits' distances. Electrical uncoupling by the action of either 1 mM 2–4-dinitrophenol (DNP), or 3.5 mMn-Heptan-1-ol (heptanol), induced a decrease of the particle's diameter, which amounted to –0.69±0.01 nm (mean ±se) in the quickly frozen preparations and –0.71±0.01 nm in the chemically fixed ones. The particles' distance was decreased by –0.96±0.04 nm in the quickly frozen samples and by –0.90 ±0.03 nm in the chemically fixed ones and the E-face pits' distance was similarly reduced. All differences were statistically significant (P<0.001 for all dimensions). Electrical recoupling after the heptanol effect promoted a return of these gap junctional dimensions towards normal values, which was about 50% complete within 20 min. It is concluded that very similar morphological alterations of the gap junctional structure are induced in the mammalian heart by different treatments promoting electrical uncoupling and that these conformational changes appear independently of the preparative procedure. The suggestion that the observed decrease of the particles' diameter is genuinely related to the closing mechanism of the unit cell-to-cell channel set in thei centers is thus confirmed.     

Single crystals of dextran: High temperature polymorph

Lamellar single crystals of a high temperature polymorph of synthetic dextran were prepared at temperatures ranging from 120 to 200°C in a mixture of water and polyethylene glycol. Individual crystals with lath-like shapes gave well resolved electron diffraction diagrams from which the reciprocal unit cell parameters a^*, b^* and γ^* could be measured. The direct cell parameters were then determined from a series of electron diffraction diagrams obtained by sequential tilting of the crystal about the b^* axis. This gave a = 0·922 ± 0·001 nm, b = 0·922 ± 0·001 nm, c (chain axis) = 0·78 ± 0·01 nm, α = γ = 90° and β = 91·3° ± 0·5°. The crystal symmetry was P2₁ with b as the unique monoclinic axis. These data coupled with the observed density of the crystals, indicated that the unit cell contained two antiparallel dextran chains of two residues each. When the crystals were grown at temperatures between 90 and 120°C, a percentage of crystals containing both low and high temperature polymorphs were obtained. These mixed crystals had most likely grown in syntaxy.     

A double coil chromatin sub-unit model

A model is proposed for the structure of DNA in chromatin sub-units. Each sub-unit is proposed to contain two turns of an inner coil, with a pitch of about 40and an external diameter of 70 . Around the inner coil is wound, in opposite handedness, a slightly larger amount of DNA at a diameter of about 150 . The total contour length consistent with the electron micrographs and X-ray scattering is 600–700 , or about 200 base pairs. It is suggested that the inner coil is protein rich and contains all of the histones except H1, which is associated with the outer coil. The double-coil model is consistent with previous biochemical and biophysical studies of chromatin. The existence of 200 and 100 base pair digestion fragments and a 6 to 1 DNA compaction are readily explained.This model is based upon the electron microscopic observation of replicas of frozen chromatin and X-ray and neutron scattering. Structural details of 25are preserved and visualized by the freeze fracture electron microscopy techniques employed.     

Propylene oxide: mutagenesis, carcinogenesis and molecular dose

The results from mutagenic and carcinogenic studies of propylene oxide (PO) and the current efforts to develop molecular dosimetry methods for PO–DNA adducts are reviewed. PO has been shown to be active in several bacterial and mammalian mutagenicity tests and induces site of contact tumors in rodents after long-term administration. Quantitation of N7-(2-hydroxypropyl)guanine (7-HPG) in nasal and hepatic tissues of male F344 rats exposed to 500 ppm PO (6 h/day; 5 days/week for 4 weeks) by inhalation was performed to evaluate the potential of high concentrations of PO to produce adducts in the DNA of rodent tissues and to obtain information necessary for the design of molecular dosimetry studies. The persistence of 7-HPG in nasal and hepatic tissues was studied in rats killed three days after cessation of a 4-week exposure period. DNA samples from exposed and untreated animals were analyzed for 7-HPG by two different methods. The first method consisted of separation of the adduct from DNA by neutral thermal hydrolysis, followed by electrophoretic derivatization of the adduct and gas chromatography-high resolution mass spectrometry (GC–HRMS) analysis. The second method utilized ³²P-postlabeling to quantitate the amount of this adduct in rat tissues. Adducts present in tissues from rats killed immediately after cessation of exposure were 835.4±80.1 (respiratory), 396.8±53.1 (olfactory) and 34.6±3.0 (liver) pmol adduct/μmol guanine using GC–HRMS. Lower values, 592.7±53.3, 296.5±32.6 and 23.2±0.6 pmol adduct/μmol guanine were found in respiratory, olfactory and hepatic tissues of rats killed after three days of recovery. Analysis of the tissues by ³²P-postlabeling yielded the following values: 445.7±8.0 (respiratory), 301.6±49.2 (olfactory) and 20.6±1.8 (liver) pmol adduct/μmol guanine in DNA of rats killed immediately after exposure cessation and 327.1±21.7 (respiratory), 185.3±29.2 (olfactory) and 15.7±0.9 (liver) pmol adduct/μmol guanine after recovery. Current methods of quantitation did not provide evidence for the endogenous formation of this adduct in control animals. These studies demonstrated that the target tissue for carcinogenesis has much greater alkylation of DNA than liver, a tissue that did not exhibit a carcinogenic response.     

Magnetic nanoparticles supported ionic liquids for lipase immobilization: Enzyme activity in catalyzing esterification

Candida rugosa lipase was immobilized on magnetic nanoparticles supported ionic liquids having different cation chain length (C₁, C₄ and C₈) and anions (Cl⁻, BF₄⁻ and PF₆⁻). Magnetic nanoparticles supported ionic liquids were obtained by covalent bonding of ionic liquids–silane on magnetic silica nanoparticles. The particles are superparamagnetic with diameter of about 55 nm. Large amount of lipase (63.89 mg/(100 mg carrier)) was loaded on the support through ionic adsorption. Activity of the immobilized lipase was examined by the catalysis of esterification between oleic acid and butanol. The activity of bound lipase was 118.3% compared to that of the native lipase. Immobilized lipase maintained 60% of its initial activity even when the temperature was up to 80 °C. In addition, immobilized lipase retained 60% of its initial activity after 8 repeated batches reaction, while no activity was detected after 6 cycles for the free enzyme.     

Responses of Tradescantia stamen hairs and pollen to UV-B irradiation

Clones 02 and 4430 of Tradescantia were tested in field, greenhouse and controlled environment chambers as monitors for the potentially hazardous UV-B irradiation increase that could result from stratospheric ozone decrease. In addition to about 16 hr of solar emissions at about 2100 micro-einsteins·m⁻²·s⁻¹ (400–700 nm) and 15 hr at about 1800 micro-einsteins·m⁻²·s⁻¹ in the field and greenhouse, respectively, plants were given 7 hr of supplemental UV-B irradiation per day for 27 days. After the first 7 days of UV-B irradiation exposure, cumulative data were recorded for 20 days. Cuttings of Tradescantia plants in controlled-environment, exposed to 16 hr of simulated solar emission of about 800 micro-einsteins·m⁻²·s⁻¹ (400–700 nm), were also exposed to 10 hr of supplemental UV-B irradiation per day for 1 or 2 days. All plants were checked for somatic aberrations (color changes in the flower petals and stamen hairs), number of hairs per stamen, and cells per hair. Pollen germination and pollen tube growth were noted after a 90-min UV-B irradiation period.Somatic aberrations occurred infrequently in the petals and were judged unreliable criteria for use in monitoring enhanced UV-B irradiation environments. The number of aberrant events within stamen hairs, however, was significantly increased by the UV-B irradiation treatments. while pollen germination and pollen tube growth were significantly reduced. These data indicate that color changes in stamen hairs and pollen viability are useful criteria for monitoring UV-B irradiation changes.     

Aggregation of mono- and dinucleosomes into chromatin-like fibers

Three classes of chicken erythrocyte chromatin particles differing in their content of lysine-rich histones and/or spacer DNA have been studied in order to determine their ability to aggregate into complexes resembling those observed in native chromatin. The complexes have been obtained in the presence of MgCl₂ and NaCl and studied by electron microscopy. Mononucleosomes, containing spacer DNA and histones H1 and H5, give rise to thick (about 70 nm) ellipsoidal particles in the presence of 0.5 mM MgCl₂. These particles are disrupted by the addition of small amounts of NaCl (5–20 mM). On the other hand in 0.5 mM MgCl₂ dinucleosomes give rise to regular fibrous complexes of about 40 nm in diameter which are very similar to native chromatin fibers. These complexes are much more stable when NaCl is added. We conclude that for the stability of nucleosomal aggregates, similar to native chromatin fibers, a continuity of DNA structure is not required, but the presence of divalent cations, spacer DNA and lysine-rich histones is essential.     

High affinity estrogen binding by rabbit liver

Macromolecular binding components for [³H]estradiol-17β are present to cytosol prepared from rabbit liver. When cytosol from sexually mature male liver was incubated with [³H]estradiol and analyzed for binding on low ionic strength sucrose gradients, two peaks of binding activity were detected. One peak had a sedimentation coefficient of 4–5 S and the other had a sedimentation coefficient of 8–9 S. The two components differed from each other regarding steroid specicity and various physiocochemical parameters. [³H]-estradiol binding to the 4–5 S component was not inhibited by estrogens, 5α-dihydrotestosterone, progesterone or cortisol. Binding to this component did not appera to be saturable and lavel was rapidly stripped from it by cahrcoal. Estradiol bindng to the 8–9 S component was estrogen specific, saturable and of high affinity. The specific binder dissociates on high ionic strength sucrose gradients and sediments as a 4–5 S moiety. The specific binding protein has a K_d of 3.05 · 10⁻¹⁰ M and a dissociation half-time of 33 h and there are 35.2 fmol of binding sites/mg cytosol protein. Estrogen binders are also present in liver cytosol from sexually mature female and sexually immature male rabbits. During prolonged incbuation of [³H]estradiol with mature male liver cytosol at 0–5°C polar metabolites of estradiol are produced.     

Studies on the fine structure of the mitochondrial derivative in spermatozoa of a gastropod

Spermatozoa of Limax sp. were studied by electron microscopy following thin section and freeze-fracture techniques. Mature spermatozoa were seen to be helically shaped, 150 μm long cells. A single mitochondrion extends the entire length of the spermatozoon. Its helical turn is the same as that of the spermatozoon. Freezefracture images of the spermatozoon reveal that the EF and PF, plasmalemmal faces contain scattered, 7–9 nm size particles, and that the PF, outer mitochondrial membrane face contains 8–10 nm size particles. The corresponding EF, outer mitochondrial face contains matching pits. A paracrystalline complex is situated between the inner and outer mitochondrial membranes. The complex is constructed of a series of 8–9 nm thick, 35 nm wide, helically orientated, tripartate elements which extend the full length of the spermatozoon. The helical tilt angle is approximately 55 °. Each element is composed of tightly approximated (interspace distance 10 nm), strands of particles 8–9 nm in diameter. Speculations as to the significance of this complex, and its location between inner and outer mitochondrial membranes are made. It is concluded that the paracrystalline order of the complex either reflects the molecular packing of enzyme systems present in the mitochondrion, or some other unknown function.     

Macromolecular conformation of chitosan in dilute solution: A new global hydrodynamic approach

Chitosans of different molar masses were prepared by storing freshly prepared samples for up to 6 months at either 4, 25 or 40 °C. The weight-average molar masses, M_w and intrinsic viscosities, [η] were then measured using size exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALLS) and a “rolling ball” viscometer, respectively.The solution conformation of chitosan was then estimated from:

(a) the Mark–Houwink–Kuhn–Sakurada (MHKS) power law relationship [η] = kM_w^a and

(b) the persistence length, L_p calculated from a new approach based on equivalent radii [Ortega, A., & Garcia de la Torre, J. (2007). Equivalent radii and ratios of radii from solution properties as indicators of macromolecular conformation, shape, and flexibility. Biomacromolecules, 8, 2464–2475].

Both the MHKS power law exponent (a = 0.95 ± 0.01) and the persistence length (L_p = 16 ± 2 nm) are consistent with a semi-flexible rod type (or stiff coil) conformation for all 33 chitosans studied. A semi-flexible rod conformation was further supported by the Wales–van Holde ratio, the translational frictional ratio and sedimentation conformation zoning.