Thermodynamic parameters of helix-coil transition in polypeptide chains. II. Poly-L-lysine

The helix–coil transitions for poly-L -lysine (PL) were investigated by the methods of spectropolarimetry, viscometry and potentiometric titration in 0.2M NaCl at different temperatures as well as in 0.2MNaBr, 1MKCl, and in mixtures of 0.2MNaCl or NaBr with methanol at room temperature. The enthalpy and entropy differences between the helical and coillike states of uncharged PL molecules in 0.2.M NaCl were determined from the potentiometric titration curves. The cooperativity parameters σ for PL in different solvents were determined by two methods (from the sharpness of the transition and from the dependence of the intrinsic viscosity on the helical content in the transition region). In 0.2MNaCl σ has a value of (2.3 ± 0.5) × 10^?4 and does not depend on temperature, i.e., the cooperativity of the helix-coil transition, as for PGA, is mainly of an entropy origin (the initiating of the helical region is accompanied by the entropy decrease ΔS_i = ?12 eu/mole of helical regions). A comparison of the obtained results for PGA and PL with the molecular theories of the helix-coil transitions shows that the role of dipole-dipole interactions of nonneighboring peptide groups is greatly overestimated in these theories, leading to a considerable enthalpy contribution to the free energy of initiating helical regions which is not observed in the experiment.     

Molecular weight estimation using sodium dodecyl sulfate-pore gradient electrophoresis

Pore gradient electrophoresis (PGE) in the presence of sodium dodecyl sulfate (SDS) provides a means for high resolution fractionation of multicomponent protein systems and permits estimation of molecular weights for macromolecules ranging from 10³ to 10⁶. We have evaluated the performance of several methods used to construct calibration curves for estimation of molecular weights using SDS-PGE. A linear relationship between the logarithm of molecular weight, log (M_r), and the logarithm of the relative mobility, log (R_l), can be obtained for a 30-fold range of molecular weights. However, this range of linearity depends on the choice of the concentration gradient, the degree of crosslinking of the gel, and on the nature of the underlying relationship between the retardation coefficient, K_R, and the molecular weight. An empirical relationship, first introduced by Lambin et al. (1976, Anal. Biochem.74, 567) between log (M_r) and the logarithm of the gel concentration at the position reached by the protein, log (%T), provides better linearity over a wider molecular weight range than does the use of log (R_l). We have compared these relatienships by experimental analysis of 10 standard proteins and by a theoretical analysis of an idealized model system. A computer program has been developed which provides appropriate statistical estimation of the molecular weight for an unknown protein, together with its standard error and 95% confidence limits. A new method has also been developed for analysis of nonlinear calibration curves in terms of molecular weight versus distance migrated, based on a theoretically justifiable, physical-chemical model. This model implies that either the relationship between log (M_r) and log (R_l) or the one between log (M_r) and log (%T) will become nonlinear as the range of molecular weight is extended. We suggest that the use of a nonlinear least-squares curve-fitting procedure provides an optimal method for molecular weight estimation when sufficient data are available. Based on these findings, a general strategy is presented for estimation of molecular weights by polyacrylamide gel electrophoresis.     

Thermodynamic parameters of helix-coil transition in polypeptide chains. I. Poly-(L-glutamic acid)

The helix-coil transitions for poly(L -glutamic acid) (PGA) in 0.2M NaCl and in its mixture with dioxane were studied by the methods of spectropolarimetry, viscometry, and potentiometric titration at different temperatures from 8 to 50°C. The enthalpy and entropy differences between the helical and coillike states of uncharged PGA molecules were determined from the curves of potentiometric titration. The temperature dependence of the cooperativity parameter σ was determined by two methods: from the sharpness of transition and from the dependence of the intrinsic viscosity on the helical content in the transition region. In 0.2MNaCl, σ= (2.5 ± 0.5) × 10^?3 and practically does not depend on temperature, i.e., the cooperativity of the helix-coil transition is connected mainly with the entropy decrease in initiating helical regions (ΔS_i ≈ ?12 is mole of helical regions). On the contrary, initiation of a helical region in the water-organic solvent mixture is accompanied by a considerable enthalpy increase.     

Renaturation of triple helical polysaccharide lentinan in water-diluted dimethylsulfoxide solution

Triple helical lentinan, a β-(1→3)-d-glucan from Lentinus edodes, was denatured in dimethylsulfoxide (DMSO) into single random coils. The randomly coiled lentinan/DMSO solutions were diluted with pure water to w_H (the weight fraction of water in the mixed solvent) of 95%, and their intrinsic viscosity [η], weight-average molecular weight M_w, radius of gyration R_g, and hydrodynamic radius R_h were investigated at 25 °C after over 5-day storage. The [η] and M_w values, especially the conformation parameter ρ (≡R_g/R_h), of the renatured lentinan were close to those of the originally extracted one, suggesting that random lentinan chains in DMSO were reassembled into triple helical structures. Moreover, the renatured lentinan in 95% water/5% DMSO solution exhibited a unique behavior of triple helical glucans that shear modulus G′ decreased sharply at temperature from 8.4 °C to 13.3 °C with increasing temperature, which was ascribed to the intramolecular conformation transition from ordered triple helical I to disordered triple helical II. The AFM images gave was suggested intuitively evidence that the renatured lentinan mainly existed as rod-like chains, supporting that formation of triple helical structure. The optimal lentinan concentration for triple helical configuration formation was estimated to be over 0.04%. The time dependence of R_h and UV absorption of the water-diluted lentinan/DMSO solution with an indicator of azo dye of Congo red suggested that renaturation of triple helix was a very rapid process. Moreover, the blue-shift of UV-vis absorption spectra suggested that the dye molecules of Congo red were assembled into supramolecular structure in the hydrophobic cavity of the renatured triple helical lentinan. All the results showed that the triple helical structure formed once the randomly coiled lentinan/DMSO was diluted to the final water content of 95%.     

Interactions of adenosine triphosphate with snake hemoglobins. Studies in Liophis miliaris,Boa constrictor and Bothrops alternatus

The hemoglobins of three snake species: Liophis miliaris, Bothrops alternatus and Boa constrictor present a single ATP binding site per tetramer. The ATP association constant values for the deoxyhemoglobins at pH 7.5 were about K_D ≅ 10⁶ M⁻¹ (10⁷ M⁻¹ for B. contrictor), three to four orders of magnitude higher than the respective values for oxyhemoglobin of about K_O ≅ 10² M⁻¹. The deoxyhemoglobin constant values markedly decrease as a function of pH, becoming, at pH 8.5, about K_D ≅ 10³ M⁻¹ whereas for the oxyhemoglobin the constants remain of about the same, K_O ≅ 10² M⁻¹, at the pH range studied. The high ATP binding affinity constants, compared to those of human hemoglobin A, were explained from a molecular structural standpoint, considering L. miliaris hemoglobin, whose complete primary sequence is known. Two distinct amino acid residue differences were found in the β-chain, one being Trp (NA3) (more hydrophobic) in the snake hemoglobin which substitutes the Leu (NA3) in human hemoglobin, and the second being Val 101 β (G3) instead of Glu 101 β (G3). The substitutions could provide an un-neutralized, positively charged, residue Lys-104β and, taking into account its high pK value, the pH dependence of ATP binding affinity for the snake hemoglobin would originate from pH-dependent ionization of phosphate groups of the allosteric effector. The physiological implications of the high ATP binding constant, as well as the possible protective role of the nucleotide binding against the effect of high environmental temperatures on the oxygen dissociation curves, are discussed.     

Analysis of low angle light scattering results from T7 DNA

Selected light scattering data, obtained in earlier studies on T7 DNA in 0.195 M Na⁺, are analyzed by comparison with calculations from the theory of wormlike coils, both with and without excluded volume effects. The results confirm the conclusion from an earlier criticism, that linear extrapolations of data from the 10° to 20° angular range give incorrect values for the limiting molecular weight, M_T, and for the limiting root-mean-square radius, R_T. Further, it is shown that the excluded volume parameter, ?, must be used to provide a proper fit of calculated curves to experimental data. The revised analysis gives the following parameters for T7 DNA: M_T = 25.5 × 10⁶ ;R_T= 587 nm; ? = 0.08; and the statistical segment length, 1/λ = 120 nm. These parameters agree well with other values in the literature. The method of analysis, therefore, provides reliable results from light scattering data on high-molecular-weight, native DNA.     

Mathematical Model of Thermal Destruction of Bacillus stearothermophilus Spores

The experimental survival curves of Bacillus stearothermophilus spores in aqueous suspension, for six constant temperatures ranging from 105 to 130°C, displayed an initial shoulder before a linear decline. To interpret these observations, we supposed that, before the heat treatment, the designated spore suspension contained a countable and mortal N₀ population of activated spores and an M₀ population of dormant spores which remained masked during spore counting and had to be activated before being destroyed by heat. We also hypothesized that the mechanisms of both activation and destruction are, at constant temperature, ruled by first-order kinetics, with velocity constants k_A and k_D, respectively. Mathematical analysis showed that this model could represent not only our experimental survival curves, but also all other shapes (linear and biphasic) of survival curves found in the literature; also, there is an inherent symmetry in the model formulation between the activation and destruction reactions, and we showed that the dormancy rate (τ = M₀/N₀) is the only parameter which permits a distinction between the two reactions. By applying the model to our experimental data and considering that the dormancy rate is not dependent on the treatment temperature, we showed that, for the studied suspension, the limiting reaction was the activation reaction.     

Reversed-phase thin-layer chromatography in the parametrization of lipophilicity of some series of arylaliphatic acids

In the series of arylacetic acid and β-aryl-n-butyric acids, chromatography was carried out on a thin layer of silica gel impregnated with silicone oil, with 50% acetone as mobile phase. A separation mechanism in this system was evaluated using relationships between R_M values and the concentration of the lipophilic solvent in the silica-gel layer. It was found, that both partition and adsorption mechanisms participate, and that the adsorption effect increases with decreasing lipophilicity of the acids. The dichotomy of the mechanism manifests itself in the non-linear course of the relationships between R_M values and π parameters, or fragmental constants f derived from the partition system n-octanol—water. Such relationships can be expressed by a single quadratic dependence between lipophilic parameters and R_M values, or by two separate linear expressions with different slopes for different regions of substituent lipophilicities. The linear dependence between R_M and π at the lower range of lipophilicity is most probably made possible by significant linear dependence between π parameters and molecular surface areas of the substituents.     

Molecular weight separation of proteins and peptides with a new high-pressure liquid chromatography column.

A new high-pressure liquid molecular weight chromatography column was evaluated for its ability to separate proteins and peptides. The column was able to give a linear separation of compounds between 5,000–700,000 M_r. Chromatography of posterior pituitary extracts, tumor-associated fetal antigens, and estrogen receptors demonstrated the ability of the column to separate biological samples.     

A complete hyaluronan hydrodynamic characterization using a size exclusion chromatography-triple detector array system during in vitro enzymatic degradation

Size exclusion chromatography coupled with triple detection (online laser light scattering, refractometry, and viscosimetry) (SEC-TDA) was applied for the study of hyaluronan (HA) fragments produced during hydrolysis catalyzed by bovine testicular hyaluronidase (BTH). The main advantage this approach provides is the complete hydrodynamic characterization without requiring further experiments. HA was hydrolyzed using several BTH amounts and for increasing incubation times. Fragments were characterized in terms of weight and number average molecular weights (M_w and M_n, respectively), polydispersity index (M_w/M_n), hydrodynamic radius (R_h), and intrinsic viscosity ([η]). The Mark-Houwink-Sakurada (MHS) curves (log [η] versus log M_w) were then derived directly. Fragments covering a whole range of M_w (10-900 kDa) and size (R_h = 4-81 nm) and presenting a rather narrow distribution of molar masses (M_w/M_n = 1.6-1.7) were produced. From the MHS curves, HA conformation resulted in a change from a random coil toward a rigid rod structure while decreasing the M_w. HA enzymatic hydrolysis in the presence of a BTH inhibitor was also monitored, revealing that inhibition profiles are affected by ionic strength. Finally, a comparison of the kinetic data derived from SEC-TDA with the data from rheological measurements suggested different strengths of the two methods in the determination of the depolymerization rate depending on the hydrolysis conditions.     

Electron microscopical and biochemical studies on the oligomeric states of human erythrocyte tripeptidyl peptidase—II

The oligomeric structure of native and low ionic strength-dissociated human erythrocyte tripeptidyl peptidase II (TPP-II) have been investigated by PAGE (without SDS) and transmission electron microscopy. The native TPP-II complex has a very high molecular mass (>10⁶) and has a ‘double bow’ or short double helical conformation, 50 nm in length and 20 nm wide. This complex dissociates into a range of smaller oligomers upon dialysis against a dilute Tris-HCl buffer, with an M_r 270,000 dimer having some tendency to predominate during short (1 to 4 h) dialysis times. Small 2-dimensional arrays of the dissociation products have been produced using the negative staining-carbon film technique when polythylene glycol (M_r 10,000) is included, within which the M_r 270,000 dimer of TPP-II appears to be the repeating unit.     

Molecular weight estimation of proteins by electrophoresis in linear polyacrylamide gradient gels in the absence of denaturing agents.

It was shown that in linear polyacrylamide gradient gels migration distance of a given protein increases as a function of the square root of the time of electrophoresis. The linearity between these two parameters is demonstrated by the statistical analysis of experimental data obtained with proteins of different shapes and a wide range of electrophoretic mobility. The slopes of the regression lines calculated by this method can be utilized to determine the molecular weight of a nondenatured protein. In fact, there is a linear relationship between the log of the molecular weights and the log of the slopes for proteins with M_rs between 20,000 and 950,000.     

Preparation and characterization of molecular weight fractions of glycosaminoglycan from sea cucumber Thelenata ananas using free radical depolymerization

A glycosaminoglycan from sea cucumber Thelenata anana (THG) was isolated as a polymer of molecular weight of around 70 kDa. Its low molecular weight derivatives were first prepared by free radical depolymerization with hydrogen peroxide in the presence of copper(II) ion. The parameters of the process were investigated by a high-performance gel permeation chromatography. Analyses of chemical composition and molecular weight distribution indicated that the fragmentation of the main-chain of THG occurred randomly, obeyed pseudo first-order kinetics, and produced species with rather narrow and unimodal distribution of molar mass. The characterization of different molecular weight fractions was investigated by using viscometry and atomic force microscopy (AFM). Analysis of molecular weight and intrinsic viscosity in terms of the known theories for unperturbed wormlike cylinder yielded 1201 ± 110 nm⁻¹, 15.3 ± 1.5 nm, and 1.5 ± 0.3 nm for molar mass per unit contour length M_L, persistence length q, and diameter d, respectively. The M_L and d values were approximately consistent with those observed by AFM. The present data suggest that THG may dissolve in 0.1 M aqueous NaCl as single-stranded helical chains.     

DNA binding of dinuclear iron(II) metallosupramolecular cylinders. DNA unwinding and sequence preference

[Fe₂L₃]⁴⁺ (L = C₂₅H₂₀N₄) is a synthetic tetracationic supramolecular cylinder (with a triple helical architecture) that targets the major groove of DNA and can bind to DNA Y-shaped junctions. To explore the DNA-binding mode of [Fe₂L₃]⁴⁺, we examine herein the interactions of pure enantiomers of this cylinder with DNA by biochemical and molecular biology methods. The results have revealed that, in addition to the previously reported bending of DNA, the enantiomers extensively unwind DNA, with the M enantiomer being the more efficient at unwinding, and exhibit preferential binding to regular alternating purine–pyrimidine sequences, with the M enantiomer showing a greater preference. Also, interestingly, the DNA binding of bulky cylinders [Fe₂(L-CF₃)₃]⁴⁺ and [Fe₂(L-Ph)₃]⁴⁺ results in no DNA unwinding and also no sequence preference of their DNA binding was observed. The observation of sequence-preference in the binding of these supramolecular cylinders suggests that a concept based on the use of metallosupramolecular cylinders might result in molecular designs that recognize the genetic code in a sequence-dependent manner with a potential ability to affect the processing of the genetic code.     

Liver protein phosphatases: studies of the presumptive native forms of phosphorylase phosphatase activity in liver extracts and their dissociation to a catalytic subunit of Mr 35,000.

Several aspects of the properties of phosphorylase phosphatase in crude rat liver extracts were investigated. Treatment of tissue extracts with either trypsin, ethanol, or urea was found to dissociate phosphorylase phosphatase activity to a form of M_r 35,000. The M_r 35,000 enzyme form was derived from three native enzyme forms. The major cytosolic form of phosphorylase phosphatase had a molecular weight of 260,000 as determined by gel filtration and was dissociated to a M_r 35,000 form by treatment with either ethanol or urea. Treatment of the M_r 260,000 form with trypsin led to its conversion to M_r 225,000 and a M_r 35,000 form. A minor cytosolic form of M_r 200,000 was also present. This minor activity was latent until activated by trypsin treatment and was converted to a M_r 35,000 form by such treatment. The third form was found to chromatograph as a form of molecular weight greater than 500,000 on gel filtration and, like the M_r 200,000 form, was only detected after activation with trypsin. Subsequent to this treatment, it too behaved as a M_r 35,000 enzyme. Although a single major enzyme form was present in the cytosol, multiple molecular weight forms could be generated in crude extracts simply by the use of vigorous mechanical homogenization procedures. This suggested that artifactual forms of enzyme may readily be produced, possibly by proteolytic cleavage of the native enzyme.     

Studies of DNA dumbbells. V. A DNA triplex formed between a 28 base-pair DNA dumbbell substrate and a 16 base linear single strand

CD spectra and melting curves were collected for a 28 base-pair DNA fragment in the form of a DNA dumbbell (linked on both ends by T₄ single-strand loops) and the same DNA sequence in the linear form (without end loops). The central 16 base pairs (bp) of the 28-bp duplex region is the poly(pu) sequence: 5′-AGGAAGGAGGAAAGAG-3′. Mixtures of the dumbbell and linear DNAs with the 16-base single-strand sequence 5′-TCCTTCCTCCTTTCTC-3′ were also prepared and studied. At 22°C, CD measurements of the mixtures in 950 mM NaCl, 10 mM sodium acetate, 1 mM EDTA, pH 5.5, at a duplex concentration of 1.8 μM, provided evidence for triplex formation. Spectroscopic features of the triplexes formed with either a dumbbell or linear substrate were quite similar. Melting curves of the duplex molecules alone and in mixtures with the third strand were collected as a function of duplex concentration from 0.16 to 2.15 μM. Melting curves of the dumbbell alone and mixtures with the third strand were entirely independent of DNA concentration. In contrast, melting curves of the linear duplex alone or mixed with the third strand were concentration dependent. At identical duplex concentrations, the dumbbell alone melts ～20°C higher than the linear duplex. The curve of the linear duplex displayed a significant pretransition probably due to end fraying. On melting curves of mixtures of the dumbbell or linear duplex with the third strand, a low temperature transition with much lower relative hyperchromicity change (～ 5%) was observed. This transition was attributed to the melting of a new molecular species, e.g., the triplex formed between the duplex and single-strand DNA molecules. In the case of the dumbbell/single-strand mixture, these melting transitions of the triplex and the dumbbell were entirely resolvable. In contrast, the melting transitions of the linear duplex and the triplex overlapped, thereby preventing their clear distinction. To analyze the data, a three-state equilibrium model is presented. The analysis utilizes differences in relative absorbance vs temperature curves of dumbbells (or linear molecules) alone and in mixtures with the third strand. From the model analysis a straightforward derivation of f_T(T), the fraction of triplex as a function of temperature, was obtained. Analysis of f_T vs temperature curves, in effect melting curves of the triplexes, provided evaluation of thermodynamic parameters of the melting transition. For the triplex formed with the dumbbell substrate, the total transition enthalpy is ΔH_T = 118.4 ± 12.8 kcal/mol (7.4 ± 0.8 kcal/mol per triplet unit) and the total transition entropy is ΔS_T = 344 ± 36.8 cal/K · mol (eu) (21.5 ± 2.3 eu per triple unit). The transition curves of the triplex formed with the linear duplex substrate displayed two distinct regions. A broad pretransition region from f_T = 0 to 0.55 and a higher, sharper transition above f_T = 0.55. The transition parameters derived from the lower temperature region of the curve are ΔH′_T = 44.8 ± 9.6 kcal/mol and ΔS′_T = 112 ± 33.6 eu (or ΔH′ = 2.8 ± 0.6 kcal/mol and ΔS′ = 7.0 ± 2.1 eu per triplet). These values are probably too small to correspond to actual melting of the triplex but instead likely reveal effects of end fraying of the duplex substrate on triplex stability. Transition parameters of the upper transition are ΔH′_T = 128.0 ± 2.3 kcal/mol and ΔS′_T = 379.2 ± 6.4 eu (ΔH′ = 8.0 ± 0.2 kcal/mol and ΔS′ = 23.7 ± 0.4 eu per triplet) in good agreement (within experimental error) with the transition parameters of the triplex formed with the dumbbell substrate. Supposing this upper transition reflects actual dissociation of the third strand from the linear duplex substrate this triplex is comparable in thermodynamic stability to the triplex formed with a dumbbell substrate. Even so, the biphasic melting character of the linear triplex obscures the whole analysis, casting doubt on its absolute reliability. Apparently triplexes formed with a dumbbell substrate offer technical advantages over triplexes formed from linear or hairpin duplex substrates for studies of DNA triplex stability. © 1993 John Wiley & Sons, Inc.     

On the interaction of caffeine with nucleic acids: III. 1NMR studies of caffeine-5'-adenosine monophosphate and caffeine-poly(riboadenylate) interactions

1) The self-association of both caffeine (Cf) and 5'-adenosine monophosphate (AMP) in aqueous solution has been reinvestigated by ¹H NMR. The self-association process is characterized by an isodesmic model. The apparent self-association constants of the vertical stacking process are K_Cf= (10.6 ± 1.0) M^?1 and k_amp = (1.67 ± 0.17) M^?1. The arrangement of the monomeric units in the stacked aggregates is discussed in terms of isoshielding curves theoretically calculated by Giessner-Prettre and Pullman. Models are proposed which are consistent with these and further previous NMR data. 2) The interaction of Cf and AMP has been studied by ^?1 H NMR. The apparent association constant of the complex Cf-AMP is K_C-A = (7.3 ± 1.2) M^?1.Two models of the mutual arrangement of AMP and Cf in the complex are proposed on the basis of the calculated isoshielding curves considering both ring current and local atomic diamagnetic anisotropy effects. 3) The interaction of Cf and poly(riboadenylate), (rA)_n is indicated by a downfield shift of the H-8 line but an upfield shift of the H-2 line in the ¹H NMR spectra of (rA)_n. The concentration dependence of the ¹H NMR shifts of both Cf and (rA)_n can be explained by the existence of two binding mechanisms. We suggest (i) partial insertion of Cf between adjacent base residues of ordered single-stranded regions of (rA)_n and (ii) outside binding of Cf in form of monomeric Cf as well as of self-associated aggregates. The complex geometry of insertion proposed on the basis of the calculated isoshielding curves is characterized by a stronger overlapping of the Cf ring and the H-2 proton of (rA)_n as compared to the H-8 proton.     

A novel water-soluble β-(1→6)-d-glucan isolated from the fruit bodies of Bulgaria inquinans (Fries)

A low molecular-weight polysaccharide named BIWP2 was purified from the fruit bodies of Bulgaria Inquinans (Fries) via hot-water extraction, followed by freeze-thawing and gel filtration chromatography on Sephadex G-75. Monosaccharide composition analysis revealed that BIWP2 contained exclusively glucose. High performance size exclusion chromatography (HPSEC) showed that it was a homogeneous polysaccharide fraction. Its molecular weight was estimated to be 2.6 KD and the polydispersity index (M_w/M_n) was calculated to be 1.4. Periodate oxidation, methylation, and NMR analyses indicated that BIWP2 was a linear β-(1→6)-d-glucan without side chains. This is the first time to report a linear β-(1→6)-d-glucan with low molecular weight in non-lichenized ascomycete.     

The molecular transformation of rabbit testis proacrosin into acrosin.

Partially purified rabbit testis proacrosin formed only one acrosin of 73,000 ± 3000 apparent molecular weight (M_r) during the early phase of “autoactivation” at pH 8. Complete “autoactivation” then converted this acrosin to a 38,000 ± 3000 M_r, acrosin. These results suggest the existence of a proacrosin dimer (73,000) or a dimer of proacrosin and an acrosomal membrane protein which were converted first to an acrosin dimer (73,000 M_r) or to an acrosin-membrane protein dimer and then to the acrosin monomer (38,000). The formation of a 73,000 ± 3000 M_r acrosin from a 73,000 ± 3000 proacrosin is explainable by assuming that either a small activation pertide(s) is released or none at all.