Protein Brownian Rotation at the Glass Transition Temperature of a Freeze-Concentrated Buffer Probed by Superparamagnetic Nanoparticles

For applications from food science to the freeze-thawing of proteins it is important to understand the often complex freezing behavior of solutions of biomolecules. Here we use a magnetic method to monitor the Brownian rotation of a quasi-spherical cage-shaped protein, apoferritin, approaching the glass transition T_g in a freeze-concentrated buffer (Tris-HCl). The protein incorporates a synthetic magnetic nanoparticle (Co-doped Fe₃O₄ (magnetite)). We use the magnetic signal from the nanoparticles to monitor the protein orientation. As T decreases toward T_g of the buffer solution the protein’s rotational relaxation time increases exponentially, taking values in the range from a few seconds up to thousands of seconds, i.e., orders of magnitude greater than usually accessed, e.g., by NMR. The longest relaxation times measured correspond to estimated viscosities >2 MPa s. As well as being a means to study low-temperature, high-viscosity environments, our method provides evidence that, for the cooling protocol used, the following applies: 1), the concentration of the freeze-concentrated buffer at T_g is independent of its initial concentration; 2), little protein adsorption takes place at the interface between ice and buffer; and 3), the protein is free to rotate even at temperatures as low as 207 K.     

Adverse effect of cake collapse on the functional integrity of freeze-dried bull spermatozoa

Under optimal freeze-drying conditions, solutions exhibit a cake-like porous structure. However, if the solution temperature is higher than the glass transition temperature of the maximally freeze-concentrated phase (T_g′) during drying phase, the glassy matrix undergoes viscous flow, resulting in cake collapse. The purpose of the present study was to investigate the effect of cake collapse on the integrity of freeze-dried bull spermatozoa. In a preliminary experiment, factors affecting the T_g′ of conventional EGTA buffer (consisting of Tris–HCl, EGTA and NaCl) were investigated in order to establish the main experimental protocol because EGTA buffer T_g′ was too low (−45.0 °C) to suppress collapse. Modification of the EGTA buffer composition by complete removal of NaCl and addition of trehalose (mEGTA buffer) resulted in an increase of T_g′ up to −27.7 °C. In the main experiment, blastocyst yields after ooplasmic injection of freeze-dried sperm preserved in collapsed cakes (drying temperature: 0 or −15 °C) were significantly lower than those of sperm preserved in non-collapsed cake (drying temperature: −30 °C). In conclusion, freeze-dried cake collapse may be undesirable for maintaining sperm functions to support embryonic development, and can be inhibited by controlling both T_g′ of freeze-drying buffer and temperature during the drying phase.     

The State of Water in Muscle Tissue as Determined by Proton Nuclear Magnetic Resonance

The nuclear magnetic resonance (NMR) of water protons in live and glycerinated muscle, suspensions of glycerinated myofibrils, and solutions of several muscle proteins has been studied. T₁ and T₂, measured on partially hydrated proteins by pulsed spin-echo techniques, decreased as the ratio of water to protein decreased, showing that the water which is tightly bound by the protein has short relaxation times. In live muscle fibers the pulse techniques showed that, after either a 180 or a 90° pulse, the relaxation of the magnetization is described by a single exponential. This is direct evidence that a fast exchange of protons occurs among the phases of the intracellular water. The data can be fitted with a model in which the bulk of the muscle water is in a phase which has properties similar to those of a dilute salt solution, while less than 4-5% of the total water is bound to the protein surface and has short relaxation times. Measurements of T₁ and T₂ in protein solutions showed that no change in the proton relaxation times occurred when heavy meromyosin was bound to actin, when myofibrils were contracted with adenosine triphosphate (ATP), or when globular actin was polymerized.     

Relaxation Kinetic Study of Eudragit? NM30D Film Based on Complex Modulus Formalism

This study is aimed at resolving and characterizing the primary (α) and secondary relaxations (β) in Eudragit® NM30D film based on apparent activation energies derived from complex modulus formalism using dielectric analysis (DEA). The glass transition (T_g) of the film was determined using differential scanning calorimetry (DSC). The α relaxation corresponding to T_g and the β relaxations occurring below T_g were probed using DEA. The occurrence of α and β relaxations in Eudragit® NM30D film was elucidated using the complex modulus of the dielectric response employing loss modulus and permittivity data. Activation energies of these relaxations and the fundamental frequency so determined support the assignment of the relaxation pattern in the Eudragit® NM30D film. DEA methodology of the complex modulus formalism is a useful tool for differentiating the α and β relaxation kinetics in Eudragits® not easily studied using traditional thermal methods such as DSC. The kinetics associated with α and β relaxations so determined will provide formulation design support for solid orals that incorporate Eudragit® polymers. As mobility changes can affect stability and diffusion, the dipolar α and β relaxations revealed through DEA analysis may enable a better correlation to functionality of Eudragit® based pharmaceutical dosage forms.KEY WORDS: activation energy, loss modulus, permittivity, primary relaxation, secondary relaxation     

Design Maps for the Hyperthermic Treatment of Tumors with Superparamagnetic Nanoparticles

A plethora of magnetic nanoparticles has been developed and investigated under different alternating magnetic fields (AMF) for the hyperthermic treatment of malignant tissues. Yet, clinical applications of magnetic hyperthermia are sporadic, mostly due to the low energy conversion efficiency of the metallic nanoparticles and the high tissue concentrations required. Here, we study the hyperthermic performance of commercially available formulations of superparamagnetic iron oxide nanoparticles (SPIOs), with core diameter of 5, 7 and 14 nm, in terms of absolute temperature increase ΔT and specific absorption rate (SAR). These nanoparticles are operated under a broad range of AMF conditions, with frequency f varying between 0.2 and 30 MHz; field strength H ranging from 4 to 10 kA m⁻¹; and concentration c_MNP varying from 0.02 to 3.5 mg ml⁻¹. At high frequency field (∼30 MHz), non specific heating dominates and ΔT correlates with the electrical conductivity of the medium. At low frequency field (<1 MHz), non specific heating is negligible and the relaxation of the SPIO within the AMF is the sole energy source. We show that the ΔT of the medium grows linearly with c_MNP, whereas the SAR_MNP of the magnetic nanoparticles is independent of c_MNP and varies linearly with f and H². Using a computational model for heat transport in a biological tissue, the minimum requirements for local hyperthermia (T_tissue >42°C) and thermal ablation (T_tissue >50°C) are derived in terms of c_MNP, operating AMF conditions and blood perfusion. The resulting maps can be used to rationally design hyperthermic treatments and identifying the proper route of administration – systemic versus intratumor injection – depending on the magnetic and biodistribution properties of the nanoparticles.     

Amide Proton Spin-Lattice Relaxation in Polypeptides: A Field-Dependence Study of the Proton and Nitrogen Dipolar Interactions in Alumichrome

The proton nuclear magnetic resonance (NMR) spin-lattice relaxation of all six amides of deferriferrichrome and of various alumichromes dissolved in hexadeutero-dimethylsulfoxide have been investigated at 100, 220, and 360 MHz. We find that, depending on the type of residue (glycyl or ornithyl), the amide proton relaxation rates are rather uniform in the metal-free cyclohexapeptide. In contrast, the ¹H spinlattice relaxation times (T₁'s) are distinct in the Al³⁺-coordination derivative. Similar patterns are observed in a number of isomorphic alumichrome homologues that differ in single-site residue substitutions, indicating that the spin-lattice relaxation rate is mainly determined by dipole-dipole interactions within a rigid molecular framework rather than by the specific primary structures. Analysis of the data in terms of ¹H—¹H distances (r) calculated from X-ray coordinates yields a satisfactory linear fit between T₁^-1 and Σr^-6 at the three magnetic fields. Considering the very sensitive r-dependence of T₁, the agreement gives confidence, at a quantitative level, both on the fitness of the crystallographic model to represent the alumichromes' solution conformation and on the validity of assuming isotropic rotational motion for the globular metallopeptides. An extra contribution to the amide proton T₁^-1 is proposed to mainly originate from the ¹H-¹⁴N dipolar interaction: this was supported by comparison with measurements on an ¹⁵N-enriched peptide. The nitrogen dipolar contribution to the peptide proton relaxation is discussed in the context of {¹H}—¹H nuclear Overhauser enhancement (NOE) studies because, especially at high fields, it can be dominant in determining the amide proton relaxation rates and hence result in a decreased effectiveness for the ¹H—¹H dipolar mechanism to cause NOE's. From the slope and intersect values of T₁^-1 vs. Σr^-6 linear plots, a number of independent estimates of τ_r, the rotational correlation time, were derived. These and the field-dependence of the T₁'s yield a best estimate <τ_r> ≈ 0.37 ns, in good agreement with 0.38 ns [unk] <τ_r> [unk] 0.41 ns, previously determined from ¹³C and ¹⁵N spin-lattice relaxation data.     

300 MHz proton NMR study of the differentiation of diploid human epidermal keratinocytes

The nuclear magnetic resonance spin-lattice (T₁) and spin-spin (T₂) relaxation times are closely related to the molecular motions of the molecules in a liquid sample. T₁ and T₂ of human epidermal cells were measured at 300 MHz as functions of harvesting methods (i.e., scraping vs trypsinization) and age in culture. It was found that T₁ and T₂ values have smaller variances when the cell is harvested by trypsinization rather than scraping. The correlation coefficients for both T₁ and T₂, obtained from cells harvested by trypsinization, are much higher than those obtained from cells harvested by scraping. More importantly, this is the first report to monitor in vitro aging through relaxation times measurement. There is a significant increase in the values of T₁ and T₂ from the third to seventh passages. Human keratinocytes slowed down and even ceased to grow the seventh passage. Therefore, the cellular water molecules of human keratinocytes have higher mobility in a more differentiated state. The factors contributing to the change in relaxation times as cells progress toward senescence are discussed.     

Simultaneous Determination of Glass Transition Temperatures of Several Polymers

Aims

A simple and easy optical method is proposed for the determination of glass transition temperature (T_g) of polymers.

Methods & Results

T_g was determined using the technique of microsphere imaging to monitor the variation of the refractive index of polymer microsphere as a function of temperature. It was demonstrated that the method can eliminate most thermal lag and has sensitivity about six fold higher than the conventional method in T_g determination. So the determined T_g is more accurate and varies less with cooling/heating rate than that obtained by conventional methods. The most attractive character of the method is that it can simultaneously determine the T_g of several polymers in a single experiment, so it can greatly save experimental time and heating energy.

Conclusion

The method is not only applicable for polymer microspheres, but also for the materials with arbitrary shapes. Therefore, it is expected to be broadly applied to different fundamental researches and practical applications of polymers.     

Ribonucleic acids from barley leaves

1. The total RNA and the RNA present in 27000g pellet (probably composed of chloroplasts, nuclei and mitochondria) and in 27000g supernatant (probably composed of microsomes and soluble proteins) fractions (separated by centrifugation at 27000g of a leaf homogenate prepared in 0·5m-sucrose–0·02m-tris–HCl, pH7·6) of barley leaves were extracted by phenol–sodium lauryl sulphate and their elution profiles on Sephadex G-200 and on ECTEOLA-cellulose anion-exchanger were examined and their nucleotide compositions and the melting curves were determined. 2. The pellet and the supernatant fractions contained respectively about 55% and 20% of the total RNA, whereas 25% of the total RNA was lost during homogenization of the leaf tissue with sucrose–buffer. 3. The total RNA or the RNA from pellet or supernatant fractions, which by its behaviour on Sephadex G-200 columns was found to be predominantly of high molecular weight (i.e. of ribosomal origin), produced about 13 peaks on ECTEOLA-cellulose columns. The RNA species in the pellet and supernatant fractions probably resembled each other in molecular size or secondary structure or both. However, they were present in relatively different amounts in these fractions. 4. The T_m (i.e. the temperature at which 50% of the maximal increase in extinction had occurred) of total RNA and of RNA from pellet fraction was 64·5° whereas T_m of RNA from the supernatant fraction was 73°. The total RNA and the RNA from pellet fraction also resembled each other in nucleotide composition, and the RNA from the supernatant fraction in accordance with its high T_m had a high GMP+CMP content.     

Molecular mobilities in chromaffin granules magnetic field dependence of proton T1 relaxation times

The magnetic field dependence of the NMR spin-lattice relaxation time of water protons in intact bovine chromaffin vesicles has been studied over the range 1.00–23.49 kG. The T₁ relaxation time shows a dispersion a t field values near 20 kG. The observed proton resonance arises mainly from solvent protons (¹H₂O), but the relaxation rate, which is a weighted average over all sites with which the solvent protons rapidly exchange (i.e., NH and OH protons), is dominated by exchangeable protons in the most slowly moving soluble component. The field dependence of the T₁ dispersion demonstrates the existence of a site of exchangeable protons for which τ_r = 1.9±0.5 ns at 3°C. This site is assigned to ATP and cationic groups to which its phosphate esters are complexed, since previously measured correlation times of epinephrine and the chromogranin backbone are nearly an order of magnitude too short to explain the T₁ dispersion. Quantitative estimates of the relative numbers of exchangeable protons on the different soluble components support this interpretation. The temperature dependence of T₁ of the peak due to exchangeable protons has also been measured over a temperature range ?3 to 25°C. T₁ lengthens by about 30% over this range and exhibits no discontinuous behavior, as would be expected if a gel transition or structural alterations in the storage complex occurred. T₁ lengthens by less than 10% in chromaffin granule pastes that have been maintained at 25°C for 24 h, indicating considerable thermal stability in the storage complex. Possible effects on the solvent T₁ due to paramagnetic ions have been considered with the conclusion that they are probably negligible or of minor significance.     

Thermal Properties of Freeze-Concentrated Sugar-Phosphate Solutions

In order to understand the effect of phosphate salts on the freeze-concentrated glass-like transition temperature (T _g′) of aqueous sugar solutions, two types of sugar (glucose and maltose) and five types of phosphate salts (Na₃PO₄, Na₄P₂O₇, Na₅P₃O₁₀, K₃PO₄, and K₄P₂O₇) were employed, and the thermal properties of various sugar-phosphate aqueous systems were investigated using differential scanning calorimetry. The T _g′ of glucose increased with increasing sodium phosphates up to a certain phosphate ratio, decreasing thereafter. The maximum T _g′ value was slightly higher in the order of Na₃PO₄ > Na₄P₂O₇ ≥ Na₅P₃O₁₀. Maltose-sodium phosphate also showed a similar trend as glucose-sodium phosphate samples. However, the degree of T _g′-rise of maltose systems was much less than that of glucose. It is thought that the T _g′ elevated by the molecular interaction between sugar and phosphate ions will be reduced by hydrated sodium ions. In comparisons between potassium phosphate and sodium phosphate, it was found that sugar-potassium phosphates showed the lower maximum T _g′ at a lower phosphate ratio than sugar-sodium phosphates. In addition, the T _g′ of potassium phosphates dropped sharply in comparison with sodium phosphates at the high phosphate ratio. These results suggest that potassium phosphates are lower T _g′ than sodium phosphates, and that potassium ion plays a better plasticizer than sodium ion. A certain amount of sodium phosphates (Na₃PO₄ and Na₄P₂O₇) caused devitrification. Potassium phosphates, however, did not show devitrification which can be explained by the fact that potassium ion can be dynamically restricted by sugar.     

A broad glass transition in hydrated proteins

We performed Raman and Brillouin scattering measurements to estimate glass transition temperature, T_g, of hydrated protein. The measurements reveal very broad glass transition in hydrated lysozyme with approximate T_g ∼ 180 ± 15 K. This result agrees with a broad range of T_g ∼ 160–200 K reported in literature for hydrated globular proteins and stresses the difference between behavior of hydrated biomolecules and simple glass-forming systems. Moreover, the main structural relaxation of the hydrated protein system that freezes at T_g ∼ 180 K remains unknown. We emphasize the difference between the “dynamic transition”, known as a sharp rise in mean-squared atomic displacement <r²> at temperatures around T_D ∼ 200–230 K, and the glass transition. They have different physical origin and should not be confused.     

The protein glass transition as measured by dielectric spectroscopy and differential scanning calorimetry

The glass transition and its related dynamics of myoglobin in water and in a water–glycerol mixture have been investigated by dielectric spectroscopy and differential scanning calorimetry (DSC). For all samples, the DSC measurements display a glass transition that extends over a large temperature range. Both the temperature of the transition and its broadness decrease rapidly with increasing amount of solvent in the system. The dielectric measurements show several dynamical processes, due to both protein and solvent relaxations, and in the case of pure water as solvent the main protein process (which most likely is due to conformational changes of the protein structure) exhibits a dynamic glass transition (i.e. reaches a relaxation time of 100 s) at about the same temperature as the calorimetric glass transition temperature T_g is found. This glass transition is most likely caused by the dynamic crossover and the associated vanishing of the α-relaxation of the main water relaxation, although it does not contribute to the calorimetric T_g. This is in contrast to myoglobin in water–glycerol, where the main solvent relaxation makes the strongest contribution to the calorimetric glass transition. For all samples it is clear that several proteins processes are involved in the calorimetric glass transition and the broadness of the transition depends on how much these different relaxations are separated in time.     

Glass transition temperatures of a ready to eat breakfast cereal formulation and its main components determined by DSC and DMTA

The effect of water content on the glass transition temperatures of a ready to eat cereal formulation was determined, as well as for its major components, oat flour, rice flour and an oat–rice flour blend, in the same ratio as they are present in the formulation. All samples were compression moulded at high temperature and were moisture conditioned in a 10–22% interval (dry basis). Glass transition temperatures (T_g) were measured by differential scanning calorimetry (DSC) and the main mechanical relaxation temperatures (T_α), measured by dynamic mechanical thermal analysis (DMTA). The relaxation temperatures taken at tan δ peaks, were found 20–30 °C larger than T_g. Besides the plasticizing effect of water adequately described by the Gordon–Taylor equation, no differences of T_g (and T_α) values between the major components were obtained at a constant moisture content. The T_g and T_α values of the RTE formulation were found to be about 30 °C lower than its components, a result which was attributed to the plasticizing effect of the minor components in the formulation (sugar and malt extract).     

The amorphous state of spray-dried maltodextrin: sub-sub-Tg enthalpy relaxation and impact of temperature and water annealing

The annealing behaviour of a spray-dried maltodextrin was investigated by differential scanning calorimetry. Special attention was paid to the effect of temperature and humidity on the annealing process. Comparison was also made with the glassy state of the same compound prepared by various cooling processes. The presence of a very pronounced sub-T_g peak upon ageing reveals the specificities of the glass and the complexity of the relaxation spectrum of the spray-dried material. This peak seems actually to correspond to a partial ergodicity recovery that may be attributed to onset of molecular mobility occurring below T_g. The position of the sub-T_g peak with regard to the conventional T_g was systematically studied. It clearly showed the difference between the effect of temperature and water plasticization on the relaxations occurring in the glassy state of materials prepared by spray-drying.     

Self-association of oxyhaemoglobin. A nuclear magnetic relaxation study in H2O/D2O solutions

The proton and deuterium longitudinal relaxation rates were Studied at room temperature up to the highest protein concentrations in oxyhaemoglobin solutions of different H₂O/D₂O composition. The deuterium relaxation rates followed the experimentally well known single linear dependence on protein concentration, the slopes being little influenced by solvent (D₂O/H₂O) composition. The proton ralaxation rates show two different liner dependences on haemoglobin concentration. The entire concentration range is described by two straight lines with the threshold concentration about 11 mM (in haem), The ratio of the slopes is 1.6 (high-to-low Hb-conc.). Only in the higher concentration range two T₁'s were observed if the solvent contained more than half of D₂O. The slow relaxation phase of protons has T₁'s similar to those measured in solutions with less than half of D₂O. The relaxation of the other phase was ten times faster. The ratio of the proton populations in these two phases was equal to 2 (slow-to-fast) and independent of protein concentration. The fast relaxing protons are attributed to water molecules encaged within two or more haemoglobin molecules which associate for times long enough on the PMR time-scale.     

P relaxation responses associated with n(2)/o(2) diffusion in soybean nodule cortical cells and excised cortical tissue

N₂-fixing Bradyrhizobium japonicum nodules and cortical tissue derived from these nodules were examined in vivo by ³¹P nuclear magnetic resonance (NMR) spectroscopy. Perfusion of the viable nodules and excised cortical tissue with O₂ followed by N₂ or Ar caused a loss of orthophosphate (Pi) resonance magnetization associated with the major portion of acidic Pi (δ 0.9 ppm, pH 5.5) residing in the cortical cells. Resumption of O₂ perfusion restored approximately 80% of the intensity of this peak. Detailed examination of the nuclear relaxation processes, spin-lattice relaxation time (T₁), and spin-spin relaxation time (T₂), under perfusion with N₂ or Ar as opposed to O₂, indicated that loss of signal was due to T₁ saturation of the acidic Pi signal under the rapid-pulsed NMR recycling conditions. In excised cortical tissue, Pi T₁, values derived from biexponential relaxation processes under perfusing O₂ were 59% 3.72 ± 0.93 s and 41% 0.2 ± 0.08 s, whereas under N₂ these values were 85% 7.07 ± 1.36 s and 15% 0.39 ± 0.07 s. The T₁ relaxation behavior of whole nodule vacuolar Pi showed the same trend, but the overall values were somewhat shorter. T₂ values for cortical tissue were also biexponential but were essentially the same under O₂ (38% 0.066 ± 0.01 s and 63% 0.41 ± 0.08 s) and N₂ (39% 0.07 ± 0.01 s and 61% 0.37 ± 0.01 s) perfusion. Soybean (Glycine max) root tissue as well as Pi solutions exhibited single exponential T₁ decay values that were not altered by changes in the perfusing gas. These data indicate that oxygen induces a change in the physical environment of phosphate in the cortical cell tissue. Although under certain conditions oxygen has been observed to act as a paramagnetic relaxation agent, model T₁ experiments demonstrate that O₂ does not significantly influence Pi relaxation in this manner. Alternatively, we suggest that an increase in solution viscosity brought on by the production of an occlusion glycoprotein (under O₂ perfusion) is responsible for the observed relaxation changes.     

Faster Metabolite 1H Transverse Relaxation in the Elder Human Brain

¹H magnetic resonance spectroscopy (MRS) is unique among imaging modalities because signals from several metabolites are measured during a single examination period. Each metabolite reflects a distinct intracellular process. Furthermore transverse (T₂) relaxation times probe the viability of the cell microenvironment, e.g., the viscosity of the cellular fluids, the microscopic susceptibility distribution within the cells, and the iron content. In this study, T₂s of brain metabolites were measured in the occipital lobe of eighteen young and fourteen elderly subjects at a field strength of 4 tesla. The T₂s of N-acetylaspartate, total creatine, and total choline were 23%, 16% and 10% shorter in elderly than in young subjects. The findings of this study suggest that noninvasive detection of T₂ provides useful biological information on changes in the cellular microenvironment that take place during aging.