The rate of reaction between cytochrome C peroxidase and hydrogen peroxide is not diffusion limited.

The apparent biomolecular rate constant for the cytochrome C peroxidase (EC 1.11.1.5)-hydrogen peroxide reaction has been measured as a function of temperature between 5 and 25 degree C at pH 4,5.5, and 7 and as a function of viscosity over a fifteen-fold range. From the independence of the rate constant on the viscosity, it is concluded that the reaction is not diffusion limited.     

Effect of protein dynamics upon reactions that occur in the heme pocket of horseradish peroxidase

Free base and Pd porphyrin derivatives of horseradish peroxidase show long-lived excited states that are quenched by the presence of the peroxidase inhibitor, benzhydroxamic acid. The relaxation times of the excited-state luminescence and the rates of the quenching reaction for these derivatives of peroxidase were monitored as a function of pH, temperature, and viscosity with the view of examining how protein dynamics affect the quenching reaction. As solvent viscosity increases, the rate decreases, but at the limit of very high viscosity (i.e., high glycerol or sugar glass) the quenching still occurs. A model is presented that is consistent with the known structure of the enzyme-inhibitor complex. It is considered that the inhibitor is held at an established position but that solvent-dependent and independent motions allow a limited diffusion of the two reactants. Since there is a steep dependence upon distance and orientation, the diffusion toward the favorable position for reaction enhances the reaction rate. The solvent viscosity dependent and independent effects were separated and analyzed. The importance of internal reaction dynamics is demonstrated in the observation that rigidity of solvent imposed by incorporating the protein into glass at room temperature allows the reaction to occur, while the reaction is inhibited at low temperature. The results emphasize that protein dynamics plays a role in determining reaction rates.     

Efficient synthesis of 5′-O-laurate of 1-β-d-arabinofuranosylcytosine via highly regioselective enzymatic acylation in binary solvent mixtures

Regioselective enzymatic acylations of 1-β-d-arabinofuranosylcytosine (ara-C) with vinyl laurate (VL) in binary organic solvents were explored for the preparation of 5′-O-laurate of ara-C. Among the nine kinds of enzymes, Novozym 435 showed the highest regioselectivity (>99.9%) towards the 5′-OH of ara-C. This lipase showed higher catalytic activity in hexane–pyridine than in other tested solvent mixtures. The most suitable VL to ara-C molar ratio, initial water activity, and reaction temperature were shown to be 15:1, 0.07, and 50 °C, respectively, under which the initial reaction rate and the maximum substrate conversion were as high as 84.0 mmol L^?1 h^?1 and 98.1%, respectively. The product of Novozym 435-catalyzed acylation was characterized by ¹³C NMR and confirmed to be 5′-O-laurate of ara-C.     

Acylation of Antioxidant of Bamboo Leaves with Fatty Acids by Lipase and the Acylated Derivatives’ Efficiency in the Inhibition of Acrylamide Formation in Fried Potato Crisps

This study selectively acylated the primary hydroxyl groups on flavonoids in antioxidant of bamboo leaves (AOB) using lauric acid with Candida antarctica lipase B in tert-amyl-alcohol. The separation and isolation of acylated derivatives were performed using silica gel column chromatography with a mixture of dichloromethane/diethyl ether/methanol as eluents. Both thin layer chromatography and high-performance liquid chromatography analyses confirmed the high efficiency of the isolation process with the purified orientin-6″-laurate, isoorientin-6″-laurate, vitexin-6″-laurate, and isovitexin-6″-laurate that were obtained. The addition of AOB and acylated AOB reduced acrylamide formation in fried potato crisps. Results showed that 0.05% AOB and 0.05% and 0.1% acylated AOB groups significantly (p < 0.05) reduced the content of acrylamide in potato crisps by 30.7%, 44.5%, and 46.9%, respectively.     

Stopped-flow ESR study on the reactivity of vitamin E, vitamin C and its lipophilic derivatives towards Fremy's salt in micellar systems

The reaction between Fremy's salt and alpha-tocopherol (VE), ascorbic acid (VC) and its lipophilic derivatives ascorbyl-6-caprylate (VC-8), 6-laurate (VC-12) and 6-palmitate (VC-16) were studied by stopped-flow ESR spectroscopy in cetyl trimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS) micelles, as a model reaction of these antioxidants with alkyl peroxy radicals in biological systems. The second order rate constants for the reaction of Fremy's salt with VE in CTAB and SDS micelles were found to be 7.9 x 10(3) and 2.2 M-1 s-1, respectively, with as high as a 3600-fold variation. Rate constants for VC, VC-8, VC-12 and VC-16 are 4.3, 35, 53 and 56 x 10(3) M-1 s-1 and 3.3, 2.7, 1.2 and 0.86 x 10(3) M-1 s-1 in CTAB and SDS micelles, respectively. The results demonstrate remarkable effects of the charge type of the micelles and the side-chain of the antioxidants on the antioxidation reactivity in the micelles. It reveals that the inter-micellar diffusion may be the rate-limiting step for antioxidation carried out in micelles.     

Synthesis and rheological properties of responsive thickeners based on polysaccharide architectures

New thermothickening copolymers were synthesized by grafting responsive poly(ethylene oxide-co-propylene oxide) [PEPO] onto three different polysaccharide backbones: carboxymethylcellulose [CMC], alginate [ALG], and carboxylated dextran [DEX]. The coupling reaction between carboxylic groups of biopolymers and the terminal amine of PEPO was activated at low temperature ( T < 10 degrees C) in water by using carbodiimide and N-hydroxysuccinimide. In these conditions it was shown that the formation of amide bonds strongly depends on the concentration of reactive groups, which is limited by the viscosity of the polymer sample. While a full conversion was obtained for the low molecular weight dextran, the efficiency of grafting remains low (between 30 to 40%) for CMC and alginate, which give a solution of high viscosity even at low concentration. When studied in the semidilute regime, all the copolymer solutions clearly exhibit thermothickening behavior with a large and reversible increase of viscosity upon heating. The association temperature and the gelation threshold were shown to depend on polymer concentration as it is expected from the phase diagram of PEPO precursor. Similarly, the influence of added salt on PEPO solubility in water has been used to control the self-assembling behavior of copolymer formulations. The relative comparison between the three copolymers reveals that the amplitude of the viscosity jump induced by heating mainly depends on the proportion of responsive material inside the macromolecular architecture rather than the dimensions of the main chain. The high increase of viscosity, which can reach several orders of magnitude between 20 degrees C and body temperature, clearly demonstrates the potentiality of these copolymers in biomedical applications like injectable gels for tissue engineering.     

The diffusion-controlled reaction kinetics of the binding of CO and O2 to myoglobin in glycerol-water mixtures of high viscosity

The kinetics of the reaction of myoglobin (Mb) with CO and O₂ have been studied as a function of temperature by flash photolysis in mixtures of glycerol and water of high viscosities. This was done in order to examine the importance of diffusion-controlled kinetics on protein-ligand reactions. The apparent activation enthalpies of the binding reaction show changes with temperature consistent with a change from chemical activation control of the reaction at higher temperatures to diffusion control at the lower temperatures and higher viscosities. The activation enthalpies for ligand binding in the diffusion-controlled temperature region are similar in value to the viscosity activation energies of the particular solvent mixture as might be expected for a diffusion-controlled reaction. Curve fitting of the rate-temperature data yields factors by which the diffusion-controlled reaction departs from that predicted for reaction between spherically symmetric, uniformly reactive molecules of equal radii. This factor is between 0.1 and 6, depending both upon the solvent mixture and the ligand. Various models for diffusion-controlled reaction with steric requirements are examined in order to rationalize these results. The existence of a linear correlation between ΔH3 and ΔS3 for the chemica activation-controlled portions of reaction yield isokinetic temperatures of 305 and 288 °K for the CO and O₂ reactions, respectively.     

Pilot plant scale extraction of alginates from Macrocystis pyrifera 4. Conversion of alginic acid to sodium alginate, drying and milling

The last three steps of the alginate production process were studied:conversion of alginic acid to sodium alginate, drying, and milling. Threemethods were used to follow the conversion reaction: measuring the pH (a) intheethanol-water liquid of the reaction mixture, (b) after dissolving a sample ofthe fiber taken from the reaction mixture, (c) after dissolving the driedsodiumalginate obtained from the reaction. To obtain a neutral dried sodium alginate,in the first method the pH should be adjusted to 9, and in the second the pHshould be adjusted to 8. The best method to control the reaction was todissolvea sample of the fiber and adjust the pH to 8. The best proportion to reach thecritical point, where pH just begins to rise, was 0.25 parts of sodiumcarbonateto 1 part of alginate in the initial dry algae. A pH above 7 may produce abreakdown of the molecule, reducing significantly the viscosity of the finalalginate. Four different temperatures were used to dry the alginate: 50, 60,70,and 80 °C. Drying time to reach 12% moisture ranged from 1.5h at 80 °C to 3 h at 50°C. The best drying temperature was 60 °C for2.5 h. The effect of drying temperature on alginate viscosity wasdependent on the alginate type. Low and medium viscosity alginates were notsignificantly affected, but alginate with high viscosity was reduced by 40 to54% using the temperature range of 60 to 80 °C. A fixed hammermill was used to reduce the particle size of the dried sodium alginate.Particlesize measurements showed that after a first milling the product contained 76%large particles (20–60 mesh) and 24% fine particles (80–120 mesh).After a third milling the product still contained 42.9% large particles. Nosignificant effect was found on alginate viscosity because of the millingsteps.     

Elongation of actin filaments is a diffusion-limited reaction at the barbed end and is accelerated by inert macromolecules

We used a fluorescence method to measure the rate constants for the elongation of pyrene-labeled actin filaments in a number of different solvents. The absolute values of the rate constants were established by electron microscopy. Using glycerol, sucrose, or ethylene glycol to vary the solution viscosity, the association rate constant (k+) was 10(7) M-1 s-1 viscosity-1 (in centipoise). Consequently, plots of 1/k+ versus viscosity are linear and extrapolate to near the origin as expected for a diffusion-limited reaction where the rate constant approaches infinity at zero viscosity. By electron microscopy, we found that this inhibitory effect of glycerol is almost entirely at the fast growing, barbed end. For the pointed end, plots of 1/k+ versus viscosity extrapolate to a maximum rate of about 10(6) M-1 s-1 at zero viscosity, so that elongation at the pointed is not limited by diffusion. In contrast to these small molecules, polyethylene glycol, dextran, and ovalbumin all cause a concentration (and therefore viscosity)-dependent increase in k+. At any given viscosity, their effects are similar to each other. For example, at 3 centipoise, k+ = 2.2 X 10(7) M-1 s-1. We presume that this is due to an excluded volume effect that causes an increase in the thermodynamic activity of the actin. If the proteins in the cytoplasmic matrix have a similar effect, the association reactions of actin in cells may be much faster than expected from experiments done in dilute buffers.     

The clinical significance of whole blood viscosity in (cardio)vascular medicine

Whole blood is a non-Newtonian fluid, which means that its viscosity depends on shear rate. At low shear, blood cells aggregate, which induces a sharp increase in viscosity, whereas at higher shear blood cells disaggregate, deform and align in the direction of flow. Other important determinants of blood viscosity are the haematocrit, the presence of macro-molecules in the medium, temperature and, especially at high shear, the deformability of red blood cells. At the sites of severe atherosclerotic obstructions or at vasospastic locations, when change of vessel diameter is limited, blood viscosity contributes to stenotic resistance thereby jeopardising tissue perfusion. However, blood viscosity plays its most important role in the microcirculation where it contributes significantly to peripheral resistance and may cause sludging in the postcapillary venules. Apart from the direct haemodynamic significance, an increase in blood viscosity at low shear by red blood cell aggregation is also associated with increased thrombotic risk, as has been demonstrated in atrial fibrillation. Furthermore, as increased red blood cell aggregation is a reflection of inflammation, hyperviscosity has been shown to be a marker of inflammatory activity. Thus, because of its potential role in haemodynamics, thrombosis and inflammation, determination of whole blood viscosity could provide useful information for diagnostics and therapy of (cardio)vascular disease.     

The effect of solvent viscosity on the rate-determining step of fatty acid synthetase

The overall activity of an animal fatty acid synthetase at the saturation level of substrate concentration decreased when the solvent viscosity, eta, of the reaction mixture was increased with viscogens such as glycerol, sucrose, and polyethylene glycol. The activity of the enzyme changed roughly proportional to eta-P, where p = 1.0 for glycerol, p = 0.66 for sucrose, and p less than 0.6 for polyethylene glycol with different molecular sizes. The thioesterase activity, which catalyzes the final partial reaction in the multifunctional enzyme, was not affected by 5-fold increase of solvent viscosity with sucrose. These results suggested that the rate-determining step of the enzyme other than the thioesterase reaction involves a microscopic transport step, the rate of which is influenced by the solvent viscosity. The microscopic transport step may be related to the transfer of the reaction intermediate from one active site to another or to the motion of a larger part of the enzyme requisite for the catalytic reaction. In the solution containing glycerol, the rate-determining motion was primarily diffusion limited since the inverse of the initial rate was proportional to eta, i.e., p = 1. Since the substrate concentration was at a saturation level in this experiment, the viscosity-dependent step cannot be the encounter between the enzyme and substrates, but must be intramolecular in origin, most probably the reaction catalyzed by beta-ketoacyl synthetase. In solutions containing other viscogens, however, p was less than 1.0, indicating a significant involvement of chemical steps in the rate-determining step as well. Bovine serum albumin, when used as a proteinic viscogen, also decreased the initial rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of solvent viscosity and different guanidine salts on the kinetics of ribonuclease A chain folding

The kinetics of folding of the two forms of unfolded ribonuclease A have been measured as a function of solvent viscosity by adding either glycerol or sucrose. The aim is to find out if either reaction is rate limited by segmental motion whose rate depends on external friction. The fast folding reaction (U₂ ? N) is known to be the direct folding process, and the slow folding reaction (U₁ ? N) is known to be rate limited by an interconversion between two forms (U₁ ? U₂) which are present after unfolding in strongly denaturing conditions. No dependence on solvent viscosity is found, either for the direct folding reaction or for the interconversion reaction. Each folding reaction has also been tested to see if its rate depends on the concentration of one or more partly folded intermediates, by adding denaturants destabilize any partly folded structures. Different guanidine salts are used as denaturants to vary the denaturing effectiveness of the salt while holding the guanidinium ion concentration constant. The rates both of the direct folding reaction and of the interconversion reaction decrease in relation to the denaturing effectiveness of the salt. However, there is a basic difference between the responses of the fast and slow folding reactions to low concentrations of denaturants. Although each folding reaction produces native protein, there is an 800-fold decrease in the rate of the fast folding reaction in 1M guanidine thiocyanate and only a 13-fold decrease in the rate of the slow folding reaction. This is consistent with the fast reaction being the direct folding process and the slow reaction being rate limited by the initial conversion of the slowrefolding to the fast-refolding form. Both the lack of viscosity dependence and the effects of denaturants indicate that the formation of structure is rate limiting in the direct folding reaction, U₂ ? N. The failure to find a viscosity dependence for the interconversion reaction, U₁ ? U₂, indicates that in this reaction also friction-limited segmental motion is not the rate-limiting process. Since the U₁ ? U₂ interconversion still occurs when the polypeptide chain is completely unfolded, the surprising result is that its rate in refolding conditions depends significantly on a reaction intermediate which is “denatured” by guanidine salts.     

Adenosine deaminase: viscosity studies and the mechanism of binding of substrate and of ground- and transition-state analogue inhibitors

We have studied the effects of viscosogenic agents, sucrose and ficoll, on (1) the hydrolysis of adenosine and of 6-methoxypurine riboside catalyzed by adenosine deaminase and (2) the rates of association and dissociation of ground-state and transition-state analogue inhibitors. For adenosine, Vmax/Km is found to be inversely proportional to the relative viscosity with sucrose, an agent affecting the microscopic viscosity, while no effect is found with ficoll, an agent affecting the macroscopic viscosity. Viscosogenic agents have no effect on the kinetic constants for 6-methoxypurine riboside. Thus, the bimolecular rate constant, Vmax/Km = 11.2 +/- 0.8 microM-1 s-1, for the reaction with adenosine is found to be at the encounter-controlled limit while that for the reaction with the poor substrate 6-methoxypurine riboside, 0.040 +/- 0.004 microM-1 s-1, is limited by some other process. Viscosity-dependent processes do not make a significant (less than 10%) contribution to Vmax. The dissociation constants for inhibitors are unaffected by viscosity. The ground-state analogue inhibitor purine riboside appears to bind at a rate comparable to that of adenosine. However, the slower rates of association (0.16-2.5 microM-1 s-1) and dissociation (5 X 10(-6) to 12 s-1) of transition-state analogue inhibitors are affected by the viscosity of the medium to approximately the same extent as the encounter-controlled rates of association and dissociation of adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Physical Factors on Reactions of Horse-Radish Peroxidase Complexes with Reduced Cytochrome c

This investigation concerns the effect of certain physical factors—viscosity, dielectric constant, ionic strength, and temperature of the medium—on the reaction of hydrogen peroxide and ferrocytochrome c in the presence of the enzyme horse-radish peroxidase. From study of the effects of viscosity and dielectric constant, it was concluded that the reaction between the secondary complex of hydrogen peroxide and enzyme on the one hand and ferrocytochrome c on the other is controlled by diffusion in media of high viscosity and by electrostatic effects at low viscosities. With respect to ionic strength, the data at pH 4.7 indicated a dipole-dipole interreaction. The temperature dependence of the over-all reaction had a Q₁₀ of 1.25.     

结实期相对高温对稻米淀粉粘滞性谱及镁、钾含量的影响

选用武育粳3号(粳稻)和扬稻6号(籼稻)两个水稻品种,利用人工气候室在水稻灌浆结实期进行控温试验,设置全结实期相对高温、结实前期相对高温、结实后期相对高温、全结实期适温(对照,日均温23 ℃)对照4个处理,探讨结实期不同时段相对高温(日均温度-30 ℃)对稻米淀粉粘滞性谱和Mg、K等元素含量的影响.结果表明：结实期相对高温使武育粳3号和扬稻6号淀粉粘滞性谱特征值发生改变,其中糊化开始温度、冷胶粘度、回复值和消减值升高,最高粘度、热浆粘度和崩解值下降;结实期相对高温促进了米粉中Mg、K、N含量的提高,特别是K含量的大幅提高,使稻米中Mg/K和Mg/(N·AC·K)明显降低;而2个水稻品种的直链淀粉含量（AC）对相对高温的反应截然相反,武育粳3号的AC降低、扬稻6号的AC升高.全结实期相对高温对稻米淀粉粘滞性谱和Mg、K等元素含量的影响最大,结实前期其次,结实后期的影响较小,开花后20 d内是稻米淀粉粘滞性谱和Mg、K等元素含量受温度影响的关键时期.Mg/K和Mg/(N·AC·K)与淀粉粘滞性谱特征值显著相关,可以作为稻米食味品质评价的参考指标.     

Investigation of Preparation and Mechanisms of a Dispersed Particle Gel Formed from a Polymer Gel at Room Temperature

A dispersed particle gel (DPG) was successfully prepared from a polymer gel at room temperature. The polymer gel system, morphology, viscosity changes, size distribution, and zeta potential of DPG particles were investigated. The results showed that zirconium gel systems with different strengths can be cross-linked within 2.5 h at low temperature. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) results showed that the particles were polygonal particles with nano-size distribution. According to the viscosity changes, the whole preparation process can be divided into two major stages: the bulk gel cross-linking reaction period and the DPG particle preparation period. A polymer gel with a 3-dimensional network was formed in the bulk gel cross-linking reaction period whereas shearing force and frictional force were the main driving forces for the preparation of DPG particles, and thus affected the morphology of DPG particles. High shearing force and frictional force reduced the particle size distribution, and then decreased the zeta potential (absolute value). The whole preparation process could be completed within 3 h at room temperature. It could be an efficient and energy-saving technology for preparation of DPG particles.     

Characterization of extremely thermostable enzymatic breakers (alpha-1,6-galactosidase and beta-1,4-mannanase) from the hyperthermophilic bacterium Thermotoga neapolitana 5068 for hydrolysis of guar gum

An alpha-galactosidase and a beta-mannanase produced by the hyperthermophilic bacterium, Thermotoga neapolitana 5068 (TN5068), separately and together, were evaluated for their ability to hydrolyze guar gum in relation to viscosity reduction of guar-based hydraulic fracturing fluids used in oil and gas well stimulation. In such applications, premature guar gum hydrolysis at lower temperatures before the fracturing process is completed is undesirable, whereas thermostability and thermoactivity are advantageous. Hyperthermophilic enzymes presumably possess both characteristics. The purified alpha-galactosidase was found to have a temperature optimum of 100-105 degrees C with a half-life of 130 minutes at 90 degrees C and 3 min at 100 degrees C, while the purified beta-mannanase was found to have a temperature optimum of 91 degrees C and a half-life of 13h at this temperature and 35 min at 100 degrees C.These represent the most thermostable versions of these enzymes yet reported. At 25 degrees C, TN5068 culture supernatants, containing the two enzyme activities, reduced viscosity of a 0.7% (wt) guar gum solution by a factor of 1.4 after a 1.5-h incubation period and by a factor of 2.4 after 5 h. This is in contrast to a viscosity reduction of 100-fold after 1.5 h and 375-fold after 5 h for a commercial preparation of these enzymes from Aspergillus niger. In contrast, at 85 degrees C, the TN5068 enzymes reduced viscosity by 30-fold after 1.5 h and 100-fold after 5 h compared to a 2.5-fold reduction after 5 h for the control. The A. niger enzymes were less effective at 85 degrees C (1.6-fold reduction after 1.5 h and a 4.2-fold reduction after 5 h), presumably due to their thermal lability at this temperature. Furthermore, it was determined that the purified beta-mannanase alone can substantially reduce viscosity of guar solutions, while the alpha-galactosidase alone had limited viscosity reduction activity. However, the alpha-galactosidase appeared to minimize residual particulate matter when used in conjunction with the beta-mannanase. This could be the result of extensive hydrolysis of the alpha-1,6 linkages between mannose and galactose units in guar, allowing more extensive hydrolysis of the mannan chain by the beta-mannanase. The use of thermostable enzymatic breakers from hyperthermophiles in hydraulic fracturing could be used to improve well stimulation and oil and gas recovery. (c) 1996 John Wiley & Sons, Inc.     

Viscosity-dependent structural fluctuations in enzyme catalysis.

The effect of viscosity on the rate of catalysis of carboxypeptidase A has been tested. By use of the tripeptide carbobenzoxy-l-alanyl-l-alanyl-l-alanine [Z(L-Ala)3] as substrate, it was shown that most of the effect on the hydrolysis rate caused by the presence of 30 or 40% methanol or glycerol in aqueous solution can be ascribed to a contribution of viscosity to the catalytic rate constant, kcat. Arrhenius plots of kcat in 30 and 40% glycerol or methanol are linear and almost parallel. When the rate constants are "corrected" for the viscosity of various media, the difference between the various Arrhenius plots is considerably reduced; it vanishes, within experimental error, when the effect of the dielectric constant of the solutions is taken into account as well. It is proposed that the viscosity of the medium can influence the rate-limiting step of the enzymic reaction, which is the rate of transitions over the energy barrier preceding product formation. According to the suggested mechanism, the enzyme--substrate complex can overcome this energy barrier by viscosity-dependent structural fluctuations. The quantitative agreement between the theory and the experimental results suggests that (a) due to the temperature dependence of the viscosity of the solution, the potential energy barrier of the reaction is about 5 kcal/mol lower than the observed activation energy and (b) information about the structural flexibility of the complex can be obtained by kinetic measurements.     

Application of a novel apparatus, the quartz chemical analyzer, to the determination of endotoxin in blood.

A novel apparatus called a quartz chemical analyzer (QCA) has been developed using a quartz crystal resonator. This apparatus measures sample viscosity changes based on resonant frequency changes of the quartz crystal. The apparatus was used to determine bacterial endotoxin concentrations by monitoring the gelation reaction of Limulus amebocyte lysate. The QCA determined endotoxin concentrations with good accuracy and reproducibility in the range of 0.001-3 EU/ml for endotoxin standard (JP XII). For endotoxin determination in human whole blood and plasma samples, the inhibitory reaction was eliminated by pretreatment of a fourfold dilution at 60 degrees C and incubation for 30 min. There are many advantages of the QCA method compared with the turbidimetric and chromogenic methods. For example, QCA can measure sample viscosity changes with high sensitivity and accuracy because QCA detects minor resonant frequency changes and the frequency data give a numerical value for easy quantitation. QCA can examine turbid samples, and the required quantities of samples and reagents are small, since the quartz crystal detects sample viscosity changes directly. The endotoxin determination time may be shortened by raising the reaction temperature, and QCA can detect other types of coagulation reactions.     

Kinetics of hemoglobin S gelation followed by continuously sensitive low-shear viscosity: Changes in viscosity and volume on aggregation

The kinetics of gelation of deoxyhemoglobin S were investigated as a function of temperature, concentration of hemoglobin, and solvent composition. Measurements were made by continuously monitoring the changes in viscosity with time, after polymerization had been induced by rapidly raising the temperature. A specially constructed low-shear viscometer was used. The solution density was also measured continuously to determine whether a volume change accompanied aggregation.The results confirm earlier work in showing that the time-dependence of the viscosity is composed of a variable latent period (several minutes to tens of hours) during which there is only a slight and very gradual increase in viscosity, followed by a stage in which the viscosity rises very sharply within a very short time. The length of the initial latent period is highly dependent upon the HbS³ concentration (33rd ± 6 power) and temperature. If the duration is interpreted as the inverse of a reaction rate, the activation energy is 96 ± 10 kcal/mol for solutions containing inosital hexaphosphate. Unlike measurements reported by others, no dependence of the latent period on shear rate was observed at the low shear rate employed. When IHP is omitted from the hemoglobin solutions, qualitatively similar results are obtained; however, the latent period depends on the 26th ± 6 power of the deoxyhemoglobin S concentration and yields an average activation energy of 125 ± 10 kcal/mol. The length of the latent period is increased 40-fold. Tris is known to prevent gelation but the inhibition can be partly reversed by adding IHP. When this is done, highly concentration-dependent latent periods are again observed. The results may be interpreted in terms of nucleation kinetic theories: a critical nucleus composed of approximately 30 hemoglobin molecules is required for gelation; and the energy barrier (which is larger in the absence of IHP) to the formation of this critical aggregate is approximately 100 kcal/mol.Gelation is not accompanied by a detectable volume change (limits 5 × 10^?5 g/ml). This indicates that the volume change of the reaction must be less than + 60 cm³/mol when the aggregates represent one half of the HbS available for polymerization.