Search

The development of a magnetic suspension densimeter that has been built for measurement of the density of compressed liquid at pressures up to 30 MPa in the temperature range 20 to 150°C is described. The densimeter was first built by the author and his coworkers at NIST. We describe here further improvements made on a second system built at NMIJ based on the same principle. The densimeter uses a small coil suspended from an electronic balance. Within the coil is placed a sample cell in which the pressurized sample and a buoy, which is a permanent magnet, are enclosed. For measurement of density, balance readings are recorded (1) with the buoy at rest and (2) with the buoy in magnetic suspension. The measurement procedure is basically a hydrostatic weighing, which is simpler than those of conventional magnetic densimetry. As an example, measurements of toluene density performed as part of an inter-laboratory comparison are presented. The data agreed with reliable literature values to within a few hundredths of a per cent.

For an independent determination of the Avogadro constant, the density of a 1-kg crystal silicon sphere was determined by direct measurements of its mass and volume. A scanning-type interferometer was used to measure the diameters, and the volume was calculated from the mean of uniformly distributed diameters. The sphere was weighted using a balance for the prototype kilogram at the National Research Laboratory of Metrology (NRLM), and the mass was determined accurately by direct measurements of the buoyancy force acting on the sphere. The total uncertainty of the density is estimated to be 0.34 ppm. Effects of a thin oxide layer and impurities on the density of a pure crystal are evaluated. >

A new interferometer has been developed for the accurate determination of the density of a silicon crystal, in which a single‐crystal silicon sphere of nearly perfect geometry is placed in a Fabry–Perot etalon of accurately known plate distance, and the diameters are obtained by measuring the two gaps between the etalon and the adjacent surface of the sphere. A new method is used to measure the sum of the length of the two gaps by scanning the etalon against the sphere. Two wavelengths, 633 nm from a frequency‐stabilized He–Ne laser and 441 nm from a free‐running He–Cd laser, are used to determine the order of interference by applying the method of exact fractions. The diameter of about 94 mm has been measured with a resolution of 0.5 nm. Diameter measurements from uniformly distributed directions have shown that the mean diameter has been determined with a standard deviation of 8.6 nm, corresponding to 0.28 ppm in the volume determination. The total uncertainty of the volume is estimated to be 0.34 ppm. Effects of a thin oxide layer and impurities on the bulk density are discussed.

The thermal expansion of pure water having natural isotopic abundance was determined by the dilatometric method in a temperature range from 0 to 85 °C and under a pressure of 101 325 Pa. The following equation was obtained: ρ(t)/ρmax = 1 - [(t - 3,98152)2 (t + 396,18534) (t + 32,28853)/609 628,6(t + 83,12333) (t + 30,24455)] where ρ(t) is the density of water at temperature t, which is expressed in terms of the ITS-90, and ρmax the maximum density. The density ratio which the above equation gives is estimated to have an uncertainty of approximately 1 × 10-6.

Interactions of Escherichia coli RecA protein with ATP and its analogues in the absence of DNA were studied by circular dichroic (CD) spectroscopy. The binding of RecA protein to ATP increased the CD band of ATP at around 260 nm. The positive CD band of the RecA protein-ATP complex suggested that the bound ATP was in the anti conformation, in accord with X-ray crystallographic data [Story, R.M. and Steitz, T.A. (1992) Nature 355, 374-376]. At pH 7.5 and at 25 degrees C the dissociation constant (Kd) and thermodynamic parameters for the binding of ATP to RecA protein were 18 microM (delta G = -6.5 kcal.mol-1), delta H = 0 kcal.mol-1, and delta S = 22 cal.mol-1.K-1. A non-hydrolyzable ATP analogue, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), gave a spectral change similar to that of ATP. The Kd for this analogue, 22 microM, was very close to the Km of ATP. These results in the absence of single-stranded DNA were different from those obtained by kinetic analysis [Weinstock, G.M. et al. (1981) J. Biol. Chem. 256, 8850-8855], which indicated that the inhibition constant of ATP gamma S was much smaller than the Km of ATP in the presence of DNA. For other ATP analogues (dATP, ADP, and dADP), similar spectral changes were observed, and their Kd values ranged from 19 to 54 microM. UTP, dUTP, and TTP also gave CD spectral changes, but not AMP, GTP, dGTP, CTP, and dCTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is the most complicated enzyme in the respiratory chain and is composed of at least 26 distinct polypeptides. Two hydrophilic subfractions of bovine heart complex I were systematically resolved into individual polypeptides by chromatography. Three polypeptides (51, 24, and 9 kDa) were isolated from the flavoprotein fraction (FP) of complex I, and the complete amino acid sequence of the 9 kDa polypeptide was determined. The 9 kDa polypeptide is composed of 75 amino acids with a molecular weight of 8,437. This protein exhibits no obvious sequence similarity to other proteins. The iron-sulfur protein fraction (IP) of complex I was separated into eight polypeptides, 75, 49, 30, 20, 18, 15, 13 kDa-A, and 13 kDa-B. The 20 kDa polypeptide was recognized as a novel component of IP for the first time. The N-terminal and several peptide sequences of the 20 kDa polypeptide were determined. Comparison of the sequences revealed significant sequence similarities of the 20 kDa polypeptide to the psbG gene products encoded in the chloroplast genome. The conserved sequence in these proteins was also found in the small subunit of the nickel-containing hydrogenases. These results suggest that complex I is related to other redox enzyme complexes.

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is the most complicated system in the respiratory chain. It consists of many subunits, some of which hold iron-sulfur clusters, but structural information is still limited. The amino acid sequences of two 13 kDa polypeptides, 13 kDa-A and 13 kDa-B polypeptides, of iron-sulfur protein fraction (IP) of bovine heart mitochondrial complex I were determined by a combination of protease digestion, Edman degradation, and carboxypeptidase digestion. The 13 kDa-A polypeptide was composed of 96 amino acids with a molecular weight of 10,536. The 13 kDa-B polypeptide consisted of 114 amino acids and had an acetylated amino terminus. The molecular weight of this protein was calculated to be 13,130 including the acetyl group. These proteins had no obvious sequence similarity to other known proteins. The partial amino acid sequence of 30 kDa-B polypeptide of IP was also determined to reveal a characteristic arrangement of cysteine residues that could be involved in iron-sulfur cluster formation.

A magnetic suspension densimeter is described that has been built for measuring the density of compressed liquids at pressures up to 15 MPa in the temperature range 20°–200°C with an uncertainty of 0.1%. The densimeter combines the principle of magnetic levitation of a buoy with that of liquid density determination by hydrostatic weighing. To accomplish this, the support coil is suspended from an electronic balance, and the balance readings are recorded (1) with the buoy at rest, and (2) with the buoy in magnetic suspension. Details are given of the construction of the cell, coil, buoy, and thermostat. The procedure is described by which cell and buoy are aligned so that the suspended buoy does not touch the cell wall. Test data on the densities of seven different liquids were obtained at room temperature. They agree with reliable literature values to within 0.1%. In a separate experiment, the bulk thermal expansion coefficient of the buoy material was determined. This experiment and its results are also given here.