Laser Light-scattering Photometry Research Articles

Improving Methodology of Particulate Measurement in Periodic Technical Inspection with High-Sensitivity Techniques: Laser Light Scattering Photometry and Particle Number Method

First, the validity of the current test procedure for particle measurement adopted in a periodic technical inspection (PTI) was evaluated by comparing test results obtained with the PTI and a type approval test (TAT) procedure using a Euro V level diesel truck with an intentionally damaged diesel particulate filter (DPF). PM and particle number (PN) with the TAT increased with increasing DPF damage ratio, and the PTI results well reproduced those in the TAT. However, the regulation limit of the PTI was so loose that even a 100% damaged DPF resulted in emission well below the PTI limit, although 0.5% and 5% damage ratios resulted in values exceeding the PN and PM limits in TAT, respectively. Then, we evaluated three different techniques for particle detection with the PTI procedure, such as an opacity meter, which is currently used in PTI, laser light scattering photometry (LLSP), and the PN method. For the detecting DPF failure, opacity meters did not have sufficient sensitivity. On the other hand, the LLSP was sensitive enough for detecting DPF failure, but the results varied among LLSP devices. This variation was due to the test procedure of PTI, which is strongly transient (no load racing). The LLSP and PN measurement devices were sensitive enough to detect DPF failure at idle, which was quite steady, and the variation observed in the PTI procedure did not occur. PN counting of particles over 15 nm was more sensitive than LLSP and was sensitive enough to detect DPF failure, which did not result in the PM limit in TAT being exceeded.

Oligomeric State of the Escherichia coli Metal Transporter YiiP

YiiP is a 32.9-kDa metal transporter found in the plasma membrane of Escherichia coli (Chao, Y., and Fu, D. (2004) J. Biol. Chem. 279, 17173-17180). Here we report the determination of the YiiP oligomeric state in detergent-lipid micelles and in membranes. Molecular masses of YiiP solubilized with dodecyl-, undecyl-, decyl-, or nonyl-beta-d-maltoside were measured directly using size-exclusion chromatography coupled with laser light-scattering photometry, yielding a mass distribution of YiiP homo-oligomers within a narrow range (68.0-68.8 kDa) that equals the predicted mass of a YiiP dimer within experimental error. The detergent-lipid masses associated with YiiP in the mixed micelles were found to increase from 135.5 to 232.6 kDa, with an apparent correlation with the alkyl chain length of the maltoside detergents. Cross-linking the detergent-solubilized YiiP with 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) resulted in a dimeric cross-linked product in an EDC concentration-dependent manner. The oligomeric state of the purified YiiP in reconstituted membranes was determined by electron microscopic analysis of two-dimensional YiiP crystals in negative stain. A projection structure calculated from measurable optical diffractions to 25 A revealed a pseudo-2-fold symmetry within a molecular boundary of approximately 75 x 40 A, indicative of the presence of YiiP dimers in membranes. These data provide direct structural evidence for a dimeric association of YiiP both in detergent-lipid micelles and in the reconstituted lipid bilayer. The functional relevance of the dimeric association in YiiP is discussed.

Exhaustive test for diesel vehicles

Australia's environmental protection agency is pilot-testing a new method for evaluating diesel vehicle emissions. The technique which uses laser light-scattering photometry to give on the spot pollutant readings has attracted international attention thanks to its speed and low cost. The new method can determine exhaust NO2 and particulates in only 10 minutes in a drive-up testing station. This short item describes the new Australian test, and how it could be used.

Direct and Indirect Methods for Molar-Mass Analysis of Fragments of the Capsular Polysaccharide ofHaemophilus influenzaeType b

Two methods are described for direct molar-mass measurement of low-molar-mass fragments obtained by oxidative cleavage of the capsular polysaccharide ofHaemophilus influenzaetype b. Absolute molar masses were determined by size-exclusion chromatography (SEC) with detection by multiangle laserlight-scattering photometry (MALLS) and differential refractometry (RI). The end-group structure of the polysaccharide fragments allowed the direct measurement of average chain length by quantitative1H NMR, from which molar masses were derived. Variation between the molar masses obtained by the two methods ranged from 5 to 7%. When molar masses estimated by indirect methods were compared to SEC-MALLS/RI data, significant deviations were observed. Analysis by SEC with secondary calibration with dextran standards gave molar masses that exceeded the SEC-MALLS/RI data by as much as 2.5-fold. Molar masses estimated by a combination of colorimetric assays varied from the SEC-MALLS/RI data by as much as 50%. These results demonstrated the applicability and superior accuracy of the direct methods of molar-mass determination of the polysaccharide fragments.

Conformation of [formula omitted] in aqueous solution

The conformation and dilute solution properties of (2→1)-β- d-fructan in aqueous solution were studied by gel permeation chromatography, low-angle laser light-scattering photometry, viscometry, small-angle X-ray scattering and electron microscopy. Fractions covering a broad range of weight-average molecular weights ( M w) from 1.49 × 10 4 to 5.29 × 10 6 were obtained from a native sample by ultrasonic degradation and fractional precipitation. For M w < 4 × 10 4, the intrinsic viscosity [η] varies with M w 0.71, indicating that the fructan chain behaves as a random coil expanded by an excluded-volume effect in this molecular weight region. For M w > 10 5, [η] exhibits an unusually weak dependence on M w and finally becomes almost independent of molecular weight. This behaviour is interpreted in terms of a globular conformation of the high-molecular-weight fructan molecules. Small-angle X-ray-scattering measurements and electron microscopic observations support this interpretation of the values of [η] observed.

Change in oligomeric structure of solubilized Na + /K +-ATPase induced by octaethylene glycol dodecyl ether, phosphatidylserine and ATP

Membrane-bound Na + /K +-ATPase purified from dog kidney was solubilized with octaethylene glycol dodecyl ether (C 12E 8), and the resultant solubilized enzyme was chromatographed on a TSKgel G4000SW XL or G3000SW XL column equilibrated with elution buffers containing various ligands affecting oligomerization of the enzyme. Weight-averaged molecular weight ( M w ) values for the main protein components eluted were estimated by low-angle laser light-scattering photometry. With increasing concentration of C 12E 8 included in the elution buffer from 0.1 to 5 mg/ml, the M w decreased from 230 000 to 153 000, indicating that C 12E 8 induced dissociation of the enzyme. In contrast, the M w of the protein component increased up to 1.44 · 10 6 as the concentration of phosphatidylserine (PS) added to the elution buffer containing a fixed concentration of 0.3 mg/ml C 12E 8 was increased to 120 μg/ml. The association and/or aggregation were reversible by removal of the PS by rechromatography. Addition of PS to the elution buffer also allowed the solubilized enzyme to exhibit ATPase activity comparable to that of the membrane-bound enzyme during passage through the column. This was also the case with phosphatidylglycerol (PG) and phosphatidylinositol, but not with phosphatidylcholine or phosphatidylethanolamine. The specific refractive index increment d n / d c p ) of the solubilized enzyme was increased by addition of exogenous PG or PS, strongly suggesting that the phospolipid became bound to the enzyme, and that it induced association of the enzyme. The association induced by PS was inhibited by ATP and ADP, but not AMP. The concentrations for half-maximal inhibition were 0.44 mM for ATP and 0.88 mM for ADP. The PS-induced associated enzyme isolated by chromatography in the presence of 120 μg/ml PS was dissociated by ATP with K 0.5 of 0.16 mM. The dissociating effect of C 12E 8, ATP and ADP and the associating effect of PS on the solubilized enzyme are consistent with the reports that C 12E 8 mimics the effect of regulatory ATP at the low-affinity site on the conformationl transition from E 2 to E 1, and that phospholipids are essential for the reverse transition from E 1 to E 2. The results can be explained by assuming that the enzyme takes the form of a loosely associated diprotomer in the E 1 state and a tightly associated one in the E 2 state.

Interfacing gradient elution ion-exchange chromatography and low-angle laser light-scattering photometry for analysis of proteins

Molecular weights (MWs) of different proteins were determined by interfacing gradient elution ion-exchange chromatography and low-angle laser light-scattering photometry (IEC-LALLS). A high-performance strong cation-exchange column was used to elute proteins using fast (5 min) and conventional (15-30 min) gradients. The eluted proteins were characterized on-line by determining their MWs using LALLS. The specific refractive index (RI) increment (d n/d c) and the RI of the solvent used over the gradient range were determined off-line and used to calculate the absolute weight-average MWs. Four proteins, ribonuclease A, α-chymotrypsinogen A, trypsinogen and β-lactoglobulin A (β-LACT) were studied. Accurate MWs were obtained for all the proteins using fast and conventional gradients, except for β-LACT, which aggregated as a function of the gradient employed. The degree of aggregation of β-LACT increased as the rapidity of the gradient was increased over a fixed gradient range. This study indicated that it is possible to separate and characterize proteins rapidly using IEC-LALLS.

Characterization of carbonic anhydrase isozyme CA2, which is the CAH2 gene product, in Chlamydomonas reinhardtii.

From high-CO2 (5% CO2) grown unicellular green alga, Chlamydomonas reinhardtii, carbonic anhydrase (CA) was isolated by affinity chromatography and characterized. Isolated CA was identified as an isozyme (CA2) which is the product from the second gene CAH2 by peptide sequencing. The CA2 was inactivated by dithiothreitol. This treatment caused dissociation of CA2 into the large (38 kDa) and small subunits (4243 Da). The molecular mass of the CA2 holoenzyme measured by low-angle laser light-scattering photometry and precision differential refractometry combined with gel-filtration HPLC was 87.9 kDa. These results and gene structure indicate that CA2 is a heterotetramer consisting of two large and two small subunits linked by disulfide bonds like CA1, which is the CAH1 gene product. The specific activity of CA2 purified by anion-exchange HPLC was 3300 units per mg protein, which was approximately 1.6 times higher than that of CA1. Therefore, it was concluded that two structurally related isozymes, CA1 and CA2, are present in the wild type cells of C. reinhardtii and differentially regulated by the atmospheric CO2 concentration.

Active and monomeric human red cell glucose transporter after high performance molecular-sieve chromatography in the presence of octyl glucoside and phosphatidylserine or phosphatidylcholine

The human red cell glucose transporter (Glut 1) was purified by ion-exchange chromatography in the presence of octyl glucoside. The state of association of the protein was studied, and the transport activity was determined after exchange of copurified membrane lipids for phosphatidylserine (PS) or phosphatidylcholine (PC). The purpose was to analyze the Glut 1 preparation for homogeneity and activity prior to attempts at crystallization. Analyses by high performance molecular-sieve chromatography showed that the Glut 1 was monomeric immediately after the ion-exchange purification: the M r of the Glut 1 polypeptide was estimated to be 49 000 ± 6000 by TSKgel G3000SW chromatography monitored by low-angle laser light-scattering photometry, differential refractometry and UV photometry. This required determination of the absorption coefficient of the Glut 1, which was measured to be 1.13 ± 0.03ml mg −1 cm −1 at 280 nm, referring to the polypeptide concentration. The M r value is consistent with the cDNA-deduced M r 54117 of the very similar HepG2 glucose transporter polypeptide. At 2°C, pH 7 and an ionic strength of 0.06 M, the Glut 1 associated gradually during three days to form oligomers. These formed much more rapidly at room temperature or at high ionic strength. Freshly prepared Glut 1 retained high activity after separation from membrane lipids on a TSKgel G3000SW column in the presence of 40 mM octyl glucoside and 1 mM PS or PC. In contrast, most of the activity was lost when the membrane lipids were separated from the protein in the absence of eluent lipids. The presence of a phospholipid was thus essential for retention of high activity of the Glut 1 in octyl glucoside and PC was nearly as effective as PS.

Use of on-line laser light-scattering photometry coupled to chromatographic separations for determination of biopolymer molecular mass, branching, size and shape distributions

Use of on-line laser light-scattering photometry coupled to chromatographic separations for determination of biopolymer molecular mass, branching, size and shape distributions

Subunit constitution of carbonic anhydrase from Chlamydomonas reinhardtii

Carbonic anhydrase purified from the cell surface of Chlamydomonas reinhardtii was inactivated by treatment with dithiothreitol. This treatment caused dissociation of the holoenzyme into 35-kDa (A) and 4-kDa (B) subunits as revealed by SDS/PAGE. The 35-kDa subunit was further separated into two components A1 (35 kDa) and A2 (36.5 kDa) by SDS/PAGE using a gradient gel. These two components have the same amino acid sequence up to at least the 10th amino acid from the N-terminus. The molecular masses were estimated at 76 kDa and 35 kDa for the holoenzyme and the large subunit, respectively, and the molar ratio of the former to the latter at 1:2, by using the techniques of low-angle laser light-scattering photometry and precision differential refractometry combined with gel-filtration HPLC. The molar ratio of the 35-kDa/4-kDa subunits was estimated at 1:1 the gel-filtration HPLC monitored with precision differential refractometry. Atomic-absorption spectrophotometry revealed that the holoenzyme contains two atoms of zinc. These results suggest that the holoenzyme is a heterotetramer composed of two large subunits (A1 and A2) and two small subunits (B).

Size-exclusion chromatography—low-angle laser light-scattering photometry of lignite macromolecules

The determination of molecular weight distribution in humic acids, derived from lignite, has been successfully carried out by the application of size-exclusion chromatography, coupled with low-angle laser light-scattering photometry. Reduction of soluble derivatives with zinc in acetic anhydride was required to reduce the absorbance so that a linear scattering response was obtained. A benzyl derivative and a methyl derivative exhibited the best chromatographic results, giving weight average molecular weights of about 1 500 000.

Gel permeation chromatography : XII. Computer-assisted gel permeation chromatography and low-angle laser light-scattering photometry

An automated gel permeation chromatograph-low-angle laser light-scattering system capable of measuring the molecular-weight distribution of soluble polymers without the necessity of calibration is described. The automation system eliminates most of the tediousness associated with molecular-weight distribution measurement. The molecular-weight distributions and molecular-weight averages of a broad range of polystyrene standards were determined using this technique. The results obtained are shown to agree closely with literature data.