Determination of molecular weight and related hydrodynamic parameters of high molecular weight protein fraction from a few oilseeds

The hydrodynamic parameters of the major protein fraction, viz. arachin from groundnut, alpha-globulin from sesame seed, brassin (M) from mustard seed and helianthinin from sunflower seed, have been determined in a single solvent system (0.05 M Tris-HCl buffer, pH 7.5 containing 0.5 M sodium chloride): sedimentation coefficient (s0(20,w)) and diffusion coefficient (D0(20,w)) by analytical ultracentrifugation, intrinsic viscosity [eta] by Ostwald viscometry and partial specific volume (V) by densimetry. The molecular weights (M) of the four proteins, calculated using the sedimentation-viscosity and sedimentation-diffusion coefficient methods, were found close to each other. The values have been compared with those in the literature and the reasons for discrepancies have been discussed.     

Critical examination for the presence of a low molecular weight fraction in serum iron

Native human pool serum and individual sera were ultrafiltered by Pellicon ultrafilters (Millipore) and the ultrafiltrates were extracted by an ammonium pyrrolidinedithicarbamate/methylisobutylketone system after addition of different internal iron standards to three of four identical ultrafiltrates. The extracts were examined for iron content by atomic absorption spectrometry. During ultrafiltration pH 7.4 was miantained by a constant atmosphere of a CO₂/air mixture.Low molecular weigth iron in native human sera from normal, normal orally iron substituted and siderotic individuals was found to be less than 0.05 μg/100 ml. Elevating serum citrate to 3-fold normal had no effect on this result.More iron became ultrafiltrable if the serum pH were lowered by citric acid as compared with hydrochloric acid.     

Molecular weight distribution of carrageenans by size exclusion chromatography and low angle laser light scattering

A procedure to determine the absolute weight average molecular weight and molecular weight distribution of carrageenans by high pressure aqueous size exclusion chromatography coupled with low angle laser light scattering is described. Experimental parameters are successively discussed, particular attention being focused on the absence of shear degradation during elution. The distribution curves were highly reproducible in time and weight average molecular weights integrated along the chromatogram were in good agreement with static light scattering results. A large difference in the molecular weight range between native (food-grade) and acidic degraded carrageenan samples was observed. Weight average molecular weights were found to be in good correlation with viscosity values, for degraded as well as undegraded products. It is also shown that the method described can help people using carrageenans in pharmacological studies by providing information on the real molecular weight distribution of the products they are employing.     

Enrichment of low molecular weight fraction of serum for MS analysis of peptides associated with hepatocellular carcinoma

A challenging aspect of biomarker discovery in serum is the interference of abundant proteins with identification of disease-related proteins and peptides. This study describes enrichment of serum by denaturing ultrafiltration, which enables an efficient profiling and identification of peptides up to 5 kDa. We consistently detect several hundred peptide-peaks in MALDI-TOF and SELDI-TOF spectra of enriched serum. The sample preparation is fast and reproducible with an average CV for all 276 peaks in the MALDI-TOF spectrum of 11%. Compared to unenriched serum, the number of peaks in enriched spectra is 4 times higher at an S/N ratio of 5 and 20 times higher at an S/N ratio of 10. To demonstrate utility of the methods, we compared 20 enriched sera of patients with hepatocellular carcinoma (HCC) and 20 age-matched controls using MALDI-TOF. The comparison of 332 peaks at p < 0.001 identified 45 differentially abundant peaks that classified HCC with 90% accuracy in this small pilot study. Direct TOF/TOF sequencing of the most abundant peptide matches with high probability des-Ala-fibrinopeptide A. This study shows that enrichment of the low molecular weight fraction of serum facilitates an efficient discovery of peptides that could serve as biomarkers for detection of HCC as well as other diseases.     

功能性低分子量岩藻多糖的研究进展

低分子量岩藻多糖来源于褐藻,是一类含有硫酸基团的多糖,具有多种生物学功能,如抗凝血、抗病毒、抗血栓、抗肿瘤等功能,因此可被广泛地应用于医药、食品等领域。着重介绍了低分子量岩藻多糖的制备及其生物学功能的研究进展。     

A high molecular weight RNA fraction in chromatin.

Utilizing a new chromatin isolation and fractionation technique we have obtained a high molecular weight RNA fraction from L-929 cell chromatin. The synthesis of this RNA is not greatly inhibited by concentrations of 0.04 mug/ml actinomycin D in the medium. Its synthesis appears to be strongly inhibited by 2 mug/ml of alpha-amanitin. The RNA appears to be quickly degraded (or removed from the chromatin) and does not contain a poly(A) sequence at its 3'-OH terminal end. Our working hypothesis is that this RNA is "nascent" heterogenous nuclear RNA partially transcribed from regions of the chromatin.     

Radioimmunoassay of low molecular weight human kininogen

A radioimmunoassay for low molecular weight (LMW) human Kininogen has been carried out. The first step was to prepare LMW Kininogen from human plasma. The proposed method allowed to get chemically pure and biologically active LMW Kininogen. This preparation was used to induce antibody. Optimal conditions for labelling and incubation were determined. This method may be applied to the assay of Kininogen in human plasma.     

Effect of molecular weight on the antioxidant property of low molecular weight alginate from Laminaria japonica

In this study, three alginate fractions with different molecular weights and ratios of mannuronic acid (M) to guluronic acid (G) were prepared by enzymatic hydrolysis and ultrafiltration to assess the antioxidant property of alginates from Laminaria japonica with molecular weight below 10 kDa. The antioxidant properties of different molecular weight alginates were evaluated by determining the scavenging abilities on superoxide, hydroxyl, and hypochlorous acid and inhibitory effect on Fe²⁺-induced lipid peroxidation in yolk homogenate. The results showed that low molecular weight alginates exhibited high scavenging capacities on superoxide, hydroxyl, and hypochlorous acid radicals and good inhibition of Fe²⁺-induced lipid peroxidation in yolk. By comparison, alginate A1 with molecular weight below 1 kDa and M/G of 1.84 had better scavenging activity on superoxide, hydroxyl, and hypochlorous acid radicals in vitro than A2 (1–6 kDa), A3 (6–10 kDa), ascorbic acid, and carnosine. With similar M/G ratio, A2 exhibited better antioxidant activity on superoxide and hypochlorous acid radicals than A3. However, fraction A3 with molecular weight of 6–10 kDa exhibited higher inhibitory ability on lipid peroxidation in yolk in vitro than A1 and A2. The results indicated that molecular weight played a more important role than M/G ratio on alginate to determine the antioxidant ability. By comparison, low molecular weight alginates composed of guluronic acid and mannuronic acid exhibited better antioxidant ability on oxygen free radicals than sulfated polysaccharides from L. japonica in our previous study and represent a good source of marine polysaccharide with potential application as natural antioxidant.     

Characterization of the low molecular weight human serum proteome

Serum potentially carries an archive of important histological information whose determination could serve to improve early disease detection. The analysis of serum, however, is analytically challenging due to the high dynamic concentration range of constituent protein/peptide species, necessitating extensive fractionation prior to mass spectrometric analyses. The low molecular weight (LMW) serum proteome is that protein/peptide fraction from which high molecular weight proteins, such as albumin, immunoglobulins, transferrin, and lipoproteins, have been removed. This LMW fraction is made up of several classes of physiologically important proteins such as cytokines, chemokines, peptide hormones, as well as proteolytic fragments of larger proteins. Centrifugal ultrafiltration of serum was used to remove the large constituent proteins resulting in the enrichment of the LMW proteins/peptides. Because albumin is known to bind and transport small molecules and peptides within the circulatory system, the centrifugal ultrafiltration was conducted under solvent conditions effecting the disruption of protein-protein interactions. The LMW serum proteome sample was digested with trypsin, fractionated by strong cation exchange chromatography, and analyzed by microcapillary reversed-phase liquid chromatography coupled on-line with electrospray ionization tandem mass spectrometry. Analysis of the tandem mass spectra resulted in the identification of over 340 human serum proteins; however, not a single peptide from serum albumin was observed. The large number of proteins identified demonstrates the efficacy of this method for the removal of large abundant proteins and the enrichment of the LMW serum proteome.     

Determination of molecular weight by neutron scattering

Theoretical and practical advantages of neutron scattering for the determination of molecular weights of particles in solution are discussed. The method presented does not use known particles for calibration and is applicable to a wide molecular weight range (10⁴–10⁹); it is not sensitive to assumptions about the partial specific volume of the particles, and the experiments are performed quite easily in standard spectrophotometer cells using 100–500 μg of material. The method is nondestructive and the sample can be recovered totally. The use of D₂O in solvents has particular advantages especially for multicomponent particles, for which the in situ molecular weight of each component can be determined separately.     

Determination of molecular weight from protein crystals

Different methods for the determination, of molecular weight from protein crystals are summarized, and in some cases extended. The use of crystal density measurements is discussed, and several approaches based on such measurements, including density extrapolation (Colman &; Matthews, 1971) and crosslinking (Cornick et al., 1973), are described and illustrated by density measurements of crystals of γ-chymotrypsin and β-amylase. The importance of allowing for “bound water” in molecular weight estimation is illustrated by examples, and a summary is given of information which may be gained from densities of crystals soaked in heavy water.The determination of molecular weight from measurement of the loss of weight of crystals on drying is discussed and illustrated using North's (1959) data.Also the method of molecular weight estimation from “crystal volume and protein content” (Love, 1957; Heidner et al., 1971) is reviewed, and a new method based on “crystal mass and protein content” is proposed.     

Synthesis of low molecular weight mimetics of heparin

Natural hexosaminoglycan heparin remains the most commonly prescribed anticoagulant in hospitalized patients. However its administration could induce side clinical events, including thrombocytopenia and bleeding. This explaines the need of development of alternative anticoagulant drugs based on modified heparin and polyanionic oligo- and polysaccharide derivatives, such as sulfated glucans, phosphomannans and fucoidans. Here we review the works on the synthesis of oligosaccharides related to low molecular weight hepain fragments and their derivatives, as well as oligosaccharides, which imitate parts of heparin chain responsible for biological activity. These works were aimed to develop the pharmaceutical preparations lacking ofheparin disadvantages.     

Stimulation of sperm motility and oxygen consumption of fowl spermatozoa by a low molecular weight fraction of seminal plasma

Washed fowl spermatozoa were incubated in a phosphate buffer containing various concentrations of fowl seminal plasma at 41 degrees C, normal body temperature, and the motility and oxygen consumption of spermatozoa were determined. Immediately after the incubation, spermatozoa showed good motility in the various diluents. However, with concentrations of seminal plasma at or below 20%, spermatozoa quickly became immotile. In contrast, at concentrations higher than 40% seminal plasma, spermatozoa were motile even after 15 min. As the concentration of seminal plasma was increased, oxygen consumption of spermatozoa also increased. A filtrate of the seminal plasma, obtained by passing the fluid through an Amicon YM-2 ultra-filtration membrane (Mr less than 1000), also stimulated the motility and oxygen consumption of spermatozoa. These results suggest that some low molecular weight factor(s) in fowl seminal plasma stimulated motility and oxygen consumption of fowl spermatozoa at 41 degrees C. A physiological role of this factor(s) may be to assist passage of spermatozoa through the vagina after natural mating.