Actual Molecular Weight Research Articles

Determination of Block Length Distributions of Poly(oxypropylene) and Poly(oxyethylene) Block Copolymers by MALDI-FTICR Mass Spectrometry

Matrix-assisted laser desorption/ionization (MALDI) was performed on an external ion source Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS) to analyze the block length distributions of triblock polymers of poly(oxypropylene) and poly(oxyethylene). The first series of results presented demonstrate that the apparent molecular weight distributions are distorted. This distortion is induced by the flight-time-induced mass discrimination inherent in the experimental technique, the variation of isotopic patterns over the measured mass range, and the overlap of peaks in the spectrum. Subsequently, a method for the treatment of molecular weight distributions measured by MALDI on an external ion source FTICR-MS is developed to yield the actual molecular weight distribution and, from that, the individual block length distributions. For the first time, detailed and accurate molecular weight data were obtained on a complex sample using this methodology, which independently validates the data provided by the manufacturer. The experimentally verified random coupling hypothesis proves the validity of the methodology.

Conjugated Polymers with Main Chain Chirality. 3. Synthesis of Optically Active Poly(aryleneethynylene)s

Binaphthyl-based chiral poly(aryleneethynylene)s of different molecular weights have been synthesized. An optically active poly(aryleneethynylene), (R)-9c, is obtained by coupling an optically active binaphthyl molecule, (R)-2,2‘-bis(octadecyloxy)-6,6‘-diethynyl-1,1‘-binaphthyl, (R)-8, with 1,4-diiodobenzene in the presence of tetrakis(triphenylphosphine)palladium(0) and cuprous iodide catalysts. GPC analysis of (R)-9c shows Mw = 29 000 and Mn = 12 000 (PDI = 2.4). Its specific optical rotation is [α]D = −272.2° (c = 0.5, CH2Cl2). Coupling of (R)-8 with 4,4‘-diiodobiphenyl using tetrakis(triphenylphosphine)palladium(0) and cuprous iodide catalysts also generates an optically active poly(aryleneethynylene), (R)-10c. GPC analysis of (R)-10c shows Mw = 37 000 and Mn = 18 000 (PDI = 2.1). The specific optical rotation of (R)-10c is [α]D = −281.7° (c = 0.18, CH2Cl2). A laser light scattering study of these polymers shows that the actual molecular weights (Mw) of these materials are higher (1.4−2.5 times) than those obtained from GPC analysis vs polystyrene standards. The repeat units of these polymers have been synthesized. UV spectroscopic studies of the repeat units and of the polymers demonstrate that the conjugation of the polymers is determined by the conjugation in their repeat units.

Novel degradation pathway of glycated amino acids into free fructosamine by a Pseudomonas sp. soil strain extract.

A Pseudomonas sp. soil strain, selected for its ability to grow on epsilon-(1-deoxyfructosyl) aminocaproic acid, was induced to express a membrane-bound enzymatic activity which oxidatively degrades Amadori products into free fructosamine. Apparent Km values for fructosyl aminocaproate, epsilon-fructosyl lysine, fructosyl glycine, and ribated lysine were 0.21 mM, 2.73 mM, 3.52 mM, and 1.57 mM, respectively. The enzyme was also active against alpha-fructosyl lysine and borohydride-reduced Amadori product, weakly active with ribated and glycated polylysine, and inactive with reducing sugars, amino acids, and glycated proteins. The enzymatic activity was highest at pH 6.5 and 25 degrees C in 0.1 M sodium phosphate, while over 80% of the activity was lost above 65 degrees C. Complete inhibition was observed by HgCl2, NaN3, and NaCN suggesting a role for SH groups and copper in the enzymatic activity. The reaction products were characterized by 1H NMR, 13C NMR, and GC/MS and found to correspond to 1-deoxy-1-aminofructose, i.e. free "fructosamine," and adipic acid. Confirmation of the free fructosamine structure was based on the complete spectroscopic identity of the borohydride reduction product with commercially available glucamine (1-amino-1-deoxyglucitol). The new enzyme is provisorily classified as fructosyl N-alkyl amino acid oxidase (EC 1.5.3) (fructosyl-amino acid:oxygen oxidoreductase) and may thus belong to a novel class of "Amadoriases" which deglycate Amadori products oxidatively. In contrast, however, the new enzyme acts on the alkylamine bond rather than the ketoamine bond of the Amadori product.

PCR template-DNA isolated quickly from monocot and dicot leaves without tissue homogenization.

Nucleic Acids Research, 1994, Vol. 22, No. 10 PCR template-DNA isolated quickly from monocot and dicot leaves without tissue homogenization Christie E.Williams* and Pamela C.Ronald Department of Plant Pathology, University of California, Davis, CA 95616, USA Received April 5, 1994; Accepted April 15, 1994 * To whom correspondence should be addressed for 5 min. Hot samples were transferred directly to a speed-vac and vacuum infiltrated for 2 min (rice, bean, corn and barley) or 6 min (oat, triticale, wheat and durum). After treatment, the leaf tissue was somewhat translucent and the buffer noticeably a. kb b. kb C. kb m 1 2 3 4 5 6 Figure 1. Separation of PCR products on gels containing 1 % Synergel (Diversified Biotech) and 0.6% agarose (BRL) in 0.5x TBE buffer. A) Lane 1 and 2 contain DNA amplified from large-scale isolations of parental plant DNA. Subsequent lanes contain DNA amplified from PEX isolations of DNA from F 2 plants. Amplification with 24-base STS primers B7 and B8 (7) resulted in single PCR products from parental classes of homozygotes (lanes 1, 2 and 6) and the two parental plus one heteroduplex product from heterozygotes (lanes 3, 4 and 5). The heteroduplex is a PCR artifact that does not migrate in accordance with its actual molecular weight (12). B) Lanes are in the same order as those in A. Amplification with 10-base RAPD primer A02 (Operon, Inc.) produced no polymorphisms among the samples. C) RAPD primer AB09 generated discrete amplification products from bean (lane 1), barley (lane 2), oat (lane 3), com (lane 4), rice (lane 5) and triticale Oane 6). Lane m contains 1 kb ladder. Downloaded from nar.oxfordjournals.org at University of California, Davis on October 26, 2010 A protocol is presented that utilizes potassium ethyl xanthogenate to liberate DNA from leaves without the need for tissue homogenization. This quick, single-tube method requires only 1/3 cm 2 of tissue and yields enough DNA to serve as template for 20 polymerase chain reactions. The resulting amplification patterns are indistinguishable from those generated from rigorous large-scale DNA extractions. Because 100—200 samples can be processed per day by a single person, this method allows for the rapid screening of large mapping populations. This protocol was developed for use with rice, but also works well with other species including barley, oat, corn and bean. Although many quick protocols have been published for small- scale isolation of DNA, certain characteristics of rice and other monocots result in inconsistent yields and poor amplification with the polymerase chain reaction (PCR). Because rice leaves are very fibrous, protocols specifying homogenization of leaf tissue (1) are laborious and undependable when scaled down. Quick protocols that amplify DNA directly from leaf tissue (2, 3, 4) have failed to rupture the tough cell wall of rice leaves sufficiently to liberate template DNA. Fast methods for the extraction of DNA from single seeds of cotton (5) rye and barley (6) and rice (7) have been published. In our hands, these procedures did yield sufficient quantities of rice DNA for PCR (about 100 ng per half- seed). However, PCR amplification was inconsistent, presumably due to the presence of inhibitors like starch. As a result, we designed a quick, small-scale prep that utilizes potassium ethyl xanthogenate (PEX; 8) to dissolve cell walls, degrade proteins and inhibit DNase activity (8). No tissue homogenization is required in order to yield enough DNA for 20 PCR amplifications from a small amount of leaf tissue. We tested our protocol on young leaves from bean (Phaseolus vulgaris), barley (Hordeum vulgare), corn (Zea mays), oat (Avena sativa), rice (Oryza sativa), triticale (a wheat/rye hybrid), wheat (Jriticum aestivum) and durum (T.durum). Upon emergence from the soil, the first true leaf was collected on ice in the greenhouse and subsequently stored at -80°C. Older rice tissue, up to 8 weeks after emergence, also was collected. Frozen segments of leaf totalling 1/3 cm 2 were selected and placed in the bottom of a small microcentrifuge tube containing 100 /tl of extraction buffer (8). This buffer consisted of 6.25 mM PEX (potassium ethyl xanthogenate or carbonodithioic acid, o-ethyl, potassium salt from Fluka), 100 mM Tris-HCl (pH 7.5), 700 mM NaCl, 10 mM EDTA (pH 8). Dipping the hydrophobic leaf tissue in 70% ethanol prior to placing it in buffer helped to keep it fully submerged. The samples were incubated in a water bath at 65 C C

Molecular weight viscosity relationships for PVC

AbstractThe inherent viscosity of PVC resin in compounds containing fillers and additives can be calculated accurately and conveniently from the weight average molecular weight or the peak molecular weight as measured by High Performance‐Size Exclusion Chromatography (HP‐SEC). Molecular weight/inherent viscosity relationships are shown for typical production PVC resins. The utility of HP‐SEC instrumentation for the determination of IV for samples of commercial interest is demonstrated. Mark‐Houwink coefficients from the literature were used to calculate a convenient conversion factor of 0.612, which can be used to convert the molecular weight measured relative to polystyrene by HP‐SEC to the actual molecular weight for PVC.

Molecular size and solvation of low molecular weight poly(ethylene oxide) and phenol–formaldehyde resols in different solvents

AbstractSeveral solvent systems are presently used to characterize the molecular weight of phenol–for‐maldehyde resins. However, results reported in the literature for different molecular weights may not be representative of true molecular weight, but rather may be distorted by aggregation and solvation. In this report an effort to clarify this situation was conducted, first by using a suitable calibration with poly(ethylene glycol) and then using this polymer as a molecular size standard to determine the size of phenol–formaldehyde oligomers in solution. In the calculation of the molecular sizes of phenol–formaldehyde resols, proper accounting of the variation of the Mark–Houwink parameters with molecular weight for low degrees of polymerization must be made for poly(ethylene glycol). The Mark–Houwink constants for poly(ethylene glycol) are very similar in presence or absence of salts, and are considered to be unaffected by solution ionic strength. It is not the case for phenol–formaldehyde, whose apparent molecular size varies with the nature of the solvent. The actual molecular weight and molecular dimension distribution are discussed for different type A resols used as adhesives in the wood composite industry.

The N-terminus and a 4,000-MW peptide from the midportion of the N-terminus of the atrial natriuretic factor prohormone each circulate in humans and increase in congestive heart failure.

Two peptides consisting of amino acids 1-30 and 31-67 of the N-terminus of the prohormone of atrial natriuretic factor (pro-ANF) that have vasodilatory and natriuretic properties were investigated to determine if they circulate in humans. Specific and sensitive radioimmunoassays were developed to amino acids 1-30, 31-67, and 99-126 of pro-ANF. Evaluation of human plasma that had been subjected to reverse-phase high-pressure liquid chromatography suggested that pro-ANFs 1-30 and 31-67 as well as ANF were distinct peaks in human plasma corresponding exactly to pure synthetic peaks of these peptides on high-pressure liquid chromatography. Molecular weight determination of the endogenous immunoreactive peptides measured in plasma by G-50 Sephadex gel permeation chromatography revealed that the pro-ANF 1-30 radioimmunoassay recognized a peptide of 10,000 MW, which is consistent with it measuring the whole N-terminus of pro-ANF (amino acids 1-98) but without ANF (C-terminus) attached to it. The pro-ANF 31-67 radioimmunoassay recognized mainly (more than 95%) a peptide of 3,900-4,000 MW, which corresponds closely with its actual molecular weight of 3,878. Our ANF radioimmunoassay recognizes a peptide in plasma of 3,000 MW with the known molecular weight of ANF being 3,081. The mean circulating concentrations of immunoreactive pro-ANF 1-98, pro-ANF 31-67, and ANF in 54 control subjects were 531 +/- 25, 371 +/- 18, and 22 +/- 1 fmol/ml (+/- SEM), respectively. Thirty patients with varying severity of congestive heart failure were also studied. The N-terminus, C-terminus, and pro-ANF 31-67 increased: twofold for New York Heart Association functional Class II, threefold to ninefold for Class III, and 10- to 20-fold for Class IV patients with congestive heart failure. Thus, the N-terminus and a 4,000-MW peptide from the midportion of the N-terminus of pro-ANF as well as ANF circulate normally and increased proportionately to the increasing severity of congestive heart failure. However, because the pro-ANF 31-67 radioimmunoassay was the only assay that discriminated between patients with Class I congestive heart failure and control subjects, this assay may be the most useful to accurately classify the severity of congestive heart failure.

The influence of molecular weight on the Resonant Raman Scattering of polyacetylene

For the first time, polyacetylene, (CH) x, films of known number average molecular weights, M n, have been examined using Resonance Raman Scattering techniques. (CH) x samples of 500, 5300, 10500, and 100,000 Daltons were synthesized using the classical titanium tetra-n-butoxide/triethyl aluminum catalyst/cocatalyst system. After thermal isomerization, modeling of the RRS spectra using the Brivio, Mulazzi model indicate that the 100,000 Dalton polymer is composed principally of long trans conjugated segments. In contrast, polymers of 10500 M n and below are seen to contain significantly larger fractions of short trans conjugated segments. For samples below 10500, no clear relationship between actual polymer molecular weight and G, the ratio of long to short segments, is observed. These results suggest that not until very long chains are obtained can samples containing a large fraction of long conjugated segments be obtained. Ambient, short term oxidation of 100,000 M n polymer shows an increase in satellite band intensities at ω c−c and ω c=c, where ω is frequency, which corresponds to a decrease in G. Low level chain oxidation or doping is shown to preferentially occur within long conjugated segments.

The interleukin 1 receptor

The diverse biological activities of interleukin 1 (IL-1) have attracted interest from a wide range of biological disciplines ~ After systemic injection into a variety of animals, it seems that IL-1 affects nearly every organ system. Interested parties fall into two groups: those studying the potential beneficial effects of IL-1 on host defense mechanisms, particularly the hemopoietic stem cell, and those looking at ways to block the production and/or activity of IL-1 since many of its biological activi- ties are associated with a pathogenic disease process such as tissue degradation in arthritis and diabetes. Some groups are now preparing to use IL-1 in humans to bolster the stem cell's reactivity to colony-stimulating factors (CSFs)I while other groups in clinical trials with claims that new drugs for pain and arLhriti.~ work by reducing production of IL-1. No doubt the cloning of an IL-1 receptor molecule is of vital importance to both areas of investigation. Cloning of the 80 kDa IL-l-binding protein An 80 kDa IL-l-specific binding protein has been observed on a variety of cell lines 2-7. It recognized both IL-loL and IL-113. Treatment of this 80 kDa protein with glycanases reduces its size to about 65 kDa, indicating that the variability in molecular weight reported by several investigators may be due to differential glycosy- lation. In addition, some experiments using tunicamycin and plant lectins suggest that the glycosylation sites of the 80 kDa IL-1 receptor may play a role in receptor affinity a. The 80 kDa IL-l-binding protein has been purified to homogeneity from the murine cell line EL4 6.1 C10 and the 26 amino-terminal amino acids have beer, determinedg~ Using a direct in-situ binding assay, the 80 kDa IL-l-binding protein has been cloned 1° and the amino acid sequence derived from the cDNA matches that of the purified protein. The actual molecular weight of the 80 kDa IL-l-binding protein is 64.598 kDa. This protein, which is not homologous to other proteins presently reported in the various databases, is the IL-1 receptor. The question remains whether this is the sole IL-1 receptor responsible for the multitude of biological activities of IL-1. If other IL-l-binding proteins exist, do they function alone or in combination with the 80 kDa protein to form the IL-1 receptor? The II.-1 receptor is a member of the immunoglobulin superfamily Several groups have been actively involved in studies on the binding of IL-1 to the plasma membranes of various cells and cell lines. The report by Sims etal. ~° is a welcome addition to these studies and their effo~-ts will certainly help to advance the :lnderstanding of the biology of IL-1. The murine IL-1 receptor is comprised of three domains: a 319 amino acid ~-xtracellular portion, a 21 amino acid hydrophobic trar,~,membrane segment, and a 217 amino acid piece probably located in the cytoplasm. The sequence of the cytoplasmic segment does not show any similarities to those of protein Departments of Medicine, Pediatrics and Physiology, Tufts University School of Medicine and New England Medical Center, Boston, MA 0211 I, USA.

Characterization of star-shaped polystyrene samples by size exclusion chromatography

Well defined star-shaped polystyrene samples have been studied by means of size exclusion chromatography (GPC) apparatus fitted with three detectors in series, viz. low angle light scattering photometer, continuous viscometer, and differential refractometer. Thus, the light scattering intensity, the limiting viscosity number and the concentration are measured continuously on the eluate of the chromatographic column. The agreement between the molecular weight averages computed from these GPC data and those measured by standard techniques on the overall sample has been found quite satisfactory. The viscometric data arising from the GPC set-up are somewhat less accurate, but they clearly indicate the highly branched character of the samples studied. Access to the actual molecular weight distribution for a sample of star polymer is rendered possible by this method. Even the presence of linear contaminants does not interfere provided the elution curves for stars and for the linear constituents do not overlap.

Resonant Raman scattering of controlled molecular weight polyacetylene

Polyacetylene, (CH)x, films of 500, 5300, 10 500, and 100 000 Daltons number average molecular weights (Mn ) were synthesized using the titanium tetra-n-butoxide/triethyl aluminum-catalyst/cocatalyst system and examined using resonant Raman scattering techniques. Before isomerization, trans segments are found to exist mainly as short, isolated sequences independent of Mn. After thermal isomerization, theoretical analysis of the RRS spectra using the Brivio, Mulazzi model indicate the ratio of long trans conjugated segments (N≥30) to short trans conjugated segments (N≤30) is significantly larger for 100 000 Dalton polymer in comparison to polymer of 10 500 Mn and below. For samples below 10 500 Daltons, no clear relationship between actual polymer molecular weight and G is observed. Optimization of the isomerization conditions for 100 000 Dalton polymer results in trans-(CH)x with a G=0.80. These results suggest that not until very long molecular chains are obtained can samples composed principally of long conjugated segments be obtained. It is proposed that defects which arise during and after the polymerization limit the content of long segments. Ambient, short term oxidation of 100 000 Mn polymer shows a decrease in G from 0.80 to 0.70. Low level chain oxidation or doping is shown to preferentially occur within long conjugated segments.

Pulsed radiation studies of photodynamic sensitisers: the nature of DHE.

Abstract Laser flash photolysis has previously been used to study the nature of DHE via measurements of photophysical parameters which are dependent on the molecular weight of the system being studied. These results to date allow only a lower limit to be established for DHE which imply that in some environments such as detergents more than two porphyrin units are linked. We have now determined the triplet extinction coefficient of DHE by the pulse radiolysis technique via an energy transfer method which allows the triplet extinction of DHE to be estimated independent of the molecular weight. The combined techniques allow the actual molecular weight of DHE to be established at about 4200. Laser flash techniques have also now been used to determine, for a number of potential photodynamic sensitisers, the quantum yield of triplet state formation (θT) and, using the direct luminescence of singlet oxygen at 1270 nm, the quantum yield of singlet oxygen formation (θδ). For many of the porphyrins studied θδ is less than θT. For DHE itself there is a substantial increase in θδ in detergent compared to buffer. The θδ yields for a number of related systems including 'simple’systems such as haematoporphyrin, for linked porphyrin‐chlorin systems, (including DHE in which the end porphyrin is reduced to a chlorin–DHEC), and for phthalocyanines are compared. For the DHEC the θδ is close to that of DHE itself which may imply that such chlorins could be of use in photodynamic therapy (PDT).

Relationship between curved DNA conformations and slow gel migration.

We propose some specific DNA conformations that explain, in terms of molecular conformations, the anomalous gel electrophoretic behavior of the sequences (VA4T4X), and (V2A3T3X2)i where V and X are either G or C. Previously (J. Biomole. Struct. Dyn. 4, 41, 1986) we considered hydrophobic interactions among aliphatic hydrocarbon groups in A/T sequences. In the sequences (T)n.(A)n, the T's are slightly bent to yield structures with tightly stacked methyl groups along one side of the major groove. By folding together the two pairs of stacked methyls on the opposite sides of the major groove. TTAA might yield a relatively sharp bend. On this basis, we show below that the sequences (VT4A4X)i might form a very tightly coiled super-helix whereas the sequences (VA4T4X)i form a broad super-helix of radius approximately 120 A for i = 25. The sequence (V2A3T3X2)i forms a slightly smaller radius super-helix. The time of passage through the gel has been taken to be inversely proportional to the smallest dimension of the molecule. Specifically we are taking the ratio of the apparent molecular weight to the actual molecular weight to be related to the moment of inertia I1 about the smallest principal axis of the molecular conformation. We find a good fit to the experimental gel mobility data of Hagerman (2) if we assume this ratio to be proportional to (I1)1/5.

Molecular weight distribution of a bulk ultra‐high molecular weight polyethylene product—impax 5M+ UHMW–NAT

A bulk ultra-high molecular weight polyethylene product, IMPAX 5M + UHMW-NAT, was fractionated using an increasing-temperature sequential-extraction technique. In the presence of an inert atmosphere and an antioxidant, 5 g of material were methodically dissolved in decahydronaphthalene over the temperature range, 80-191 degrees C. Initially, intrinsic viscosities were measured in decalin at 135 degrees C by single point capillary viscometry. From this data the viscosity average molecular weights were estimated using a logarithmic expression. Results showed that the intrinsic viscosities (molecular weights) increased linearly with extraction temperature over a range from 10 to 40 dL/g (1 to 8 X 10(6)) and that the molecular weight distribution was log normal. Mass balances both before and after extraction indicated that less than 0.3% of the material had a molecular weight less than 10(6) and that less than 0.1% of the material was gel. Zero shear viscometry of bulk fractionated polymer and powdered whole polymers confirmed that capillary viscometry increasingly underestimates the true intrinsic viscosities as the polymer chain lengths (molecular shear forces) increase. Indeed, the actual molecular weights ranged from about 2-14 X 10(6). Knowledge of the molecular weight distribution of bulk products and the presence of either volatiles or crosslinked networks is critical for the continued design and development of superior wearing and fatigue-resistant implants.

Isolation of galactosyltransferase from human milk and the determination of its N-terminal amino acid sequence

Galactosyltransferase (EC 2.4.1.22), purified to homogeneity from human milk by affinity chromatography, had an apparent molecular weight of 53,000 as determined by denaturing polyacrylamide gel electrophoresis. Subtration of the estimated contribution of the oligosaccharide portion of the molecule leaves a M r of 47,000. An N-terminal amino acid sequence analysis of the isolated protein revealed a sequence similar to that found near the 5′ end of a cDNA clone isolated by Shaper et al (11), which encodes a 35,500 molecular weight protein. Either the molecular weight of galactosyltransferase, has been overestimated, or a discrepancy exists between the actual molecular weight of galactosyltransferase and that predicted by the bovine cDNA clone isolated by Shaper et al (11).

Physical characterization of PBCV virus by sedimentation field flow fractionation

A virus (PBCV) from the symbiotic Chlorella-like algae of Paramecium bursaria has been characterized by sedimentation field flow fractionation (SdFFF). The method yields effective molecular weight M′ (molecular weight × buoyancy factor) with a precision of better than 5% under a variety of experimental conditions. The value for M′ is used to calculate true molecular weight values through combination with density values; densities were obtained from outside sources, from ultrasonic densitometry, and from sedimentation coefficients determined independently. The actual molecular weight is also determined through SdFFF analysis of the virus in carriers of different densities. Values for the particle diameter are obtained from SdFFF zone broadening studies. The various SdFFF characterization results are compared with molecular weight, particle size, and density values determined by conventional methods.

Separation of complexes containing protein A and IgG or Fcγ fragments by high-performance liquid chromatography

Protein A of Staphylococcus aureus is a bivalent Fc receptor that can form complexes with immunoglobulin G (IgG) or Fcγ fragments that activate humoral (e.g., complement) and cellular (e.g., lymphocyte) components of the immune system both in vitro and in vivo. To obtain complexes formed between protein A of Staphylococcus aureus (SpA) and rabbit IgG or Fcγ fragments for purposes of characterizing their compositions and studying their biological activities, we have used high-performance liquid chromatography to separate complexes in 20 min. Complexes were prepared with trace amounts of 125I-SpA and 131I-IgG or 131I-Fcγ to simplify the analyses. With excess molar amounts of IgG or Fcγ the complexes have the molecular formulas [(IgG) 2SpA] 2 or [(Fcγ) 2SpA] 2. With excess SpA, complexes corresponding to (IgG)(SpA) or (Fcγ)(SpA) are formed, perhaps with other complexes that have different ratios of components. Since SpA is a rod-shaped molecule it elutes at a molecular weight corresponding to 240,000 rather than the true value of 42,000. This behavior is reflected in the elution of certain complexes at shorter retention times than expected on the basis of actual molecular weights, and facilitates separation of complexes from free IgG or Fcγ. The true molecular weights and molecular formulas of complexes isolated by HPLC were verified by ultracentrifugation. This HPLC method was used to study the interconversion and stability of complexes.

Molecular weight measurement of human satietin

Attempts were made to estimate the actual molecular weight of satietin, an endogenous food intake inhibitor, by means of classical and high-performance gel filtration and gel electrophoresis. Satietin, which proved to be a glycoprotein, was isolated from human plasma. Sephacryl S-300 gel and TSK-GEL G 3000 SW columns were used as media for gel filtration and gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE), respectivley. A relatively good correlation was established between the molecular weight measurements carried out by different methods, although satietin as a carbohydrate-rich glycoprotein gave an underestimated value by gel filtration when the calibration graphs were constructed with simple proteins. The molecular weight of satietin was determined to be about 43,000 daltons by SDS-PAGE and 29,000 daltons by gel filtration when using a buffer containing 1% of sodium dodecyl sulphate.

Gel high-performance liquid chromatographic studies on the elution behavior of chemically deglycosylated human chorionic gonadotropin and its subunits

The elution behavior of human chorionic gonadotropin and its subunits, and their desialylated and deglycosylated derivatives was studied on gel high-performance liquid chromatography. Using three TSK G-SW columns, excellent separations were achieved for eleven human chorionic gonadotropin derivatives. In a comparison of the native (30% carbohydrate content), asialo (24%) and HF-deglycosylated (8%) subunits, the estimated values of molecular weights on the basis of a series of reference globular proteins deviated in various degrees from the actual molecular weight. The extent of deviation depended on the carbohydrate content and possibly the location of carbohydrate chains. The data from recombination studies suggest that the β-subunit is the dominant determinant for their expanded molecular size.

Anomalous Electrophoretic Behavior of a Cyanogen Bromide Peptide from Type III Collagen

The relationship between relative mobility on sodium dodecyl sulfate-polyacrylamide gels and the logarithm of molecular weight was linear for peptides derived by CNBr cleavage from purified collagen chains. One striking exception was found: when the CNBr peptide alpha 1(III)CB-8 was subjected to electrophoresis on a polyacrylamide slab gel calibrated with other peptides derived from the type III collagen chain, its relative mobility corresponded to a molecular weight of approximately 8,000. On a gel calibrated with globular proteins, its apparent molecular weight was 12,000. Its actual molecular weight, as determined by gel filtration and by amino acid analysis, is approximately 12,000. The dodecyl sulfate-protein binding ratio for this peptide did not differ from that of other collagenous peptides or globular proteins. Free electrophoretic mobilities and retardation coefficients were determined for selected collagenous peptides and globular proteins by electrophoresis on tube gels of different concentrations of acrylamide. By these indices, alpha 1(III)CB-8 resembled a globular protein rather than a collagenous peptide. Presumably, some difference in primary structure of this peptide results in conformational changes of its complex with dodecyl sulfate, such that the peptide-dodecyl sulfate complex behaves more like a globular protein than a collagenous peptide on polyacrylamide gels.