scholarly journals Computer simulation of protein self-association during small-zone gel filtration. Estimation of equilibrium constants

1981 ◽  
Vol 195 (1) ◽  
pp. 213-219 ◽  
Author(s):  
F J Stevens ◽  
M Schiffer
Keyword(s):  
Computer Simulation ◽  
Association Studies ◽  
Equilibrium Constants ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Light Chains ◽  
Immunoglobulin Light Chains ◽  
Bence Jones Protein ◽  
Self Association ◽  
Individual Column

A simulation is developed that qualitatively describes the small-zone-gel-filtration behaviour of a reversibly associating protein. The results reflect the dependence of the apparent molecular weight of a reversibly associating protein on the equilibrium constant (KD) and initial concentration of the protein as well as the column length. The behaviour of a protein on an individual column is characterized and thus a means is provided for estimation of KD. The procedure is extended to describe the behaviour of a mixture of two proteins capable of heterologous as well as homologous association. This computer simulation has been applied in association studies of immunoglobulin light chains [Stevens, Westholm, Solomon & Schiffer (1980) Proc. Natl. Acad. Sci. 77, 1144--1148]. The KD value determined for the Bence--Jones protein Au (10(5) M-1) is close to the value (6.6 X 10(4) M-1) determined by other methods [Maeda, Steffen & Engel (1978) Biophys. Chem. 9, 57-64].

The development of chromatography for the characterization of protein interactions: a personal perspective

2003 ◽  
Vol 31 (5) ◽  
pp. 1010-1014 ◽  
Author(s):  
D.J. Winzor
Keyword(s):  
Ligand Binding ◽  
Protein Interactions ◽  
Equilibrium Constants ◽  
Gel Filtration ◽  
Personal Perspective ◽  
Self Association ◽  
Quantitative Procedure ◽  
Insight Into

This article reviews the progress of a personal endeavour to develop chromatography as a quantitative procedure for the determination of reaction stoichiometries and equilibrium constants governing protein interactions. As well as affording insight into an aspect of chromatography with which many protein chemists are unfamiliar, it shows the way in which minor adaptations of conventional chromatographic practices have rendered the technique one of the most powerful methods available for the characterization of interactions. That pathway towards quantification is followed from the introduction of frontal gel filtration for the study of protein self-association to the characterization of ligand binding by the biosensor variant of quantitative affinity chromatography.

Historical perspectives in clinical pathology: Bence Jones protein—early urine chemistry and the impact on modern day diagnostics

2020 ◽  
pp. jclinpath-2020-206675
Author(s):  
Sheromna Sewpersad ◽  
Tahir S Pillay
Keyword(s):  
Light Chain ◽  
Clinical Pathology ◽  
Light Chains ◽  
Immunoglobulin Light Chains ◽  
Bence Jones Protein ◽  
Historical Perspectives ◽  
Serum Free Light Chain ◽  
The Impact ◽  
Made In ◽  
Bence Jones Proteins

This is the third in the series of historical articles dealing with developments in clinical pathology. Bence Jones proteins are immunoglobulin light chains found in excessive quantities in urine in multiple myeloma and are believed to be one of the first tumour markers ever discovered . Dr Henry Bence Jones is credited with the discovery of this protein in 1847 that bears his name and he can also be regarded as the first chemical pathologist/clinical chemist. Since then, numerous advances and refinements have been made in the measurement and detection of urine light chain proteins which have resulted in the current sensitive serum free light chain assays used today.

Interference of polyclonal free light chains with identification of Bence Jones proteins

1993 ◽  
Vol 39 (8) ◽  
pp. 1734-1738 ◽  
Author(s):  
P P Hess ◽  
W Mastropaolo ◽  
G D Thompson ◽  
S S Levinson
Keyword(s):  
Gel Filtration ◽  
Double Diffusion ◽  
Light Chains ◽  
Gel Chromatography ◽  
Bence Jones Protein ◽  
Free Light Chains ◽  
Isoelectric Points ◽  
Immunofixation Electrophoresis ◽  
Sample Dilution ◽  
Bence Jones Proteins

Abstract We present a case in which kappa free light chains caused difficulty in interpreting classical urinary immunoelectrophoresis, but immunofixation electrophoresis (IFE) demonstrated the presence of a lambda-Bence Jones protein. Analysis of the urine by Ouchterlony double diffusion and IFE after gel-filtration chromatography showed that the difficulty was caused by the presence of large amounts of polyclonal free light chains. The workup also demonstrated that although IFE is the more sensitive and specific technique, IFE performed on concentrated urinary samples is especially subject to misinterpretation unless densely staining patterns are diluted and reassayed. This process of sample dilution provides a means for titrating antigen and antibody concentrations such that condition-specific patterns become visible on the gel. This workup also shows that, at some dilutions, polyclonal free light chains may migrate in the same manner as an oligoclonal band in a so-called ladder configuration. These bands were observed from both monomeric and dimeric fractions isolated by gel chromatography, consistent with reports that this pattern is largely linked to the isoelectric points of the molecules. We speculate that, in rare instances, the distinction between polyclonal and monoclonal kappa free light chains migrating as a ladder-banding pattern may be equivocal.

Restricted electrophoretic heterogeneity of immunoglobulin light chains in urine: a cause for confusion with Bence Jones protein

1991 ◽  
Vol 37 (9) ◽  
pp. 1570-1574 ◽  
Author(s):  
E M MacNamara ◽  
F Aguzzi ◽  
C Petrini ◽  
J Higginson ◽  
C Gasparro ◽  
...  
Keyword(s):  
Light Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Light Chains ◽  
Immunoglobulin Light Chains ◽  
Healthy Individuals ◽  
Tubular Proteinuria ◽  
Bence Jones Protein ◽  
Protein Reabsorption ◽  
Renal Tubular

Abstract The detection of Bence Jones protein, an important part of the investigation of suspected myeloma, is most commonly done by agarose or cellulose nitrate electrophoresis followed by immunofixation. Bence Jones protein is recognized as single or multiple bands of one type of light chain. Unfortunately, improvements in sensitivity of these techniques (use of high-affinity antisera and higher resolution electrophoresis) frequently allow detection of multiple light chain bands in the urine of patients who do not have a B-cell dyscrasia. The bands are usually kappa, although they may be accompanied by lambda bands. This pattern may lead to the misdiagnosis of Bence Jones protein and oligoclonal light chain production in patients. Here we show that this pattern is produced by polyclonal light chains; it is present in the urine of all patients with a tubular proteinuria of any etiology and may be induced in healthy individuals by blocking their renal tubular protein reabsorption. Polyclonal light chains separate into monomers and dimers on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and into four major bands with many minor bands by isoelectric focusing. This difference in charge and possibly size results in the banding pattern seen on good-quality electrophoresis and immunofixation.

Physico-Chemical Properties of Recombinant Desulphatohirudin

1990 ◽  
Vol 63 (03) ◽  
pp. 499-504 ◽  
Author(s):  
A Electricwala ◽  
L Irons ◽  
R Wait ◽  
R J G Carr ◽  
R J Ling ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Chemical Properties ◽  
Alpha Helix ◽  
Apparent Molecular Weight ◽  
Correlation Spectroscopy ◽  
Nmr Studies ◽  
Recombinant Hirudin ◽  
Physico Chemical ◽  
Recombinant Molecule

SummaryPhysico-chemical properties of recombinant desulphatohirudin expressed in yeast (CIBA GEIGY code No. CGP 39393) were reinvestigated. As previously reported for natural hirudin, the recombinant molecule exhibited abnormal behaviour by gel filtration with an apparent molecular weight greater than that based on the primary structure. However, molecular weight estimation by SDS gel electrophoresis, FAB-mass spectrometry and Photon Correlation Spectroscopy were in agreement with the theoretical molecular weight, with little suggestion of dimer or aggregate formation. Circular dichroism studies of the recombinant molecule show similar spectra at different pH values but are markedly different from that reported by Konno et al. (13) for a natural hirudin-variant. Our CD studies indicate the presence of about 60% beta sheet and the absence of alpha helix in the secondary structure of recombinant hirudin, in agreement with the conformation determined by NMR studies (17)

Mechanism of Inhibitory Action on Platelet Activation of a Phospholipase A2 Isolated from Lachesis muta (Bushmaster) Snake Venom

1997 ◽  
Vol 78 (05) ◽  
pp. 1372-1380 ◽  
Author(s):  
André L Fuly ◽  
Olga L T Machado ◽  
Elias W Alves ◽  
Célia R Carlinis
Keyword(s):  
Platelet Aggregation ◽  
Enzymatic Activity ◽  
Phospholipase A2 ◽  
Inhibitory Activity ◽  
Thermal Inactivation ◽  
Anticoagulant Activity ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Crude Venom ◽  
Lachesis Muta

SummaryCrude venom from Lachesis muta exhibited procoagulant, proteolytic and phospholipase A2 activities. A phospholipase A2, denoted LM-PLA2 was purified from L. muta venom to homogeneity, through a combination of chromatographic steps involving gel-filtration on Sephacryl S-200 HR and reverse phase chromatography on a C2/C18 column. LM-PLA2 presented a single polypeptide chain with an isoelectric point at pH 4.7 and apparent molecular weight of 17 kDa. Partial aminoacid sequence indicated a high degree of homology for LM-PLA2 with other PLA2 from different sources.LM-PLA2 displayed a potent enzymatic activity as measured by indirect hemolysis of red blood cells but it was neither lethal when injected i.p. into mice nor did it present anticoagulant activity. Furthermore, LM-PLA2 displayed a moderate inhibitory activity on the aggregation of rabbit platelets induced by low levels of ADP, thrombin and arachidonate. In contrast, platelet aggregation induced by high doses of collagen was strongly inhibited by LM-PLA2 as well as ATP-release. Treatment of the protein with p-bromophenacyl bromide or 2-mercapto-ethanol, as well as thermal inactivation studies, suggested that the platelet inhibitory effect of LM-PLA2 is dependent on its enzymatic activity. Thus, the platelet inhibitory activity of LM-PLA2 was shown to be dependent on the hydrolysis of plasma phospholipids and/or lipoproteins, most probably those rich in phosphatidylcholine. Surprisingly, lyso-phosphatidylcholine released by LM-PLA2 from plasma was shown to preferentially inhibited collagen-induced platelet aggregation, in contrast to other PLA2s, whose plasma hydrolytic products indistinctly affect platelet’s response to several agonists.

Diversity of expressed V and J regions of immunoglobulin light chains in Xenopus laevis

1993 ◽  
Vol 23 (8) ◽  
pp. 1980-1986 ◽  
Author(s):  
Sue E. Stewart ◽  
Louis Du Pasquier ◽  
Lisa A. Steiner
Keyword(s):  
Xenopus Laevis ◽  
Light Chains ◽  
Immunoglobulin Light Chains
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