Effects of the isolation methodology on protein profiles of blood trypomastigotes of Trypanosoma cruzi

Parasitology ◽  
2003 ◽  
Vol 126 (1) ◽  
pp. 41-51 ◽  
Author(s):  
C. HÖLSCHER ◽  
R. HARTMANN ◽  
H. MOSSMANN ◽  
G. A. SCHAUB
Keyword(s):  
Molecular Weight ◽  
Bovine Serum Albumin ◽  
Trypanosoma Cruzi ◽  
Serum Albumin ◽  
High Molecular Weight ◽  
Centrifugal Force ◽  
Bovine Serum ◽  
Exchange Chromatography ◽  
Protein Supplementation ◽  
Surface Proteins

Blood trypomastigotes of Trypanosoma cruzi were isolated from infected athymic rnu/rnu rats and purified by an improved procedure of DEAE-Sephacel ion-exchange chromatography. Elution into a buffer supplemented with bovine serum albumin avoided column-induced changes on the surface of the parasites. Biotin-labelled bovine serum albumin, fluorescence microscopy, flow cytometry and Western blot analysis revealed a very intense binding of albumin to the parasite. Incubation and washing of cells without protein supplementation did not result in any damage or lysis of parasites but it did cause extensive shedding of cellular and surface proteins into the supernatant which could be prevented by using the protein-supplemented buffer. A decreasing yield of high molecular weight cellular proteins in relation to centrifugal force was a general phenomenon observed in scanning densitometry of SDS gels after isolation in either protein-supplemented buffer or protein-free buffer. The quantity of shed cellular components increased with increasing centrifugal force. In contrast, quantities of high molecular weight, biotin-labelled surface proteins increased with greater centrifugal force, indicating labelling of otherwise inaccessible residues. These data emphasize the importance of protein supplementation of buffers with proteins and of choosing low centrifugation forces (<400 g) during investigations of T. cruzi.

scholarly journals Effect of surfaces on fluid-phase prekallikrein activation

Blood ◽  
1981 ◽  
Vol 57 (3) ◽  
pp. 553-560
Author(s):  
CF Scott ◽  
EP Kirby ◽  
PK Schick ◽  
RW Colman
Keyword(s):  
Molecular Weight ◽  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
High Molecular Weight ◽  
Bovine Serum ◽  
Plasma Proteins ◽  
Fluid Phase ◽  
Factor Xii ◽  
Cold Insoluble Globulin ◽  
Kallikrein Activity

The activation of prekallikrein by factor XII fragments (XIIf), during incubation in plastic tubes was previously noted to be increased by high molecular weight (HMW) kininogen as well as other plasma proteins. In this report, we investigated the mechanism responsible for this increase. Although we confirmed that HMW kininogen, bovine serum albumin, fibrinogen, cold insoluble globulin, and mixed phospholipids apparently increased prekallikrein activation, we found that the product of prekallikrein activation (kallikrein) lost substantial activity in less than 0.5 min after exposure to a variety of fresh surfaces. This loss was partially prevented by the presence of various proteins and phospholipids. Similar protection against inactivation of XIIf, the enzyme in this reaction, was also found. In contrast, no loss of the substrate, prekallikrein, was observed during incubation. The loss of kallikrein activity was found to be proportional to the surface area of the incubation vessel as well as the concentration of kallikrein. Further loss of kallikrein activity could also be prevented by pretreating the vessel with kallikrein. We therefore conclude that various substances apparently affect prekallikrein activation in a purified system by preventing the enzyme and product in the reaction mixture from losing activity due to adsorption to a surface.

scholarly journals Effect of surfaces on fluid-phase prekallikrein activation

Blood ◽  
1981 ◽  
Vol 57 (3) ◽  
pp. 553-560 ◽  
Author(s):  
CF Scott ◽  
EP Kirby ◽  
PK Schick ◽  
RW Colman
Keyword(s):  
Molecular Weight ◽  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
High Molecular Weight ◽  
Bovine Serum ◽  
Plasma Proteins ◽  
Fluid Phase ◽  
Factor Xii ◽  
Cold Insoluble Globulin ◽  
Kallikrein Activity

Abstract The activation of prekallikrein by factor XII fragments (XIIf), during incubation in plastic tubes was previously noted to be increased by high molecular weight (HMW) kininogen as well as other plasma proteins. In this report, we investigated the mechanism responsible for this increase. Although we confirmed that HMW kininogen, bovine serum albumin, fibrinogen, cold insoluble globulin, and mixed phospholipids apparently increased prekallikrein activation, we found that the product of prekallikrein activation (kallikrein) lost substantial activity in less than 0.5 min after exposure to a variety of fresh surfaces. This loss was partially prevented by the presence of various proteins and phospholipids. Similar protection against inactivation of XIIf, the enzyme in this reaction, was also found. In contrast, no loss of the substrate, prekallikrein, was observed during incubation. The loss of kallikrein activity was found to be proportional to the surface area of the incubation vessel as well as the concentration of kallikrein. Further loss of kallikrein activity could also be prevented by pretreating the vessel with kallikrein. We therefore conclude that various substances apparently affect prekallikrein activation in a purified system by preventing the enzyme and product in the reaction mixture from losing activity due to adsorption to a surface.

scholarly journals REAGINIC ACTIVITY ASSOCIATED WITH IGG IMMUNOGLOBULIN

1966 ◽  
Vol 123 (5) ◽  
pp. 845-858 ◽  
Author(s):  
Robert T. Reid ◽  
Percy Minden ◽  
Richard S. Farr
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Anion Exchange ◽  
Bovine Serum ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Unique Property ◽  
Serum Fractions ◽  
Human Sera

Five human sera with reaginic activity to a number of allergens were fractionated using anion exchange chromatography. In each serum, fractions which contained detectable IgG and no detectable IgA had capacity to fix to skin and subsequently elicit a P-K reaction. Four of these sera had reaginic activity about equally distributed between fractions containing only IgG and fractions containing mixtures of IgG and IgA. A fifth serum contained reaginic activity to crystalline bovine serum albumin (BSA) and most of the activity was associated with the fraction which contained only IgG. This serum was extensively studied using a variety of techniques and it was confirmed that most of the reagin to BSA in this serum was in those fractions containing only IgG. Since reaginic activity can no longer be considered a unique property of IgA the implications of finding antibody with reaginic qualities in immunoglobulins other than IgA are discussed.

The production of polyclonal antibodies against the mycotoxin derivative patulin hemiglutarate

1993 ◽  
Vol 39 (9) ◽  
pp. 861-863 ◽  
Author(s):  
L. J. McElroy ◽  
C. M. Weiss
Keyword(s):  
Molecular Weight ◽  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Antibody Titer ◽  
Bovine Serum ◽  
Polyclonal Antibodies ◽  
Competitive Elisa ◽  
Low Molecular Weight ◽  
Specific Antibodies ◽  
Indirect Competitive Elisa

The mycotoxin patulin is a toxic, carcinogenic, unsaturated lactone produced by a number of molds. Polyclonal antibodies against patulin hemiglutarate were produced. Specific antibodies against patulin alone, however, were not clearly demonstrated. Because of its low molecular weight, patulin required conjugation to bovine serum albumin (BSA) to increase its immunogenicity. Anti-patulin-hemiglutarate-BSA antibody titer and specificity were determined using indirect and indirect competitive ELISA, respectively. Immunoassays would facilitate detection and quantitation of patulin.Key words: patulin, mycotoxin, antibodies.

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