scholarly journals Iodine-mediated inactivation of lipid- and nonlipid-enveloped viruses in human antithrombin III concentrate

Blood ◽  
1995 ◽  
Vol 86 (2) ◽  
pp. 791-796 ◽  
Author(s):  
F Highsmith ◽  
H Xue ◽  
X Chen ◽  
L Benade ◽  
J Owens ◽  
...  
Keyword(s):  
Sindbis Virus ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Albumin ◽  
Enveloped Viruses ◽  
Complete Inactivation ◽  
Ensure Patient Safety ◽  
Sds Page ◽  
At Iii

Human plasma-derived protein concentrates intended for clinical use must be treated for viral inactivation to ensure patient safety. This study explored the use of liquid iodine for inactivation of several lipid- and nonlipid-enveloped viruses in an antithrombin III (AT-III) concentrate. Iodine at levels of 0.01% to 0.02% caused between 43% and 94% loss of AT-III activity, as well as degradation of AT-III as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. However, addition of up to 0.1% human albumin protected the AT-III against both inactivation and fragmentation. At albumin levels sufficient to retain greater than 75% of AT-III activity, greater than 6 logs of sindbis, encephalomyocarditis, and vesicular stomatitis viruses, greater than 4 logs of pseudorabies, and greater than 3 logs of human immunodeficiency virus were inactivated. Except with sindbis virus, this represented complete inactivation of all the viruses spiked into the AT-III concentrate.

scholarly journals Antithrombin Milano: a new variant with monomeric and dimeric inactive antithrombin III

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 496-500 ◽  
Author(s):  
M Wolf ◽  
C Boyer ◽  
A Tripodi ◽  
D Meyer ◽  
MJ Larrieu ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Two Dimensional ◽  
Western Blots ◽  
Sds Page ◽  
New Variant ◽  
At Iii ◽  
Monospecific Antisera

Abstract A qualitative defect of antithrombin III (AT III) has been demonstrated over three generations in eight members of an Italian family by the discrepancy between a normal amount of antigen and decreased antithrombin and anti-Xa activity in the presence or in the absence of heparin. By two-dimensional immunoelectrophoresis in the absence of heparin, two peaks of AT III were present in all patients' plasma. AT III was purified from normal and propositus plasma by sulfate dextran precipitation followed by heparin affinity chromatography. The elution profile of the patient's AT III was abnormal and allowed the separation of two populations of AT III, normal and abnormal. The first fraction (normal AT III) contained AT III activity, migrated as a single peak by two-dimensional immunoelectrophoresis and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), demonstrated a single band with a molecular weight (mol wt) identical to that of normal AT III (60,000). Conversely, the last fraction, devoid of AT III activity, migrated as a single abnormal peak by two-dimensional immunoelectrophoresis in the absence of heparin. By SDS-PAGE, two bands were observed: one with a mol wt of 60,000 and a second one with a mol wt of 120,000. Western blots clearly demonstrated cross-reactivity of the 120,000 and 60,000 mol wt bands with monospecific antisera to human AT III. Reduction of the 120,000 mol wt band converted it to a single 60,000 mol wt band, suggesting the presence of an abnormal dimeric form of AT III. The name AT III Milano is proposed for this new variant.

scholarly journals Antithrombin Milano: a new variant with monomeric and dimeric inactive antithrombin III

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 496-500
Author(s):  
M Wolf ◽  
C Boyer ◽  
A Tripodi ◽  
D Meyer ◽  
MJ Larrieu ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Two Dimensional ◽  
Western Blots ◽  
Sds Page ◽  
New Variant ◽  
At Iii ◽  
Monospecific Antisera

A qualitative defect of antithrombin III (AT III) has been demonstrated over three generations in eight members of an Italian family by the discrepancy between a normal amount of antigen and decreased antithrombin and anti-Xa activity in the presence or in the absence of heparin. By two-dimensional immunoelectrophoresis in the absence of heparin, two peaks of AT III were present in all patients' plasma. AT III was purified from normal and propositus plasma by sulfate dextran precipitation followed by heparin affinity chromatography. The elution profile of the patient's AT III was abnormal and allowed the separation of two populations of AT III, normal and abnormal. The first fraction (normal AT III) contained AT III activity, migrated as a single peak by two-dimensional immunoelectrophoresis and by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), demonstrated a single band with a molecular weight (mol wt) identical to that of normal AT III (60,000). Conversely, the last fraction, devoid of AT III activity, migrated as a single abnormal peak by two-dimensional immunoelectrophoresis in the absence of heparin. By SDS-PAGE, two bands were observed: one with a mol wt of 60,000 and a second one with a mol wt of 120,000. Western blots clearly demonstrated cross-reactivity of the 120,000 and 60,000 mol wt bands with monospecific antisera to human AT III. Reduction of the 120,000 mol wt band converted it to a single 60,000 mol wt band, suggesting the presence of an abnormal dimeric form of AT III. The name AT III Milano is proposed for this new variant.

scholarly journals Purified radiolabeled antithrombin III metabolism in three families with hereditary AT III deficiency: application of a three-compartment model

Blood ◽  
1986 ◽  
Vol 67 (1) ◽  
pp. 93-98
Author(s):  
EA Knot ◽  
E de Jong ◽  
JW ten Cate ◽  
AH Iburg ◽  
CP Henny ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Compartment Model ◽  
Control Group ◽  
Surface Binding ◽  
Catabolic Rate ◽  
At Iii ◽  
Three Compartment Model ◽  
The Mean

Purified human radioiodinated antithrombin III (125I-AT III) was used to study its metabolism in six members from three different families with a known hereditary AT III deficiency. Six healthy volunteers served as a control group. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and crossed immunoelectrophoresis (CIE) showed the purified AT III to be homogeneous. Amino acid analysis of the protein revealed a composition identical to a highly purified internal standard. The specific activity was 5.6 U/mg. Analysis of plasma radioactivity data was performed, using a three-compartment model. Neither plasma disappearance half-times nor fractional catabolic rate constants differed significantly between patients and control subjects. The mean absolute catabolic rate in the patient group was significantly lower than that of the control group at 2.57 +/- 0.44 and 4.46 +/- 0.80 mg/kg/day, respectively. In addition, the mean patient alpha 1-phase, flux ratio (k1,2 and k2,1) of the second compartment alpha 2-phase and influx (k3,1) of the third compartment were significantly reduced as compared with control values. It has been tentatively concluded that the observed reduction in the second compartment may be caused by a decrease in endothelial cell surface binding.

scholarly journals Antithrombin III Budapest: a single amino acid substitution (429Pro to Leu) in a region highly conserved in the serpin family

Blood ◽  
1992 ◽  
Vol 79 (5) ◽  
pp. 1206-1212 ◽  
Author(s):  
RJ Olds ◽  
DA Lane ◽  
R Caso ◽  
M Panico ◽  
HR Morris ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Sds Page ◽  
Additional Support ◽  
Variant Protein

Abstract Antithrombin III (AT) is a major plasma serine protease inhibitor and a member of the serpin family of proteins. We have characterized the molecular and genetic basis of AT Budapest, an inherited variant of AT that is associated with thrombotic disease in affected family members. A single amino acid substitution, 429Pro to Leu, was identified, occurring in a region of the molecule that is highly conserved in members of the serpin family. Two forms of variant protein were present in approximately equal amounts in the plasma of the propositus, who is homozygous for the mutation. One form, which had apparently normal Mr, bound heparin strongly and retained some residual thrombin inhibitory activity. The other form had only weak heparin affinity and no antiproteinase activity, and had slightly decreased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions; this normalized in the presence of a reducing agent, suggesting it was caused by a change in conformation. Additional support for a difference in conformation of the two forms of variant was provided by the finding that the fraction that bound heparin- Sepharose was recognized by a monoclonal antibody raised against normal AT, whereas the weak-affinity fraction was not.

scholarly journals Antithrombin III Budapest: a single amino acid substitution (429Pro to Leu) in a region highly conserved in the serpin family

Blood ◽  
1992 ◽  
Vol 79 (5) ◽  
pp. 1206-1212 ◽  
Author(s):  
RJ Olds ◽  
DA Lane ◽  
R Caso ◽  
M Panico ◽  
HR Morris ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Sds Page ◽  
Additional Support ◽  
Variant Protein

Antithrombin III (AT) is a major plasma serine protease inhibitor and a member of the serpin family of proteins. We have characterized the molecular and genetic basis of AT Budapest, an inherited variant of AT that is associated with thrombotic disease in affected family members. A single amino acid substitution, 429Pro to Leu, was identified, occurring in a region of the molecule that is highly conserved in members of the serpin family. Two forms of variant protein were present in approximately equal amounts in the plasma of the propositus, who is homozygous for the mutation. One form, which had apparently normal Mr, bound heparin strongly and retained some residual thrombin inhibitory activity. The other form had only weak heparin affinity and no antiproteinase activity, and had slightly decreased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions; this normalized in the presence of a reducing agent, suggesting it was caused by a change in conformation. Additional support for a difference in conformation of the two forms of variant was provided by the finding that the fraction that bound heparin- Sepharose was recognized by a monoclonal antibody raised against normal AT, whereas the weak-affinity fraction was not.

Mechanism Of The Inactivation Of Trypsin By Antithrombin III

1981 ◽  
Author(s):  
A Danielsson ◽  
I Björk
Keyword(s):  
Disulfide Bonds ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
General Mechanism ◽  
Reducing Conditions ◽  
Sds Page ◽  
Activity Measurements ◽  
General Aspects ◽  
Modified Forms

The reaction between bovine antithrombin III (AT) and bovine trypsin was'studied and compared to the inactivation of thrombin by the inhibitor in order to elucidate general aspects of the mechanism of AT action. AT and trypsin formed inactive 1:1 complexes, as determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/ PAGE) and trypsin activity measurements. In the absence of heparin, the reaction was about 100-fold faster than the AT-thrombin reaction. The reaction rate increased when AT was preincubated with heparin before trypsin was added. Purified AT-trypsin complex dissociated at pH 10 into free enzyme and two proteolytically modified forms of AT, while no intact AT appeared. In SDS/PAGE, the two modified inhibitors gave bands corresponding to apparent molecular weights of 52000 and 48000 under reducing conditions, while both forms co-migrated with intact AT (mol wt 56000) under non-reducing conditions. This indicates that each of the two modified forms of AT had been cleaved into two or more chains held together by disulfide bonds. Under reducing conditions, the larger of the modified AT chains co-migrated grated with the large chain of thrombin-modified AT, i.e. the form of AT which dissociates from the AT-thrombin complex and which is cleaved at the reactive Arg-Ser bond of the inhibitor. Control experiments showed that the smaller of the two chains was formed by tryptic cleavage of the larger chain. Antisera specific for thrombin-modified AT reacted with purified AT-trypsin complex, demonstrating that the inhibitor was present in the complex in a form immunologically identical to thrombin-modified AT. An analogous finding has been reported earlier for the anti thrombin- thrombin complex. Together, these results suggest the same general mechanism for inhibition of trypsin and thrombin by AT.

scholarly journals Prothrombin Tokushima: characterization of dysfunctional thrombin derived from a variant of human prothrombin

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 565-569
Author(s):  
T Inomoto ◽  
A Shirakami ◽  
S Kawauchi ◽  
T Shigekiyo ◽  
S Saito ◽  
...  
Keyword(s):  
Active Site ◽  
Binding Sites ◽  
Gel Electrophoresis ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor Xa ◽  
Molecular Defect ◽  
Sds Page

A mutant prothrombin, designated prothrombin Tokushima, was purified from plasma of a proband with 12% of normal plasma clotting activity and 42% of normal prothrombin antigen. The purified preparation gave a single band with the same mobility as that of “prothrombin” by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The factor Xa-catalyzed proteolysis of prothrombin Tokushima examined by SDS-PAGE was found to be identical to that of “prothrombin.” Subsequently thrombin Tokushima was prepared by CM-Sepharose CL-6B column chromatography after prothrombin activation by factor Xa. The molecular weight of thrombin Tokushima estimated by SDS-PAGE was identical to that of “thrombin.” Thrombin Tokushima exhibited less than 22% of normal clotting activity, and the value of kcat/Km (mumol/L-1 second-1) was less than one tenth of that of “thrombin” when Boc-Val- Pro-Arg-4-methylcoumaryl-7-amide was used as a substrate. However, active site titration using p-nitrophenyl-p′-guanidinobenzoate failed to detect any difference between the two. Thrombin Tokushima was 2.5% as effective as “thrombin” in inducing platelet aggregation. Interaction of thrombin Tokushima with antithrombin III was much slower than “thrombin” when followed by SDS-PAGE. Based on the residual thrombin activity, it was 33% as effective as “thrombin” in forming a complex with antithrombin III. These results indicate that the molecular defect resides in the thrombin portion of prothrombin Tokushima and that the binding sites for various substrates appear to be greatly impaired.

scholarly journals Prothrombin Tokushima: characterization of dysfunctional thrombin derived from a variant of human prothrombin

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 565-569 ◽  
Author(s):  
T Inomoto ◽  
A Shirakami ◽  
S Kawauchi ◽  
T Shigekiyo ◽  
S Saito ◽  
...  
Keyword(s):  
Active Site ◽  
Binding Sites ◽  
Gel Electrophoresis ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor Xa ◽  
Molecular Defect ◽  
Sds Page

Abstract A mutant prothrombin, designated prothrombin Tokushima, was purified from plasma of a proband with 12% of normal plasma clotting activity and 42% of normal prothrombin antigen. The purified preparation gave a single band with the same mobility as that of “prothrombin” by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The factor Xa-catalyzed proteolysis of prothrombin Tokushima examined by SDS-PAGE was found to be identical to that of “prothrombin.” Subsequently thrombin Tokushima was prepared by CM-Sepharose CL-6B column chromatography after prothrombin activation by factor Xa. The molecular weight of thrombin Tokushima estimated by SDS-PAGE was identical to that of “thrombin.” Thrombin Tokushima exhibited less than 22% of normal clotting activity, and the value of kcat/Km (mumol/L-1 second-1) was less than one tenth of that of “thrombin” when Boc-Val- Pro-Arg-4-methylcoumaryl-7-amide was used as a substrate. However, active site titration using p-nitrophenyl-p′-guanidinobenzoate failed to detect any difference between the two. Thrombin Tokushima was 2.5% as effective as “thrombin” in inducing platelet aggregation. Interaction of thrombin Tokushima with antithrombin III was much slower than “thrombin” when followed by SDS-PAGE. Based on the residual thrombin activity, it was 33% as effective as “thrombin” in forming a complex with antithrombin III. These results indicate that the molecular defect resides in the thrombin portion of prothrombin Tokushima and that the binding sites for various substrates appear to be greatly impaired.

scholarly journals Expression in a cell-free system of normal and variant forms of human antithrombin III. Ability to bind heparin and react with alpha-thrombin

Blood ◽  
1990 ◽  
Vol 76 (8) ◽  
pp. 1521-1529
Author(s):  
RC Austin ◽  
RA Rachubinski ◽  
F Fernandez-Rachubinski ◽  
MA Blajchman
Keyword(s):  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Translation Product ◽  
Free System ◽  
Reactive Center ◽  
At Iii ◽  
Free Translation

Human antithrombin III (AT-III) cDNA was cloned into the cell-free expression phagemid vector pGEM-3Zf(+) and site-directed mutagenesis was used to remove nucleotides encoding the signal peptide. AT-III messenger RNA (mRNA) transcripts derived from this construct were translated in an mRNA-dependent rabbit reticulocyte lysate (RRL) system containing (35S)methionine. Immunoprecipitation of the cell-free translation mixture with rabbit polyclonal antibodies to AT-III showed, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE), a 47-Kd polypeptide which is the non-glycosylated mature form of plasma AT-III. Densitometric scanning showed that this polypeptide constitutes greater than 90% of the radiolabeled polypeptides produced in this system. Heparin-Sepharose chromatography resulted in the elution of cell-free derived AT-III as a broad peak between 0.2 and 0.7 mol/L NaCl. The cell-free derived AT-III also reacted with human alpha- thrombin. In 2 minutes approximately 20% of the AT-III was found associated with a higher molecular weight species, consistent with the formation of a 1:1 stoichiometric covalent complex between alpha- thrombin and AT-III. Unfractionated heparin accelerated the rate of formation of such complexes. When Ser394 was mutated to Leu to form the AT-III Denver mutant, the cell-free translation product of this mutation did not show any significant complex formation when reacted with alpha-thrombin. A truncated form of AT-III (Met251-Lys432), containing only the putative thrombin-binding domain, was synthesized independently. This 21-Kd polypeptide did not bind heparin; however, it was cleaved by alpha-thrombin presumably at the reactive center Arg393- Ser394. When Ser394 was mutated to Leu the cell-free translation product of this truncated AT-III mutation did not react with alpha- thrombin at the reactive center. This simple cell-free approach, along with site-directed mutagenesis, should allow for the rapid and accurate mapping of the functional domains of human AT-III.

scholarly journals A purified lectin with larvicidal activity from a woodland mushroom, Agaricus semotus Fr.

2021 ◽  
Vol 65 (1) ◽  
pp. 65-73
Author(s):  
Isaiah O. Adedoyin ◽  
Taiwo S. Adewole ◽  
Titilayo O. Agunbiade ◽  
Francis B. Adewoyin ◽  
Adenike Kuku
Keyword(s):  
Larvicidal Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Complete Inactivation ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Effects Of Temperature

This study investigated the larvicidal activity on Culex quinquefasciatus of lectin purified from fresh fruiting bodies of woodland mushroom, Agaricus semotus. A. semotus lectin (ASL) was purified via ion-exchange chromatography on DEAE-cellulose A-25 and size exclusion chromatography on Sephadex G-100 matrix. Molecular weight (16.6 kDa) was estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The effects of temperature, pH, metal chelation- and larvicidal activity of ASL were also investigated. The ASL indifferently agglutinated the erythrocytes of the human ABO blood system and was stable at acidic pH and below 50 °C whereas 66% of its activity was lost at 60 °C with complete inactivation at 70 °C. ASL is a metalloprotein requiring barium ion as chelation of metals by 50 mM EDTA rendered the lectin inactive, while the addition of BaCl2, among other metal salts, restored the activity. ASL showed larvicidal activity against C. quinquefasciatus larvae after 24 h with a mortality of 5 and 95% at 5 and 25 mg/mL respectively, and LC50 of 13.80 mg/mL. This study concluded that purified A. semotus lectin showed impressive larvicidal activity, which could be exploited in its development as an insecticidal agent.

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