Detection and Affinity Purification of Oxidant-Sensitive Proteins Using Biotinylated Glutathione Ethyl Ester

Monoclonal antibodies to human acrosin were required for studies of immunological interference with fertilization. Since human acrosin was not available in adequate amounts, monoclonal antibodies have been raised in mice against purified bovine acrosin and screened for cross-reaction with human sperm cells. Two of these antibodies are described, B4F6 and C2E5. Data from enzyme-linked immunosorbent assays, immunoblots, immunoprecipitation, and indirect immunofluorescence on sperm cells indicate that B4F6 binds only to bovine acrosin, and that C2E5 binds both to bovine and to human acrosin at a conformationally determined epitope. The antibodies do not inhibit the hydrolysis of benzoylarginine ethyl ester by acrosin, but C2E5 did inhibit the dissolution of the hamster zona pellucida by purified human acrosin. The antibodies have also been used for affinity purification of acrosin and proacrosin.

Download Full-text

Isolation of Human Fibrinogen and its Derivatives by Affinity Chromatography on Gly-Pro-Arg-Pro-Lys-Fractogel

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645062 ◽

1990 ◽

Vol 63 (03) ◽

pp. 439-444 ◽

Cited By ~ 19

Author(s):

C Kuyas ◽

A Haeberli ◽

P Walder ◽

P W Straub

Keyword(s):

Gel Electrophoresis ◽

Affinity Purification ◽

Polyacrylamide Gel Electrophoresis ◽

Ease Of Use ◽

Terminal Sequence ◽

Purification Method ◽

Human Fibrinogen ◽

Isolation Methods ◽

One Step ◽

Complementary Binding

SummaryWith an immobilized synthetic pentapeptide GlyProArgProLys comprising the N-terminal sequence GlyProArg of the α-chain of fibrin, a new affinity method for the quantitative isolation of fibrinogen out of anticoagulated plasma was developed. The method proved to be superior to all known isolation methods in respect to ease of use and yield, since fibrinogen could be isolated in one step out of plasma with a recovery of more than 95% when compared to the immunologically measurable amounts of fibrinogen. Moreover the amounts of contaminating proteins such as fibronectin, factor XIII or plasminogen were negligible and the purity of the isolated fibrinogen was higher than 95% as measured by polyacrylamide gel electrophoresis. The clottability was 90% and more. Another advantage of this affinity purification method is the possibility to isolate fibrinogen quantitatively out of small plasma samples (<5 ml). Further, abnormal fibrinogen molecules, provided their complementary binding site for GlyProArg is preserved, may also be quantitatively isolated independent of any solubility differences as compared to normal fibrinogen. In addition fibrin(ogcn) fragments originating from plasmic digestion can be separated on the basis of their affinity to GlyProArg. The described affinity gel can be used more than 50 times without any loss of capacity.

Download Full-text

Isolation of Multiple Types of Plasminogen Activator Inhibitors from Vascular Smooth Muscle Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646626 ◽

1989 ◽

Vol 61 (03) ◽

pp. 517-521 ◽

Cited By ~ 15

Author(s):

Walter E Laug ◽

Ruedi Aebersold ◽

Ambrose Jong ◽

Willian Rideout ◽

Barbara L Bergman ◽

...

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Affinity Purification ◽

Muscle Cells ◽

Exchange Chromatography ◽

Natural Resistance ◽

Protease Nexin ◽

Pai 1

SummaryLarge arteries have a natural resistance to tumor cell invasion thought to be due to the production of protease inhibitors. Vascular smooth muscle cells (VSMC) representing the major cellular part of arteries were isolated from human aortas and grown in tissue culture. These cells were found to produce large amounts of inhibitors of plasminogen activators (PA). Fractionation of VSMC-conditioned medium by heparin-affigel chromatography separated three immunologically and functionally distinct PA inhibitors (PAI), namely PAI-1, PAI-2 and protease-nexin I. The three inhibitors were characterized by functional assays and immunoblotting. PA inhibitor 2 (PAI-2) had little affinity for heparin, whereas PA inhibitor 1 (PAI-l) bound to heparin and was eluted from the column at NaCl concentrations of 0. 1 to 0.35 M. Protease-nexin I, eluted at NaCl concentrations of 0.5 M and higher. Most of the PAI-1 was present in the latent, inactive form. PAI-1 was further purified by ion exchange chromatography on a Mono-Q column. Partial sequencing of the purified PAI-1 confirmed its nature by matching completely with the sequence deduced from the cDNA nucleotide sequence of endothelial cell PAI-1. Thus, human VSMC produce all three presently known PAI and these can be separated in a single heparin affinity purification step.

Download Full-text