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Prothrombin

DOMAIN STRUCTURE OF PROTHROMBIN The domain structure of prothrombin is represented, where: GLA = region containing γ-carboxyglutamic acid residues, KRINGLE = regions of internal sequence homology, CATALYTIC DOMAIN = region containing the serine protease catalytic triad. Arrows indicate the sites which are proteolytically cleaved by factor Xa during activation of the zymogen.

 

PURCHASING AND PRODUCT INFORMATION

 

Catalog Number

HCP-0010

HCP1-0010

HCP12-0010

HCP2-0010

HCP1-0011

HCP2-0011*

BCP-1010

BCP1-1010

MCP-5010

Description

Human Prothrombin

Human Prothrombin Frag 1

Human Prothrombin Frag 1.2

Human Prothrombin Frag 2

Human Prethrombin-1

Human Prethrombin-2*

Bovine Prothrombin

Bovine Prothrombin Frag 1

Mouse Prothrombin

Size

2 mg

1 mg

1 mg

1 mg

1 mg

1 mg

2 mg 

1 mg

100 g

Formulation

50% (vol/vol) glycerol/H2O

50% (vol/vol) glycerol/H2O

50% (vol/vol) glycerol/H2O

50% (vol/vol) glycerol/H2O

50% (vol/vol) glycerol/H2O

50% (vol/vol) glycerol/H2O

50% (vol/vol) glycerol/H2O

50% (vol/vol) glycerol/H2O

Hepes buffered saline

* Note:  Naja cleavage of human II is between E262 and D263.  This cleavage yields a "pre-2" product that is 9 amino acids longer than native pre-2.

Storage

-20oC (except mouse prothrombin which is -80oC)

Purity

>95% by SDS-PAGE

NOT tissue/cell culture grade. Not tested for endotoxin.

Activity Determination

clotting assay (for standard Prothrombin only)

Shelf Life (properly stored)

12 months

Sample Gel Information:

Gel: Novex 4-12% Bis-Tris

Load: 1 g per lane; purified human prothrombin

Buffer: MOPS

Standard: SeeBluePlus 2; Myosin (191 kDa), Phosphorylase B (97 kDa), BSA (64 kDa), Glutamic Dehydrogenase (51 kDa), Alcohol Dehydrogenase (39 kDa), Carbonic Anhydrase (28 kDa), Myoglobin Red (19 kDa), Lysozyme (14 kDa)

 

Sample publications referencing our Prothrombin:

  1. Krarup A, Wallis R, Presanis JS, Gal P, Sim RB (2007) Simultaneous Activation of Complement and Coagulation by MBL-Associated Serine Protease 2. PLoS ONE 2(7): e623. doi:10.1371/journal.pone.0000623

This publication list is not all encompassing, and is only meant to provide limited examples of how Haematologic Technologies' products are used.  We encourage you to search the literature for other examples pertinent to your experimentation, and to contact us with any technical questions.

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SAMPLE DATA SHEET

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Overview of Prothrombin

Prothrombin is a vitamin K-dependent plasma protein which is synthesized in the liver (1). Prior to secretion into plasma, prothrombin undergoes post-translational modification by a vitamin K-dependent carboxylase which converts ten specific glutamic acid residues to γ-carboxyglutamic acid (gla). The ten gla residues are located within the first 40 amino acids of the mature protein and contribute to the ability of prothrombin to bind to negatively charged phospholipid membranes. Prothrombin contains two regions of internal homology which are referred to as "kringle" structures. These regions of conspicuous secondary structure are located between residues 40 and 270 of the mature plasma protein and replace the growth factor domains found in several other plasma serine proteases. Thus far, no function has been ascribed to these regions, but there is suspicion that they may play a role in one of several binary protein interactions involving prothrombin. The mature single chain protein circulates in plasma as a zymogen and, during coagulation, is proteolytically activated to the potent serine protease α-thrombin. This proteolysis is catalyzed by the prothrombinase enzyme complex. During activation, prothrombin is cleaved at Arg271-Thr272 (human) / Arg273-Thr274 (bovine) and at Arg320-Ser321 (human) / Arg323-Ser324 (bovine) to a "pro" fragment (fragment 1.2) and thrombin, the latter of which is composed of two chains covalently linked by a disulfide bond.  In the case of human prothrombin/thrombin, there is an additional thrombin feed-back cleavage at Arg284-Thr285 resulting in an additional 13 amino acids being removed from the mature thrombin A chain. 


Human prothrombin is prepared from fresh frozen human plasma as described by Bajaj and coworkers (2). Bovine prothrombin is prepared from fresh bovine plasma using a modification of the procedure described by Owen and coworkers (3). Purified prothrombin is supplied in 50% (vol/vol) glycerol/H2O and should be stored at -20oC. Purity is determined by SDS-PAGE analysis, and activity is measured by clotting and/or chromogenic substrate assay, following conversion of prothrombin to thrombin. 

Properties of Prothrombin

Localization: Plasma
Plasma concentration: 100 g/ml (1)
Mode of action: Zymogen; precursor to the serine protease a-thrombin
Molecular weight: 72,000 (1,4,5). 

Fragments:

Prothrombin Fragment 1 - 21,700

Prothrombin Fragment 2 - 12,866

Prothrombin Fragment 1.2 - 34,566

Prethrombin Fragement 1 - 49,900

Prethrombin Fragment 2 - 37,580

Extinction coefficient:
E
1 %
1 c m, 280 nm
= 13.8 (human) (4)
= 14.4 (bovine) (1)
Isoelectric point: 4.7-4.9 (human) (6)
4.4-4.9 (bovine) (6)
Structure: single chain, NH2-terminal gla domain, two kringle regions
Percent carbohydrate: 8.2 % (human) (4)
10.0 % (bovine) (5)
Post-translational modifications: ten gla residues (4,5)

 References 

1. Mann, K.G., et al., Methods in Enzymology, 45, 156 (1976). 
2. Bajaj, S.P., et al., Prep. Biochem., 11, 397 (1981). 
3. Owen, W.G., et al., J. Biol. Chem., 249, 594 (1974). 
4. Kisiel, W., et al., Biochem. Biophys. Acta, 304, 103 (1973). 
5. Magnusson, S., et al., In Proteases in Biological Control, ed. E. Reich, D.B. Rifkin, E. Shaw, pp. 123-149. New York: Cold Spring Harbor Laboratories, 1975. 
6. Discipio, R.G., et al., Biochemistry, 18, 899 (1979). 

 

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