2 Plasma Derived and Enzymes

Despite their complexity, blood and plasma are abundant biological resources for the discovery of drug targets and biomarkers for human . It is estimated that plasma may contain as many as 40,000 different proteins from about 500 products. Sigma-Aldrich has been isolating and purifying plasma and blood derived proteins for more than 40 years. As an original manufacturer, we offer the world's largest portfolio of proteins for research applications. Many of these proteins are produced on a large-scale custom basis under GMP for our manufacturing customers. For more extensive information on individual products and additional technical resources on plasma and blood related proteins and enzymes visit the Enzyme Explorer at sigma-aldrich.com/enzymeexplorer.

Plasma Composition Proteins Plasma and Blood Related Protein Levels in Normal Plasma Protein Levels in Normal Plasma continued Amount in Normal Plasma Amount in Normal Plasma Protein (mg/100 mL) Protein (mg/100 mL) α1-Acid 55–140 3.1 S Leucine-rich α2-glycoprotein 2–3 3500–5000 3.8 S Histidine-rich α2-glycoprotein 5–15 α1-Antichymotrypsin 30–60 4 S α2, β1-Glycoprotein Trace Antihemophilic factor (Factor VIII) (1–2) 8 S α3-Glycoprotein 3–5 III (20–40) Zn-α2-Glycoprotein 2–15 α1-Antitrypsin 200–400 α1B-Glycoprotein 15–30 Basic protein B1 – α1T-Glycoprotein 5–12 Basic protein B2 <1 α2HS-Glycoprotein 40–85 γ Bence Jones protein Trace 9.5 S α1-Glycoprotein (serum P protein) 3–8 κ Bence Jones protein Trace β2-Glycoprotein I 15–30 15–60 β2-Glycoprotein II 12–30 Cholinesterase 0.5–1.5 β2-Glycoprotein III 5–15 Complement C1 Esterase inhibitor 15–35 Type 2–1 160–300 Complement C1r Component – Haptoglobin Type 1–1 100–220 Complement C4 Binding Protein – Haptoglobin Type 2–2 120–260 Complement C8 Component 8 50–115 Complement C9 Component 23 Immunoglobulin A 90–450 Complement Factor B Immunoglobulin D <15 Complement Factor D Immunoglobulin E <0.06 Complement Factor H Immunoglobulin G 800–1800 Complement C1q Component 10–25 Immunoglobulin M 60–250 Complement C1s Component 1–2 Inter-α- inhibitor 20–70 Complement C3 Component 55–120 Lysozyme 0.5–1.5 Complement C4 Component 20–50 α2- 150–420 Complement C6 Component 7 α1- 4–9 ComplementC2 Component 2–3 β2-Microglobulin Trace ComplementC5 Component 4–15 Plasminogen (6–25) ComplementC7 Component 6 Post γ- – C-Reactive Protein <1 Prealbumin, thyroxine-binding 10–40 α1-Fetoprotein ~1µg Pregnancy-associated 2-glycoprotein Trace 200–450 2–3 -stabilizing factor (Factor XIII) (1–4) Prothrombin (5–10) 25–40 Retinol-binding protein 3–6 Galactoglycoprotein Thyroxine-binding globulin 1–2 GC Globulin 20–55 ~7 0.6 S γ2-Globulin <1 Transferrin 200–320 2 S γ2-Globulin 0.1 From “The Plasma Proteins”, Volume IV, 2nd Edition, 1984, Frank W. Putnam. sigma-aldrich.com/biofiles

4 Plasma Derived Proteins and Enzymes Pathway Proteins

α 2 Antithrombin III from bovine plasma (Human Sequence) EC No. 2905164 lyophilized powder, activity: 200‑400 units/mg protein 1% ...... (E280 = 6.5) When reconstituted with 1.0 mL of deionized water, solution will contain a minimum of the stated activity in 0.1 M sodium chloride and 0.02 M Tris, pH 7.5. 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 One unit is the activity present in 0.1 mL of normal human pooled plasma tested in the presence of 0.1 unit of . Homogeneous by gel electrophoresis. B DRY ICE A9141-10UN 10 units A9141-50UN 50 units α -Antiplasmin from human plasma 2 Antithrombin III from rat plasma ≥ lyophilized powder, activity: 5 inhibitor Units/mg protein 1% Lyophilized from 25 mM ammonium bicarbonate lyophilized powder, activity: >200 units/mg protein (E280 = 6.5) When reconstituted with 1.0 ml of deionized water, solution will

Proteins Purified by a modification of the method of Wiman. One unit will reduce the activity of two units of by 50% at contain a minimum of the stated activity in 0.1 M sodium chloride, and 37 °C, pH 7.2, using Val-Leu-Lys-p-nitroanilide. 0.02 M Tris, pH 7.5. 1% 1 One unit is the activity present in 0.1 mL of normal human pooled Protein determined by E280 = 6.7 B plasma tested in the presence of 0.1 unit of heparin. A0914 Homogeneous by gel electrophoresis. NB DRY ICE A6397-10UN 10 units Antithrombin III (Human Sequence) Antithrombin III p N-Linked Glycans

Plasma and Blood Related Ecarin from Echis carinatus venom ...... ...... [55466‑26‑7] vial of ~50 units 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 Prothrombin activator One unit will activate prothrombin to produce one unit of amidolytic Signal Heparin Binding activity at pH 8.4 at 37 °C. One amidolytic unit will hydrolyze 1.0 μmole Peptide of N-p-Tosyl-Gly-Pro-Arg p-nitroanilide per min at pH 8.4 at 37 °C. L R: 42/43 S: 22-26-28-36/37/39-45 B q Antithrombin III E0504-1VL 1 vial

Factor Xa inhibitor; Heparin cofactor, AT III [90170‑80‑2] Activating Enzyme from Russell's viper venom inhibitor; Inhibitor of and activated [65522‑14‑7] E.C. 3.4.21.95 (Xa). Effective concentration equimolar with proteinase. lyophilized powder, activity: 20 units/mg protein Not to be confused with thrombin antiserum. Stabilized in albumin and sodium chloride. EC No. 290-516-4 RTECS # CD4071900 One unit will increase the Factor V activity in 0.1 mL of fresh normal Antithrombin III from human plasma human plasma by at least 2× after incubation for 3 min at 37 °C. B ≥ lyophilized powder, 95% (SDS-PAGE) F3380-100UN 100 units Lyophilized from 20 mM Tris, pH 8.0 One unit is the activity present in 0.1 mL of normal human pooled plasma tested in the presence of 0.1 unit of heparin. I B A2221-25UG 25 μg A2221-125UG 125 μg sigma-aldrich.com/biofiles 5

Factor IX human Factor X in vivo Processing (Bovine Sequence)

Christmas factor; Plasma thromboplastin component Factor X (Precursor) ‑ ‑ [9001 28 9] N-Linked Glycan .... lyophilized powder .... Disulfides Purified Factor IX shows one band at molecular weight of 55 kDa using SDS gel electrophoresis under non-denatured, non-reduced conditions. 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 Signal Peptidase S1 Lyophilized from 20 mM Tris buffered saline, pH 7.4, containing 0.02% Peptide sodium azide. Propeptides

Factor IX is purified from a concentrate of pooled normal human O-Linked Glycans plasma using an Anti-Factor IX-Agarose column followed by gel Factor X (Zymogen) filtration. 1 vial = 100 μg protein. Factor IXa or VII cleavage ...... Disulfides Reconstitute with 1 mL deionized water. B DRY ICE 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 Proteins Related Blood and Plasma F0650-1VL 1 vial Peptidase S1

Propeptides Light Chain Heavy Chain

Factor Xaα

Disulfides

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 Peptidase S1 Propeptide Light Chain Heavy Chain (not required for activity)

Active site Factor Xaβ

Disulfides

20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 Peptidase S1

Light Chain Heavy Chain

Active site

Factor X Activated (Xa) from bovine plasma

Factor Xa [9002‑05‑5] E.C. 3.4.21.6 aqueous glycerol solution Factor Xa catalyzes the hydrolysis of the Arg-Thr and then Arg-Ile bonds in prothrombin to yield active thrombin. The fairly strict recognition sequence is Ile-Glu (or Asp)-Gly-Arg-↓-X. It may sometimes cleave at other basic residues, depending on the conformation of the target protein. Factor Xa will not cleave if a proline residue follows the of the recognition sequence. pH Optimum: 7.6‑8.0 Temperature Optimum: 37 °C Fusion proteins are commonly expressed with a factor Xa cleavable Ile- Glu (or Asp)-Gly-Arg-↓-X sequence. Typically 1 mg of fusion protein can be incubated with 10 μg of factor Xa for 2.5 hours at 37 °C. One unit of activated Factor X will liberate 1.0 μmole of p-nitroanilide from N-benzoyl-L-isoleucyl-L-glutamyl-L-glycyl-L-arginine-p-nitroaniline per minute at pH 8.3 at 37 °C. B WET ICE sigma-aldrich.com/biofiles F9302-50UG 50 μg

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Fibrin from human plasma  ~60% protein (>80% of protein is clottable) Contains ~15% sodium citrate and ~25% sodium chloride. [9001‑31‑4] B Prepared by clotting fibrinogen to yield a crosslinked fibrin F3879-100MG 100 mg EC No. 232-597-0 I F3879-250MG 250 mg F3879-1G F5386-1G 1g 1g F3879-5G 5g q Fibrinogen  35‑65% protein (~95% of protein is clottable). Factor I Non-protein content is sodium citrate and sodium chloride. ‑ ‑ [9001 32 5] Essentially plasmin(ogen) free Soluble dimer mol wt 340 kDa solubility α chain mol wt 63.5 kDa 0.9% NaCl ...... soluble β chain mol wt 56 kDa B γ Proteins Related Blood and Plasma chain mol wt 47 kDa (about 4% carbohydrate content) F4883-500MG 500 mg Fibrinogen from bovine plasma F4883-1G 1g F4883-5G 5g EC No. 2325986 Fibrinogen is an acute phase protein that is part of the coagulation  ~65% protein (>60% of protein is clottable) cascade of proteins. The end result of the cascade is the production of Contains ~15% sodium citrate and ~20% sodium chloride. thrombin that converts fibrinogen to fibrin. Thrombin rapidly B proteolyzes fibrinogen, releasing fibrinopeptide A. The loss of this small F4129-1G 1g peptide is not sufficient to make the resulting fibrin molecule insoluble, F4129-5G 5g but it tends to form complexes with adjacent fibrin and fibrinogen molecules. Thrombin then cleaves a second peptide, fibrinopeptide B, Fibrinogen from murine plasma from fibrin and the fibrin monomers formed then polymerize spontaneously to form an insoluble gel. The polymerized fibrin is held ~50% protein (>80% of protein clottable) together by noncovalent and electrostatic forces and stabilized by the Contains ~20% sodium citrate and ~30% sodium chloride. transamidating enzyme, factor XIIIa, that is produced by the action of Protein determined by biuret thrombin on factor XIII. The insoluble fibrin aggregates (clots) and EC No. 232-598-6 B aggregated then block the damaged and prevent F4385-5MG 5mg further bleeding. The amount of fibrinogen in the plasma can serve as F4385-25MG a nonspecific indicator of whether or not an inflammatory process is 25 mg F4385-100MG present in the body. Fibrinogen from any mammalian source will be 100 mg cleaved by thrombin from any mammalian source. Protein determined by biuret Fibrinogen from porcine plasma EC No. 232-598-6  Type I-S, ~75% protein (≥75% of protein is clottable) ~70% protein (>70% of protein is clottable) Contains ~10% sodium citrate and ~15% sodium chloride. Contains ~12% sodium citrate and ~18% sodium chloride. B Protein determined by biuret B F8630-1G 1g EC No. 232-598-6 F2629-5G F8630-5G 5g 5g F8630-10G 10 g F8630-25G 25 g Fibrinogen from rat plasma

~70% protein (>60% of protein is clottable)  ~60% protein (~95% of protein is clottable) Contains ~12% sodium citrate and ~18% sodium chloride. Contains ~15% sodium citrate and ~25% sodium chloride. B Protein determined by biuret. EC No. 232-598-6 B F4753-250MG 250 mg F6755-25MG 25 mg F4753-1G 1g F6755-100MG 100 mg F4753-5G 5g F4753-10G 10 g Fibrinogen from sheep plasma

Fibrinogen from human plasma ~75% protein (>70% of protein is clottable) sigma-aldrich.com/biofiles Contains ~10% sodium citrate and ~15% sodium chloride. EC No. 2325986 Protein determined by biuret. Protein determined by biuret EC No. 232-598-6 B I F9754-1G 1g F9754-5G 5g

Fibrinogen p