Human α-Thrombin Preparations

Abstract

Thrombin production from human Cohn fraction III paste (>20 g; 340 ± 110 mg/kg paste, N = 65) has yielded predominantly α-thrombin (97 ± 3%, N = 29), with high clotting and esterase activities (2.8 + 0.4 US “NIH” units/μg protein; 88 ± 8% esterase active, N = 65), free of detectable plasmin(ogen) and Factors II, VII, IX(a) or X(a). Preparations in 0.75 M NaCl (pH/v6) at 2-7 mg/ml (A250nm = 1,83 m-1 cm-1 cm 0.10 M NaOH) retain activities frozen at <-50°C for >1 year. The enzyme (My 36,600 + 800, N = 9) consists of two polypeptides (A chain Mr 4,600 ± 200, N = l6; B chain Mp 32,000 ± 1,000, N = 22), is an isoionically heterogeneous glycoprotein (pI 7.0, 7.3, 7.6; carbohydrate-staining B chain), displays sparing solubility (e.g., in 0.15 M NaCl, pH~7; precipitates in H2O), is easily denatured (low I; pH <5 or >10; T1/2 53°C), and autolyzes to non-clotting derivative forms (β- followed by 7-thrombin; both <1% clotting of α-thrombin). Chloride salts (I 3, pH 8.3) of Group I (except Li) enhanced a-thrombin (1 mg/ml) thermal stability (T1/2 60-61 vs 49°C for Li+), whereas Group II salts reduced its stability (T1/2 40, 44, 48°C for Ca2+, Ba2+, Sr2+, respectively). Autolytic loss of clotting activity (22°C, 48 h) followed the series: Na+ (>90% loss) > K+ (90$) > Rb+ (70%) >Cs+ (30%) > Li+ (10%)~Ca2+~Sr2+~Mg2+ > Ba2+ (<1%). Clotting activity was correlated with enzymic homogeneity of α-thrombin (3.09 ± 0.57 US units/μg extrapolated pure-active enzyme, N = 29). (Supported in part by USPHS Grant HL I3160)