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On the quaternary structure of human thrombin
UKIEF
NOTES
493
On the quaternary structure of human thrombin Recently, bovine thrombin has been the subject of a number of studies. HOWever, the data published are controversial and do not allow definitive conclusions as to the structure of thrombin and the size of its molecuIei-5. Even less is known about human thrombin. The present communication reports some of the results of our studies on th- structure of human thrombin. The enzyme was prepared by the method of JIagnusson6, after the activation of human prothrombin with 250/b sodium citrate solution. The preparation obtained had a specific activity of 2000 conventional activity units per mg of protein. The purified enzyme was studied by disc electrophoresis in polyacrylamide gel at pH 9.5 (Trisglycine electrode buffer) and at pH 4.5 (p- a 1anine-acetate buffer)‘. Electrophoresis at pH 9.5 gave only one “acid” component (Fig. I, a). However, when the enzyme was allowed to stand in I m&I B-mercaptoethanol solution, pH S.o, for I h, the electrophoretic pattern was significantly changed. On disc electrophoresis at pH 9.5, a single “acid” fraction was seen, possessing much liigher electrophoretic mobility. (Fig. I, b). Furthermore, electrophoresis in the acid medium, pH 4.5, gave a single
Fig. I. Pol~acr?;lamide ,ocl elcctrophoresis of human thrombin at pH 9.5 (a, b) and 4.5 (c, d). a, pattern of native thrombin; b and c, patterns of thrcnnbin after treatment with I mA1 ,%mercaptoethanol: b ar.d d, puttcrns of thrombin after treatment with I 31 P-mcrcaptoethanol.
“alkaline” fraction, which was not usually seen in native thrombin (Fig. I, c). The enzyme was eluted from the gel and its activity was determined. The “acid” fraction was found to possess only esterase activity, while the “allkaline” fraction had both clotting and esterase activities. In I M /?-mercaptoethanol solution the “alkaline” fraction broke down into two components. One of these components possessed comparatively high electrophoretic mobility (it migrated with the front); the other was characterized with lower electrophoretic mobility than the initial (Fig. I, d). These phenomena were accompanied by a complete loss of clotting activity. Treatment of thrombin with performic acid gave similar results. Thus, in the presence of high concentrations of /Gmercaptoethanol and after oxidation with performic acid, thrombin broke down into 3 fractions, detected on disc electrophoresis. The fractions were designated, depending on their positions in polyacrvlamide gel, as “acid”, “alkaline” and “neutral”. The amino acid composition of each fraction was determined. The acid fraction
was characterized by high content of dicarboxylic amino acids. It had 35’j/b of dicarboxylic amino acids and 79d of diamino monocarboxylic amino acids. The alkaline fraction contained up to 5o”/;, of diamino monocarboxylic amino acids and ION; of dicarboxylic amino acids; the neutral fraction contained more or (24(,,,) less equal quantities of dicarboxylic (28:/,) and diamino monocarboxylic‘ amino acids. On the basis of the amino acid analysis the minimal molecular wciglrt of each fraction was calculated. It was found to be I~OOO for the acid fraction and hooo 7ooo for both alkaline and neutral fractions. The molecular weight of the performic acidtreated thrombin was determined b\- electrophoresis in polyacrylamide gel in the presence of sodium dodecvlsulfate”. Positions of the two components in the gel corresponded to positions of proteins with molecular weights of hooo and 15000. Thus, on tile basis of the stud& reported here, it might be suggested that human thrombin consists of 3 polypeptide chains with different charges, having molecular weights of 6000 and 15000. Specitic clotting activity could be assigned to the alkaline fraction of thrombin. The acid fraction is probably necessaq’ for conformational stability of the human thrombin molecule. The possibility cannot be excluded that this fraction is responsible for sornr~ other function which is unknown at present. Institute of Biological and Medical Chemistry, U.S.S.R. dcade?pa_yqf Medical Scic~mxs, .%~oscow (U.S.S.R.)
T. 11. CHITLKOVA 1:. N. OREKHOVICH
ERRATA In de 1471 February issue, \‘ol. 31 (z), in the summary on page 363, the last line (with undiluted urine) should be deleted. and materials” : In the 1971 May issue, Vol. 32 (3), correct in “Reagents (I) Bromide-bromate solution: add 166 mg of KBrO, and 714 of KBr to a Ioo-ml volumetric flask. Dissolve in water and dilute to IOO ml. (4) Brucine acetate IO,:, : dissolve 2500 mg of brucine in 1.5 ml of glacial acetic and IO ml of water in a 250 ml volumetric flask. Dilute to 250 ml. In the 1971 June issue, Vol. 33 (I), on page 180, the second last line of Exper. I, in Table I on page 181 and in Table TI and TIT on 183, for disint. /min/hmole read disint. /tnjn/nmole.
NOTES
493
On the quaternary structure of human thrombin Recently, bovine thrombin has been the subject of a number of studies. HOWever, the data published are controversial and do not allow definitive conclusions as to the structure of thrombin and the size of its molecuIei-5. Even less is known about human thrombin. The present communication reports some of the results of our studies on th- structure of human thrombin. The enzyme was prepared by the method of JIagnusson6, after the activation of human prothrombin with 250/b sodium citrate solution. The preparation obtained had a specific activity of 2000 conventional activity units per mg of protein. The purified enzyme was studied by disc electrophoresis in polyacrylamide gel at pH 9.5 (Trisglycine electrode buffer) and at pH 4.5 (p- a 1anine-acetate buffer)‘. Electrophoresis at pH 9.5 gave only one “acid” component (Fig. I, a). However, when the enzyme was allowed to stand in I m&I B-mercaptoethanol solution, pH S.o, for I h, the electrophoretic pattern was significantly changed. On disc electrophoresis at pH 9.5, a single “acid” fraction was seen, possessing much liigher electrophoretic mobility. (Fig. I, b). Furthermore, electrophoresis in the acid medium, pH 4.5, gave a single
Fig. I. Pol~acr?;lamide ,ocl elcctrophoresis of human thrombin at pH 9.5 (a, b) and 4.5 (c, d). a, pattern of native thrombin; b and c, patterns of thrcnnbin after treatment with I mA1 ,%mercaptoethanol: b ar.d d, puttcrns of thrombin after treatment with I 31 P-mcrcaptoethanol.
“alkaline” fraction, which was not usually seen in native thrombin (Fig. I, c). The enzyme was eluted from the gel and its activity was determined. The “acid” fraction was found to possess only esterase activity, while the “allkaline” fraction had both clotting and esterase activities. In I M /?-mercaptoethanol solution the “alkaline” fraction broke down into two components. One of these components possessed comparatively high electrophoretic mobility (it migrated with the front); the other was characterized with lower electrophoretic mobility than the initial (Fig. I, d). These phenomena were accompanied by a complete loss of clotting activity. Treatment of thrombin with performic acid gave similar results. Thus, in the presence of high concentrations of /Gmercaptoethanol and after oxidation with performic acid, thrombin broke down into 3 fractions, detected on disc electrophoresis. The fractions were designated, depending on their positions in polyacrvlamide gel, as “acid”, “alkaline” and “neutral”. The amino acid composition of each fraction was determined. The acid fraction
was characterized by high content of dicarboxylic amino acids. It had 35’j/b of dicarboxylic amino acids and 79d of diamino monocarboxylic amino acids. The alkaline fraction contained up to 5o”/;, of diamino monocarboxylic amino acids and ION; of dicarboxylic amino acids; the neutral fraction contained more or (24(,,,) less equal quantities of dicarboxylic (28:/,) and diamino monocarboxylic‘ amino acids. On the basis of the amino acid analysis the minimal molecular wciglrt of each fraction was calculated. It was found to be I~OOO for the acid fraction and hooo 7ooo for both alkaline and neutral fractions. The molecular weight of the performic acidtreated thrombin was determined b\- electrophoresis in polyacrylamide gel in the presence of sodium dodecvlsulfate”. Positions of the two components in the gel corresponded to positions of proteins with molecular weights of hooo and 15000. Thus, on tile basis of the stud& reported here, it might be suggested that human thrombin consists of 3 polypeptide chains with different charges, having molecular weights of 6000 and 15000. Specitic clotting activity could be assigned to the alkaline fraction of thrombin. The acid fraction is probably necessaq’ for conformational stability of the human thrombin molecule. The possibility cannot be excluded that this fraction is responsible for sornr~ other function which is unknown at present. Institute of Biological and Medical Chemistry, U.S.S.R. dcade?pa_yqf Medical Scic~mxs, .%~oscow (U.S.S.R.)
T. 11. CHITLKOVA 1:. N. OREKHOVICH
ERRATA In de 1471 February issue, \‘ol. 31 (z), in the summary on page 363, the last line (with undiluted urine) should be deleted. and materials” : In the 1971 May issue, Vol. 32 (3), correct in “Reagents (I) Bromide-bromate solution: add 166 mg of KBrO, and 714 of KBr to a Ioo-ml volumetric flask. Dissolve in water and dilute to IOO ml. (4) Brucine acetate IO,:, : dissolve 2500 mg of brucine in 1.5 ml of glacial acetic and IO ml of water in a 250 ml volumetric flask. Dilute to 250 ml. In the 1971 June issue, Vol. 33 (I), on page 180, the second last line of Exper. I, in Table I on page 181 and in Table TI and TIT on 183, for disint. /min/hmole read disint. /tnjn/nmole.