Physicochemical studies on tryptic digestion of bovine fibrinogen.

The high molecular weight fragments observed during tryptic digestion of bovine fibrinogen and the variation of their relative proportion with time has been studied. Separation of the different molecular species was carried out by gel filtration and the molecular weights of the isolated fragments were determined by sedimentation equilibrium and from their electrophoretic mobilities in sodium dodecylsulfate-polyacrylamide gels. The fibrinogen is degraded by trypsin into distinct fragments, with molecular weights of 270 000, 170 000, 90 000 and 50 000 accompanied by a series of smaller fragments whose properties were not investigated. The relative proportion of the components was estimated from area measurements on scans of the stained gels obtained after electrophoresis in the presence of sodium dodecylsulfate. The relative concentration and the molecular weight of each component established its molar concentration in each of the digestion mixtures obtained after varying incubation times (1-60 min). These data were used for a kinetic analysis of the process. The kinetic model derived on the basis of the trinodular model of fibrinogen (see Appendix) gave a very good representation of all the experimental results.     

Rabbit cardiac myosin. II. Proteolytic fragmentation with insolubilized papain.

The substructure of the cardiac myosin molecule was examined by the limited proteolytic digestion of the parent molecule with (dialdehyde starch)-methylenedianiline-mercuripapain, S-MDA-mercuripapain, at low temperatures and neutral pH, using moderate enzyme to myosin rations. Pertinent properties of the insoluble enzyme complex were also examined. Kinetic, ultracentrifugal, and chromatographic observations of the fragmentation process revealed that a single type of lytic reaction occurs during the early stages, predominately releasing heavy meromyosin subfragment 1 (HMM-S1) and myosin rods. With further time digestion, the rods are additionally cleaved yielding light meromyosin and HMM-S2, and HMM-S1 is found to be partially degraded. The major proteolytic subfragments were isolated, purified, and characterized with respect to their enzymatic, optical, amino acid, and physicochemical properties. Only HMM-S1 exhibited Ca-2+-activated ATPase activity, and at a level three- to fourfold higher than that of native myosin. Moreover, its hydrohynamic properties suggest that it is globular in structure. On the other hand, light meromyosin-A (LMM-A) (which consists mainly of rods), and HMM-S2 appear to be highly asymmetric, rigid, alpha-helical molecules devoid of the amino acid proline. Strong similarities were evident in all aspects upon comparison of these results with documented information concerning the skeletal system. On the basis of the physical and chemical properties of the proteolytic subfragments relative to that of native myosin, it was further concluded that the cardiac myosin molecule is a double-stranded, alpha-helical rod ending in tow subfragment 1 globules, of which only one may be enzymatically active at a time.     

Inhibition of plasmin by fibrinogen.

The kinetics of inhibition of the amidolytic activity of plasmin on D-Val-L-Leu-L-Lys p-nitroanilide hydrochloride (S-2251) by fibrinogen and fibrin were determined. Reciprocal (1/v versus 1/[S]) plots of plasmin inhibition by 0.50 microM-fibrinogen showed a non-linear downward curve. The Hill coefficient (h) was 0.68, suggesting negative co-operativity. By contrast, fibrin produced a simple competitive inhibition of plasmin (Ki = 12 micrograms/ml). Addition of 0.1 mM-6-aminohexanoic acid shifted the non-linear curve obtained in the presence of fibrinogen to a straight line as for controls, indicating that 6-aminohexanoic acid abolishes the fibrinogen-induced inhibition. Transient exposure of the enzyme to pH 1.0 abrogates the ability of fibrinogen to inhibit plasmin activity. Acidification had no effect on the Vmax but increased the Km of plasmin. The present evidence for modulation of plasmin reveals a novel mechanism for control of fibrinolysis by fibrinogen, a component of the coagulation system and the precursor of the physiological substrate of plasmin.     

Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor. II. Crystallographic refinement at 1.9 A resolution

The crystal structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor has been refined with data to 1.9 Å resolution, using a procedure described by Deisenhofer &; Steigemann (1974) in their refinement of the crystal structure of the free inhibitor. This procedure involves cycles consisting of phase calculation using the current atomic model, Fourier synthesis using these phases and the observed structure factor amplitudes and Diamond's real-space refinement (Diamond, 1971,1974). At various stages, difference Fourier syntheses are calculated to detect and correct gross errors in the model and to localize solvent molecules.The refinement progressed smoothly, starting with the model obtained from the isomorphous Fourier map at 2.6 Å resolution. The R-factor is 0.23 for 20,500 significantly measured reflections to 1.9 Å resolution, using an over-all temperature factor of 20 Ų. The estimated standard deviation of atomic positions is 0.09 Å.An objective assessment of the upper limit of the error in the atomic coordinates of the final model is possible by comparing the inhibitor component in the model of the complex with the refined structure of the free inhibitor (Deisenhofer &; Steigemann, 1974). The mean deviation of main-chain atoms of the two molecular models in internal segments is 0.25 Å, of main-chain dihedral angles 5.1 ° and side-chain dihedral angles 6.5 °.A comparison of the trypsin component with α-chymotrypsin (Birktoft &; Blow, 1972) showed a mean deviation of main-chain atoms of 0.75 Å. The structures are closely similar and the various deletions and insertions cause local structural differences only.     

胰蛋白酶消化法测定牛胶原蛋白三股螺旋结构的含量

本研究建立了一种测定胶原蛋白的三股螺旋结构含量的方法。该方法通过使用柱前衍生高效液相色谱(HPLC)法表征经胰蛋白酶酶解后胶原蛋白羟脯氨酸(Hyp)质量浓度的变化,进而对胶原蛋白的三股螺旋结构进行定量。探讨了不同的酶解时间(0~48h)、酶与底物的比例(1∶100、1∶50和1∶20)和温度(20、25、30、37℃)对明胶降解率的影响。获得了酶解的最佳条件——当胰蛋白酶与底物的比例为1∶50时,25℃酶解3h。使用该方法对明胶胶原蛋白混合液检测,结果表明,该方法能灵敏(RSD<10%)的测定胶原蛋白三股螺旋结构的含量。该方法不仅可用于生物组织研究领域,也可用于胶原蛋白食品、保健品和组织工程产品质量的评价。     

Proteolytic fragmentation and peptide mapping of human carboxyamidomethylated tracheobronchial mucin

Human tracheobronchial mucin was isolated from lung mucosal gel by chromatography on Sepharose 4B in the presence of dissociating and reducing agents, and its thiol residues were carboxyamidomethylated with iodo[1(-14)C]acetamide. The 14C-carboxyamido-methylated mucin was purified by chromatography on Sepharose 2B. No low molecular weight components were detected by molecular sieve chromatography or polyacrylamide gel electrophoresis in the presence of dissociating and reducing agents or by analytical density centrifugation in CsCl/guanidinium chloride. After digestion of the purified 14C-mucin with trypsin-L-1-tosylamido-2-phenylethyl chloromethyl ketone, three fractions (TR-1, TR-2, and TR-3) were observed by chromatography on Sepharose 4B. TR-1, a 260-kDa mucin glycopeptide fragment, contained all of the neutral hexose and blood group activity and 20% of the radioactivity in the undigested mucin. TR-1 was refractory to a second incubation with trypsin but could be digested by papain or Pronase to a smaller mucin glycopeptide fraction, as judged by the slight decrease in apparent molecular weight on Sepharose CL-4B. These mucin glycopeptides contained approximately 50% of the radioactivity in the TR-1 fraction, indicating that the glycosylated domains of carboxyamidomethylated tracheobronchial mucin contained thiol residues. The remainder of the radioactivity from papain or Pronase digests of TR-1 eluted, like the TR-3 fractions, in the salt fraction on Sepharose CL-4B. Peptide mapping of the nonglycosylated TR-3 fraction by TLC and high voltage electrophoresis yielded six principal and several less intensely stained ninhydrin reactive components, with the radiolabel concentrated in one of the latter peptides. Peptide purification of the TR-3 fraction by high pressure liquid chromatography on a C18 reverse phase column demonstrated the presence of four major peptides, with TR-3A being the dominant component. The TR-3D peptide contained S-carboxy-aminomethylcysteine and had 69% sequence similarity to the sgs-7 salivary glue protein of Drosophila.     

The interaction in human plasma of antiplasmin, the fast-reacting plasmin inhibitor, with plasmin, thrombin, trypsin and chymotrypsin.

The inhibition of plasmin, (EC 3.4.21.7), thrombin (EC 3.4.21.5), trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) by antiplasmin, the recently described fast-reacting plasmin inhibitor of human plasma, was studied. To determine the quantitative importance of antiplasmin relative to the other plasma protease inhibitors, enzyme inhibition assays were performed on whole plasma and on plasma specifically depleted in antiplasmin, after addition of excess enzyme. Plasmin was the only enzyme for which the inhibitory capacity of antiplasmin-depleted plasma was lower than that of normal plasma. To determine the affinity of the enzymes for antiplasmin, as compared to the other inhibitors, various amounts of enzymes were added to normal plasma and the formation of enzyme-antiplasmin complexes studied by crossed immunoelectrophoresis using specific antisera against antiplasmin. Plasmin and trypsin, but not thrombin or chymotrypsin formed complexes with antiplasmin. It is concluded that antiplasmin is the only fast-reacting plasmin inhibitor of human plasma. It is also a fast-reacting inhibitor of trypsin but only accounts for a very small part of the fast-reacting trypsin-inhibitory activity of plasma. This can be explained by the low concentration of antiplasmin (1 muM) in normal plasma, compared to the other inhibitors (e.g. alpha1-antitrypsin: 40-80 muM).     

Comparative kinetic studies of the primary specificity of bovine and salmon trypsin]

A comparative kinetic analysis of Pacific salmon and bovine trypsins revealed that the former hydrolyzes p-nitroanilide-N,L-benzoyl-D,L-arginine (BApNA) with a far greater efficiency in comparison with bovine trypsin due to the decrease in Km. The inhibition constants for the BApNA hydrolysis by bovine and salmon trypsin with glycine, beta-alanine, L-lysine, L-arginine and benzamidine were determined. With an increase in the length of the hydrocarbon chain in the inhibitor molecule (i.e., in the order of glycine-beta-alanine-L-lysine) the inhibiting effect increased both with salmon and bovine trypsins. The Ki values for benzamidine and L-arginine appeared to be by one order of magnitude higher with salmon trypsin than with bovine trypsin. L-arginine was a much more effective inhibitor compared to L-lysine when both salmon and bovine trypsins were used.