Metal-sensitive and thermostable trypsin from the crevalle jack (<Emphasis Type="Italic">Caranx hippos</Emphasis>) pyloric caeca: purification and characterization

Background

Over the past decades, the economic development and world population growth has led to increased for food demand. Increasing the fish production is considered one of the alternatives to meet the increased food demand, but the processing of fish leads to by-products such as skin, bones and viscera, a source of environmental contamination. Fish viscera have been reported as an important source of digestive proteases with interesting characteristics for biotechnological processes. Thus, the aim of this study was to purify and to characterize a trypsin from the processing by-products of crevalle jack (Caranx hippos) fish.

Results

A 27.5 kDa trypsin with N-terminal amino acid sequence IVGGFECTPHVFAYQ was easily purified from the pyloric caeca of the crevalle jack. Its physicochemical and kinetic properties were evaluated using N-α-benzoyl-_DL-arginine-p-nitroanilide (BApNA) as substrate. In addition, the effects of various metal ions and specific protease inhibitors on trypsin activity were determined. Optimum pH and temperature were 8.0 and 50°C, respectively. After incubation at 50°C for 30 min the enzyme lost only 20% of its activity. K _m , k_cat, and k _cat /K _m values using BApNA as substrate were 0.689 mM, 6.9 s^-1, and 10 s^-1 mM^-1, respectively. High inhibition of trypsin activity was observed after incubation with Cd²⁺, Al³⁺, Zn²⁺, Cu²⁺, Pb²⁺, and Hg²⁺ at 1 mM, revealing high sensitivity of the enzyme to metal ions.

Conclusions

Extraction of a thermostable trypsin from by-products of the fishery industry confirms the potential of these materials as an alternative source of these biomolecules. Furthermore, the results suggest that this trypsin-like enzyme presents interesting biotechnological properties for industrial applications.

     

Kinetics of copper incorporation into a biosynthetic purple Cu(A) azurin: characterization of red, blue, and a new intermediate species

Evolutionary links between type 1 blue copper (T1 Cu), type 2 red copper (T2 Cu), and purple Cu(A) cupredoxins have been proposed, but the structural features and mechanism responsible for such links as well as for assembly of Cu(A) sites in vivo are poorly understood, even though recent evidence demonstrated that the Cu(II) oxidation state plays an important role in this process. In this study, we examined the kinetics of Cu(II) incorporation into the Cu(A) site of a biosynthetic Cu(A) model, Cu(A) azurin (Cu(A)Az) and found that both T1 Cu and T2 Cu intermediates form on the path to final Cu(A) reconstitution in a pH-dependent manner, with slower kinetics and greater accumulation of the intermediates as the pH is raised from 5.0 to 7.0. While these results are similar to those observed previously in the native Cu(A) center of nitrous oxide reductase, the faster kinetics of copper incorporation into Cu(A)Az allowed us to use lower copper equivalents to reveal a new pathway of copper incorporation, including a novel intermediate that has not been reported in cupredoxins before, with intense electronic absorption maxima at ~410 and 760 nm. We discovered that this new intermediate underwent reduction to Cu(I), and proposed that it is a Cu(II)-dithiolate species. Oxygen-dependence studies demonstrated that the T1 Cu species only formed in the presence of molecular oxygen, suggesting the T1 Cu intermediate is a one-electron oxidation product of a Cu(I) species. By studying Cu(A)Az variants where the Cys and His ligands are mutated, we have identified the T2 Cu intermediate as a capture complex with Cys116 and the T1 Cu intermediate as a complex with Cys112 and His120. These results led to a unified mechanism of copper incorporation and new insights regarding the evolutionary link between all cupredoxin sites as well as the in vivo assembly of Cu(A) centers.     

Synthesis and characterization of copper-, zinc-, manganese-, and cobalt-substituted dimeric heteropolyanions, [(alpha-XW9O33)2M3(H2O)3](n-) (n = 12, X =As(III), Sb(III), M = Cu(2+), Zn(2+); n = 10, X = Se(IV), Te(IV), M = Cu(2+) and [(alpha-AsW9O33)2WO(H2O)M2(H2O)2](10-) (M = Zn(2+), Mn(2+), Co(2+)

Interaction of the lacunary [alpha-XW9O33](9-) (X = As(III), Sb(III)) with Cu(2+) and Zn(2+) ions in neutral, aqueous medium leads to the formation of dimeric polyoxoanions, [(alpha-XW9O33)2M3(H2O)3](12-) (M = Cu(2+), Zn(2+); X = As(III), Sb(III)), in high yield. The selenium and tellurium analogues of the copper-containing heteropolyanions are also reported: [(alpha-XW9O33)2Cu3(H2O)3](10-) (X = Se(IV), Te(IV)). The polyanions consist of two [alpha-XW9O33] units joined by three equivalent Cu(2+) (X = As, Sb, Se, Te) or Zn(2+) (X = As, Sb) ions. All copper and zinc ions have one terminal water molecule resulting in square-pyramidal coordination geometry. Therefore, the title anions have idealized D3h symmetry. The space between the three transition metal ions is occupied by three sodium ions (M = Cu(2+), Zn(2+); X = As(III), Sb(III)) or potassium ions (M = Cu(2+); X = Se(IV), Te(IV)) leading to a central belt of six metal atoms alternating in position. Reaction of [alpha-AsW9O33](9-) with Zn(2+), Co(2+), and Mn(2+) ions in acidic medium (pH = 4-5) results in the same structural type but with a lower degree of transition-metal substitution, [(alpha-AsW9O33)2WO(H2O)M2(H2O)2](10-) (M = Zn(2+), Co(2+), Mn(2+)). All nine compounds are characterized by single-crystal X-ray diffraction, IR spectroscopy, and elemental analysis. The solution properties of [(alpha-XW9O33)2Zn3(H2O)3](12-) (X = As(III), Sb(III)) were also studied by 183W-NMR spectroscopy.     

Nuclear orbiting and anomalies in nuclear reactions

In this paper, we report our measurements of back-angle oxygen and carbon particle yields from ¹⁶O+⁸⁹Y, ¹²C+⁹³Nb reactions forming the same compound nucleus ¹⁰⁵Ag at the same excitation energy and spin distribution. We find anomalously large oxygen yield and entrance channel dependence at high excitation energies from ¹⁶O+⁸⁹Y reaction implying formation of a dinuclear orbiting complex. Possible connection between nuclear orbiting and fast fission is also discussed.