Kinetic aspects of human placental alkaline phosphatase enzyme membrane.
The crosslinking of alkaline phosphatase of human placenta with human serum albumin has been optimized. During the physico-chemical characterization of this immobilized biocatalyst, special attention was paid to attributes such as the irreversibility of the enzyme support bonding, the stability of the catalytic activity, and the effects of pH and temperature on this activity. Regarding stability, patterns of denaturation are proposed, to account for inactivation curves over time and under storage/operation conditions. These patterns, in some cases, indicate the existence of different populations of immobilized enzyme molecules, with a different degree of sensitivity to denaturation. The activity vs pH profiles are clearly modified by the immobilization process. This is because the pH of the free homogeneous solution, measurable with a pH-meter, differs from the real pH of the immediate microenvironment of the immobilized enzyme molecules due to the effects of proton accumulation in the microenvironment (in the reaction catalysed by alkaline phosphatase, protons are produced), to limitations to the free diffusion of H+ and to the possible partition effects of H+ due to polar interactions with residues or molecules of the enzyme membrane. In the experimental working conditions, the apparent optimum temperatures are centered at 40 degrees C, inactivation (thermal denaturation) occurring above this temperature. In the temperature range 10-40 degrees C, the kinetic control over the overall activity of the immobilized enzyme was observed, causing the Arrhenius profiles to be linear.
Roig
M G
MG
Departamento de Quimica Fisica, Facultad de Farmacia, Universidad de Salamanca, Spain.
Serrano
M A
MA
Bello
J F
JF
Cachaza
J M
JM
Kennedy
J F
JF
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D005976:Glutaral / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010920:Placenta / Q000201:enzymology*; D012709:Serum Albumin / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D012709:Serum Albumin; D000469:Alkaline Phosphatase; D005976:Glutaral