Effect of pH, Dietary Proteins and Trypsin Inhibitors on the Hydrolytic Rate of Human Granulocyte Colony-Stimulating Factor (G-CSF) by Rat Digestive Enzymes

The effects of pH, dietary proteins and trypsin inhibitors on the hydrolytic rate of recombinant human granulocyte colony-stimulating factor (G-CSF) by digestive enzymes were studied by in vitro incubation experiments. The bile obtained by cannulation into the rat common bile duct and the eluates obtained by infusing distilled water into the gastrointestinal tract were used as sources of digestive enzymes. Some proteins including dietary proteins such as casein and ovalbumin, and two kinds of trypsin inhibitors, chicken egg white and soybean ones, were used to examine the protective action on the hydrolysis of G-CSF by digestive enzymes. With an experiment using the digestive enzyme fluids obtained after centrifugation of bile by ultra-filters with a molecular weight (Mr) cut off of 30000, 10000 and 5000, the proteolytic activity to G-CSF decreased as the cut off Mr decreased. The enzyme solution obtained with a membrane with a Mr cut off of 5000 still had an enzyme activity against G-CSF. The protease activity was mainly ascribed to the pancreatic fluid, but the hepatic bile still had an enzyme activity. The hydrolytic rate of G-CSF was dependent on the pH of the enzyme fluid, and the intestinal fluid. The hydrolytic rate of G-CSF was studied at pHs of 1.68, 4.01, 6.86 and 9.18. Especially, as the pH decreased to 1.68, the hydrolytic rate of G-CSF considerably decreased. Some proteins including dietary proteins also affected the hydrolytic rate of G-CSF. The strength of the inhibitory effect of the proteins is the following order ; ovalbumin > casein > mucin > keratin. Trypsin inhibitors considerably decreased the hydrolytic rate of G-CSF by the digestive enzymes. The inhibitory effect of trypsin inhibitors on the hydrolytic rate of G-CSF is predominantly stronger than that of the dietary proteins.