Study On The Properties Of Pancreatic A-amylase

The biochemical properties of pancreatic a-amylase (E.C.3.2.1.1) was studied. A survey of effect of metal ions on the a-amylase showed that the activity of the enzyme was significantly boosted by Pb²⁺,Cd²⁺ and Ca²⁺when the ions concentration between 2～10umol/L, but when the concentration of Pb²⁺,Cd²⁺ and Ca²⁺ are surpass 9 or 10umol/L,the activity of enzyme was obsulutely inhibited. The inbition showed that the action was in the order: Pb²⁺＞Cd²⁺. But,Ca²⁺ always enhance the activity of pancreatic a-amylase whin the concentration of 0～50mmol/L,the inhibition was not appeared. The inhibition order of Mn²⁺,Co²⁺ to pancreatic a-amylase is: Mn²⁺＞Co²⁺,The hihest level of Mn²⁺ inhibition can reach 80ï¼…,However,in the same concentrtion, The inhibition of Co²⁺ was only 31.2ï¼….Study on the effects of Mn²⁺,Cd²⁺,Co²⁺and Pb²⁺ on UV differential spectrum and fluorescent spectrum of the pancreatic a-amylase found: UV differential spectrum of a-amylase denatured respectively by these four metal ions showed absorption peaks appeared at 230nm,270nm. The peaks of UV differential spectrum suggested that the conformation of enzyme molecule changed from ordered structure to random coils. The characteristic peak of the fluorescence emission of pancreatic a-amylase presented at 342nm when the fluorescence excitation spectrum was at 278 nm Mn²⁺,Cd²⁺,Co²⁺and Pb²⁺led to decrease in fluorescence intensity while the maximum emission peak remained unchangable. The results indicated these four ions influenced the conformation of pancreatic a-amylase. The characteristic peak of the fluorescence emission of pancreatic a-amylase presented at 342 nm when the fluorescence excitation spectrum was at 278 nm,Which was increased by the Tpy residue exposed from the inside of protein,but there are a little contribution from Tyr. In this experiment, four ions all led to decrease in fluorescence intensity in different degree,The main reason is that the mental ions interfere the active site of the pancreatic a-amylase,which causes the changes of enzyme's conformation.The rutin inhibits pancreatic a-amylase in an noncompetitive manner.The interaction between rutin and a-amylase has been studied by fluorescence and UV spectroscopy.In the condition of pH6.9,Rutin treatment led to the increasing of UV absorbtion and the quenching of intrinsic fluorescence of pancreatic a-amylase.The formation of constants was 0.44×10⁵.Study on the effection of some polyhydroxy compounds,such as xanthan gum,glycerol,sucrose,chondroitin sulfate on the thermostability of pancreatic a-amylase in different conditions of concentration and time.The 0.2ï¼…xanthan gum,2ï¼…sucrose,4ï¼…glycerol,0.2ï¼…chondroitin sulfate were best concentration selected,The protection was in the order: chondroitin sulfate＞sucros＞glycerol＞xanthan gum.The ultraviolet spectra and fluorescence spectrum of native a-amylase which adding stabilizers were studied. The ultraviolet spectra and fluorescence spectrum of native a-amylase were all greater than the enzyme which added stabilizers,it suggests that,The stabilizer can prevent or weak the deactivation of the native enzyme when hot treatment condition,moreover can stop the conformational changes.so the stabilizer can keep the natural structure of native pancreatic a-amylase.From the ultraviolet spectra and fluorescence spectrum,we can also conclude that molecular peptide chain of pancreatic a-amylase was unfold step by step under the hot denaturation condition.