Interaction Between Hydroxypropyl-β-cyclodextrin And Five Kinds Of Protein

Cyclodextrins is a non-toxic and cyclic oligosaccharide, which is composed of six toeight D-glucose monomers linked by α-1,4-glucosidic bonds. As a derivatives ofCyclodextrins, Hydroxypropyl-β-cyclodextrins (HP-β-CD) has a hydrophobic centralcavity and hydrophilic outer surface, which makes it easy to form host-guest complexes withhydrophobic molecules of appropriate sizes and shapes. Since it is easy to produce HP-β-CDand the cost is low, HP-β-CD could be treated as a new modifier of zymoprotein to inductfurther research. According to the traits of enzymatic reaction respectively, uv-vis analysis orfluorescence analysis could be used to study the activity of enzyme before and after themodification. Fluorescence analysis was also applied to study the changed inner fluorescentof zymoprotein which was modified by HP-β-CD at different temperatures. In addition, themolecular docking tests was employed to study the molecular structure of zymoprotein andthe optimization of HP-β-CD. The most likely interaction sites and modes between HP-β-CDand biological macromolecules could be find out.The results of this paper mainly included the following aspects:1. Interactions between HP-β-CD and superoxide dismutase(SOD)The thesis compared the inhibitions of SOD on the auto-oxidative rate of pyrogallolbefore and after the modification of HP-β-CD. The results showed that the modified SOD’scomplex enhanced the inhibition rate of pyrogallol auto-oxidation. The enzymatic activities ofSOD was increased approximately by27%. Fluorescence spectrometry was employed toresearch the fluorescence sensitization of SOD which was modified by HP-β-CD. It wasfound that the inclusion ratio between SOD and HP-β-CD was1:1, and the inclusion complexformed Spontaneously under natural conditions. In addition, the results of sychronousfluorescence showed that the launch peaks of tyrosine (Tyr) and tryptophan (Trp) wereabout at280nm and340nm respectively, and the sychronous fluorescence of inclusioncomplexs were intensified. The results of molecular docking were that: there were10aminoacid residues involved in the hydrogen bond formation which were LYS9, ASP11, GLN15and ASN51of B chain; LYS9, ASP11, GLN15and GLY54of G chain; LYS9andTHR34of O chain.2. Interactions between HP-β-CD and penicillinaseThe influences on the decomposition rate of penicillin sodium brought by penicillinasebefore and after modification were compared.. It could be found that the decomposition rateof penicillin sodium increased to2.12times when penicillinase was modified. Fluorescenceanalysis showed that HP-β-CD enhanced the fluorescence of penicillinase. The inclusion ratiobetween penicillinase and HP-β-CD was1:1, and the inclusion complex formedSpontaneously. The results of sychronous fluorescence revealed that the sychronousfluorescence of inclusion complexes were stronger than penicillinase; Penicillinase’sfluorescence was mainly derived from tyrosine(Tyr). The results of molecular dockingshowed that there were only three aminoacid residues involved in the hydrogen bondformation. They were VAL39, ASN180and ASP183.3. Interactions between HP-β-CD and L-lactate dehydrogenaseThe thesis compared the enzymatic reaction rate of L-lactate dehydrogenase before andafter modification. The result was that the enzymatic reaction rate of modified L-lactatedehydrogenase was about2.48times of the original. Fluorescence spectrum analysis showedthat HP-β-CD could enhance the fluorescence of L-lactate dehydrogenase. The inclusioncomplex ratio of L-lactate dehydrogenase and HP-β-CD was1:1, and the inclusion complexformed Spontaneously at the temperature of293K. In addition, L-lactate dehydrogenasefluorescence mainly derived from tyrosine(Tyr).The effect of HP-β-CD on residues’sfluorescence was not obvious. The results of molecular docking showed that there were onlytwo residues involved in the hydrogen bond formation. They were SER232and ARG336, andformed two groups hydrogen bonding respectively.4. Interactions between HP-β-CD and alkaline phosphataseThe method of Fluorescence was applied to study the changes of enzymatic reaction rateof alkaline phosphatase before and after modification. The result showed that the promotionof HP-β-CD to the enzymatic reaction rate was not obvious. The analysis of Fluorescencespectrum found that HP-β-CD could enhance the fluorescence of alkaline phosphatase. Theinclusion complex ratio of alkaline phosphatase and HP-β-CD was1:1, and the inclusion complex formed Spontaneously. The analysis of sychronous fluorescence exhibited that thefluorescence of tyrosine(Tyr) and tryptophan(Trp) increased apparently, when alkalinephosphatase was modified by HP-β-CD. The results of molecular docking showed that: therewere five aminoacid residues involved in the hydrogen bond formation. They were GLU15,THR50, ARG53, PRO65and PHE74. To sum up, the main interaction forces betweenHP-β-CD and alkaline phosphatase were hydrophobic force and hydrogen bonding force.5. Interactions between HP-β-CD and elastaseThe analysis of Fluorescence spectrum found that HP-β-CD could enhance thefluorescence of elastase. The inclusion complex ratio of elastase and HP-β-CD was1:1, andthe inclusion complex formed Spontaneously. The results of sychronous fluorescencerevealed that the fluorescence of elastase was mainly derived from tyrosine(Tyr), and the peakposition had slightly blue shift. The results of molecular docking were that: there were fiveaminoacid residues involved in the hydrogen bond formation. They were ARG61, GLN192,HIS57and SER195. To sum up, the interaction force between HP-β-CD and elastase had beenslightly weakened, hydrogen bonding force was the main force.