Study Of Several Enzyme Reaction Inhibitors: Fabrication And Assay With New Methodologies

Enzyme is biological catalyst, which catalyzes a variety of biochemical reactions with high efficiency. Almost all chemical reactions involved in the metabolic processes are catalyzed by enzyme. Only when the relevant enzymes exist, corresponding life activities can be carried out. On the other hand, life activities of organism reflect the ordering of its interior chemical reaction mechanism. When this ordering controlled by many factors is destroyed, metabolic disturbance, disease, and even death will happen. Studies have shown that many human diseases are related with the change of some certain enzyme activity. Regulation of enzyme reaction activity can improve and cure disease, and many active materials may play a key role in inhibiting the enzyme activity. Therefore, the explorations for new molecules with inhibition effect on enzyme reaction and establishment of new method to detect inhibitor have significant scientific values.In this dissertation, we have not only prepared two materials, i.e. starch–pectin conjugates and starch-soluble dietary fiber conjugates, with inhbiting effect on α-amylase activity, but have also found that lignin can inhibit α-glucosidase activity. Moreover, we have proposed and established electrochemical methods to detect the inhibitors of dipeptidyl peptidase-IV and NAD(P)H : quinone oxidoreductase 1. The main research contents of this dissertation are as follows:1. Study of the inhibition effect of starch-pectin conjugate on α-amylase hydrolysis reactionThe inhibitor of α-amylase which is the target enzyme for diabetes treatment can alleviate the symptoms of diabetes and promote human health. In this part of the dissertation, we have prepared a new type of chemically modified resistant starch, i.e. the starch-pectin conjugate, through cross-linking between starch and pectin using sodium trimetaphosphate. Experimental results show that the starch-pectin conjugate can well resist the hydrolysis catalyzed by α-amylase. Further studies reveal that the degree of crystallinity is increased, although the crystal type of starch in the conjugates does not change. Moreover, thermal stability and structural homogeneity of the conjugates are positively correlated with double-helical order in the crystalline region. So, we have not only obtained a new type of resistant starch(starch-pectin conjugates) that may inhibit α-amylase hydrolysis, but have also proposed a new method for the preparation of enzyme reaction inhibitors.2. Study of the inhibition effect of starch-soluble dietary fiber conjugate on α-amylase hydrolysis reactionIn this part of the dissertation, resistant starch and soluble dietary fiber(SDF) with different physiological properties are cross-linked by using a linker, sodium trimetaphosphate, and different kinds of starch-SDF conjugates are obtained under the treatment of both conventional heat-moisture treatment(CHMT) and microwave heat-moisture treatment(MHMT). Experimental results show that the starch-SDF conjugate prepared under the treatment of CHMT can almost resist the hydrolysis of the α-amylase completely. However, the resistance of the starch-SDF conjugate prepared under the treatment by MHMT has nearly no effect. Further studies reveal that SDF is combined into the outer layer of the overall starch granule by the linking with 2, 3, or 6-OH of glucose in the amorphous region in the treatment of CHMT. This combination shows a large protective effect on the whole starch granules, resulting in the inhibition effect of the conjugate on enzymatic hydrolysis. Whereas, in the treatment of MHMT, SDF is linked into the amorphous part of starch blocks derived from destructive starch granule, resulting in the conjugates with high homogeneity and its reluctant inhibition on enzyme hydrolysis. So, we have not only successfully prepared the starch-SDF conjugates which can almost inhibit α-amylase hydrolysis completely, but have also revealed the inhibition mechanism of the conjugate.3. Study of the inhibitory effect of lignin on α-glucosidase As the target enzyme of diabetes treatment, the discovery of α-glucosidase inhibitor has significant clinical value. In this part of the dissertation, we have found that lignin is an effective inhibitor against α-glucosidase with minimized IC50 value of 0.076 μM, which is much lower than that of acarbose(0.66 m M), a market diabetes healer. Further studies show that lignin can competitively inhibit the enzymatic activity with Ki value of 0.3×10-5 M. Meanwhile, it is known that lignin can interact with α-glucosidase to form 1:1 complex with the binding constant of 1.39 × 106 M-1 at 293 K. The binding of lignin to α-glucosidase is mainly driven by hydrophobic interaction and hydrogen bond, with binding distance of 3.54 nm. The formation of lignin-α-glucosidase complex may result in the alteration of α-helix structure and aromatic amino sides as well as the increase of protein granule volume. Furthermore, this study reveals that the binding of lignin to α-glucosidase conforms to the first order exponential decay function. This work would provide a scientific basis for the development of lignin as an active substance for diabetes treatment.4. An electrochemical method for dipeptidyl peptidase-IV inhibitor screeningDipeptidyl peptidase-IV(DPP-IV) is a target enzyme for diabetes drug therapy. Considering the defects of the current methods for DPP-IV inhibitor screening, we have proposed an electrochemical method for the determination of its activity and screening of its inhibitors. In this method, an enzyme substrate(Fc-peptide) is immobilized on the surface of a gold electrode, and double signal amplification is accomplished via an additional layer consisting of phenyl rings and gold nanoparticles. By using this method, the activity of DPP-IV can be determined at levels as low as 39 n U/m L over a linear detection range as wide as from 0.5 μU/m L to 2.5 m U/m L. In the meanwhile, the inhibitory effect of diprotin A and the His-Leu dipeptide on the activity of DPP-IV has also been tested by using this new method, and the IC50 values have been known to be 93.5 and 95.5 μM, respectively. This method is also known to be rapid, accurate, and selective. It can be extended for the assay of other peptidases and, possibly, proteases, and the screening of their inhibitors.5. An electrochemical method for NAD(P)H:quinone oxidoreductase 1 inhibitor screeningA simple and rapid electrochemical method is proposed in this part of the dissertation to assay the activity of NAD(P)H:quinone oxidoreductase 1(NQO1), a potential target for anticancer therapy. Firstly, a quinone derivative, the substrate of NQO1, is designed to be covalently immobilized on the surface of gold electrode. In the presence of the enzyme, the quinone derivative transforms to the corresponding hydroquinone derivative, followed by the expected cyclizative cleavage reaction via the gem-dialkyl effect. So, the hydroquinone derivative species are released from the electrode surface into the bulk solution. As the electrochemical probe, quinone derivative can generate one redox wave in aqueous buffer and its peak current is correlated with the activity of NQO1, so an electrochemical method to determine the enzyme activity can be proposed. Under the optimized conditions, NQO1 activity can be assayed by using this new method in the range from 0.1 to 10 U/m L with a detection limit of 0.22 U/m L. Moreover, resveratrol, an antitumor compound, is evaluated by using this method with the maximum inhibition rate of 95% and IC50 value of 23.70 μM. Therefore, with wide detection range, high sensitivity, acceptable reliability and rapid response, the established method can be used for not only the investigation of NQO1 activity but also the screening of the enzyme inhibitor.