Study On The Interaction Between Several Terpenoids And Bovine Serum Albumin Or Keratin

Isodon plants are widely and plenty distributed over our country and contains anumber of kinds of diterpene and triterpene compounds. Our laboratory isolated fourkinds correspondence-kaurane diterpenoids from Vesey Isodon and Blue calyxwhich produced in Gansu，which including Compound1, Compound2, Compound3and Compound4, two kinds correspondence-kaurane triterpene which includingUrsolic acid and2-α-hudroxy-ursolic acid. In this thesis we used UV spectra（UV）,Fluorescence Spectroscopy（FS）, Circular Dichroism（CD）, Resonance LightScattering Spectroscopy（RLS）and other methods to study the interactions betweenseveral terpene compounds and Bovine Serum Albumin（BSA），another interactionbetween several terpene compounds and keratin. Hoping to provide some significancereference to development of anticancer drugs after studying interactions betweenseveral kinds of terpene compounds and protein.The main results are as follows:1. Ursolic acid and2-α-hudroxy-ursolic acid are pentacyclic triterpenoid. Theyhave similar chemical structure. There are different functional groups in C-2location.When interaction with bovine serum albumin, binding constant K_UA-BSA(0.6934×10³L·mol^-1)more than K_{2-α-hudroxy-UA-BSA}(0.1538×10³L·mol^-1), Ursolic acid interact withBSA combining ability stronger than2-α-hudroxy-ursolic acid interact with BSA.Efficiency of energy transfer EUA-BSA（0.036）less than E_{2-α-hudroxy-UA-BSA}（0.059）,combined with the distance rUA-BSA（1.43）more than r_{2-α-hudroxy-UA-BSA}（1.32）. Hudroxyin C-2location let2-α-hudroxy-ursolic acid interaction with BSA energy easier totransfer, combined with a shorter distance. Synchronous fluorescence results showthat Ursolic acid and2-α-hudroxy-ursolic acid interaction with BSA is thecontribution of tryptophan residues. Ultraviolet spectrum shows that Ursolic acid and2-α-hudroxy-ursolic acid interaction with BSA have better effect. It due to the BSAmolecules within the peptide chain stretching, hydrophobic groups in naked andhydrophobic effect weakened. CD results show that Ursolic acid changes ofsecondary structure of BSA, α-helix reduced from53.40%to39.08%.RLS results show that new complex intermolecular aggregation.2. FS results show that Compound1interaction with BSA at6∶1²0∶1molarconcentration ratio. Compound2、 Compound3、 Compound4interaction with BSAat30∶1⁷0∶1molar concentration ratio.Compound1is more likely to interactwith proteins than other diterpenoids. Binding constant KCompound1-BSA(7.207×10⁵L·mol^-1)、KCompound2-BSA(4.681×10⁶L·mol^-1)、KCompound3-BSA(0.987×10⁵L·mol^-1)，KCompound4-BSA(1.680×10⁵L·mol^-1). Binding site number nCompound1-BSA（1.6859）、nCompound2-BSA（1.9355）、nCompound3-BSA（1.5325），nCompound4-BSA（1.6032）. It’smean that Diterpenoid interaction with BSA ability stronger than triterpenoid.Compound1and Compound2interaction with BSA ability stronger than which,Compound3and Epinodosi interaction with BSA combining ability. Because of thedifference of functional groups.3. Ultraviolet spectrum shows that4kinds of diterpenoid interaction with BSA,all show the hyperchromic effect, BSA intramolecular aromatic heterocyclichydrophobic groups between the hydrophobic effect. FS shows that4kinds ofditerpenoid interaction with BSA, led to the static quenching with not generatedfluorescent compounds. Combined distance，rCompound1–BSA（1.462）、rCompound2–BSA（1.476）、rCompound3-BSA（1.566），rCompound4-BSA（1.571）. Efficiency of energytransfer，ECompound1-BSA（0.0302）、ECompound2-BSA（0.0303）、ECompound3-BSA（0.0212），ECompound4-BSA（0.0208）. Compound1, Compound2interaction with BSA,energy transfer efficiency and combined with distance are similar，easier to transferenergy，combined with a shorter distance. Because of Compound1and Compound2have similar structure， Compound3and Compound4have similar structure.Diterpenoid’s energy transfer efficiency is smaller than triterpenoid, but combinedistance longer. Synchronous fluorescence spectroscopy show that Compound1andCompound4interaction with BSA fluorescence quenching are contribution byresidual tryptophan and tyrosine residues, Compound2and Compound3interactionwith BSA fluorescence quenching by the contribution by tryptophan residues.4. Keratin were isolated from ox tongue which ultraviolet absorption peak arenear235nm， excitation peak at260nm and emission peak at285nm. Compound1has emission peak at330nm,342nm,361nm. The biggest fluorescence intensity at 342nm. Keratin’s ultraviolet absorption increase when Compound1concentrationincrease, hyperchromic effect. It’s mean keratin amino acid residues interaction withCompound1. FS result show that Keratin interaction with Compound1, generatednew compounds which themselves don’t launch fluorescent. As Compound1concentration increased, fluorescence intensity is weaken. Binding constant KCompound1-BSA(0.2062×103mol·L^-1), binding site number nCompound1-BSA（0.42）, efficiency ofenergy transfer ECompound1-BSA（0.098）, conbined distance rCompound1-BSA（1.21nm）.Compared with the Compound1interaction with BSA, binding constant and bindingsite number are small, combined distance is shorter, energy transfer efficiency is low.