肌红蛋白中二级结构检测方案(圆二色光谱仪)

智能文字提取功能测试中

Contact：Jasco， Inc.8649 Commerce Dr.Easton， MD 21601800-333-5272 Fax：410-822-7526www.jascoinc.com CD APPLICATION NOTE 02-03CD detection of Myoglobin StructureDuring an Automated pH Titration Circular dichroism spectroscopy (CD) is verysensitive to the secondary structure ofpolypeptides and proteins. Circular dichroism is aform of light absorption spectroscopy thatmeasures the difference in absorbance of right-and left-circularly polarized light (rather than thecommonly used absorbance of isotropic light) by asubstance. It has been shown that CD spectrabetween 260 and approximately 180 nm can beanalyzed for the different secondary structuretypes： alpha helix， beta sheet， beta turn， randomcoils， etc. Secondary structure determination byCD is reported to achieve accuracies of 0.97 forhelices， 0.75 for beta sheet， 0.50 for turns， and 0.89for other structure types. In this application CDwill be used to follow the denaturation ofmyoglobin during an automated pH titration withacid. Introduction A protein can be thought of as a hetero-polymer， oftenhundreds of units long， composed of twenty differentamino acids. The sequence of amino acids dictates thewell-defined native 3-D structure， which often includeshelix， sheet， turn， and random coil regions. Althoughthe backbone of amino acids is covalently linked，usually non-covalent interactions are responsible forthe folded 3-D structure. In the native protein， thehydrophobic amino acid residues are generallyysequestered in the interior while the hydrophili：cresidues are exposed to the solvent. The protein canbe unfolded (denatured) by heating， changing the pH，and adding reagents such as detergents， urea， orguanidine hydrochloride. Myoglobin is a commonmodel protein used to investigate the structuralchanges that can occur upon pH titration. Myoglobin is an extremely compact hemeprotein (MW~17，800)， found primarily in cardiac andred skeletal muscles， which functions in the storage ofoxygen and facilitates the transport of oxygen to themitochondria for oxidative phosphorylation. Myoglobinis particularly abundant in diving mammals includingthe whale， seal， and porpoise that are able to remainsubmerged for long periods due to the storage ofoxygen by muscle myoglobin. Myoglobin consists of asingle polypeptide chain of about 153 amino acids.Approximately 70% of the main chain is folded intoeight major， right-handed alpha helices. The majorityof the rest of the chain forms turns between helicesdevoid of symmetry. Four of the helices are terminatedwith a proline residue， whose five-membered ring doesnot fit within a straight stretch of the alpha helix， thereby disrupting it. Hydrophobic attractions are thedriving force behind protein folding. The conformations of the threephysiologically pertinent forms of myoglobin-deoxymyoglobin， oxymyoglobin， and metmyoglobin (ferrimyoglobin)-arevery similar except at the sixth coordination position.Methods including fluorescence， circular dichroism(CD)， nuclear magnetic resonance (NMR)， andelectrometric titrations have been utilized to investigatethe conformations of myoglobin. After loss of the heme，unfolding occurs in two stages： partial unfolding of thenative apoprotein to a molten globule intermediate andthen complete disruption of all the helical segments.In the structural model for myoglobindenaturation， ferrimyoglobin oxidizes and loses heme，yielding apomyoglobin in the N state. The heme groupdissociates from the protein at very low pH. The N-formobserved under neutral and mildly acidic conditions(pH 4.5-7.0) has ~80% alpha helical content.Likewise， the N-form observed under neutral and mildlyacidic conditions has ~55% alpha helix content. The B，C， and E helices which make up the heme pocket thenunfold to give the molten globule state in which the A，G， and H helices are still intact. The last step is theconversion of the intermediate to the completelyunfolded U state. The U-forms have a small residualalpha helix content and high intrinsic viscosity underhighly acidic conditions (pH <2.0) indicative of a random-coil conformation. The resistance of holomyoglobin to denaturation is a function of both the intrinsic stability of the apoprotein tertiary structure and the strength of the interactions with the prosthetic group. An 18 u g/mL solution of myoglobin was prepared bydissolving horse skeletal musclemyoglobinindeionized water. Chemical denaturation of the proteinwas initiated by the addition of 0.1M Sulfuric Acid usingan automated titrator (ATS-429). The protein unfoldingwas followed using a JASCo J-810)CDspectropolarimeter. The sample was contained in a1cm quartz： cuvette： using a magnetic：stirrer.Myoglobin CD spectra were automatically measured at0.05 mL intervals using Spectra Manager Software.(Figure 1)The totally automated study was completedin just under an hour. CD spectra were collected from260/180 nm with a data pitch of 0.1 nm. A band widthof 1 nm was used with a detector response time of 4sec. and scanning speed of 50nm/min. Data wasanalyzed using Jasco Interval Scan Analysis. Tirant Table Titrant Times Sample Conc. Titrant Con. Titrank Volume Cel Volume 18 0 017.561 239.024 0.0517.1429 466.667 0.0516.7442 683.721 0.0516.3536 890909 0.0516 1088.89 0.0515.6522 1278.26 0.0515.3191 1459.57 0.0515 1633.33 0.05 14.6939 1800 0.05 14.4 1960 0.05 刚 Total Titrank Times ： Tolel Titrant Volume： 0.5mL Figure 1. The titration table automatically calculatesthe change in sample and titrant concentration that willoccur with each subsequent titration. Results and Discussion The pH Titration Sulfuric acid was chosen for the titration since thechloride ions in an acid like HCl interfere with the CDspectrum. Several concentrations of acid wereevaluated with0.1M chosen.Concentrations higherthan this caused immediate denaturation upon the firstdelivery. As shown in Figure 1， the Spectra Managersoftware automatically calculates the concentration ofboth the titrant (H2SO4) and the myoglobin after eachaliquot.The software can perform the calculation tomaintain a constant volume in the cuvette. As shown in Figure 2， the CD of the myoglobinchanged as the pH of the solution decreased. Theintensity of the band centered at 193nm began agradual decrease until 0.5mL of acid had been addedand the pH lowered to approximately 2. At this pointthe helices which comprise the heme pocket haveunfolded and lost some of their alpha helical shape. Figure 3 shows a screen shot of the Cross-sectional analysis program. Using this feature awavelength can be selected and the change in the CDsignal followed with increasing concentration of thetitrant. Using this feature one can clearly see that theprotein began to dissociate at an acid concentration of125ppm and that the denaturation is finished once theacid concentration reaches 200ppm. This clearly demonstrates the loss of the alpha helical structure ofthe myoglobin. Figure 2. CD spectra demonstrating the spectralchanges that occur with pH titration. Figure 3.Cross section analysis of the spectralchanges that occur at 193nm. Circular dichroism spectroscopy (CD) is very  sensitive to the secondary structure of  polypeptides and proteins. Circular dichroism is a  form of light absorption spectroscopy that  measures the difference in absorbance of rightand left-circularly polarized light (rather than the  commonly used absorbance of isotropic light) by a  substance. It has been shown that CD spectra  between 260 and approximately 180 nm can be  analyzed for the different secondary structure  types: alpha helix, beta sheet, beta turn, random  coils, etc. Secondary structure determination by  CD is reported to achieve accuracies of 0.97 for  helices, 0.75 for beta sheet, 0.50 for turns, and 0.89  for other structure types. In this application CD  will be used to follow the denaturation of  myoglobin during an automated pH titration with  acid.