The experiment mainly focusing on what kinds of effects collagen have when environment changed, the environment including buffer, pH and ionic nature. Using a variety of testing methods to search collagen’s morphological change, as well as the essential reason that environmental impact to collagen structure, stability, mechanical properties, biological properties(crosslinking effect)

Sui, Mengmeng

Theseus

    Effect of Solution on the isoelectric point of collagen            Guide professor: Yunqiao Ding Email address: 673659149@qq.com Student name: Mengmeng Sui              Date: 13.06.2016 Qilu University of Technology   Target and significance: My experiment mainly focusing on what kinds of effects collagen have when environment changed, the environment including buffer, pH and ionic nature. Using a variety of testing methods to search collagen’s morphological change, as well as the essential reason that environmental impact to collagen structure, stability, mechanical properties, biological properties(crosslinking effect). By studying the structure of collagen to presume the location of a number of diseases; By studying self-assembly of collagen laying a solid foundation for the development of new biomaterials; researching of Chromium can help researchers find better pollution-free leather additives for leather tanning industry.  Works and results: Part1. The collagen I used is extracted from crucian and grass carp by student Chenggong Yang. Using infrared spectrometer to Characterize collagen with amid band. Circular Dichroism is used to characterize the integrity of collagen triple helix. 4000 3000 2000 10000.70.80.91.0  T%Wavenumber/cm-1 Collagen from crucian collagen from grass carp Pic1-1：crucian carp/grass carp collagen-IR  180 200 220 240 260-80-400  thetawavelength/nm crucian carp grass carp Pic1-2：crucian carp/grass carp collagen-CD Part2. In the first part, I used Fluorescence Spectroscopy detect fluorescence intensity of collagen in different concentration and buffer.  4 5 6 7 840455055  Fluorescence intensitypH(0.1AcH-AcNa, 0.2Na2HPO4-0.3NaH2PO4)collagen from crucian0.001mg/ml0.01mg/ml Pic1-3: AcH(0.1M)-AcNa(0.1M)/[Na2HPO4(0.2M)-NaH2PO4(0.3M)](buffer)-crucian collagen 4 6 8801001201400.01mg/mL  Fluorescence intensitypH (0.01Ac-AcNa,0.02Na2HPO4-0.03NaH2PO4)Collagen from crucian0.001mg/mL Pic1-4: AcH(0.01M)-AcNa(0.01M)/[Na2HPO4(0.02M)-NaH2PO4(0.03M)](buffer)-crucian collagen 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.54550556065707580  0.001mg.mLFluorescence intensitypH (0.01Citric acid-0.02Na2HPO4)0.01mg.mLCollagen from crucian Pic1-5: citric acid（0.01M）- Na2HPO4（0.02M）(buffer)- crucian collagen 3 4 5 6 7 8 9100120140  Fluorescence intensitypH(0.1AcH-AcNa, 0.2Na2HPO4-0.3NaH2PO4)collagen from grass carp0.01mg/ml0.001mg/ml Pic1-6: AcH(0.1M)-AcNa(0.1M)/[Na2HPO4(0.2M)-NaH2PO4(0.3M)](buffer)- grass collagen 3 4 5 6 7 8100110120  Fluorescence intensitypH(0.01AcH-AcNa, 0.02Na2HPO4-0.03NaH2PO4)collagen from grass carp0.001mg/ml0.01mg/ml Pic1-7: AcH(0.01M)-AcNa(0.01M)/[Na2HPO4(0.02M)-NaH2PO4(0.03M)](buffer)- grass collagen 2 4 6 81121201280.01mg/mL  Fluorescence intensitypH (0.02Na2HPO4-0.01Citric acid)Collagen from grass carp0.001mg/mL Pic1-8: citric acid（0.01M）- Na2HPO4（0.02M）(buffer)- grass collagen Typically, the higher the concentration of collagen, the fluorescence intensity should be stronger, but a small range of reversion can be found according to the experimental data. The fluorescence intensity has relation with both collagen solution and pH as well as concentration of buffer, at the same time, salt ions in buffer also produce a specific effect on the fluorescence intensity of collagen, so this experiment will be explored in the next part : salt ions effect on collagen solutions.  Part3. I used 15 kinds of slats in this part of experiment(CH3COOH、CH3CONa、NaH2PO4、Na2HPO4、NaCl、MgCl、CrCl、CaCl2、CH3COOK、Ca(CH3COO)2、Mg(CH3COO)2、CH3COONa、Cr2(SO4)3、K2SO4、Na2SO4、MgSO4、Cr(CH3COO)3、KCl、CrO3) Then I used Fluorescence Spectroscopy detect fluorescence intensity of collagen in different situation.  . Table1-1: The pH and FL in salts(40mmol/l)- crucian collagen(0.05mg/ml) The pH of the collagen solution 3.14 The pH of collagen-salty solution and FL pH FL NaCl 2.96 438.4/5.692 Na2SO4 3.34 436.8/5.745 NaCH3COO 4.74 438.4/5.737 KCl 3.11 438.4/6.441 K2SO4 3.36 436.6/6.773  After adding salts, there are 4 ~ 6nm blue shifts in the collagen solution, and the absorption intensity is greatly reduced, which illustrates the structure of the collagen had changed after the metal ions is added. The reason that varying degrees for the decrease in fluorescence intensity is that there are differences in the way the metal ion binding with collagen.  There is fluorescence quenching in Cr(III). Transition metal ion chromium (III) is a very good fluorescence quencher may be related to fluorescence quenching in collagen molecules carboxyl complexation formation.  Pic1-9 fluorescence intensity of Cr2(SO)4 Summary: There are some elements have special effect to collagen’s isoelectric point as well as fluorescence intensity, such as buffer, pH and salty ions. They affect amount of application function of collagen. This essay mainly use fluophotometer to measure fluorescence intensity of collagen in different conditions. The result shows that there is a reversion of a certain pH in fluorescence intensity under ultra dilute solution. KCH3COO 4.75 437.8/6.740 MgCl2 2.97 439.2/4.935 MgSO4 3.25 436.2/5.029 Mg(CH3COO)2 3.34 436.8/5.745 CaCl2 2.79 437.2/7.384 CaSO4   Ca(CH3COO)2 4.84 438.8/6.383 CrCl3 2.78  Cr2(SO4)3 2.90  Cr(CH3COO)2 3.80  Collagen’s medification happens during adding slats, and there is a coordination reaction between Tyr and metal ions. 4~6nm blue shift in fluorescence intensity happened during it, and the intensity becomes lower dramatically.  Future work: The topic of this thesis is ‘Effect of Solution on the isoelectric point of collagen’, but indeed, professor Ding and I found it was more meaningful to focus a small part of it, so I mainly focusing about ‘the effect of solution on Spectroscopic Properties of collagen’, so in the next research, I will fulfill the whole experiment.  Lacking of time could not allow me to complete the whole experiment and get the final reasons for those experimental phenomena, so in the next experiment, we are focusing on why those phenomena happen.  Relating to the current equipment, the sensitivity cannot detect sample solution in very dilute state, so in the next study, we are going to study the theoretical calculation and simulation methods, obtained under special circumstances change in fluorescence intensity of collagen conclusions.  In our experiment, it also lacks the structure of collagen and collagen extracted salt solution analysis (such as CD spectroscopy, atomic force microscopy, etc.), rheological testing. So we will fulfill them in the future. 

Effect of solution on the isoelectric point of collagen

http://www.theseus.fi/bitstream/10024/115589/1/Mengmeng_Sui.pdf

Effect of solution on the isoelectric point of collagen

Abstract

Similar works

Full text

Available Versions

Theseus