Poliakrilamid-bakır-protein üçlü komplekslerinin oluşumu ve fizikokimyasal özellikleri
The Formation of ternary polyacrylamide-copper-protein coplexes and their physicochemical properties
- Tez No: 39661
- Danışmanlar: PROF.DR. A. SEZAİ SARAÇ
- Tez Türü: Doktora
- Konular: Kimya, Chemistry
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 1994
- Dil: Türkçe
- Üniversite: İstanbul Teknik Üniversitesi
- Enstitü: Fen Bilimleri Enstitüsü
- Ana Bilim Dalı: Belirtilmemiş.
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: 61
Özet
ÖZET Bu çalışmada seryum(lV) sülfatın çok hızlı elektron transferi Özelliğinden yararlanarak amino asit uç gruplu poliakrilamid elde e- dilmesinde baslatıcı olarak kullanılmıştır. Bu amaçla seryum(IV)-glisin redoks reaksiyonunda oluşan glisin radikali aracılığı ile poliakrilamid polimerizasyonu gerçekleştirilmiştir. Baslatıcı miktarının, sıcaklığın ve zamanın polimerizasyon verimine ve polimerin molekül ağırlığına etkisi araştırılmıştır. Bu yöntemle elde edilen polimerlerin metal ile oluşturduğu komplekslerin oluşumu ve fizikokimyasal davranışları incelenmiştir. Ayrıca polimerlerin metallerle kompleks yapma özelliği ve bunların proteinlerle oluşturduğu üçlü komplekslerin biyolojik ve farma kolojik önemi nedeniyle sıcaklık, baslatıcı ve zaman etkisiyle sentezlenen farklı molekül ağırlığındaki polimerlerin bakır ile oluşturacağı kompleksler fizikokimyasal metodlarla incelenmiş ve oluşan metal polimer komplekslerinin proteinle oluşturduğu üçlü kompleksler spektrofotometrik metodla incelenmiştir. Polimer-metal çözeltilerine farklı miktarlarda protein içeren protein çözeltileri ilave edildiğinde, üçlü karışımdaki protein miktarı artarken suda çözünmeyen kompleks oluşumu artmakta (ngs^/npjvjK^l) ve maksimum değere ulaşmakta (nBSA^PMK^) ve ^aha faz^a protein ila ve ettiğimizde (siBSA/^MK^) sistemin tekrar suda çözünür hale geldi ği 400 nm deki spektrofotometrik ölçümlerin sonuçları ile bulunmuştur, ikili kompleksin aksine üçlü kompleksin oluşumunun zamana bağlı olarak değiştiği gözlenmiştir. Bu olay suda çözünmeyen üçlü komplekslerin oluşum kinetiğinin farklı reaksiyon koşullarında spektrofotometrik olarak araştırılmasına olanak sağlamıştır. Bu nedenle suda çözünmeyen üçlü kompleksin oluşum kinetiği- bileşenleri hazırlama yöntemi ne, bileşenlerin karışımlarının oranına, düşük molekül ağırlıklı tuzlara, sıcaklığa ve polielektrolitin molekül ağırlığına bağlı olarak incelenmiştir.
Özet (Çeviri)
THE FORMATION OF TERNARY POLYACRYLAMIDE-COPpER-PROTEIN COMPLEXES AND THEIR PHYSICOCHEMICAL PROPERTIES SUMMARY Cerium(IV) sulphate, cerium(IV) perchlorate and cerium(IV) nit rate have been commonly used for free radical polymerization. The redox reaction between cerium(lV) sulphate and organic reducing com pound form a radical for initiation, of vinyl polymerization. Ce(IV) is reduced to Ce(III) and oxidized compound starts the polymerization. If HLH represents organic molecules and M represents monomer, the polymerization reaction has been shown as following. HLH + Ce(IV)- > HL. + Ce(III) + H+ ki HL. + M -U. HL-M. Initiation k HL-M. nM - ^> HL-M, Propagation k n ' tl 2HL - M, «- Polymer Termination n+ 1 k HL - Mn+1 + Ce(IV) -^* Polymer + Ce(III) + Hf Termination It is well known that these eerie salts also form very effective redox systems in the presence of organic reducing agent such as alco hols, aldehydes, carboxylic acids. This method was also used for the preparation of graft copolymers of vinyl monomers such as acrylonit- ri le and acryl amide. In this work the polymerization of acrylamide initiated by the eerie sulphate and glycine. The dependence polymerization yields and molecular weights of polymers on the ratio of molecules of acry lamide to the molecules of glycine, polymerization time and tempera ture. During all reactions, the concentration of sulphuric acid of polymer solution was constant and 0.05 M. It was observed that the decrease on the ratio of the molecules of acrylamide to the molecules of glycine resulted an increase in the molecular weight, but a decrease in the yield of the polymers of acrylamide containing glycine end group (PE). The effect of polyme rization temperature and time on the yield and molecular weight of vipolymer were studied. Following proposed chemical structures of PE synthesised whether the termination of polymerization is occured by mutual or by Ce(IV). Where n=280 (PE1)9 630 (PE2) and 3200 (PE3), X - (AAL - X or (AA)“ - X n n and X group have following formuleas ^COOH -CH. - CH - CH *? i -v. I NH2 From the FT-IR results of our PE at the 1390-1410 cm”1, the presence of carboxylic groups was observed unlike the homopolyacryla- mide. The titration of end carboxylic groups in PE was performed by using NaOH and molecular weight of polymers (Mn) was calculated as following. 3 Equiv of Carboxyl end groups/ 10“ g = - ^- - ! - m Mn. LîJ°l equiv. of carboxyl ends/10 g Where V, N, m- the volumes normality of titrant and weight of polymer respectively. The average molecular weight (Mv) was obtained by measuring viscosity of solution at 30°C by using following equation a °-66 [qj - 6.8 x 10”My Specific conductivity of PE at definite weight was measured by using E 518 Metro hm Harisau Conductometer. The result have been shown in Table 1. VllTable 1 Molecular Weight and Conductivities of Polyelectrolyte Polyelectrolyte fn“I Mv Mn Mv/Mn n= &. â K x 106 L VJ M»» (nhm-lrn ^AA (ohm-lcnH) PE1 0,44 20 000 19 000 1.05 280 380 PE2K 0.80 45 000 22 200 2.02 630 296 PE3- 2.34 230 000 - - 3200 168 a: The spesific conductance of 0,3 g polymer dissolved in 50 ml water. B: Since poller have high molecular weight» carboxylic acid groups were difficult to determine For the synthesis of polymer metal complexes (PMC), Polymer was dissolved in neutral water at pH 7 and CUSO4.5H2O was dissolved in water in acidic medium (pH 4) and the solution of CUŞO4 was added to polymer solution while stirring at 20°C then pH of solution was adjus ted to about 7 and viscosity, spectrophotometri c properties at 254 nm and surface tension of solution were determined The synthesis of ternary complexes of PE-metal -protein, protein (bovine serum albumin, BSA, M=70.000) was dissolved in water at pH 7 and added to polymer metal complex solution, then pH of solution was also adjusted to 7. Molar ratio of CUSO4 to polymer was kept constant at 0.1 (ncuS04/nAA=0-1 » nCuS04 and nAA are tne number of tne moles in one volume of solution). For spectrophotometri c measurement (Shimadzu UV-160 A), UV-vi- sible was used at the 200-1000 nm. FT-IR spectrum of substances was taken by Jasko 5300 spectrophotometer 1- The Preparation of The Polymer Metal Complex Addition of metal ion to polymer (PE) starts to give an homo geneous solution for the certain value of r)QU/n^ at pH 7. CUSO4 is not soluble at pH 7, but its mixture with PE is soluble in neutral water system this result gives an idea the formation of the soluble polymer metal complex. In fact UV-visible spectrum of FE support the idea of complex formation at 254 nm. The absorbans increase linearly with increasing concentration of the cupper ions. This implies that cupric ions form complexes with PE molecules. The infrared spectra of PE and PMC have been taken as compared with PE spectra in the case PMC spectra, we have observed new peaks vmat 592, 610, 800 and 1100 cm”1 and increase a peak at the 2800 cm“1. This result suggest that the cupric ion is bound to -NH2» -C=0, -C00H groups in PE. The viscosity of PE and its metal complex showed that the reduced viscosity against concentration of the cupric ion passes through a minimum. This effect may be readly understood, by the interaction of the polyion with metal ions in two ways. In the small concentration of metal ions, intramolecular interaction have been possible. This effect leads to compactization of macromo- lecular coils. The higher concentration of metal ions take place intermolecular interaction of polymer coils. The divalent cupric ions act as ”fasteners“ between the different polymer coils. Ternary complexes of metal-polymer complexes with bovine serum albumin have also investigated. 1> The Polymer Metal Complexes of BSA 2.1. Ternary Complex Formation For the ternary complexes, soluble polymer metal complexes were used. The physico-chemical parameters of the used PMC have been shown in Table 2. Table 2. Molecular Weight and Conductance of PMC PMC MV(PE) -nCu/nAA ~ ( - ) Spesific conductance c g (cm- 1 ohm-1) x 1Ü6 PMC1 20 000 0.1 0.552 986 PMC2 45 000 0.1 0.823 981 PMC3 230 000 0.1 2.558 931 a: The interaction of BSA was studied at this concentration ratio In the neutral water medium, the solution of PE was mixed with the solution of BSA at about pH 7 without cupric ions. In the wide range ratio of component, the system remains homogeneous. This system was investigated with spectrophotometry measurements of optical density (Apgg) °f tne mixture of protein and PE1 on the amount of added protein at constant concentration of the PEL The value of A28O increases with increasing the protein concentration in the system. But in all case the values practically are the same with ^280 obtained for the free protein solution and polymer protein mix tures. These results suggest that there is no interaction between PE and BSA. But when we add protein solution to PMC solution in the same condition, the value of A28O strongly changed with increasing of the concentration of BSA. i xPF.-Ctr -BSA was prepared mixture with adding different pro tein concentration to PMC solution. At tire begifiing of reaction,, alî systems were water soluble and homogeneous' then time to time the dependence on the ratio of these mixtures, the homogenity was lost and insoluble particles appeared in the system. Aftfcr 24 hours, the optical densities of the mixtures were measured. The dependence of the optical density in 400 nm (I\/\qq) on the molecules of protein glohuls per one polymer molecules (”BSA^pMr) passes through a maxi mum. Starting with very low values of nusA/npMC in the system phase separation takes place, which indicates the formation of an insoluble complex. On further increase in nus/^/npMc- %() increases and gives a limiting value, depending on the protein concentration in the“ mixture, partial or complete prevention of phase separation takes place. After the critical ratio of the pro tein/ polymer (uBSA/nPMCr?) the mixture again shows water soluble character, i.e. the system begins to be homogeneous. Analogically the dependence I\aq{) on the ratio np|v|r/npcn passes through a maximum when adding the PMC solution of l?SA at constant concentrations. According to both cases without dependence of the method of preparation of mixtures, in systems insoluble triple poly mer-metal-protein complexes occur. 1.1. Hie Kinetics of The fonuation of Triple Complexes 1- OSA was added to the solution of PMC 2- PMC was added to the solution of BSA In both cases, the value of A400 increases with the increasing the time of reaction and then attains a limiting value. The rate of reaction (dA/dt) was obtained from the tangent of curves of the ini tial time of react. ion mixtures. The value of the (dA/dt)j and (dA/dt)? were found 1.93 x 10~? and 1.22 x 10”2 respectively. Accord ing to these values, the reaction 1 is more rapid than reaction 2. In“ t he second system when adding PMC to BSA at the initial moment, low amount of polymer metal complex. Unlike the first system accord ing to this, the more insoluble complex forms, after adding the others amount PMC distribution of polymer chains between complex particles is difficult and therefore absorbans increases slowly in second system, 2.2.1. The Effect of The Ratio of Components PE-Ci/-+-BSA mixture was prepared by adding of BSA solution to PMC solution. The ratio nncft/np^c was chosen as the initial time of reaction (nRrA/npMr=0. 15), the maximum precipitation (nBSn/nPHc=l) and the ratio of solubility of this system (nB$/\/nFMC-2). The change of value A/jqq depending on time for the different ratio take place various characters. The rate of the reaction (dA/dt) wascalculated from the tangent, of curves mentioned above. With increas ing of the concentration of proteins at first, the rate of reaction increases to reach limit value and then decreases. By the ratio (”BSA^PMC^) of *-Me formation of soluble complexes, the value of W- dA/dt=0. 2.2.2. The Effect of The Dilution Systems The triple systems, obtained at the ratio corresponding to maximum precipitation (nps/^/npi^^l) was dissolved with neutral water and investigated by the spectrophotometry method at different time of the reaction at the constant temperature. The mixture was prepa red by adding protein solution to PMC solution. We have seen that dA/dt is a function of the degree of dilution and then attains a maximum value. When we have very high dilution of the triple mixture the value of dA/dt begin rapidly decreases and attains a minimum value. It should be accepted that by the dilution, according to decrease of concentration, the velocity of diffusion and the process of exchange with the triple particles in the system increase. Very high dilution leads to decrease of the interaction between the col loid particles. Tor this reason the velocity of the formation of the insoluble colloid particles decreases. 2.2,3. lhe Effect of Low Molecular Salts The effect of the low molecular weight salts, the mixture PMC- BSA was prepared at different concentration- of the sodium chloride by adding NaCl to triple solutions. The ratio of the npsA^PHC ^? constant and equals 1. The analysis of the systems by spectrophoto metry method showed that the concentration of NaCl acts on not only a homogeneity of the system but also the velocity of the formation of the colloid particles. It is seen that with increasing NaCl concentration, the slope of curves decrease and at higher concentra tion of NaCl, the triple system has been homogeneous and the absor- bans A/joo against time practically does not change. 2.2.1. T tie Effect of The Molecular Weight of PE The effect of the molecular weight of PE has been studied at different molecular weiolit of PE: 20.000 (PMC1), 45.000 (PMC2) and 230.000 (PMC3). In all'casos, the ratio is nCu/nAA-0.1 and np^n/npi^l. The reactions of the triple systems PE-Cu?f-BSA were studied at constant temperature by adding BSA solution to PMC solu tion. The value of optical density of triple systems depend on the molecular weight of polyelectrolyte and decrease with increasing mo lecular weight. In higher molecular weights the triple system is more homogeneous than that in low molecular weight. Morevor the change of the A/J00 with time increases more strongly in low molecular weight than in hi gh molecular weight of polyelectrolyte.
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