A study of the binding of the biologically important hematin molecule to a novel imidazole containing poly(N-isopropylacrylamide) microgel

A study of the binding of the biologically important hematin molecule to a novel imidazole containing poly(N-isopropylacrylamide) microgel
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  A study of the binding of the biologically importanthematin molecule to a novel imidazole containingpoly(  N  -isopropylacrylamide) microgel V.J. Cornelius * , M.J. Snowden, J. Silver, G.R. Fern School of Chemical and Life Sciences, Medway Campus, University of Greenwich, Central Avenue,Chatham Maritime, Kent ME4 4TB, UK  Abstract The influence of two different comonomers incorporated separately at 1 %w/w into a poly(  N  -isopropylacrylamide)microgel is investigated by photon correlation spectroscopy, transmission electron microscopy and high sensitivitydifferential scanning calorimetry. The synthesis, characterisation and subsequent binding affinity of iron protopor-phyrin IX to the microgels was also investigated. M € ossbauer and electronic absorbance spectroscopies were used tocharacterise the resulting protoporphyrin IX iron(III) polymer complexes. Co-polymer microgels containing eithersulfide or imidazole residues acted as a matrix for the binding of haems to form soluble molecular structures containingmultiple iron centres.   2003 Elsevier B.V. All rights reserved. Keywords:  Poly(NIPAM) microgel; Imidazole; Iron protoporphyrin IX; M € ossbauer spectroscopy 1. Introduction Intelligent colloidal microgels prepared from  N  -isopropylacrylamide (NIPAM) are the subject of an ever-growing number of publications [1–10].The use of further monomers [7,8], and the incor-poration of comonomers [11,12] have also beeninvestigatedtobroadenthealreadylargenumberof possible applications [13–15]. These SMART mi-crogels find extensive application since they exhibitdramatic changes in volume with external stimulisuch as changes in temperature [16–19], pH [20],and ionic strength [21]. The potential applicationsinclude catalysis [22], paints and coatings [23–25],supports for organic synthesis [26,27], stabilisers[28], nanocasting and the production of photonicbandgapmaterials [29]. Whenswollenwith solventthey are typically below 1  l m in diameter with aparticle matrix that has many interstitial spaces.Microgels have high potential as controlled releasedevices due to their inherent ability to bind mole-cules to sites within the polymer matrix.It is well documented that microgels synthesisedwith NIPAM shrink and swell reversibly as afunction of temperature in response to changing Reactive & Functional Polymers 58 (2004) 165– REACTIVE&FUNCTIONALPOLYMERS * Corresponding author. Tel.: +44-1634-88-30-42; fax: +44-1634-88-30-44. E-mail address: (V.J. Cornelius).1381-5148/$ - see front matter    2003 Elsevier B.V. All rights reserved.doi:10.1016/j.reactfunctpolym.2003.12.003  solvent quality [30]. At elevated temperatures theaffinity of the polymer for the solvent is greatlyreduced which causes a significant change in thehydrodynamic volume of the microgel particles[31]. This is a direct result of solvent exclusion,changes in dimensions of the particles and altera-tions in polymer–polymer interactions in relationto polymer–solvent interactions. It has been re-ported [32–38] that absorption of specific mole-cules to the microgel can occur when the particlesare expanded, at low temperatures, and the mole-cules are expelled from the matrix when the par-ticle collapses on heating to a temperature abovethe critical temperature. As a result of their abilityto bind and expel molecules as a function of tem-perature, microgels have found considerable use ascontrolled uptake and delivery devices for mole-cules such as a specific drug [32–37] or heavy metalions [38].Mammalian haemoglobin consists of four pep-tide chains called globins, each of which is boundto a haem. The iron in the haem is coordinated tothe four pyrrole nitrogens of protoporphryin IX,and to the imidazole nitrogen of a histidine residuefrom the globin. The sixth coordination position isavailable for binding to oxygen or other smallmolecules. In the vitally important respiratoryprocess haemoglobin binds oxygen and formsoxyhaemoglobin, HbO 2 , the oxygenated form of haemoglobin, when the oxygen is displaced bycarbon monoxide carboxyhaemoglobin, HbCO[39] is formed. In biological systems it is wellknown that iron nitrogen [40,41] and iron sulfurreactions [42,43] are common, hence the binding of iron(II/III) protoporphyrin centres (haems) tomicrogels presents an important model for mam-malian biology.Novel copolymer microgels were preparedincorporating specific functional groups topotentially enhance the binding of iron(III)protoporphyrin IX hydroxide (hematin). Thefunctional groups incorporated contain either ni-trogen or sulfur ligands in order to increase theaffinity of haem complex to the microgel. Thebinding was investigated both quantitatively andqualitatively using M € ossbauer and electronic ab-sorbance spectroscopy. The haem microgel com-plex produced may potentially find use for thereversible binding of oxygen and carbon monoxideand in catalysis. 2. Materials and methods  2.1. Microgel synthesis Reagent grade  N  -isopropylacrylamide (NI-PAM: Sigma-Aldrich),  N  0 ,  N  -methylenebisacryla-mide (BIS: Sigma-Aldrich), potassium persulfate(BDH chemicals) 1-vinyl imidazole (Sigma-Aldrich), and allyl methyl sulfide (Acros Organics)were obtained and used without further purifica-tion. Three microgels were prepared as 0.5% dis-persions using the standard surfactant freeemulsion polymerisation method adapted from theprocedure described by Crowther and Vincent [5];the homopolymer NIPAM microgel and two co-polymer microgels containing vinyl imidazole orallyl methyl sulfide, both incorporated at 1 %w/winto a poly (NIPAM) microgel.Colloidal microgel particles were produced, inthe absence of surfactant, by a free-radical poly-merisation reaction in water, at 70   C, under anitrogen atmosphere. 0.5 g l  1 potassium persul-fate initiator was placed in a three-necked round-bottomed flask and stirred continuously for 6 h at120 rpm. Pre-dissolved  N  -isopropylacrylamide andthe cross-linking agent  N  ,  N  -methylenebisacryl-amide were added to the reaction vessel, at con-centrations of 5.0 and 0.5 g l  1 , respectively. Thepreparation of both co-polymer microgel disper-sions followed the same procedure; for the vinylimidazole microgel 0.05 g l  1 was pre-dissolvedwith 4.95 g l  1 NIPAM and 0.5 g l  1  N  ,  N  -methyl-enebisacrylamide. The only difference in thepreparation of the sulfur containing microgel tothe vinyl imidazole copolymer microgel is that allylmethyl sulfide is a volatile liquid so 0.05 g l  1 wasinjected directly into the reaction mixture at thesame time as the pre-dissolved solution was added.In all cases, after 6 h at 70   C, the crude mi-crogel was cooled and repeatedly dialysed againstdeionised water (to remove unreacted monomerand ionic species) until a constant conductivityvalue (1  l S) was obtained. Typical reaction yieldswere of the order of 90%. Dry weight analysis of  166  V.J. Cornelius et al. / Reactive & Functional Polymers 58 (2004) 165–173  the microgel showed the dispersions to be of theorder 0.45±0.05 %w/w.  2.2.  57  Iron protoporphyrin(III) IX hydroxide syn-thesis Iron(III) protoporphyrin IX hydroxide wassynthesised from iron(III) protoporphyrin IXchloride, which itself was synthesised using themethod previously described [44] using concen-trated hydrochloric acid (Sigma-Aldrich),  57 Fefoil, protoporphyrin IX (Sigma-Aldrich) and so-dium chloride (Sigma-Aldrich). This was usedwithout further purification.The solid product obtained [44] was dissolved inpotassium hydroxide at pH 8, and then slowly thepHwasloweredto6.5usingverydilutesulfuricacid(0.01 mol l  1 ) both obtained from BDH chemicals.The solution was heated gently until a precipitateformed. After filtration and washing with ice colddeionised water the solid was re-dissolved in po-tassium hydroxide and the pH lowered slowly untila precipitate formed, this was repeated to yield ir-on(III) protoporphyrin IX hydroxide (hematin).The final precipitate was characterised using elec-tronic absorbance spectroscopy and M € ossbauerspectroscopy. All samples were stored at 4   C.  2.3. Haem microgel binding  At pH 7, 10 ml of iron(III) protoporphyrin IXhydroxide solution (50 mg/l) was added to 5 ml (4g/l) of each of the three microgels separately (NI-PAM homopolymer, 1% imidazole and 1% methylsulfide). They were then each sealed in a centrifugetube and degassed by 10 nitrogen/vacuum cycles.The centrifuge tubes were placed in a glove boxand left open for 12 h when sodium dithionite wasadded until the iron was reduced to iron(II) pro-toporphyrin IX. The samples were again sealedand then centrifuged at 5000 rpm for 4 h afterwhich they were opened in a glove box to decantthe supernatant. The supernatant was then analy-sed after re-oxidation using UV–Vis spectroscopy.Quantitative analysis of the haem microgelbinding was achieved by preparing 10 solutions of hematin at known concentrations in the absence of air below 50 ppm at pH 7. The Soret band and oneQ band was measured and found to follow thesame trend but only the Soret band analysis isreported for accuracy. As the solutions were at pH7 the sample was present predominately as ir-on(III) protoporphyrin IX hydroxide as opposedto [Fe(III)PPIX] 2 O. For each individual microgelthe capacity for binding was then determined usingthe described method by using consistent amountsof microgel and sodium dithionite (always in ex-cess) and varying only the concentration of haemin the centrifuge tube. After centrifugation thesupernatant was analysed by UV–Vis spectroscopyin the absence of air using matched sealed quartzcuvettes. This depletion from solution was re-peated three times for each microgel and the datawas found to be reproducible.  2.4. Transmission electron microscopy Transmission electron micrographs of the mi-crogel particles were obtained by pipetting ap-proximately 10  l l of microgel dispersion onto acarbon coated copper grid to support the particlesand allowed to dry. A JOEL JEM 200CX TEMoperated between 120 and 200 kV was used toobtain electron micrographs and the mean particlediameter was determined from measuring at least25 particles per micrograph.  2.5. Photon correlation spectroscopy Samples were prepared for analysis by photoncorrelation spectroscopy in clean stoppered vialsby approximately four-fold dilution with deionisedwater. The hydrodynamic diameter of each mi-crogel dispersion was obtained from dynamic lightscattering measurements using a   Malvern Instru-ment   Zetasizer 3000 instrument, fitted with a 5MW He–Ne laser ( k  632.8 nm) and a detectorplaced at 90  . Five measurements were taken ateach temperature at 5   C intervals over the tem-perature range of 25–60   C.  2.6. High sensitivity differential scanning calorime-try Thermodynamic analysis of the microgel dis-persions was carried out using a Microcal MC-2D V.J. Cornelius et al. / Reactive & Functional Polymers 58 (2004) 165–173  167  ultrasensitive DSC (Microcal Inc Northampton,MA, USA). Data acquisition and analysis werecarried out using the DA2 software package,supplied by the manufacturer. High sensitivitydifferential scanning calorimetry (HSDSC) deter-minations used scan rates of 60 Kh  1 and all theconformational transitions examined were repro-ducible in that the first and second heating scanswere identical.  2.7. Electronic absorbance spectroscopy A calibration curve of iron(III) protoporphyrinIX hydroxide in aqueous solution at pH 6.5 wasobtained by visible analysis at 386 nm and 25   Cusing a Hewlett Packard 8452A single beam diodearray spectrophotometer and the temperaturemaintained using a Hewlett Packard 89090A Pel-tier temperature control unit. The clean quartzcuvette used was sealed and all oxygen removed by10 vacuum/nitrogen flushes.  2.8. M  € o ssbauer spectroscopic analysis The M € ossbauer spectra of all haem containingcompounds were obtained at ambient pressure at77 K using a previously reported custom appara-tus [45]. Spectra were fitted to Lorentzian lineshapes and all the data was referenced to a 25  l mthick natural iron foil. 3. Results and discussion 3.1. Microgel characterisation The microgel particles were characterised byphoton correlation spectroscopy (PCS: Fig. 1),transmission electron microscopy (TEM: Fig. 2)and high sensitivity differential scanning calorim-etry (HSDSC: Fig. 3).Hydrodynamic size determination of the mi-crogels at 25   C showed that the polydispersitywas low for all microgel dispersions (see Table 1).The NIPAM homopolymer microgel is very simi-lar in size to that of the copolymer microgel in-corporating imidazole. The methyl sulfidemicrogel was found to be significantly bigger. Theonly difference between the synthesis of this mi-crogel was the comonomer, allyl methyl sulfide,which was not pre-dissolved but directly injectedinto the reaction vessel. Although the particles arelarger, from all other characterisation, the micro-gel dispersion behaves exactly the same as theother microgels under investigation.The size of the particles obtained by TEM wasconsistently larger than the size obtained by dy-namic light scattering. This is a consequence of theevaporation of solvent from within the microgel asthe particles are dried, which facilitates a collapseof the structure to give the appearance of an oblatespheroid. The particle size was determined by re-peatedly measuring the distance from the centresof neighbouring particles. This is consistent withdata reported by Saunders and Vincent for othersimilar microgel dispersions [15]. The monodis-perse nature of the particles from the micrographsis consistent with the low polydispersity suggestedby dynamic light scattering and the uniformity of the dispersion attributed to interparticle repulsionand surface tension.PCS was also used to study the effect of tem-perature on particle size over the temperaturerange 25–60   C. The size was measured repeatedlyat 5   intervals and the polydispersity of the parti-cles was found to be low in all cases. The volumephase transition temperatures (VPTT) were de-termined by obtaining a first derivative plot of thechange in size vs. temperature and were found tobe 34.5 (5)   C in all cases. These values comparedfavourably with the VPTT obtained by HSDSCshown in Fig. 3 (this data has not been correctedfor concentration effects). The reason that theVPTT values obtained for the copolymer micro-gels are not significantly different from the ac-cepted value of 34   C for the homopolymermicrogel of NIPAM [15] is because of the low levelof comonomer incorporated. The change in tran-sition temperature often seen on incorporating acomonomer is due to the alteration of the com-position of the polymeric backbone with respect toits hydrophobicity. As the comonomer  s incorpo-rated are not highly hydrophobic and were onlyincorporated at 1% it was not expected that therewould be a large deviation from the VPTT ob-tained for the homopolymer. 168  V.J. Cornelius et al. / Reactive & Functional Polymers 58 (2004) 165–173  Two novel copolymer microgels incorporatingeither imidazole or sulfide residues have beenprepared using standard methods and character-ised [13,15]. The particles formed have been de-termined to be monodisperse and as expected, theyare sensitive to heat with swelling ratios of 2.01,2.35 and 2.73 for the 1% imidazole, NIPAM and1% methyl sulfide microgel, respectively. The datais comparable to data reported previously onsimilar systems [13,15]. 3.2. Haem analysis The M € ossbauer spectrum of iron(III) proto-porphyrin IX hydroxide was recorded at ambientpressure at 77 K (see Figs. 4 and 5). The low Fig. 2. TEM of microgel particles. (a) NIPAM microgel, (b) 1% allyl methyl sulfide microgel, (c) 1% vinyl imidazole microgel. All atsame magnification. 02004006008001000120020 25 30 35 40 45 50 55 60 65 Temperature (˚C)    H  y   d  r  o   d  y  n  a  m   i  c   d   i  a  m  e   t  e  r   (  n  m   ) NIPAM 1% methyl sulfide 1% imidazole Fig. 1. Size data as a function of temperature by photon correlation spectroscopy. V.J. Cornelius et al. / Reactive & Functional Polymers 58 (2004) 165–173  169
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