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MY RESEARCH PROPOSALS/OBJECTIVES

My research revolves around several disciplines of chemistry, namely, bioinorganic, transition metal, physical, organic, and analytical chemistry. My research objectives are listed below:

1.    Isolation, purification, characterisation, and reactivity of purple acid phosphatase (PAP) from mammalian and plant species. 

  PAPs are a category of dinuclear metal-containing enzymes, which catalyse the hydrolysis of phosphate esters [O3P(OR)2-] in living organisms {D.F. Hunt et al., Biochem. Biophys. Res. Commun., volume 144, p. 1154 (1987)}, where R is an organic group (equation 1). It occurs in red kidney beans, soybeans, sweet potatoes, and the uterine fluids of a pregnant sow (60 days in pregnancy), to name a few. PAP, a metallo-protein, is known to contain FeII-FeIII (uterine fluids of the pregnant sow) or ZnII-FeIII (red kidney beans),

O3P(OR)2- + H2O ® HPO42- + ROH                                                               (1)

where the Fe(III) centre is linked to amino acid, tyrosine. The characteristic intense purple colour of PAPs is due a ligand to metal charge transfer (LMCT) process involving Tyrosine® FeIII. The dinuclear iron centre of the mammalian PAPs exists in one of the two oxidation states: an active reduced FeII-FeIII form (l max, 515 nm; pink) and an inactive oxidised FeIII-FeIII form (l max, 550-570 nm; purple) {J.-S. Lim et al., Inorganic Chemistry, volume 35, p. 614 (1996)}.

The important X-ray crystal structure of the ZnII-FeIII PAP from red kidney beans (Mr ~ 55 kDa per homodimer) has been reported to a resolution of 2.65 Å {N. Sträter et al., Journal of Molecular Biology, volume 259, p. 737 (1996)}. The active site structure is as shown below in Figure 1.

There has been some controversy surrounding the metal content of PAP isolated from soybeans and sweet potatoes. My aim is to isolate and purify PAP from various sources, then analyse the metal content by ICP, carry out physical measurements, grow crystals of PAP for X-ray crystallography, and to study the reactivity of this enzyme with phosphates and biological reductants and oxidants. The techniques involved in the isolation, purification, and reactivity will include chromatography, electrophoresis, dialysis, ultrafiltration, electron spin resonance (ESR), NMR, UV/Visible, and infrared spectroscopy.

As the Zn(II) and Fe(II) metal centres are labile and susceptible to substitution by other metal(II) ions, new analogues of PAP will be synthesised and their reactivities will be studied. Metal(II) ions will include Co(II), Mn(II), Hg(II), Cu(II), to name a few.

My aim also is to correlate data from inorganic reaction mechanisms involving native PAP and substituted PAP in aqueous media at physiological pHs. The main instruments used in these studies will involve the ESR and NMR spectrometers, the stopped-flow and conventional spectrophotometers.


Figure 1. ZnII active site structure of kidney bean PAP, as a model for the FeII-FeIII active site of the mammalian enzyme.


These studies will provide an interesting link to the cause of Alzheimer’s disease, where the brains of Alzheimer sufferers are said to have a low concentration of a PAP, and possibly the causes and treatment of sickle cell anaemia. I have carried out research in this area while collaborating with Professor A. Geoffrey Sykes of the University of Newcastle, United Kingdom. Funding was from the Wellcome Trust, United Kingdom.

2.    The synthesis and biomedical uses of Nitric Oxide (NOX) containing Compounds.

Nitric oxide (NO) involvement has been found in nearly every aspect of human biological processes from controlling blood pressure to neurotransmission. NO has been found to function within the immune system to fight infectious diseases, blood parasites, and tumour cells. In addition, NO has a role in heart diseases, hypertension, impotency, shock, and cognitive processes. S-nitrosothiols (RSNOs) are believed to play an important role in storing, transporting, and releasing NO. Numerous biological functions of RSNOs continue to be uncovered to date, for example, recently it has been reported that blood flow is regulated by S-nitrosohaemoglobin via the release of NO. Additionally, S-nitrosation of certain thiol groups on the calcium release channel may regulate the channel.

Work by Furchgott and Zawadzski led to the discovery of a key role in the relaxation of blood vessels for the endothelium and an endogenous substance, the endothelium derived relaxing factor (EDRF) {R. Furchgott and J.V. Zawadzski, Nature, volume 288, p. 373 (1980)}. The EDRF was identified as NO {R.M.J. Palmer et al., Nature, volume 327, p. 524 (1987) and L.J. Ignarro et al., Proc. Natl. Acad. Sci. USA, volume 84, p. 9265 (1987)}. There have been reported discrepancies between the properties of EDRF and those of NO and it has been suggested, for example, that S-nitrosothiols or an iron complex having low molecular weight thiol ligands may account for the vasodilatory properties of EDRF. The recognition of the toxicity of NO and its short half-life in vivo has led to studies suggesting protein S-nitrosothiols as possible pools of EDRF. Two well-known S-nitrosothiols are shown in Figure 2 below, along with the nitroprusside anion, a known vasodilator:


 

 Figure 2. Some S-nitrosothiols and the nitroprusside anion


The nitroprusside anion comes in the commercially available compound, sodium nitroprusside. As a prescription drug, it is known as NIPRIDE, available as a powder (50 mg) in ampoules. It is used for critical hypertension, heart failure, and controlled hypotension in open heart surgery {Prescription Drugs, Geddes and Grosset, 1997, p. 256, ISBN: 1-85534-877-2}.

S-Nitrosothiols (RSNO) are now widely believed to be important species in the storage and transport of nitric oxide in vivo {P.R. Myers et al., Nature, volume 345, p. 161 (1990)}. Since they generally show the same physiological properties as does nitric oxide itself (notably the induction of vasodilation and inhibition of platelet aggregation {M.W. Radomski et al., Br. J. Pharmacol., volume 107, p. 745 (1992)}), it has been generally assumed that RSNO species act in this way by first releasing nitric oxide. Release of NO from an RSNO can be brought about by at least three routes: (i) a spontaneous thermal reaction, which is too slow at ambient temperatures to be important, (ii) a photochemical reaction, which is also unimportant in the absence of appropriate incident radiation, and (iii) a rapid copper(II) catalysed process {D.L.H. Williams, Acc. Chem. Res., volume 32, p. 869 (1999)}. For reaction (iii) it has been shown {A.P. Dicks et al., J. Chem. Soc., Perkin Trans. 2, p. 481 (1996)} that the decomposition of RSNO is brought about by aqueous copper(I) generated by the thiol reduction of aqueous copper(II). Low levels of thiols [RSH] always present in equilibrium with RSNO {P.H. Beloso and D.L.H. Williams, Chem. Commun., p. 89 (1997)} are sufficient to bring about the reduction. All three decomposition pathways initially generate nitric oxide and the corresponding disulfide, [equation (2)] although in the

2RSNO ® RSSR + 2NO                                                                         (2)

presence of oxygen oxidation of nitric oxide occurs and the final product in aqueous buffer is the nitrite anion.

My research revolves around the synthesis, characterisation, and reactivity of S-nitrosothiols (RSNO), transition metal NO complexes, and other NO releasing compounds in aqueous media. The RSNOs currently used are S-nitrosoglutathione, S-nitroso-N-acetyl-DL-penicillamine, and S-nitrosocysteine, to name a few. Their reactivity with L-ascorbic acid, nitrogen-containing and sulphur-containing nucleophiles are in progress. Kinetics, identification of reaction products, and detection of NO are currently being carried out by my research group. Techniques involved in following the kinetics are stopped-flow and conventional spectrophotometry. Infrared spectroscopy is also used to identify the reaction products. Eventually electron spin resonance (ESR) spectroscopy will be used in the detection of free radicals produced in the release of NO from the NO releasing compounds. I will synthesise some novel copper-containing complexes then use them to react with RSNOs in order to produce NO. Physiological testing will be carried out on these compounds in the near future.

3.    Inorganic Chemistry: Reaction Mechanisms-Synthesis and characterisation, and reactivity of transition metal complexes.

My research activities are in the field of inorganic reaction mechanisms. Kinetic techniques currently used in my laboratory include conventional techniques as well as methods to investigate reactions in the milli-second, minute and hour range (stopped-flow and conventional spectrophotometry). My basic interest lies in oxidation reduction and substitution reactions of transition metal coordination compounds. Present activities are in three major areas:

The measurements of reaction rates are supplemented by structural, spectroscopic and thermodynamic studies. 95Mo NMR, ESR, 13C and 1H NMR, and other physical means are used in the structural elucidation of the transition metal coordination compounds.

Summary:

I am now concentrating on two (2) very important areas of my research, which involves inorganic reaction mechanisms in aqueous media (nitric oxide chemistry and other bioinorganic chemistry), as well as the coordination chemistry and catalytic effects (the mechanism aspects) of some selected transition metals. These catalytic effects involve the epoxidation of alkenes and well as oxidation of alkanes to alcohols

Compounds or complexes, which contain nitric oxide are of importance in their role in delivering nitric oxide, a known vasodilator, but the mechanisms involving the release of nitric oxide is not clear cut in aqueous media. We hope to complement the synthesis and reactivity (with known metallo-proteins) of these nitric oxide-containing species.

MY TEACHING PHILOSOPHY

My teaching philosophy is that all inorganic chemistry students should have training in all areas of chemistry, namely, organic, physical, and inorganic chemistry. I try to overlap these disciplines in my teaching and laboratory experiments, also aspects of safety in the laboratory is encouraged and applied. I try to make my advanced students aware of the latest developments in the areas of bioinorganic and inorganic chemistry. Applications of computers and chemistry software are encouraged and applied. Such software include MS Word, MS Excel, MS PowerPoint, Statgraphics, and Origins, just to name a few.

I am using many techniques in my dissemination of chemistry, where I am using overhead transparencies, PowerPoint, and the use of the Procter for all of my chemistry courses, especially with classes of at least 115 students. Other multimedia packages are of vital importance for the dissemination of my chemistry and will be used in the near future. I must say that I have been instructing students, both undergraduates and postgraduates, the use of the library/literature searches and the writing of good project reports.

For any undergraduate or postgraduate in my research group, the following is instilled:

11/28/01 18:37