Ei-ichiro Ochiai
University of British Columbia Voncouver 8, British Columbia, Canado
A Laboratory Program for Bioinorganic Chemistry
The recognition of the importance of inorganic chemistrv for the bioloeical sciences. includina environmental our department to establish a course chemistry, entitled "Inoreanic Chemistw for Bioloaical Sciences" which was first offered in the 1971-72 academic year. It is intended primarily for third year students of biological disciplines including biochemistry, physiology, and soil science. The course is not strictly oriented to the so-called "bioinorganic chemistry" for chemistry majors, but rather provides students of the life sciences with a basis of inorganic chemistry. I t aims to teach some fundamental inorganic chemistry with the main emphasis on coordination chemistry hut includes a discussion of certain aspects of phosphorus and sulfur chemistry, and applies these concepts in the area of biological processes. A laboratory program (one 4-hr periodlweek, 17-20 weeks) is coupled with the lectures, and is designed to illustrate some concepts outlined in the course. Phvsical methods of studvina inorganic compounds, particharly coordination cbmp~exes, are introduced in the course, and employed fairly extensively in the laboratory program. lncluded in the faboratory are visible and infrared spectroscopy, magnetic resonance (nmr), electrochemistry (polarography and conductance) and magnetic susceptibility method. ORD and esr are introduced in the lecture material. The biological students in most cases are exposed to the more modern techniques (e.g., magnetic resonance) for the first time, and this should prove beneficial in terms of applicability to future problems in their own areas. Outline of the Laboratory Stereochemistry of [Co~lll)(trien)X~] and Reaction of Coordinated Givcine Ester at Co(1ll)trien Center (trlen = ( 3 perrods, vis and ir spQClrn1 I . Prepararionof e i w - , cis-5.. and rranc-ICo~trit.n)Cl~ICi 11) 2. lhpeptide formarim at [Cortrien,)12)
The isomers listed are prepared, and characterized by visible spectra. Infrared spectra and nmr spectra are also informative. The difference in reactivity between &-aand cis-p-isomers may he demonstrated by the rate of aquation and by the rate of dipeptide formation. Such studies illustrate qualitatively how differences in structure around the active catalytic site of metalloenzymes may give rise to differences in enzyme activity. The facile dipeptide formation on the cobalt complex may exemplify the effects exerted by the catalytic metal site on a substrate in a metalloenzyme reaction, though the reaction selected is not directly relevant to a biological system. Subsequent hydrolysis of the coordinated glycylglycine
610 /Journal of Chemical Education
ester to give the glycine complex and free glycine illustrates the principle of metal ion promoted ester hydrolysis and serves as a possible model for a metal peptide system. Effectof Axial Ligand on a Tetragonal Nickel Complex (3) ( 2 periods, magnetic susceptibilityand vis spectra) 1. Preparation of Ni(Etzen)zXz (Etnen = N,N-diethylethylendi-
amine: X = CI, NCS, Nos, and I)
2. Magnetic susceptibilitymeasurement (Gouy method)
The heme complexes which are involved in hemoglobin, catalase, cytochrome c, etc., contain the iron atom in either a high or low spin state, depending on the axial ligands. Such an effect of the axial ligand on the electronic structure of a tetragonal complex may be illustrated by the magnetic moments of the series of the nickel compounds listed above. The chloride and thiocyanate complexes are blue to violet in color and paramagnetic with two unpaired electrons; while the nitrate and iodide comnlexes are vellow to orange and diamametic. More information may be obtained from measurement of the spectra and electrical conductivity. A discussion based on lieand field theory may be required as a part of the laborat:. ry report. Interaction of Pyrophosphate with Metal lons ( 4 ) ( I period, conductometrictitration) The conductometric titration of Na4P207 with MgZ+, Caz+, Mn2+, and CuZ+ salts is studied in order to demonstrate the chelating effect of the pyrophosphate group. The titration can he used to determine the compositions of the chelates. One of the most vital hiological compounds is adenosine t r i.~ h o.s ~ h a t ae .nucleotide which has H triphosphate group condensed onto the -CHzOH group of ribose; the phosphate is mostlv chelated to Me2+ in ~. cells. Polyphosphates ,pymphosphktr heing the s i ~ p l e s t ) have heen used as an ingredienr of detergents for the Dur. pose of sequestering metal ions. The function of dhosphates as detergents and their release to the environment causing the so-called eutrophication, can be discussed in terms of concepts illustrated by this experiment. interaction of Metal lons with Protein and Amino Acid
In part 1, the interaction of CuZ+ with BSA is studied spectrophotometrically in the absence and presence of HgZ+. A possible coordination site for the cupric ion may he identified from the spectra and the inhibition effect of HgZ+ can be studied. Serum albumin is considered to he a
copper carrying protein in blood. Part 2 is concerned with a rigorous quantitative determination of the coordination strength of an amino acid with a metal ion. The determination involves a pH titration procedure. A programmable calculator with a card reader can be used nicely for the complicated functions required. Oxygenation of [Co(ll)(salenJ] (7) (2 periods, vis spectra and oxygen uptake measurement) 1. Preparation of [Co(salen)];salen = bis(salicylaldehyde)ethylenediimine 2. Oxygen uptake measurements The preparation of [Co(salen)] requires an exclusion of oxygen, but can be done very easily. The oxygen uptake is studied a t constant temperature and under constant pressure, and is measured as volume change in a gas buret. The simple efficient apparatus used can he made readily by a glass-blower. The oxygen absorption is studied as a function of pressure and the thermodynamic equilibrium constant for the reaction may then be evaluated. The reversibility of the oxygenation is demonstrated by visible spectroscopy and by observing oxygen evolution. This experiment thus demonstrates the reversible oxygenation of a cobalt complex. The same phenomenon is observed in hemoglobin(Fe-complex), myoglobin(Fe), hemerythrin(Fe), and hemocyanin(Cu). The Co(II) Schiff base complex serves as very simple and perhaps useful models for the naturally occurring metalloprotein oxygen carriers. Vitamin B q 2andits Model Compounds ( 5 periods. visand rr spectra, nmr and pularography) I I'olarographv of V H n ( R I 2. S~ectruscooiestudyof some reactionsof V R I (~R I 3. V B ~ .modei eomp&nds (9) The reduction of VBlz (CN derivative) a t a dropping mercury electrode is studied in part 1. The analysis of the polarogram obtained establishes the two electron reduction. In part 2, the reduction is effected by sodium borohydride, and the methyl derivative is prepared by the reaction of methyl iodide with the Co(1) derivative. The methyl derivative is photolabile, yielding the aquocobala~ min under aerobic conditions. These reactions are studied by following changes in visible spectra. One series of the so-called VB12 model compounds is prepared in part 3; i.e., the methyl, a- and 0-cyanoethyl derivatives of cobaloxime. The isomerization of the B-isomer to the a-isomer is then studied. The isomers can be distinguished and identified hv ir and nmr spectra. The mechanism of the isomerization, and of simiiar reactions catalyzed by the enzymes requiring the VB12 coenzyme, is currently controversial. Another important reaction which has a significant environmental implication is the methylation of inorganic mercury compoun~sby the methyl derivative of of the methylation V B ~ z Or cobaloxime' An may be incorporated. Catalase and its Model compounds (10) (1period, oxygen-evolutionmeasurement) catalyzed by ~h~ decomposition of hydrogen catalase and its model compounds is studied by following the Oxygen evolution under lonstant pressure and ature. The rate data are compared in terms of the second order rate constant for catalase, Fe(III)trien, Mn(II)trien, demonCr(ln)triens and Cu(ll)trien. The strates the superiority of the enzyme over the model compounds; the order of activity is shown to be catalase >> F~ > Mn >> Cr > Cu 0. The effectiveness in catalase action of Fe and Mn among the transition metals of the first series may be discussed, and the mechanism leading to the superiority of the enzyme may be speculated upon.
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Some Other Experiments Which May be Added
Other coordination chemistry experiments may be included; for example: (1) Co-chemistry; preparation of cisand trans-[Co(en)2Clz]Cl, their characterization by vis spectra and C1 analysis, and the kinetics of the cis-trans isomerization reaction, (2) Ni-chemistry; synthesis of [Ni(en)a]Clz and [Ni(en)~Clz],and their characterization by a Job's continuous variations method, and (3) Cuchemistry; visible spectra of [CU(NH~)~(HZO)G-,,] complexes. Some other bioinorganic-oriented experiments are now under development, and these should permit students to have a wider choice to suit their interests and requirements. Concluding Remarks
The importance of inorganic chemical aspects of hiological systems has been stressed recently at a symposium (11) sponsored by American Chemical Snciety and Chemical Institute of Canada, and by a report (12) of the curriculum committee of American Chemical Society. The laboratory outlined above was implemented fully in the current 1972-73 academic year, and turned out to conform nicely to several recommendations made by the curriculum committee. As will he realized by surveying the details of the experiments, stress is placed on the inorganic chemical aspects. The orientation of the current research in bioinorganic chemistry is not fully covered by this laboratory course. More biochemical concepts and techniques should be introduced perhaps for the purpose of training "hioinorganic chemists," although the term "bioinorganic" covers broad and often ill-defined areas. Each instmctor or researcher evolves to some extent his or her own image of bioinorganic chemistry. In the broadest sense, it is concerned with any interaction of inorganic materials with biological systems. In this context, aspects of environmental chemistry could also he pursued from the viewpoint of bioinorganic chemistry, or this discipline should provide a basis for increased understanding of environmental prohlems. Some of the relevant environmental topics are dealt with in this laboratory. Those who would like additional information about the details of the experiments are invited to write to the author. The author wishes to thank Prof. C. A. McDowell, head of this department, for having given him the encouragement and opportunity to develop this laboratory, and Prof. B. R. James for useful advice and comments throughout this project. Literature cited
The experiments are based on or adapted from the following sources (1-10). Only those of direct pertinence are cited. The necessary chemicals, BSA, VBll and catalase. were purchased from Sigma Chemical Co, The popularly priced lots of these cam. oounds were not the nurest available. but were adeauate ornvided that the details of the purity were obtained. I I I Sargenon.A.M..andSearle. G. ~ . . h o nchrm.. . 6.787119671. 12) Csllman. J . P . , sndKimura, E.. J A m Chem. So?. 89.6096119671. (31 GMdgsme, D.M.. andVenan=i.L. M ..' f&m. So
