Ei-lchiro Ochiai University of British Columbia Vancouver 8, B. C., Canada
I
I
~nvironmenta~ Bioinorganic Chemistry
Living organisms are surrounded by inorganic environments and maintain complicated interactions with each other and these environments. During the long history of evolution on this planet, organisms have adapted to the environment. They have developed some protective mechanisms to harmful situations which might occur naturally. Examples are numerous; nervous systems, all sorts of homeostatic mechanisms, immunological mechanisms in higher animals, productions of inducible enzymes and subsequent metabolisms of foreign substances, etc. However. the chanee in the surroundine environment created human expioitations of nature'and technologies has been becomine" increasinelv - intolerable to life because the protective mechanisms have not been designed for the artificial situations. This is the general basis of environmental problems. Here a brief attempt is made to survey the problem from a hioinorganic chemical viewpoint.
by
Metallic Elements and Compounds Availability All metallic elements come ultimately from the crust of this planet. The distribution of elements in earth crust rock is roughly sketched in the figure and compared with that in sea water and that in human beings. Elements are usable for most organisms only when they are in aqueous media. Thus, there is some parallelism between the element distribution in sea water and that in an animal as shown in the figure. The concentrations of elements present in aqueous media are dependent on many factors (2): concentrations of anion, solubility product, pH, etc. In nature metals are carried away from rocks and soils mostly by rivers. On top of this, we are now mining out considerable quantities of metals. Table 1 gives some estimates of these amounts (3). These metals end u p in the environment in one way or another. The heavy burdens thus created must be borne by organisms. Mercury forms a very insoluble HgS and metallic mercury itself does not dissolve in aqueous media. Some anaerobic hacteria convert metallic mercury or other f o m s of mercury to methylmercury (4) as a way of detoxification for themselves. Methylmercury and its derivatives go easily into aqueous media and are accumulated in carnivorous animals higher up in the food chain. Methylation of As., Se.. and Te has also been found 14) to he effected bv some methane-forming hacteria. Some bacteria, e.g., Pseudomonas K52, on the other hand, can decompose orgauomercury compounds such as phenyl mercuric acetate to release benzene and metallic mercury (5). Modes of Interaction-Biological Levels Metallic com~oundsare absorbed into organisms and metabolized in one way or another. Some metallic ions or comoounds may be rapidly excreted through the urine or feces in animals, while some others stay longer and thus accumulate in bodies. Organisms employ many of these metallic elements as essential components, especially as the catalytic sites in enzymes or the like. Metals which have been established to be essential to man are underlined in the figure (see Ref. (6)).Fe, Zn, Cu, Mn, Mo, and Mg are essential for a variety of enzymes (7); Na, K, and
Ca are electric carriers (Na+, K+), information-carriers or triggers (CaZ+) as well as activators for certain enzymes. The only definitely known function of Co is as a constituent of Vitamin BIZ and its derivatives. Si and B are known to be essential for plants. Al, Pb, Ni,-Cr, Cd, and Hg occur ubiquitously in animals and plants; however, no definitive evidence has been produced for their essentiality. Pb, Cd, and Hg are notorious for their adverse effects. However, even essential metals could become harmful when given in large excess so that they are not removed efficientlv. This is known as "level of tolerance;" it differs from onemetal to another and from one organism to another. To eive some examples. toxic levels in animal ppm for Mn for cattle and diets are estimated to be chickens, >I00 ppm for Cu, 10 -20 ppm for Mo (depending on Cu level) and very low for Cd, Hg, Pb, Se, and As. Some marine organisms accumulate specific elements. The accumulation of carbon, nitrogen, and phosphorus by marine organisms from very dilute aqueous media are notable but understandable. Some of them concentrate calcium to form shells (CaC0.4 and others (diatoms) silicone (SiOz). Radioralians use SrS04 as skeleton material. Fe is also concentrated. All of these elements are essential for the organisms. An example of the elemental composition of a zooplankton, copepod (Calanus finmarchicus) is cited in Table 2 (8). It is interesting to note that there are certain quite rigorous specificities in the binding of metals by marine organisms. Some asidians (tunicates) concentrate 3~ X los). while other V enormouslv factor ~ ~ (enrichment ~ ~ ~ species of asiidians concentrate Nb instead 'if V (9). Molluscs are especially efficient in enriching metals from sea ~
~
Table 1. Production Rate (tonlvr) of Metallic Elements Geological
Man-Induced
rate
rate
Element
(in river)
(mining)
Fe Cu Zn
2.5 X 10' 3.8 X 10' 3.7 X lo6
3.2 X 108 4.5 X 10" 3.9 X 10'
Distribution of elements in ( a ) earth crust rock, ( b ) sea water. and (cl man. The figures are log c (c in ppm). Data are from Ref. ( 1 ) .
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water (Tahle 3); they hind even the toxic metals, Hg, Ph, and Cd. One of the reasons why these organisms accumulate metal ions selectively or nonselectively is that their mucus layers are directly exposed to sea water and act as adsorbents for metals (2). The fate of alkylmercury (RHg) in animals was traced using 203Hg (12). RHg appears in the liver and kidney first, but relatively quickly appears and accumulates in the brain and hlwd cells, while inorganic mercury compounds are mostly found in blood serum. Its excretion is far slower than inorganic Hg-compounds. It is more readily transferred to the fetus when given to a pregnant rat (13). Modes of Interaction-Molecular Levels Metal ions or compounds interact mostly with proteins. For example, Fe3+ binds with apo-transferrin; Fe3+-transferrin is carried to hone marrow where Fe is released and utilized in the synthesis of Fe-porphyrin. Fe3+ is also bound to a protein called ferritin which is helieved to store iron. Some of specific metal-protein combinations are shown in Tahle 4. One of the proteins which hinds V specifically as found in some tunicates mentioned earlier is hemovanadian; its biological function is considered to be a redox-catalysis. The interactions between essential metal ions and specific proteins in metalloenzymes or in metal-activated enzymes are well documented in a monograph (7). The effects of the three toxic metals, Hg, Cd, and Ph, have been thoroughly treated in a recent review (14). Hg2+ strongly hinds with the SH group (cysteine) in enzymes and blocks the enzymatic activity of the SH group. The SH group is known to be a t the catalytic sites in many enzymes. An example (12) will illustrate this. RHg was mentioned earlier to accumulate in blood cells. After treatment of the content of blood cells with some oroteolytic enzymes, the portion containing z03Hg was isolated and shown to he composed mainly of RHgSC2H3(NHz)COOH (R = C2H6). Cd is believed to act similarly, blocking the SH group resulting in an inhibition of enzvmatic activitv. However. Cd2+ is also verv similar to znz+ in chemical character, and hence ma; substitute Zn2+ in many Zn-enzymes and retain or in Table 2. The Elemental Comoosition of a Cone~od
Content
Element C
Enrichment factora
( P P ~ 6 . 1 X 10'
N P Fe Si Na
1.5 x 1.3 x 70 70 5.4 X 2.9 x 4.0 X 3.0 X
K Ca Mg a Enrichment factor water.
=
2.1 X 5.0 x 4.3 x 1.4 X 1.4 X 0.5 7.4 1 .0 0.2
10' lo8 10' lo3 loe 1OZ
Element
HS Pb Cd
Ti V Cv
Mn
Fe Co Cu Zn
Ref.
ment factor
Ovster enrichment factor
Metal Fe'
content ppm/eantent ppm in sea
Clam enricheontent ment (ppm) factor
.. . . .. 2.35 10V.3X 1 0 9 . 4 3 2 . 3 X 10' 3.2 X 101 . .. .. . .. . 32.7 4.5 X 10' 1.5 X 1 0 V . 3 2 2.0 X 10s 6.0 X 10' 3.27 5.6 X 10' 4.0 X 1 0 0 4 . 5 2 . 9 X 101 6.3 X 10' 0:85 3.0'; I O W . ~ ' X 10' 16.1 2 . 8 X 10' 1.1 X 1 0 8 34.0 (20) (10) 5.3 X
9.5
X 10' 8.1 X 10'
.. .
1.55 5.2 X 1.1 X
.. .
3.3 X 10' 18.5 6 . 6 X 10' 0.24 6.5 X 104 1 0 6 1.2 X 10' 36.1
.. .
10' 10s
1 . 9 X 10' 1 . 2 X lo* 2 . 1 X 10' 1.8 X 10'
8 . 5 X 1 0 4 : 3 2 3.2'; 10" 5.4 X l O V 5 . 4 3.5 X 10: 3.4 X 10, 31.2 3 . 1 X 10" (11)
236 1 Journal of Chemical Education
(11)
S~ecilicrotei in
transferrin (iran-carrier), ferritin (iron-storage), conalbumin (iron storage in chicken egg), hemoglobin (02-carrier) Cu*+ serum albumin (carrier), ceruloplasmin, hemocyanin (02-carrier) ZnP+, Cd2+ metallothionein (?) Va + hemovanadin Na +-specific ATPase Na + K+ K +-soecific ATPase +
Table 5. Examples* of Enzymes Inhibited by Heavy Metals
Corbieula mnenrichtent ment (ppm) factor 3.31
Some General Principies-Inorganic Aspects Effects of Chelations and Organometallic Deriuatiues. Many effects are brought about by chelation of metal ions and formation of organometallic derivatives. Of particular importance are those involvine soluhilitv and nermeahilitv through biomemhranes. For &le, 6e3+ tends to form insoluble hydroxides even in neutral aqueous media. Chelates formed by, e.g., hydmxamic acid derivatives (17) and others prevent precipitation and keep Fe available for Table 4. Examples of Specific Proteins for Metals
108 lo4 10' 10J 102
Table 3. Concentrations of Metals by Sea Organisms SeaUon enrich-
some cases even enhance the enzymatic activities. The binding ability of Ph2+ to the SH group is somewhat weaker than Hg2+ and Cd2+. Therefore, PhZ+ inhibits most enzymes with an active SH-group hut less readily than Hg and Cd. Some of the enzymes for which inhibitory actions of Hg, Cd, and P b have been observed are listed in Tahle 5. This inhibitory action on many important enzymatic activities is the reason why mercury compounds such as phenylmercury acetate and methylmercury have been used as fungicides for seeds, etc. A protein having an exceptionally high content of sulfur has been isolated from mammalian liver and kidney. It contains Cd and Zn in large amounts (total -8%) and is designated as metallothionein (15). Hg2+-compounds and Ag can instantaneously displace Cd and Zn. The biological function of this protein is yet to he identified. The cations Hg2+, Cd2+, and Ph2+ can also interact with polynucleic acids (DNA, RNA) and with some phospholipids. Polynucleotides have bases as well as phosphate as potent coordinating groups. Therefore, it is little wonder that some metals are found in various preparations of DNA and RNA. Examples are Cd (20 ppm) in RNA from horse kidney and Cr (1088 ppm) in nucleoprotein from beef liver (16). Various Hg- as well as Ph-compounds can produce chromosomal abnormalities. Phospholipids are one of the main constituents of various membranes of cells and subcellular particles (mitochondria, etc.). They are generally weak in binding metal ions, because they are much like hydrocarbons. However, HgZ+- and Pb2+-compounds have been shown to interact with membranes. Alkylmercury compounds (e.g., methylmercury) are known to he one order of magnitude more toxic to cells than inorganic mercury compounds. This is considered to be due to the greater affinity of alkylmercury compounds to phospholipids.
Metal ion
Enzyme inhibited
Hgz+
alkaline phosphatase, glucose-6-phosphatase, lactic dehydragenase Cda+ adenosine triphosphatase, alcohol dehydrogenase, amylase, carbonic anhydrase, carboxypeptidase (peptidase activity only), glutamic-oxaloacetic transaminase Pb'+ acetylcholin, esterase, alkaline phosphatase, adenosine triphosphatase, carbonic anhydrase, cytochrome oxidase AsS+, As + Q y r u v a t e dehydrogenase Compiled from ref. (14).
biological use. Suitable chelation also makes an electrically neutral species out of a charged metal ion, and the electrically less charged species, in general, is more permeable through biomembranes. Specificity and Interchangeability. Some important inorganic chemical concepts relevant to specificity are brieflybiscussed helow. In ihe case where i&ic or electrostatic force is predominant in the honding, the bonding strength by the electric potential of the metal ion, is which is Ze/r (Ze = electric charge and r = ionic radius). Therefore, metal ions with similar electric potential are often interchangeable. Examples are shown in Table 6. CaZ+ in many biological systems may be substituted by Eu2+ or SrZ+ as suggested in Table 6. EuZ+ can thus be utilized as a.reporter cation1 in the investigation of certain biological functions of Ca2+. SrZ+, if available, may take the place of CaZ+; thus, the radioactive Sr substitutes Ca to some extent in bones, e.g., and its radioactivity may produce some hazardous effects on the bone marrow where blood cells are produced. Be2+ may take the place of Mg2+ though being much smaller. Having a higher electric potential, i t hinds much more tightly with some proteins than Mg2+ and hence blocks their activities. The same factor also governs the coordination number and the hydration energy of a cation. The hydration enerev (kcallmole) is: Li+ 124.4: Na+ 97.0: K+ 77.0: and ~b+'?l.9. since the coordination of other ligands is substitution of water molecules by them, the hydration energy is one of the controlling factors. For example, K+ is dehydrated and coordinated to ligands, particularly hydrophorbic ligands, more readily than Na+. Another important factor is simply the size of the cation. There are known various macrocyclic ligands, biogenetic and synthetic, which bind specifically particular alkali metal ions (18, 20). For example, a macrolide called valinomycin binds K+ specifically while nonactin binds Na+; they act as antibiotics by intervening in the cation transportation. The size of the hole in a macrocycle matches the size of a cation. In many other cases (transition metals and others), effects other than electrostatic and ionic radius are operative as well, and there are a large number of potential coordination sites available in a macromolecule like protein. The potent coordinating groups in the biologically important molecules are: N-imidazole, NHz (lysine etc.), purine and pyrimidine bases (DNA, RNA) etc.; O-OH(serine, tyrosine etc.), COO- (glutamic acid, etc.), POs, etc.; S-SH(cysteine), SR (methionine etc), and others. Each metal ion seems to have a relatively strong affinity for a certain coordinating atom; e.g., Co3+ for N, Fe3+ for 0 , etc. This effect has been discussed for transition metals by Sigel and McCormick (19). However, the specificity is not very strict but rather weak in many proteins. Hence the problem is quite complex. For example, Hg2+ may bind to a protein, e.g., carbonic anhydrase in place of ZnZ+ which is the native metal, but the coordination site is not necessarily the same. A coordination of HgZ+ to the site different from the active site to be occupied by Zn2+ may, however, modify the protein structure and result in the deactivation of the enzyme. Other factors are the socalled crystal field stabilization energy (21) and the covalency. The latter is closely related to the softness-hardness and the matching of the metal ion and ligand (22). These are fundamental inorganic chemical concepts and need not be discussed here.
a
Nonmetallic Elements and Compounds
Modes of Interaction-Biological Levels Important elements are H, C, N, 0 , P, and S. Hz gas is relatively inert and difficult to be dealt with by organisms, though there are some bacteria which can convert
Table 6. Substitution of Cationa
Native cation
May be substituted by
) is the ionic radius in a The figure in ( from ref. (18) and others.
A.
Compiled
Table 7. Emissiona (ton/yr) of Inorganic Compounds into the Environment (world)
Compound CO
con
NO. N-compounds6 P-compounds~ SO* hsdrocarbons Comniled from Ref. (3). .. ~ o s t i fertilizer. y Mostly fertilizer and detergent. Hz to H z 0 and thus abstract energy from Hz. Carbon dioxide is ubiquitous and increasing steadily as we bum fossil fuel. It is used as the carbon source in the autotrophic organisms, phytoplanktons, plants, and others., Carbon monoxide is also emitted into the atmosphere in significant amounts (Table 7), e.g., from exhausts of automobile engines. It is somewhat mysterious that the content of CO in the atmosphere does not seem to be increasing despite the emission 123). It has been suggested (23) that some fungi in soil are capable of converting CO to COz. Nitrogen is one of the most inert gases, hut can be converted to NH3 and other nitrogen compounds by Azotobacter, Rhizobiurn, Clostridium pasterianun, and otbers. The Nz-transition metal complexes which have been synthesized in recent years have created a hope that the riddle of Nz-fixation by microorganisms may be clarified in the near future 124). Ammonia may he used directly by plants when the Nz-fixing bacteria is Rhizobium which is symbiotic with the host plants. Otherwise NHa will be oxidized to NO?- by the bacteria Nitrosomonas and then to NO3- by Nitrobacter. The bacteria involved obtain the energy for their sustenance from these oxidation reactions. N O 3 is absorbed by plants and reduced to NOz- and NH3. Ammonia is used for the synthesis of amino acids. The first reaction (NOSNOz-) is catalyzed by a Mocontaining enzyme, nitrate reductase, and the second NH3) by nitrite reductase. The same reaction (NOzreactions are also effected bv certain anaerobic bacteria. NOz- is quite hazardous as mentioned later. Reactions of air (NI and 0 2 ) at high temperatures. ex., in automobile engines, prodGe nitGgen oxides which -&e toxic in one way or another. Oxygen is one of the most important gases, for it lets most of the organisms on this planet, particularly heterotrophic ones, abstract energy from organic and inorganic compounds (carbohydrates, NH3, Has, Fez+, etc.) by way of oxidation. However, as a matter of course, Oz is toxic to anaerobic microorganisms. The toxicity of ozone was reviewed recently (25). Phosphate is another vital material for organisms of all kinds. In aquatic
-
-
' A cation which substitutes the native cation is called a "reporter," if it can provide some additional information which the native cation cannot, via, e.g., spectroscopic methods (epr, uv, etc.).
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environments, the concentration of phosphate is the limiting factor for the growth of phytoplanktons, including algae. Human activities (production of sewage and phosphate-containing detergents, etc.) have resulted in the sudden rise in the concentration of phosphate in some areas. The sudden and abnormal growth of algae stimulated by the increase of phosphate and its resulting dead organic material have caused the unusual population growth of heterotrophic hacteria; the net result is the deprivation of oxygen and the death of other organisms-the so-called eutrophication. Sulfur compounds (26) are converted into one another by soil hacteria in a manner similar to the conversion of nitrogen compounds mentioned above. The hacteria which oxidize elemental sulfur and a variety of sulfur compounds (metal sulfides, S Z O ~ ~etc.) -, are called Thiobacillus (27). For example, Thiobacillus concretiuorus oxidizes S or HzS to form HzS04. Most organic sulfur compounds (cysteine, etc.) in animals are oxidized by a set of enzymes and eventually excreted as S042-. Plants and certain hacteria (e.g., Desulfouibrio desulfuricans) reduce SOrZ- and synthesize cysteine and methionine. Statistics on the emission of these compounds into the environment are shown in Table 7. Modes of Interaction-Inorganic Chemistry Most of the toxic compounds are considered to interact with metal ions in enzymes and inhibit the enzymatic activities. For example, CN-, CO, and NO inhibit many of the respiratory enzymes such as: (a) Fe: hemoglobin, cytochromes (a, a3, h, hs, c, etc.), cytochrome P-450, catalase, etc.; and (h) Cu: hemocyanin, ceruloplasmin, cytochrome oxidase, etc. The CN- hinds with Fe3+ in hemeproteins more strongly than with Fez+; therefore, it inhihits the actions of the cytochromes in the electron transferring systems of mitochondria, and to a lesser degree the action of hemoglohin. CN- is also an almost nonselective and powerful inhibitor to many other metalloenzymes. CO as well as NO hinds specifically with Fez+-heme, thus inhibiting the On-carrying activity of hemoglobin and myoglohin. Table 8 shows some equilibrium constant data of hemoglohin and myoglobin with 02, CO, and NO (28). It will he seen that the affinity of CO to hemoglobin is 20400 times as great as that of Oz. NO-hemoglobin and NOmyoglobin are red in color being similar to Oz-hemoglobin and Oz-myoglohin and are so stable that this principle (formation of NO-complex) is used for the preservation of meat color (29). Fez+-hemoglobin is readily oxidized in many ways to give rise to brown Fe3+-hemoglobin (called methemoglobin), which cannot carry oxygen. One such agent is NOzwhich causes, therefore, a hazardous disease called methemoglobinemia. NOZ- is formed via bacterial actions as mentioned earlier, from inorganic nitrogen fertilizers (30). NO=- is also known to react with nucleotides and cause changes in the nucleotide sequence in DNA or RNA, thus leading to a mutation or maybe bringing about cancer. The photochemical reactions involving nitrogen oxides
238
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Journal of Chemical Education
Table 8. Equilibrium Constante K_of Hemoglobin and n Myoglobin: Hb(Mb) X XHb(Mb)
+ =
Hb
Mb
Compiled from Ref. (28). and hydrocarbons to form PAN (peroxyacylnitrate) and the importance of the latter in causing photochemical smog are well documented in any textbook on air pollution. So is SOz and sulfuric acid mist. The biological toxicities of these compounds are obvious in essence, though the details of the mechanisms are yet to he studied. Concluding Remark This article was written as a sequel to the previous report on the bioinorganic laboratory (31), and is intended to show some of the important aspects of hioinorganic chemistry. When a field emerges, it tends to he defined too narrowly, and the researchers as well as the students in the field are apt to lose the whole sight of it. The present work is an attempt to avoid such a failure. Literature Cited ill Meson B., "Principles of Geoehemi~y,"2nd Ed.. John Wiby and Sons, he.. New York. 1566. I21 Goldberg, E. D., (in1 "Chemical Oceangrsphy," (Editors: Riley, J. P., and Skirrow,G.1, Val. 1, Academic Press, New York, 1965. (3) Maffhsws, W. H., Smith, F. E.. m d Gaidborg, E. D., "Man's Impact on Tenestri. a1 and Oceanic Ecosystems," MlTRena. Cambridge. Ma-. 1971. (41 Chem. ond Eng New& July 5, p 22 (19711; Thayer, J. S., J. CHEM EDUC.. JO. 390i1973); Wmd,J. M.,sndBrown, D. G.,Str. ondBondg., lI,G(1972). (5) Su%uki,T.,Furukawa, K.,andTonomura, K., J. Fmenf. Tech, 46.1048(1968). (8) Undcnumd, E. J.. "Trace Elementa in Human and Animal Nutrition," 8 d Ed., AeademicPrc.8. hc.. New Yolk. 1971. 171 Vaiiee, B. L., and Waeker, W. E. C.. "The Proteins," 2nd Ed., Val. V. Academic Press. 1ne.New York. 1971. (8) Dietrich, G., "General Ocesnomaphy." Wiley-htemience, New York. 1561, cited in nome. R, A,, " ~ s r i n c c h e m i r ~ y wiley-hteracienco. ," N ~ W ~ o r k 1961. . (91 Cariiaie. D. B.,Notu% 181,933i1958). I101 Bmoks. R. R.. and Runsky, M. G..Limnol Ocemmgmphy, 10. 521 (1965). cited in Home, R. A,. lucRef. (81. (11) Murakami, T., Chem. andlndu. (Tokyo), 23,1297 (1970). (121 Ukita. T., Science iTokya), 41. 557 ll970): in Matsumato, F., Baunh, 0. M., and Miss* T . (Editors) "Environmental Toricoiow of Pedieides? Academic Press, he., New York, 1972. I131 Dctaiil of mataboliam and toxicity of Hg-eompounda will be found in a reconf monograph: Friberg, J.. and Voafsl, J., (Editors). "Mercury in Envimnment,"
.
.
C C.,P ncn? . .R.. . ... W ~rrsvslsnrl . ... .-, . ...
(141 Vsliee, B. L., anduiimcr, D. D.,Ann. Re". Bioehm., 41.91 (1972). (15) Msrgoshe8.M.. andVal1e.B.L.. J Arne. Chem. S a c , 79,4813 (1957). (161 Wackar, W. E.C., sndvaiiee, B.L., J. B i d Chem., 217,253 (1959). (171 Neilands. J. B.. S t r ondBondg.. I. 59 (1966). (18) Williams, R. J . P., Quart. RPU.,24,331 (1970). (191 Sigel, H.,andMeCormick, D. G.,AccounlsChem. R a , 3,201(1970). . Biophys.. 5.187 (1972). (20) Hsydan. D.A.,and Hiadky, S.B.. Q u a ~ tRou. (211 Basolo. F.. and Peerson, R. G., "Mechanism of h r q a n i e %actions? 2nd Ed.. John Wilay and Sons, New York. 1967. (22) Ahrland. S., Chstt, J., and Dauics. N. B , Quon. Re"., 12, 265 (1958); Pemon, R. G.. J. CHEM. EDUC.. 45.581.643 119681. ~ e m s ~, a y i 0p24 , (19711. (23) Inm&. R. E.. Cham. and~k. 1%) Schndler, S. W., J.CHEM. EDUC.,49.786I1972). (25) Leh,F.. J.CHEM. EDUC.50, 404i1973). (26) Leh, F.. andChan, K. M.. J. CHEM EDUC.. 5(1.246(1973). h e . , NeuYork. 1969. 127) Zsjic, J . E., "Microbial Biogeochemisfry," Academic Rp~g, (28) Antmini, E., and Brunori. M., "Hemoglobin and Myoglobin in Their Reactions wifhLigends." North-HollandPubi., Amsterdam. 1971. 1291 Russa, S. F..andSonfokko, R. B.. J. CHEM. EDUC., 50.347 (1973). 1301 Commanor, B., "The Closing Circle." Bantam Bmks, New York, 1971. 131) Oehia1,E.I.. J.CHEM. EDUC,50,610(1973).
