The interaction of cadmium and certain other metal ions with proteins and nucleic acids

Toxicology, 16 (1980) 1--37 © Elsevier/North-Holland Scientific Publishers Ltd.

Review paper

THE I N T E R A C T I O N O F CADMIUM AND CERTAIN O T H E R METAL IONS WITH PROTEINS AND NUCLEIC ACIDS*

K. BRUCE JACOBSON and J.E. T U R N E R a

Biology Division and aHealth and Safety Research Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830 (U.S.A.) (Received February 26th, 1980) (Accepted April 22nd, 1980) SUMMARY

The toxic effects of cadmium and other selected divalent cations are presumed to be related to specific chemical and physical characteristics of the ion. The chemistry o f cadmium and metal ions in general is reviewed from the viewpoint of such relevant properties as ion polarizability, electronic structure, and the hard-soft characteristics. The softness of metal ions is seen as a useful single parameter to correlate with the affinity for nucleic acids and proteins and with toxic effects. The effects of cadmium on nucleic acids and proteins are examined for a number of specific cases to illustrate the variety of interactions that are well recognized and to demonstrate the utility of soft metal ions as reagents and probes for examining the relationship o f structure and function in these macromolecules. I. INTRODUCTION

Cadmium is recognized as a non-essential element that is toxic to many plants and animals. Discovered in 1817, the metal is obtained principally as a by-product from zinc, copper, and lead ores and has important industrial applications. Cadmium is used in pigments, stabilizers for polyvinyl chloride resins, batteries, solar cells, solders, and alloys for making bearings with low friction and fatigue characteristics. It is utilized extensively in electroplating. Its special neutron-absorbing properties are used to advantage in the cadmium control rods of nuclear reactors. The technological utilization of cadmium has led to increasing levels o f *This study was sponsored by the Office o f Health and Environmental Research, U.S. Department of Energy, under contract W-7405-eng-26 w i t h the Union Carbide Corporation.

the metal in the environment and in the h u m a n body. For example, the concentrations of cadmium in wheat taken from the same location in Sweden are reported to have doubled between 1916 and 1972 [1]. Kidney samples from 19th century museum specimens were analyzed and f o u n d to contain an average of 15.1 /~g Cd/g dry weight in the renal cortex, compared with 57.1 in contemporary samples [2]. A number of useful reviews on the environmental and biological impact o f Cd 2÷ exist [ 3--6]. The ability of cadmium to react with m a n y organic compounds of biological importance has led to speculation on the ways that toxic effects are caused; however, no mechanism of toxicity for any organism has been established. Cadmium can enter the mammalian organism through the lungs as a vapor or through the digestive tract as a constituent of food and water. Certain organs are more likely than others to be damaged by this element, although the reasons for such selectivity remain obscure. A great deal of attention has been paid to the definition of toxic levels of cadmium, to the ability of certain species to concentrate this metal, and to physiological responses to its presence in the organism. Less information is available t h a t describes the reaction of the cadmium ion with compounds of biological importance. In this article we focus attention on the interaction of Cd 2÷ ions with nucleic acids and proteins. Where possible, we call attention to phenomena t h a t appear to reflect the basic chemistry and physics of Cd 2÷. We hope that a fundamental understanding of the properties of this metal will lead to the ability to predict its effects on various biological systems. In keeping with these objectives, we discuss the chemical properties of cadmium in Section II and the general physicochemical characterization of cadmium and other metal ions in aqueous solution in Section III. Section IV is devoted to studies of the interactions of cadmium with nucleic acids and their components. In Section V we discuss effects of cadmium on proteins and their constituents, including facets o f protein structure and enzymatic function. This article largely omits studies on whole organisms treated with cadmium. Our aim is to focus attention on the information t h a t can be derived from studies on nucleic acids and proteins and the various ways t h a t cadmium can bind to them and alter their normal functions. Other metal ions are considered in order to compare their properties with those of Cd 2÷. II. CHEMISTRY OF CADMIUM

The following properties of cadmium are pertinent in considerations of the mechanisms by which it enters a biological system and interacts with macromolecules. The melting point (321°C) and boiling point (765°C) of cadmium are low compared with those of most other metals; vapors are readily formed when the metal is heated. In the periodic table of elements, the congeners zinc, cadmium, and mercury form Group IIB; the inner electron shells are filled, and the outermost shell has 2 electrons. Con-

sequently, zinc and cadmium have an electrovalence of 2 as the only stable form, whereas mercury can have an electrovalence of 1 or 2. The following c o m p o u n d s of cadmium are only slightly soluble: sulfide (CdS), carbonate (CdCO3), and oxide (CdO). Both Zn(OH)2 and Cd(OH)2 are practically insoluble, while only the former is readily redissolved in excess hydroxide. An extensive discussion of the comparative solubilities of c o m p o u n d s of cadmium, zinc, calcium, and magnesium appeared previously [ 7]. Alkyl cadmium c o m p o u n d s (R2Cd and RCdC1, where R is an aliphatic hydrocarbon) have been synthesized in the laboratory and also by bacteria [8] but are unstable when exposed to air or water. In contrast to stable alkyl mercury compounds, it is n o t likely that alkyl cadmium c o m p o u n d s are hazardous to biological systems. Organic complexes are known for many metal ions, and, indeed, the coTABLE I

FORMATION CONSTANTS ORGANIC LIGANDS a M 2÷

OF VARIOUS

METAL

COMPLEXES

WITH BIDENTATE

Ligand Oxalate

Glycine

Ethylenediamine

Mercaptoacetate

Mercaptoethylamine

OC--CO

OC--CH2

H2C--CH 2

OC--CH 2

H2C--CH 2

][

- - O O-(OO)

I I

- - O NH= (ON)

II

H=N NH= (NN)

J i

--O S - (OS)

I I

- - S NH 2 (NS)

Hard ions Ca 2÷ Mg '÷ Be '÷

1.6 2.4 4.1

1.4 3.4

2.2 2.3

0.37

Intermediate ions Mn 2÷ Fe 2÷ Co 2÷ Ni 2÷ Cu ~÷ Z n 2÷

3.9 3.0 4.7 5.3 5.5 4.9

3.4 4.3 5.2 6.2 8.6 5.9

2.7 4.3 6.3 7.2 10.6 5.9

3.2 4.0 3.3

6.0 10.3 5.1

5.7 14.3

4.4 5.8 6.2 7.8

7.7 9.3 (16) 8.1

S o f t ions Cd 2÷ Hg 2÷ Pb 2+

9.4 ~24 8.5

9.9

aValues o f t h e f o r m a t i o n ~constants are s e l e c t e d f r o m Refs. 7 a n d 45. Most c o n s t a n t s were d e t e r m i n e d in 0.1--1 m o l a r salt s o l u t i o n . Values given are log ( [ M L ] / [ M ] [ L ] ) , w h e r e M is t h e divalent m e t a l ion, L is t h e organic ligand, a n d ML is t h e c o m p l e x f o r m e d b y t h e i r r e a c t i o n . T h e value s h o w n in p a r e n t h e s e s for Cu 2÷ is an estimate.

ordination structures are significant in explaining their chemical and biological properties. Metal ions are linked to biological molecules primarily through oxygen, nitrogen, and sulfur. Interactions with c o m p o u n d s conraining these elements are described b y formation (or association) constants. Formation constants for a number of metal complexes which involve bidentate coordination of model organic ligands to a selection of metal ions are given in Table I [7]. The values generally reflect "hard" and " s o f t " characteristics of the metal ions, which will be discussed in some detail in Section III. It is sufficient to point o u t here that Cd 2+, Hg 2÷, and Pb 2+ are soft metals; Ca 2+, Mg 2+, and Be 2÷ are hard metals; and the remaining metals in Table I are intermediate in character. The hard metal ions generally form the weakest complexes with these model ligands. The soft metals, Cd ~÷ and Pb 2+, form the most stable complexes with the mixed N--S donor a t o m ligand (mercaptoethylamine). For metal complexes of N--N chelating groups (ethylenedismine), Cd 2+ complexes would be less stable than those o f Ni 2÷ and Cu ~÷ and possibly Co s+ and Zn 2+. In complexes involving mixed O--N chelation (glycine), Cd 2+ would be less stable than Cu 2+, but would be more stable than Mn 2÷, Mg 2+, Ca 2÷, or Hg 2+. Cd 2+ complexes would be more stable than those of Mg 2+, Ca 2+, and possibly Fe z+ for complexes involving O--O donor a t o m chelation (oxalate). The general chemistry of cadmium suggests several characteristics for analyzing interactions of this element with biological macromolecules. First, cadmium has a high affinity for sulfur; among the ligands in Table I, for example, it reacts most strongly with sulfur. It is expected that some toxic effects o f cadmium are the result of reactions with essential sulfhydryl groups in proteins. Second, cadmium is directly below zinc in the periodic system and therefore the 2 elements show chemical similarities such that cadmium is c o m m o n l y found in zinc ores, which are the principal commercial sources of cadmium. A number of zinc~containing enzymes participate in the synthesis and function of nucleic acids. The ability of cadmium to affect the properties of some o f these enzymes will be discussed, even though detailed mechanisms are largely unknown. Third, Cd 2÷ and Ca 2+ have identical charges and almost the same physical sizes. Thus one might expect certain similarities in the behavior of these ions, although they differ in other physical respects. As will be discussed later, certain calciumcontaining proteins have been studied in forms in which cadmium has replaced the normal metal. Finally, the role of cadmium in altering the structure of nucleic acids has relevance since there are several binding configurations for metal-nucleotide complexes that are sufficiently stable to exist under normal physiological conditions. We turn n o w to a more detailed and systematic analysis of various p h y ~ ical properties of metal ions and their possible significance as reflected in biological processes. HI. PHYSICOCHE SOLUTION

CAL CHARACTERIZATION OF METAL IONS IN AQUEOUS

A. Objective The effects of cadmium in biological systems could, in principle, be under-

stood on the basis of k n o w n physical and chemical laws, given a detailed description of h o w the metal enters a biological system and proceeds to influence and interact with other chemical species in the organism. Unfortunately, the sheer complexity of even the simplest living systems precludes the practical realization of such an understanding from first physical principles. Nevertheless, certain general features of the interaction of cadmium with nucleic acids and proteins can be accounted for on the basis of the physicochemical properties of cadmium ions, particularly when compared with other metals. In this section we discuss the physicochemical characterization of Cd 2÷ and several other metal ions: the alkali and alkaline-earthmetals, N a +, M g 2+, Ca2÷; the transition-metal ion Co2+; and the remaining 2 members of Group lIB of the periodic table, Zn 2+ and Hg 2÷. B. Metal ions in aqueous solution Table II lists a number of general properties that describe metal ions in solution. The first 5 listed in the table are physical attributes of the ions themselves, while the last 4 are more indicative of the chemistry of the ion in aqueous solution. W e consider each briefly. 1. Charge and radius. The simplest quantities that describe an ion are its charge z and radius r. Ionic radius characterizes the physical extension of the electron cloud in space around the nucleus of the ion. Since this cloud is diffuse, an ion or atom does not have a precise radius. Values found in the literature vary somewhat, depending on the particular theoretical or experimental concepts applied [9]. Table III listsvalues of the radii for the ions of interest here [10]. Cd 2÷ is intermediate in size between Zn 2+ and Hg 2+. The charge/radius ratio of an ion, z/r, has the dimensions of energy per unit charge and is a numerical index that is large for small, highly charged ions. This index gives a crude indication of h o w close an ion can come to another charge and h o w large the electrostatic force will be. Based on this parameter alone, one would generally expect ions that have a large value of z/r to be able to exert strong forces on charged sites on biological macromolecules. Of the metal ions considered in Table IH, M g 2÷ has the largest value

(z/r

= 3.03). Cd 2÷ a n d Ca 2÷ have v e r y similar values. A par-

TABLE H PROPERTIES OF METAL IONS IN AQUEOUS SOLUTION

Charge, z Radius, r Ch~ge/radi~ ratio, z/r Polarizability, Electronic configuration Coordination number Stereochemistry

Hydration Hard,soft character

10. 18. 9. 17.

11 12 20 27 30 48 80

Na ÷ Mg 2+ Ca :÷ Co 2+ Z n 2+ Cd 2+ Hg 2÷

aRef. bRef. CRef. dRef.

Atomic no.

Metal ion

0.97 0.66 0.99 0.72 0.74 0.97 1.10

Radius, a r (A)

1.03 3.03 2.02 2.78 2.70 2.06 1.82

Charge/radius, z/r ( e l e c t r o n i c charges]A ) 0.25 0.1 1.1 1.2 0.8 1.8 2.9

Polarizability, b a (A 3)

SELECTED PHYSICAL DATA FOR SEVERAL METAL IONS

T A B L E III

1s22s22p += [ N e ] [Se] [ N e ] 3 s 2 3 p += J A r ] [Ar]3d ~ [ A r ] 3 d 1° [ K r l 4 d ~° [Xe]4fl,5d~ o

Electronic configuration

3 3 3 2

10" 105 l0 s × 105 x 107 x 108 × 109 9)< 1 x 4 ×

H20 exchange rate for i n n e r layer o f h y d r a t i o n c (s- 1)

0.211 0.167 0.180 0.130 0.115 0.081 0.064

Softness parameter, d Op

ticularly large value of 6.45 for Be 2+ (not shown) sets this metal apart from others. As suggested by Fernelius [11], when z/r is larger than ~ 1 . 9 there is a t e n d e n c y for an ion to become h y d r a t e d in solution. Hg 2+ has a slightly lower value and Ca 2÷ and Cd 2÷ slightly larger values; these ions are less readily h y d r a t e d than Mg 2÷, Co 2+, and Zn 2+ which have higher z/r values (Table III). Other aspects of hydration will be discussed in Subsection 5. 2. Polarizability. The polarizability a of an object (charged or uncharged) is a measure of the relative ease with which a dipole m o m e n t D can be induced in response to the presence of an external electric field E. Specifically, at low or moderate field strengths, D = aE. The dipole m o m e n t induced in an ion in response to the field of a nearby charge always contributes an attractive force in addition to any other forces that exist between the ion and the charge. In contrast to ions having low polarizability, the more polarizable ions can generally be expected to associate with polarizable macromolecular sites, which lend themselves to electron sharing. Of the ions listed in Table III, Mg 2+ has the smallest polarizability, Hg 2* the largest, and Cd 2÷ the second largest. The Mg 2+ ion is very hard, whereas the electron clouds of Cd 2+ and Hg 2÷ are more readily deformed by external electrostatic fields. The implications of these characteristics are discussed more fully in Subsection 6 on hard and soft metal ions. 3. Electronic eonfuguration. The properties of a given ion are, of course, not independent. Collectively, t h e y reflect different manifestations of the same basic physical circumstances -- a given number of atomic electrons in m o t i o n about a heavy nucleus of given charge. The individual electrons of an isolated ion interact strongly with the nucleus and with each other, but do so in a manner t h a t conserves the ion's total energy, angular mom e n t u m , and other properties. Physically, the electronic configuration is the basic property t h a t completely determines the q u a n t u m state of the ion and thereby all o f its measurable attributes. In principle, the laws o f q u a n t u m mechanics can be used to ascertain electronic structure, although analytical complexity often limits the practical extent to which this can be done w i t h o u t simplifying approximations. The electronic configurations of the ions in Table III are given in column 6. The 10 electrons of Na ÷ in its ground state occupy the 2 available (ls) q u a n t u m states in the K shell of the atom and the eight (two 2s and six 2p, indicated as 2s22p 6) states in the L shell. These shells contain their complete c o m p l e m e n t of electrons in Na* which has the very stable, noblegas configuration o f the neutral neon atom, denoted by [Ne]. Compared with Na ÷, Mg ~-÷ has the same electronic configuration, but the additional unit of nuclear charge binds the electrons more tightly, as reflected by its lower polarizability and smaller ionic radius. The next ion, Ca 2÷, follows Mg 2÷ in group IIA of the periodic table and has the noble gas electronic configuration of Ar. Co 2+ belongs to the transition metals, characterized by having vacant 3d q u a n t u m orbitals. The 10 available 3d orbitals are filled in the case of Zn 2÷, as indicated in Table III. The next 2 ions in the table,

Cd 2÷ and Hg 2÷, follow Zn 2÷ in group IIB of the periodic table, and have filled 4d and 5d electronic states. Cd 2÷ and Hg 2÷ are relatively large and polarizable ions. Although it characterizes an ion completely, the electronic configuration is of limited usefulness in building a framework for analyzing biochemical p h e n o m e n a for at least 2 reasons. First, a metal ion does n o t exist alone in a biological environment -- it interacts with water and other ligands, which have collective properties of their own that help determine the fate and disposition of the metal. Second, the physical characteristics o f potential biological bonding sites inevitably play a key role, apart from properties o f the ions. Indeed, as we shall describe in Sections IV and V, there must be a matching o f properties o f a complexed ion with properties of available interaction sites offered b y macromolecules in order for the ions to effect biochemical changes. We turn n o w to the remaining properties listed in Table II, which potentially have a more direct bearing on the chemical behavior of metal ions in aqueous solution. 4. Coordination number and 8tereochemistry. The electronic configuration of an ion can introduce constraints that might stereochemicaUy affect binding and stability. Under the assumption of pure electrostatic, noncovalent interaction, crystal field theory helps account for the coordination number and stereochemistry o f the transition metals [12]. These predictions are based on the number and symmetry properties of unfilled d-orbitals. The transition metal ions are usually paramagnetic, indicating the presence of unpaired electrons, and they often form a range of colored complexes in which the ion may have different oxidation states. Conclusions from crystal field theory afford useful insight into the coordination and symmetry requirements o f transition metal ions and the geometry of potential macromolecular sites at which they might form complexes. The elements zinc and cadmium have completely filled d~rbitals, y e t are often considered to be transition metals. 5. Hydration. At biological temperatures, extensive hydrogen bonding between neighboring water molecules tends to arrange them tetrahedrally in competition with thermal randomness. Measurements show that the oxygen--oxygen nearest-neighbor separation is 2.9 ~ in liquid water and 2.76 A in ice [13]. Because water molecules possess a permanent dipole moment, they also exert electrostatic forces on one another. In addition, t h e y are polarizable (a = 1.44 As). The charge o f a metal ion will tend to orient the dipoles of the water molecules in its vicinity. An ion with a small radius can be a c c o m m o d a t e d within the normal tetrahedral structure of water, interacting most strongly with the 4 nearest water molecules. Larger ions tend to be hexahydrated. As mentioned earlier, a z/r value of 1.9 favors hydration. The molecules of water directly surrounding an ion are often described as comprising the inner layer or inner sphere of hydration. There can also be a second, or outer, hydration layer. Hydrated metal-ion complexes exhibit a wide range of lability. Table III lists the exchange rates of water molecules from the inner hydration sphere for the ions o f principal interest here [9]. The highest exchange rates found

for any metal ion, ~ 1 0 9 S- l , correspond to substitution of a new water molecule into the inner sphere at virtually every encounter. Hg 2÷ and Na÷ exemplify this behavior, which is diffusion controlled and dependent on characteristics o f the ion only in a formal sense. The rates for Cd 2÷ and Ca 2÷ are an order of magnitude lower than those for Hg 2÷, and that for Zn 2÷ is y e t another order of magnitude lower. F o r these and the other ions in Table III, the substitution rate depends markedly on the charge, radius, and electronic configuration o f the ion, generally decreasing with increasing charge and decreasing ionic radius.When exchange rates, such as those for Co 2÷ and Mg 2÷, are in the range of 104--10 s s -1, t h e y are characteristic o f the ion with a wide variety of ligands besides water. The slowest rates occur with small, highly charged ions (e.g. ~102 s -1 for Be 2÷ and ~ 1 s -1 for AI3+). Water molecules are b o u n d so tightly that electrostatic forces can cause hydrolysis with release of H30 ÷ and binding o f the O H - to the ion before the water molecule can be exchanged. 6. Hard and soft acids and bases. A n u m b e r of diverse chemical data can be correlated on the basis of the theory o f hard and soft acids and bases, the last property listed in Table II. Ahrland et al. [14] classified electron donors and acceptors into 2 types. Class (a) ions and molecules form their most stable complexes with ligands that have electron d o n o r atoms from the first short period (N, O, F). Class (b) species form their most stable complexes with d o n o r atoms from the second (P, S, CI) or later period. Pearson [15] introduced the terminology " h a r d " and " s o f t " for the classes (a) and (b). The affinity sequences for hard ions are F >> C1 > Br > I, O ~ S Se > Te, and N >> P > As > Sb [16]. For soft ions, generally F ~ C1 < Br < I, O ~ S ~ Se ~ Te, and N ~ P > As > Sb. The sequences are the same for all hard ions, b u t some differences exist among soft ions [16,17]. The terms "intermediate" or "borderline" are also used to describe ions that can show b o t h hard and soft tendencies. Classification into these categories is n o t absolute, b u t it is distinct enough to be quite useful. Some reasons underlying the chemical preferences o f hard and soft metal ions are suggested by the data in Table III: Na ÷, Mg 2÷, and Ca 2÷ are usually classified as hard: Co x÷ and Zn 2÷ as borderline; and Cd 2÷ and Hg 2÷ as soft. The hard acceptors in Table III have noble-gas electronic configurations and very low or, in the case of Ca 2÷, intermediate poiarizabilities. The same is true of their preferred donors, the strongest ligands involving F, O, and N. A general rule is that hard acceptors prefer to bind with hard donors. The coordinating ability of hard metal ions increases regularly with increasing charge (positive oxidation state) and decreasing size. This behavior is governed by the large influence of the electrostatic forces that act b e t w e e n the metal and an oppositely charged ligand, tending to form ionic bonds. On the other hand, soft acceptors such as Cd 2÷ and Hg 2÷ (Table III) do n o t have noble-gas configurations. They prefer soft donors, and their coordinating ability does n o t increase with increasing charge and decreasing radius. Soft metal ions prefer to bind with the heavier congeners o f F, O, and N rather than these first-row atoms, with which much weaker complexes are formed. Soft acceptors, like their preferred donors, also have a high degree of polarizability. This behavior is consist-

ent with predominantly covalent bonding between soft ligands, in contrast to the ionic bonding that characterizes the association of hard d o n o r and acceptor partners. Although most chemical bonds are neither purely covalent n o t purely ionic, one can speak of the relative influence of these kinds of forces in the binding of metal ions to various ligands. The t h e o r y of hard and soft acids and bases thus provides very useful concepts and guidance for understanding certain aspects of metal-ion behavior in biological systems at the macromolecular level. Some examples are discussed in Sections IV and V. Qualitatively, deformability is a prerequisite to electron sharing and covalent bonding, as appropriately described by the term " s o f t " . It is therefore natural to associate softness of an ion with its polarizability. However, as pointed o u t by several authors [16,18,19], although softness and polarizability are roughly correlated, polarizability is more a physical, rather than chemical, quantity. Ahrland [16] has discussed various factors t h a t contribute to soft behavior. Filled or almost filled outer d-shells in acceptors favor softness. In this connection, Cd 2÷ and Zn 2÷ have the filled d l° configurations. Acceptors with less than 6 d-electrons never exhibit soft properties, and almost all acceptors with less than 5 show hard properties. Electrons from other inner orbitals can also participate in covalent bonding. Electrons outside the d-shell can increase hardness, because t h e y partially shield the d-electrons. For example, Sn 4÷ with the outer electron configuration 4d 1° is soft; Sn 2÷ with 4dl°5s 2 is n o t [16]. The presence of a large number o f d-electrons per se does n o t guarantee softness. The d-electrons must also have energies about the same as those o f other bonding electrons if t h e y are to participate in ligand formation through electron sharing. For acceptors with the same charge and outer electronic configuration, the physical polarizability increases with the number of electron shells; thus softness properties become more pronounced as one moves downward in the periodic system. In general, a soft ligand is one that has both a high polarizability and available electronic states of the " r i g h t " energies. 7. T h e r m o d y n a m i c considerations. Hard and soft characteristics of bonding can also be analyzed from a t h e r m o d y n a m i c point ot view [20]. Complex formation between ions in any system is accompanied by changes in the free energy AG, enthalpy AH, and entropy AS of the system in accordance with the requirement A G = A H - - T A S ~ O,

where T is the absolute temperature. It is instructive to examine each of these terms. When hard anions and cations form bonds, electrostatic energy is released by their closer juxtaposition. However, this energy can be more than offset by the energy required to disrupt the hydration of the separate ions, and so such a reaction is very often endothermic (AH > 0). However, the reaction proceeds because a large increase in entropy (AS > 0) results from the reduced a m o u n t of hydration when the ligands combine. For hard-hard interactions in a q u e o u s solution, T A S is usually very large, while AH is small and positive. 10

When a covalent (soft--soft) bond is formed, the reaction is exothermic and the decrease in free energy does n o t require the large increase in entropy from reduced hydration. Indeed, soft ions are generally much more weakly h y d r a t e d than hard ions. A G will have a large negative value simply from the formation o f a strong covalent bond. The electrostatic contribution to soft--soft binding will be relatively small and balanced approximately by the small dehydration energy. In stepwise complex formation with soft ligands, therefore, AH should be approximately the same at each stage providing bonds of the same character and strength are being formed. An abrupt change in AH at one stage would imply a different bonding character, such as would occur with a change in ligand coordination number. Formation of a strong complex in aqueous solution thus involves either a large gain in e n t r o p y (AS >~ 0) or a large loss in enthalpy (AH ~ 0), but never both. A large negative value of AG is due principally either to AH (soft--soft) or to TAS (hard--hard). With borderline, or intermediate ions, the 2 terms m a y make comparable contributions to AG. In this case AG will n o t be large, and the complexes will n o t be strong. Table IV shows the stepwise t h e r m o d y n a m i c changes accompanying halide ligand formation in aqueous solution at room temperature for Hg 2÷, Cd 2+, and Zn 2÷. Values of AG°n and AHn are in kilojoule/mole (kJ/mole) and those of ASn in joules/degree Kelvin (J/°K). The subscript n refers to the number of halogen ligands attached to the metal in the final state. Several features can be pointed out. First, in going from Fig2+ to Cd ~÷ to Zn 2+, the transition is in the direction from soft to hard. This is evidenced, for example, by the steady increase in AH[ within the chloride and bromide groups, the bonds becoming more endothermic due to the reduced extent of covalent bonding. Second, as expected, the extent of covalent bonding is seen to increase with the size of the halide involved, as shown by the increasingly negative values o f AH~ for Hg 2+ and Cd 2+ as one goes downward in the table. Third, Zn 2+ shows a decidedly hard character, with positive AG° values for the first 2 steps. The positive values of AH~' and AH~ indicate endothermic associations. Fourth, an increase in both AHn and ASh takes place for Zn :+ when n = 2 and for Cd :÷ when n = 3. With chloride, for example, the change in enthalpy for Zn 2÷ goes from 5.5 to 38 and the change in entropy from 15 to 120 at n = 2. These changes, which also occur for these 2 metals with the bromide, are presumably associated with changes in the coordination numbers o f the metal ions. Finally, we note that Cd 2÷ reverses itself in the nature of its stepwise acceptance of the hardest halide ion F-. The bare Cd 2+ ion is significantly h y d r a t e d and forms principally an ionic bond with F-, the first ligand acceptance being endothermic and accompanied by a large increase in entropy. The resultant CdF ÷, on the other hand, is soft and h y d r a t e d to a much lesser extent; it forms a bond with distinct covalent character when the second ligand is added. 8. Classification by softness. Several schemes have been proposed for expressing the hard--soft character of ions on a quantitative scale. Because ionic contributions to bond strength are dependent on the solvent medium, a universally valid scale for ions has been based on softness. Softness is the property more closely linked to covalent bonding, which is an inherent 11

t'O

Cd ~+

I-Ig2÷ Cd 2÷ Zn 2÷

I-Ig2÷ Cd 2÷ Zn =÷

Hg 2÷ Cd 2÷

Fluoride

Chloride

Bromide

Iodide

0.5 3

3 3 3

3 3 3

1

Molar concn.

-39.4 --3.7 2.4

-0.5

--73.45 -62.5 - 1 1 . 8 8 -- 4.0

-53.65--49.0 --10.04 "-3.3 3.3 4.7

-40.3 -9.04 1.1

-2.6

--8.5 --2.1

-7.9

(--34.5 b) -12.2 --9.2

--15.8 --5.6 --3

--4.3 --1.0 --4.3

AG:

--75.3 --9.46

-40.0 --4.10 1.5

--24.2 --0.42 5.5

5.1

-67.8 --0.8

--40.2 --2.38 42

-27.2 0.08 38

--3.0

A~

A~

AG~

AG]

AG]

hH(kJ/mole)

AG ( k J / m o l e )

aData f r o m Ref. 20. b S u m o f third and f o u r t h steps.

Metal ion

Halogen ion

~

--18.6 1.3

--6.2

(-42.0 b) --3.1 --15.9

-10.8 7.2 --8

--4.3 7.7 0

A~

--6.2 8.4

46 19.6 --5.9

54 28.9 15

26

~S~

--18 10

30 3 125

41 12.6 120

--8

AS]

AS ( J / ° K )

--34 11

5

~S:

(--25 b) 31 - - 2 3

17 43 --17

0 29 8

AS~

T H E R M O D Y N A M I C F U N C T I O N S F O R STEPWISE C O O R D I N A T I O N O F Hg 2÷, Cd 2÷, A N D Z n ~÷ WITH H A L O G E N I O N S IN A Q U E OUS S O L U T I O N a

T A B L E IV

property of electron sharing between ligand partners, independent of the solvent. In this connection, we note that water has one of the largest dielectric constants of any liquid, which greatly reduces the a m o u n t of electrostatic force between two charges relative to what they would experience in a vacuum or in the gas phase. Pearson and Mawby [21] define a softness parameter Op in terms o f the coordinate b o n d energies of a metal with the halogens F and I. Specifically, Op =

E(F) -- E(I) E(F)

where E(F) and E(I) denote the coordinate bond energies of the metal with fluoride and iodide, respectively. Numerical values of Op are given in the last column of Table III. It follows from this definition that Op is small when there is relatively little difference between the fluoride and iodide b o n d energies. Thus, small values of Op are expected to reflect the property of softness; hence Op itself can be used to express numerically the hard or soft character of a metal ion. Since b o n d energies depend strongly on the charge o f an ion, Op provides a useful comparative numerical scale of softness only for ions of a given charge. The usefulness of Op in analyzing certain data can be illustrated b y applying it to the data in Table I. The glycine (ON) and mercaptoethylamine (NS) bidentate ligands differ b y the replacement of the oxygen atom by a sulfur atom. The theory of hard and soft acids and bases would predict a greater affinity of hard ions for (ON) and soft ions for (NS). Table I shows that the formation constant for the hard ion Mg 2÷ (Op = 0.167) with (ON) is more than an order of magnitude larger than with (NS). The soft Cd 2÷ shows a difference of over 3 orders of magnitude in the opposite direction, favoring (NS) over (ON), as expected. The intermediate Zn s÷ and Co s÷ ions also prefer the sulfur ligand to the oxygen, b u t to a lesser degree than Cd s÷. A comparison between the oxalate (OO) and mercaptoacetate (OS) ligands shows a similar softness preference of Zn 2÷ and Co s+ for the sulfur ligand. Other softness parameters are described elsewhere [17,22]. Williams has suggested that softness reflects the polarizing power of a metal ion, expressed through the ionization potential I, relative to its size and charge, expressed through zS/r. The ratio rI/z s indicates the relative importance of covalent and ionic bonding in donor-acceptor systems [22--24]. These and other basic physicochemical concepts relating hard/soft character to metal ion biochemistry and toxicity are discussed in a recent review b y Nieboer and Richardson [ 24a]. IV. NUCLEIC ACIDS

The regular structures of the nucleic acids, nucleosides connected by

13

phosphodiester linkages, permit certain generalizations to be made about likely interaction sites for different metal ions. In terms of hard or soft characteristics, the phosphate residues represent negatively charged hard sites available for electrostatic binding. Electron-rich sites on the bases, on the other hand, offer soft sites for the formation of bonds more covalent than ionic in nature. Hard ions, such as Na 2*, Mg 2÷, and Ca 2÷, tend to bind almost exclusively with the phosphate groups. Indeed, such ions are essential for stabilization of the Watson-Crick double helix of DNA [25]. Mg 2÷, Mn 2÷, and Co 2÷ also function as cofactors for DNA polymerase during DNA replication and the accompanying phosphate-bond cleavage and formation [26]. Borderline ions can involve both phosphate and base coordination. The relative affinity for base coordination, relative to phosphate, for such ions has been reported as Co 2÷ = Ni 2÷ < Zn 2÷ < Mn 2÷ ~ Cd 2÷ < Cu 2÷ [27,28]. Ionic strength can drive borderline interactions one way or the other. For example, at low concentrations Zn 2÷ binds primarily with the phosphates in DNA; at higher concentrations it interacts more with the nucleoside bases [29]. Hg 2+ and Ag ÷ are examples of very soft ions, which appear to bind exclusively with the base moiety of nucleic acids [30]. Additional factors affect the interactions of the various borderline and soft metals with bases. As discussed in m a n y places [26,31], specific sites of high electron density in the purine bases are the N(1) and N(7) positions of adenine and the N(3) and N(7) of guanine; the NH2 groups of both also have high electron density. Corresponding pyrimidine sites are the N(1) position as well as the NH2 of cytosine and the 0(4) and N(1) positions of t h y m i n e and uracil. Steric factors can play a role at any of these locations. Some sites are not exposed in hydrogen-bonded double-stranded structures of nucleic acids. Unhindered atoms, such as the N(7) of the purines, can be accommodated in octahedral complexes of a metal ion. More sterically hindered sites, such as the cytosine N(1), prefer the lower coordination number of tetrahedral or square-planar complexes [31,32]. A number of general phenomena can be understood as manifestations of properties we have been discussing. Sisso~ff et al. [30] have classified metal ions according to effects on DNA structure. For example, the presence of the hard ions Na ÷, Mg2÷ and Ca 2÷ gives increased stability to the double helix. They act as counter ions to reduce the unwinding tendency due to electrostatic repulsion between the negatively charged phosphate groups on adjacent nucleotides. The borderline ion Co 2÷ has little effect on DNA stability, whereas the soft ions Cd 2÷ and Hg 2÷ decrease it. The softest ions presumably interact more strongly with bases, forming internal chelates, interstrand complexes, cross-links, and other destabilizing complexes that perturb the regular hydrogen-bonded helical structure. Many ions also form mixed chelates between phosphate groups and bases. The binding of metal ions is also influenced by pH, temperature, and, as we mentioned for Zn 2÷, ionic strength.

14

Having stated these general considerations, we turn to specific examples of the interaction of Cd 2÷ with nucleic acids and their components.

A. Purines, pyrimidines, nucleosides, and nucleotides Nuclear magnetic resonance (NMR) has been used to detect perturbations of the m o t i o n of protons in a molecule due to the presence of a metal ion. Gonda et al. [33] f o u n d t h a t Cd 2÷ and Hg 2÷ associated with certain purines and pyrimidines with constants f r o m 1 to 10 M -1. The 4 NMR signals of adenine were altered by Cd2+: those for H(2), H(8), and NH2 indicated a formation constant Ka of 1.9--2.4 M -1 and t h a t for N(7), 9.1. In the presence of Hg 2÷ the binding constant of the former 3 were all about 1 unit lower. In the case of 6-mercaptopurine the relationship of the binding constants for Hg 2÷ and Cd 2÷ was reversed from that for adenine, namely Hg 2÷ was higher (2.5) t h a n Cd 2÷ (1.8). For pyrimidine nucleosides, cytidine revealed a Ka of 1.5 for Hg 2÷ and 1.1 for Cd2*; thiouridine exhibited a constant of 2.3 for Hg 2÷ (Cd 2÷ was n o t examined). These values are the only ones available for Cd2÷-base interaction; other metal ions and bases have been examined more extensively. Martin and Mariam [34] severely criticized indiscriminate use of NMR as a means o f measuring interactions of metal ions with specific atoms of bases, nucleosides, etc.; they point o u t that line broadening in NMR measurements of IH, 13C and 3~p is often assumed to be directly related to the distance between the paramagnetic metal ion and the atom under observation. This assumption depends on the interpretation of equations t h a t are used to evaluate the transverse relaxation time; these equations contain 2 terms, one a dipolar term and the other a scaler term and the relative contribution of each term usually has not been properly evaluated. A further difficulty in interpretation of NMR results from the contribution of unpaired spin density from a paramagnetic metal ion at a carbon to both the dipolar and scaler terms. On the other hand, diamagnetic metal ions cause chemical shifts in ~H and 13C NMR that are reliable indicators of metal ion binding sites; with 31p NMR such reliability is yet to be achieved. The difficulties with paramagnetic ions are applicable to studies involving Cu2+; Cd 2÷ however is diamagnetic. The stability constants of adenosine for 6 other metal ions (Mn 2+, Co 2÷, Ni 2+, Cu 2÷, Zn 2÷, and Hg 2÷) are available as determined by means other than NMR [ 29]. For the first 5 metal ions, the stability constants are in the range of 0.1--1, whereas the constant for Hg 2+ is 104--10 s times larger. Progressing to the nucleotide adenosine 5'-monophosphate, the binding constants for the first 5 metal ions range from 500 to 5000. The presence of the phosphate increases the binding constant 2--3 orders of magnitude because this hard negative ionic site offers an additional attraction to the cation. The analysis of crystals of a cadmium derivative o f cytidine 5'-monophosphate, [Cd(5'-CMP)(H20)]n, and of a cadmium derivative of inosine 5'-monophosphate, Cd2(5'-IMP)3(H20)6, revealed a tendency of Cd 2÷ to

15

Fig. 1. Stereoscopic view of a fragment of the polymeric structure of [Cd(5'-CMP) (H20)] n. [From Ref. 35, used by permission.]

form a bond with a ring nitrogen, N(3) in the former and N(7) in the latter [35,36]. If this were to occur in a nucleic acid, the cytidine-Cd interaction would prevent normal hydrogen bonding. In the case of the 5'-CMP (Fig. 1), Cd 2÷ was shown to bond n o t only to the N(3) of the pyrimidine but also to a phosphate oxygen from each of 3 neighboring 5'-CMP molecules and to a water molecule; Cd 2÷ is therefore pentacoordinate in this instance. Each 5'-CMP is bonded to 4 cadmium atoms, and each cadmium atom is bonded to 4 nucleotides. By way of contrast, the 5'-IMP Cd 2÷ complex (Fig. 2) involved 2 independent Cd 2+ ions: 1 bound to the N(7), 2 ribose oxygen atoms, and 3 water molecules, and the second bound to 2 N(7) atoms, a phosphate oxygen, and 3 water molecules. The overall structure is stabilized by numerous hydrogen bonds: 1 to the N(1) ring nitrogen and several to oxygens in water, ribose, phosphate, and the keto group

Fig. 2. A view showing the polymeric structure of Cd2(5'-IMP)3(H~O)6. It consists of units of the type Cd~(5'-IMp2-)(5'-IMP~-)2(H20)~ arranged in a polymeric army. (There are also a number of non~oordinated water molecules of crystallization,approx, six distributed over nine positions. These play a lesser role and have been omitted for clarity). [From Ref. 36, used by permission.]

16

of the ring. This is said t o be the first report that heavy metal can bind to ribose oxygen; however, these bonds are longer and weaker than those to the phosphate oxygen. Zinc has also been crystallized with 5'-IMP [Zn(5'IMP)n, nH20] and, like cadmium, is b o n d e d to the N(7) and to 3 phosphate oxygens from 3 neighboring 5'-IMP molecules [37]. In contrast to the pentacoordinate cadmium, the zinc coordination is tetrahedral, with no water molecules b o n d e d to the metal. The cadmium--N(7) bond is distorted so that the N(7) puckers o u t o f the plane of the purine ring; the flexibility at the N(7) allows a cadmium b o n d to form and minimizes departure from parallel base stacking. D.M.L. Goodgame (personal communication) has prepared crystals o f Cd(5'-UMP)nH20 and [Cd(5'-GMP) (H20)s] " 3H20. The former is similar to the analogous cobalt c o m p o u n d studied earlier [37a] and the latter is very similar to its manganese analog [37]. A t t e m p t s t o grow good crystals o f Cd-AMP have been unrewarding. When nucleotides are crystallized with metal ions, extensive polymeric structures m a y be formed. A polymeric structure m a y be more stable than a monomeric one. The most unusual structure occurs in [Cd(5'-CMP)(H20)]n, in which cross-linked helical arrays are formed and large cylindrical channels extend through the entire crystal [34]. In some cases the ribose unit is forced into unusual conformations that m a y cause puckering. A new dinuclear cadmium complex of adenine, [(CsH6Ns)Cd(NO3)3 • H20]2, was recently obtained from reaction o f 5'-AMP with Cd(NO3)2 at pH 3.2 [38] (see Fig. 3). The analysis of the X-my diffraction pattern revealed the first k n o w n example o f 2 protonated adenine moieties chelated to 2 cadmium atoms via N(3) and N(9) sites. The molecular structure consists of a protonated adenine m o i e t y which is chelated via 2 o f its nitrogen sites to 2 cadmium ions, which in turn are coordinated to the same 2 nitrogen sites of the centrosymmetrically related, p r o t o n a t e d adenine moiety. Each metal a t o m can be considered t o be octahedraUy coordinated to 2 nitrogens of the adenine rings, 2 nitrate ions, and 2 water oxygens. Since this dinuclear complex possesses a crystallographic center of symmetry, it requires the site s y m m e t r y Ci - 1 . Each of the 8 hydrogens is hydrogenb o n d e d to neighboring nitrate oxygen atoms, thus forming a complex hydrogen-bonding network. The combined effect o f metal and proton bonding, however, does n o t seem to severely perturb the ~-electron system of the adenine ring, as evidenced in part b y the fact that it remains essentially coplanar within 0.01 A for the individual constituent atoms. The metal ions have different tendencies to accept electrons from oxygen and nitrogen donors: Zn 2÷ and Cd 2÷ interact readily with both. Certain metal ions favor bonding to nitrogen over oxygen; in comparing the first association constants of ethylenedi~mine and oxalate complexes, Martin and Mariam [34] calculated t h e following series: Mn 2÷ ~ Fe 2÷ ~ Zn 2÷ Co 2÷ ~ Cd 2÷ ~ Ni 2÷ ~ Cu 2÷ ~ I-I*. This series m a y be compared with a series constructed from effects o n melting curves for D N A [27] that showed increasing affinity for t h e base relative to phosphate sites as follows: Co 2÷ --

17

011

Fig. 3. Molecular configuration of the {[(CsH,Ns)Cd(N03)2 • H 2 0 ] ÷ ~ molecule. Primed atoms are centrosymmetrically related to the corresponding basic atoms.

18

Ni 2+ <~ Zn 2+ < Mn 2+ < Cd 2+ <~ Cu 2+. Similarities between these 2 series of metal ions are apparent; Cu ~+ has the highest affinity for nitrogen, and Cd 2+ is higher than Zn 2+. However, some marked differences are also apparent; e.g., Mn 2+ and Ni 2+ occur at different relative positions in the 2 series. The first series is based on model c o m p o u n d s n o t found in nucleic acids, whereas the latter is obtained from studies on DNA. It is generally agreed that soft metals have higher affinities for nitrogen atoms in purine and pyrirnidine rings than for phosphate oxygens. Yet other factors such as thermodynamic and steric effects must also be considered.

B. Transfer R N A Since metal ions have different affinities for the base and phosphates, various degrees of stabilization of single-stranded or helical portions of a nucleic acid b y metals can be accomplished depending on the t y p e of metal ion. The transfer R N A molecule contains: (a) helical regions due to WatsonCrick base pairs; (b) tertiary folded regions due to unusual base pairs and base triples; and (c) single-stranded regions. A chromatographic system employing a RPC-5 column resolves a mixture o f t R N A s on the basis of net charge, base composition, nucleoside modification, and tertiary structure. By comparison of chromatographic behavior in the presence or absence of Mg 2+, Cd 2+, or Zn :+, t R N A Tyr and t R N A Leu were shown to be differently affected b y each metal ion [39]. Cd 2÷ changed the properties o f t R N A T~r so that 2 isoacceptor species became inseparable. These 2 isoacceptors differ only in that one contains a guanosine in the anticodon whereas the other contains queuine a modified base derived from guanine, in the place of that guanosine and additionally a 5-methyl cytidine in place of cytidine at an unspecified location [40]. The queuine contains a positive charge which is the basis for the chromatographic difference between the 2 isc, acceptors. It is not known where Cd 2+ binds to these 2 isoacceptors, but, on doing so, it obscures their chromatographic difference. The effect o f Zn 2÷ on t R N A Tyr and t R N A Leu is to cause them to bind more tightly to the column, a positively charged matrix, so that much higher concentrations of NaC1 are required to elute them. If the Zn 2÷ caused disruption of the tertiary folded structure of the t R N A so that phosphate groups became unshielded, the t R N A would behave in t h e manner described. Since these experiments were done at pH 4.5, the hydrolysis caused by Zn 2÷ at neutral pH was avoided [27]. This chromatographic system m a y be used to make similar studies of other tRNAs and other metal ions. Because the t R N A s are detected b y the radioactive amino acid that was enzymatically attached, pure t R N A is n o t necessary for this experiment. However, it will be useful to use purified species to verify this interpretation and to examine further effects of metal ions on the t R N A structure. Crystals of t R N A have been shown to be penetrated and b o u n d b y metal ions in a specific manner [41]. The interaction o f various metals with crystalline t R N A may be studied to determine the sites at which the metal binds. If site specificity is 19

f o u n d it may be compared with the effects of the same metals on the chromatographic properties of the tRNA. C. D N A A polarographic technique was used to identify the metal ions t h a t are associated with DNA [30]. Of 8 metal ions t h a t could be detected, 5 were f o u n d in appreciable amounts: Zn 2÷, Cd 2÷, Pb 2÷, Cu 2÷, and Bi 3÷. The DNAs were obtained from bacteria, animals, and plants that had been exposed to these metals. In plant tissue DNA, cadmium was always f o u n d in the light (lower density) DNA in greater amounts than the other metals. In Nicotiana tabacum the Cd 2÷ in light DNA varied over 200-fold among tissues of this plant: leaves < stems < healthy tissue culture < tumorous tissue culture. The authors suggested that the metal ion m a y be involved in augmenting the rate of cell division. As will be mentioned in Section V, DNA polymerases and RNA polymerases often contain zinc; but it has yet to be demonstrated t h a t Cd 2÷ can stimulate their activities. Certain metal ions were shown to provide protection to DNA against damage due to radiation [42]. After calf t h y m u s DNA (dry, in a vacuum) was exposed to 6°Co radiation, the damage to the DNA was estimated by measuring: (a) carbon radicals by electron spin resonance; (b) the occurrence of chain breaks t h a t cause phosphate groups to be exposed and become subject to hydrolysis by a monoesterase; and (c) unstable neutral radicals t h a t cause tritiated water molecules in the DNA to become stabilized against exchange with external water. Various metal ions at a concentration o f 1 per 100 nucleotides, were evaluated for their ability to protect against damage. The metal ions Cr 3+, Cu 2+, and Ni 2÷ were effective in diminishing damage to the DNA by all 3 criteria In contrast, Cd 2+ provided little or no protection against the presence of radicals in the DNA, but it was effective in protecting against strand breakage. Protection against the presence of a radical was attributed to the ability of the effective metal ions to compete with the bases of DNA and capture the electrons produced by radiation, e.g. Cu 2+ -~ Cu +. The filled-shell electronic structure of Cd 2÷ is n o t well suited to electron capture; Cu 2÷ was the most effective. It is curious t h a t Cd 2+ did provide quite good protection against strand breaks even though electron capture was poor. In another system, on the other hand, strand breakage of DNA was caused b y exposing growing Escherichia coli in 3 × 10 -6 M Cd 2+ [43]. Single strand, but n o t double strand, breaks occurred t h a t could be at least partially repaired after Cd 2+ was removed. In a study by Thomason et al. [44], the binding o f benzo[a]pyrene to certain macromolecules, possibly including DNA, was altered by various di- and trivalent cations. DNA was mixed with [3H]benzo[a]pyrene along with liver microsomes prepared from rats t h a t had been injected with benzo[a]pyrene to induce the microsomal oxidation system. The a m o u n t of tritium bound to substances that are excluded by Sephadex G-200 was determined after a 30-min incubation at 37°C. The effects of metal ions on this reaction were as follows: Fe 3+ stimulated at 10 -4 M and then inhibited sigmoidally at higher concentrations; Cd 2÷, Zn 2+, Cr 3÷, and Ni 2+ 20

caused a sigmoidal inhibition; Cu 2÷ and Mn 2+ caused a progressive inhibition; and Mg 2÷ had no effect. These metal ions, which are present in asbestos, were being examined because asbestos workers are at a high risk of malignancy. The stimulatory effect of Fe 3÷ was unique among the metals tested; no a t t e m p t was made to examine the effects of mixtures of metal ions. Whether the effect of Fe 3+ is on the microsomal oxidation capacity or the DNA cannot be established from the data presented. Further studies along these lines should include efforts t o define the site of action of the cations. V. PROTEINS Proteins present a wide variety of shapes, structures, and potential binding sites for metals. Considering further interactions after one metal ion forms the first complex with a protein, the second and third formation constants are apt to depend strongly on the conformation produced by coordination of the first ion. Binding tendencies for protein often reflect in part the affinity of metal ions for amino acids [45]. Only weak binding should occur with univalent hard ions, such as Na ÷ and K ÷. These ions are attracted to singly charged oxygen donors or neutral ligands containing oxygen. Binding is stronger with divalent hard ions, such as Mg 2+ and Ca 2÷, which are involved in control mechanisms and in enzyme activation. These ions are attracted to carboxylate, phosphate, and nitrogen donors, Ca :÷ showing somewhat less affinity for these ligands than Mg2÷. In certain hydrolytic enzymes, such as bovine carboxypeptidase, Zn 2÷ is bound strongly, serving as a Lewis acid. Finally, redox catalysts, such as Fe and Co, are generally bound very strongly. The transition from evaluation of the association of metal ions with amino acids to t h a t with proteins has been dealt with by Hughes [12]. Several factors can be readily listed to illustrate some o f the variables t h a t need to be kept in mind: (a) The reaction between metal ion and amino acid usually involves the amino and carboxylate groups; thus, the degree of protonation of t h e s e groups is an important consideration. (b) For a given metal ion the formation constant for reaction with peptides is smaller than with amino acids. The terminal groups contribute only a part of the structure o f the metal complex. Crystallographic studies have demonstrated the effect of planar peptide structures in stabilizing metal complexes; deprotonated N-peptide atoms along with 2 other donors are usually b o u n d to a metal ion, such as Cu 2÷ to form a planar configuration. (c) The presence of side chains is a significant consideration. Cysteine and histidine have especially strong affinities for certain metals. (d) The stereochemistry factor can be considered from 2 viewpoints. The ligand system that reacts with the metal ion needs to be present in the proper tetrahedral, octahedral, or square-planar configuration to allow strong coordination. The stereochemical factor has to do with size of the Cd 2÷ binding site in the protein relative to the size o f the metal ion. For example, Cd 2÷ has a diameter of 1.94 £ as compared with 1.48 A for Zn2÷; t h e diameters of the h y d r a t e d 21

ions are ~ 4 . 8 and ~ 4 . 4 A, respectively. Binding sites of certain discrete sizes would accommodate the bare metal ion or the ion in various states of hydration. A review by Webb discussed several clear examples in which Cd 2÷ and other metals are b o u n d to proteins by mechanisms t h a t are readily interpretable [46]. A. A m i n o acids Each metal ion has characteristic affinities for the O, N, and S sites in the ligands to which they hind predominantly. In Section II the relative affinities for certain model compounds were discussed and it was shown that Cd 2÷ has a larger formation constant for ligands having --SH residues than any metal ion except Cu 2÷, Hg 2÷, or Pb 2÷. The affinity of Cd 2÷ for ligands having --NH2 and - - C O 0 - groups is less t h a n t h a t o f several other metal ions, but it is important to recall t h a t even a formation constant of 104 is an order of magnitude larger than t h a t for Cd 2÷ interacting with a nucleotide (Section IV). Suffur donors are soft bases, whereas nitrogen donors are hard. Cd 2÷ has a higher affinity for --SH than does Zn 2÷. Although a metal ion may react differently with a protein than it does with an equivalent mixture of amino acids, it is instructive to consider the affinity o f Cd 2÷ for various amino acids. These formation constants along with those of Zn 2÷, Hg2*, and Co 2÷ for these same ligands are presented in Table V [45]. Two association constants are presented: KI represents the reaction between the metal ion and one ligand, most likely through the ionized carboxyl groups; ~2 represents the reaction with 2 ligands. The differences between constants for Zn 2÷ and Cd 2÷, as indicated by /32, are rather consistent in t h a t the formation constants o f amino acids with Zn 2÷ are usually 10 times greater than with Cd 2÷. Values obtained by different laboratories m a y vary by an order of magnitude. Thus unless log/~2 of 1 cation is larger by at least 1 log unit than t h a t o f another cation the difference may not be significant, since these values are selected from a number of values compiled by Sill~n and MarteU and may range over several tenths for each metal. Formation constants for histidine and cysteine are especially large with all of the metal ions in Table V. Comparison of the ~2 values shows t h a t histidine binds Zn 2÷ with somewhat greater affinity than Cd 2÷, whereas in the case of cysteine the affinity for Zn :÷ exceeds t h a t for Cd 2÷ by over l 0 s. This difference between the affinities of Zn 2÷ and Cd 2÷ for cysteine binding is greater than would be predicted according to their soft base characteristics. With regard to the other amino acids, Cd 2÷ has the least affinity for lysine, tyrosine, valine, arginine, and isoleucine. As would be expected, this group of amino acids has the lowest formation constants with Zn 2÷ also. Although n o t an amino acid, noradrenaline is included for general interest in the table since it forms more stable complexes with Zn 2÷ than Cd 2÷. Dihydroxyphenylalanine (Dopa) shows only a slight preference for Zn 2÷ over Cd 2÷. With regard to the relative effectiveness of metal ions to react with amino

22

TABLE V FORI~ATION CONSTANTS FOR AMINO ACIDS AND RELATED COMPOUNDS WITH VARIOUS CATIONSa Compound

log K 1

log ~2

I-r(NH2)

Z n 2÷

Alanine

(9.9)

4.5 5.7

Aspartic acid Arginine

(9.8) (9.1)

5.96

Asparagine

(8.8)

4.51

Cysteine

(10.1)

C d 2÷

t 4.5 5.9

Co2÷

4.4

(9.5) (9.3) (9.8)

I-Iistidine Hydroxyproline Imidazole Isoleucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine Vopa Noradrenalin

(9.4) (9.7)

6.4

(7.1) 9.9 (10.5) (9.3) (9.3) (10.6) (9.2) (9.1) (9.6) (9.2) (9.6) (9.0) (8.8)

2.13

~5

5.3

4.6 4.05

{4:9

6.0

5.1

4.4

4.7 4.7

(10.4) (8.0) (8.5)

3.42

6.77

C d 2+

(7.6)

Hg2 +

18.4

8.8

(6.7) { (7.1) 8.6

(17.4)

18.2

9.9

(20.5)

(8.8) (8.4)

(7.9) (7.4)

9.2

7.9

18.2

(12.0) 9.6

(11.1) 8,2

20.6 17.7

8.2 (7.3)

6.6 (5.8)

(8.3)

(7.1)

17.6

(8.4) 9.9 (8.6) (8.6)

(7.2) 8.0 (7.4) (7.2)

(18.7) 20.5 (17.5)

(8.2)

(7.0)

(7.9) 8.2 (8.7)

(6.4) 6.7 (7.9)

2.47

{3:7

4.5

4.84 4.43

4.4 10.69

(9.1)

14.2

11910

Glutamic acid Glutamine Glycine

I-Ig2÷ Zn2+

6.52

(17.5) (17.1)

4.82

= [ML]/[M] [L], M + L ~ ML. ~, ffi [ML2]/[M] [L] 2, M + 2L ~ ML=. bValues K obtained at a ,concentration of 0--0.01 M salt; other values at in parentheses 0.01--1.0 M. Data from Ref. 45.

acids, the comparison of Hg 2÷ and Cd 2÷ is useful. The log/~2 values range from 17.1 to 20.6 for complexes between Hg 2÷ and 12 of the amino acids listed. These values are uniformly higher than those for Cd 2÷ and cover a narrower range. Certain cadmium-amino acid complexes have been crystallized. The structures obtained from X-ray diffraction patterns provide models for possible ways that Cd interacts with these residues in proteins [47]. The cadmium--asparagine and cadmium--glutamate crystals show bidentate chelation; a bond from the carbonyl oxygen to Cd 2+ and Zn 2÷ in crystals with glutamate, aspartate, cysteine, penicillamine, glutathione, and his-

23

tidine has been described [47]. There is evidence from NMR t h a t Hg 2+ interacts with the thioether in S-methylcysteine but Cd 2+, Zn 2÷, and Pb 2÷ do not. More detail on these structures is available in chapters 1--4 o f Sigel [47].

B. Sulfhydry l groups Histidine and carboxyl groups on serum albumin provide high and low affinity sites, respectively, for Cd 2+ [48]. The binding of Cd :+ to serum albumin is much stronger than t h a t o f Pb ~÷ [49]. At low pH where the carboxyl groups are n o t charged, Hg 2+ m a y bind exclusively at the albumin sulfhydryl group to produce a dimer [50,51], whereas at higher pH values other sites also seem to be available [52]. Albumin is the major protein of serum and can provide sites for a variety of metals to form complexes. The binding of Zn 2÷, Cd 2÷, and Cu 2÷ to bovine serum albumin was examined as a function of pH [53]. In the pH range 2--6 the binding of Cd 2÷ was greater t h a n t h a t o f Zn 2+ for albumin, but this was reversed for free amino acid. The sulfhydryl group in protein is clearly affected by neighboring groups. The binding site for Cd 2+ was the sulfhydryl group, and Zn 2+ bound to amino and carboxyl groups better than Cd 2÷ [54]. Cd 2+ is n o t always a reliable reagent to inhibit enzymes t h a t require --SH groups. Generally --SH reagents (Hg 2÷, Ag ÷, and iodoacetamide) inhibited both aspartate aminotransferase and L~alanine aminotransferase, but Cd 2÷ did not inhibit the former. Further differentiation of the 2 enzymes resulted from observing that, whereas cysteine inhibited both enzymes, mercaptoethanol inhibited the former but stimulated the latter enzyme [55]. The combination of Cd 2+ with a thiol might be used to see if inhibition of either enzyme was enhanced in analogy with the arsenite mechanism discussed below. Arsenite binds to sulfhydryl groups of proteins in different ways according to the substituents on the arsenite [ 56]. Inorganic and monosubstituted arsenites (AsO~ and RAsO) effectively bind to 2 - - S H groups at a time If adjacent --SH groups are unavailable, then a single --SH of the protein may be bound if an exogenous thiol source such as mercaptoethanol or cysteine is provided. Disubstituted arsenite (R2AsOH) reacts readily with single --SH groups. For certain enzymes (e.g. aldehyde dehydrogenase, ~-hydroxybutyrate dehydrogenase, acetyl-CoA carboxylase, luciferase, and glutamine synthetase) Cd 2÷ and arsenite are both effective inhibitors. It may be presumed, therefore, t h a t 2 sulfhydryl groups are adjacent and that the inhibition by Cd 2÷ may be the result o f a complex of one or both of the sulfhydryl groups with this metal. In the case of ketoacid dehydrogenases and the associated dihydrolipoyldehydrogenase, the reduced form of the lipoic acid coenzyme provides neighboring sulfhydryl groups which readily chelate divalent transition metal ions [57]. Cd 2+ and Zn 2+ form stable complexes which dissociate at lower pH values to yield the active coenzyme. On the other hand, Co 2+ and Cu ~÷ chelates are readily and irreversibly oxidized. In the ketoglutarate dehydrogenase there are 6 lipoic acids as components o f the enzyme, and 24

when they are reduced b y N A D H t h e y are readily b o u n d b y arsenite or Cd 2÷. The resulting inhibition is readily reversed b y dimercaptopropanoL As in the case with lipoic acid, the Co 2÷ and Cu 2÷ complexes are irreversible [58--60]. C. MetaUothionein Metallothionein is a protein f o u n d in liver and kidney of various animal species. The a m o u n t o f metallothionein is increased if the animal is treated with Zn 2÷, Cd 2÷, Hg 2÷, and possibly other metal ions, and the increase is due largely to the induced synthesis o f this small protein [61]. The protein has a molecular weight of 6000--8000, contains no histidine or aromatic amino acids, and is enriched in cysteine. When Cd 2÷ interacts with the thionein to produce cadmium thionein the absorbance at 250 nm increases; Cd 2+ reacting with mercaptoethanol also produces absorbance at 250 nm which is maximal when the ratio of mercaptoethanol/Cd 2÷ is a b o u t 3 [62]. Synthetic oligopeptides that contain 3 cysteines b u t no aromatic or imidazole amino acids and that have sequences similar to thionein were shown to have 5 times more capacity to bind Cd 2÷ than an equivalent a m o u n t o f cysteine SH [63]. A further indication that grouping sulfhydryl groups into 1 molecule is more effective for Cd 2÷ binding was that dithiothreitol (2 --SH groups) was better than cysteine b u t was inferior to a peptide with 3 --SH groups. These pepticIes were shown to be particularly effective in protecting mammalian cells against the toxic effects of Cd 2÷. A recent study explored the structural relationships in cadmium thionein, using H3Cd so that NMR could be used to determine the resonances which cadmium affected [63a]. Isolation of metallothionein from rabbits treated with H3Cd2+ yielded a protein with 4.4 g-atoms o f H3Cd and 2.6 g-atoms of Zn2*/mol of protein; the non-integral members are believed to be due to heterogeneity of the native metallothionein. The ~3Cd NMR spectrum consisted of 5 resonances that indicate that each Cd 2÷ ion is involved in extensive sulfur ligation, probably at least 3 sulfurs for each Cd 2÷ ion. More information was obtained by proton decoupling and replotting to minimize digital broadening. The resulting spectrum revealed considerable underlying fine structure associated with each of the ~X3Cd resonances. Two explanations were considered for the splittings that give rise to the fine structure: (1) metal ion heterogeneity of the native protein sample; or (2) spin-spin coupling that results from direct interaction of adjacent 113Cd2+ ions. By obtaining the spectrum at a higher field strength and performing homonuclear decoupling it was shown that the fine structure o f several resonances of 113Cd were altered by a decoupling pulse applied to one of the resonances. F r o m this experiment the existence o f polynuclear metal clusters in metallothionein was demonstrated. The detailed structure o f metallothionein includes, therefore, the bonding of Cd 2÷ to several sulfur atoms and the bonding of adjacent Cd 2÷ ions t o each other. The domain of each Cd 2÷, and of the Zn :÷ that are also present, m a y be unique. The complete delineation of these structures m a y require X-ray diffraction; however, the insight obtained from 1~3Cd NMR is significant.

25

D. Stabilization Metal ion stabilization of protein structure has been reviewed in some detail [64]. A quantitative expression o f such stabilization was based on the rate of release of tritium from a protein that had been equilibrated with tritium ions previously. For thionein, tritium is released rapidly; but cadmium stabilized thionein so t h a t about 20 of its hydrogens no longer were subject to rapid exchange. The metal ion apparently alters the protein configuration so t h a t a number of ionisable groups are restricted from contact with the solvent. E. Stimulation One intriguing effect of Cd 2÷ is its stimulation of the activity o f certain enzymes. A list of 25 such enzymes is given by Vallee and Ulmer [65]. In no case has the mechanism been established, but it is speculated that there are 2 or more Cd 2÷ binding sites to explain the stimulation t h a t is followed at higher concentrations by inhibitiorL One o f the most striking stimulatory effects is on a-mannosidase of mouse liver [66]; Cd 2÷ at 10 - 4 10 -3 M caused a 3--4-fold stimulation, which was blocked by the presence of stoichiometric amounts of Zn 2÷. In the range o f 10-7--10 -2 M neither Pb 2+ nor Hg 2÷ was stimulatory, Cu 2÷ (10 -4 M) caused a 1.6-fold stimulation, and Zn 2÷ was somewhat less stimulatory. At 10 -2 M all these metals caused inhibition. It should be noted that the a-mannosidase was purified 3&fold for these studies, so it is necessary to delay interpretation until artifacts due to impurities can be eliminated. Nevertheless, this enzyme offers some promise in examining the mechanism of stimulation by metal ions. Another example of Cd 2÷ stimulation is t r y p t o p h a n oxygenase from Pseudomonas acidovorans [67]. At concentrations of 10-6--10 -4 M, Cd 2÷ causes 1.6-fold stimulation followed by inhibition at 10 -3 M. The stimulation o f 5-aminolevulinate dehydratase by Cd 2÷ (110% by 1.5 × 10 -6 M) is less than that by Sn 2÷ (133% by 1.5 X 10 -6 M), Zn 2÷ (132% by 3 X 10 -6 M), or A13÷ (133% by 5 X 10 -6 M). Yet within a 4-fold range of concentration these 4 metals all cause a similar effect, whereas others (Mn 2÷, Cu 2÷, Pb 2÷) are inhibitory at 10-s--10 -6 M and y e t others (Ni 2÷, As 3*, Fe 2÷, Cr 3÷) cause no effect at concentrations from 10 -7 to 10 -4 M [68]. As has been suggested in a number of studies, the metal ion m a y cause stimulation by altering a hypothetical allosteric site on such enzymes. Oleyl-CoA desaturase from potato tubers was stimulated by Cd 2÷, but Pb 2÷ at the same concentration inhibited the enzyme by 50% [69]. At 2 ppm Cd 2÷ stimulated 1.3-fold and then caused a slight (10--30%) inhibition at 98 ppm. Hg 2÷ caused complete inhibition at 50 ppm, and Pb 2÷ caused 50% inhibition at concentrations of 2--10 ppm. In the case of Pb :÷ the inhibition declined at higher concentrations of the metal ion. In no case has the mechanism by which Cd 2* stimulates enzyme activity been defined. As mentioned, the inactivation o f a negative aUosteric site could be a plausible explanation, but to examine this possibility it will be necessary to obtain a purified enzyme. A combination of kinetic, equilibrium, and radioisotopic techniques should prove definitive. 26

F. Displacement and substitution Alcohol dehydrogenase f r o m yeast and horse liver has been shown to contain Zn 2÷ as an essential c o m p o n e n t [70,71,71a]. When the Zn 2÷ in liver alcohol dehydrogenase was replaced with Co 2+ or Cd 2÷, the activities of the enzyme in the Zn 2÷, Co 2+, or Cd 2+ form were 14, 10, and 4 min-1 mg-1 respectively [ 72]. The spectrum of the enzyme containing cadmium showed an intense absorption peak at 245 nm. The molar absorptivity (e = 10,200) approximated that of Cd-mercaptide chromophores of metallothionein (e = 14,000), so the authors suggest t h a t the metal is bound to a sulfur. A cobalt~substituted form of liver alcohol dehydrogenase [73] and yeast alcohol dehydrogenase [ 74] were described. Alkaline phosphatase (E. coli), a dimer in the native state, contains 4 zinc atoms and 1 or 2 magnesium atoms [75]. These metals can be removed with c o n c o m i t a n t loss of enzyme activity; the activity can be completely restored on the addition of 2 equivalents o f Zn 2+ per dimer. When the metal-free apoenzyme is heated it undergoes a thermally induced transition around 55°C t h a t was measured calorimetrically. This transition is suppressed by 1 equivalent of Zn 2÷ and is eliminated by 2 equivalents. One equivalent of Cd 2+ also suppresses the transition, but 1 equivalent of Mg2÷ does not. In this case, Cd 2+ and Zn 2÷ cause the same effect, and the latter at least seems to bind cooperatively to its 2 sites. When the metal is present, the stability of the protein is increased markedly. Alkaline phosphatase (E. coli) was examined further by NMR to measure resonances from 1'3Cd and 3,p [76]. Inorganic phosphate is bound by this enzyme, and the 31p signal is observed at a low field (0--10 ppm) resonance. With the metal-free enzyme, the 3~p resonance was at 2.5 ppm; when 1 equivalent o f zinc was added it split into 2 peaks at 2 and 5.5. With 2 equivalents the resonance at 5.5 became predominant, but with 4 equivalents the 2 resonances became equal in size and were located at 2 and 4 ppm. This technique seems admirably suited to exploration o f the relationship of enzyme structure at the phosphate binding site and the influence of the metal ion on this structure. When Cd 2÷ replaced Zn 2÷ on the enzyme, the 3~p resonance at 2 ppm decreased and a second resonance at 8 ppm appeared; the amplitude of the former decreased and the latter increased as Cd 2÷ was increased from 1 to 2 equivalents so that the 2 were equal in size at 2 Cd2+/mole o f enzyme dimer. One metal-binding site on each monomer was postulated, and a H3Cd resonance at 180 ppm was observed when the ratio of Cd2+/dimer was 2. When inorganic phosphate was added to the Cd 2÷ enzyme, the resonance at 170 ppm disappeared and 2 peaks appeared at 145 and 55 ppm. From these and other data, the authors conclude t h a t in the absence of inorganic phosphate, Cd 2÷ is bound to the 2 monomers and the 2 Cd 2+ are in identical environments; thus the dimer is symmetrical at least with respect to the metal ion binding sites. The presence of inorganic phosphate causes an a s y m m e t r y and allows the 2 metal sites to be measured separately. Indeed there is a negative cooperativity such t h a t when 1 metal site is occupied the other is not. Recent studies on alkaline phosphatase have demonstrated the direct interaction 27

of inorganic phosphate with the metal, 113Cd [76a]. This study was possible when the pH was lowered to 5.5 where the enzyme-phosphate complex was stabilized. The resonance due to this complex was observed using the Zn-enzyme, b u t with the Cd-enzyme the NMR resonance was split into a doublet. 1~3Cd provides a useful probe for exploring the tertiary and quaternary structure of this protein. This study may be extended to other enzymes containing zinc. In 2 calcium-binding proteins the Ca 2÷ was replaced with Cd2÷; parvalbumin [77] and troponin C [78]. In troponin C Ca 2+ is b o u n d to a low affinity site and also to a high affinity site which also binds Mg ~÷. When Ca 2+ was replaced with 1~3Cd, the NMR signal from the Cd appeared in 2 peaks at 107.5 and 111.0 ppm. Each peak varied as Mg 2+ and Ca 2+ were added back to the solution, b u t it appeared that the 2 sites exhibited very similar affinities for Cd 2÷. The use o f H3Cd as a sentinel and reporter offers much promise for studies on protein conformation. Various explanations were offered for the effects observed, so it is apparent that caution is required in interpretation o f the results. As pointed o u t above, the ionic radii of Ca 2÷ and Cd 2+ are very similar (0.99 and 0.97 A). Since the chemistry of the 2 ions is different in a number of respects, it will be o f interest to learn h o w these 2 cations can function similarly at the binding site o f the protein. G. Inhibition Oxidative phosphorylation o f mammalian mitochondria is inhibited b y low concentrations o f sulfhydryl-inactivating reagents [79]. Small amounts of Cd 2+ cause uncoupling o f oxidative phosphorylation and stimulation o f the latent ATPase; these effects are also caused b y dinitrophenol [80--83]. Larger amounts of Cd 2+ inhibit oxygen consumption [80], especially with a-ketoglutarate and pyruvate as substrates, perhaps due to the interaction of Cd 2+ on the lipoic acid coenzyme used b y these eznymes. Direct demonstration of Cd 2÷ binding to the enzyme complex in the mitochondria has n o t been shown, however. Normal rat liver mitochondria respiring on succinate experience a doubling of oxygen consumption when 10-6--10 -s M Cd 2+ is added. If the mitochondria are obtained from rats chronically poisoned with Cd 2+ there is no stimulation of oxygen uptake. Since succinate dehydrogenase possesses sulfhydryl groups that are very sensitive to heavy metals and since it is buried deeply in the inner mitochondrial membrane, it seems that the Cd 2÷ may become stably associated with this enzyme as it passes through the membrane [84].Measurement of the succinate dehydrogenase activity itself should determine whether that from Cd 2+-treated rats has enhanced activity. Alternatively, the possible influence of increased levels of metallothionine should also be evaluated. Monoamine oxidase was examined for inhibition b y a variety of trace elements at 1 mM. Only Hg 2+, Cd 2+, and Cu 2+ were significantly effective as inhibitors. Reversal of inhibition by dithiols (dithiothreitol) was demonstrated; monothiols (cysteine, homocysteine, and reduced glutathione)

28

were themselves inhibitory. These studies arose from an interest in the mechanism of hypertension [ 85]. Certain divalent metal ions were tested for their ability to inhibit a number of drug-metabolizing enzymes [86]. Cd 2÷ and Hg 2÷ had different effects on c y t o c h r o m e c reductase: Hg 2÷ was much more inhibitory than Cd 2÷. With an epoxide h y d r a t i o n activity Cd 2* was much more inhibitory than Hg 2÷. In the cases of activities t h a t cause benzo[a]pyrene h y d r o x y l a t i o n and e t h o x y c o u m a r i n deethylation, both Cd 2÷ and Hg 2÷ were inhibitory at low concentrations (~ 10 -5 M). Salts of nickel, cobalt, chromium, zinc, and lead were n o t especially inhibitory. An interpretation o f the mechanism of metal-protein interaction is complicated by the fact t h a t both microsomal and postmicrosomal fractions were used as enzyme sources. Such studies illustrate h o w drug metabolism m a y be altered, but shed little light on the mechanism of effect of the cations on the enzymes themselves. In relation to drug metabolism it should be noted t h a t c y t o c h r o m e P-450 can be denatured by Cd 2÷ to produce c y t o c h r o m e P-420, and the extent of denaturation was f o u n d to vary with Cd 2÷ concentration [87]. Alkaline phosphatase is an enzyme which contains zinc. Its activity in situ in the kidney and prostate was inhibited by Cd 2÷ but n o t by Zn 2÷ when assayed histochemically [ 88].

H. Reversal o f inhibition by another (inhibitory) metal ions The e r y t h r o c y t e enzyme 5-aminolevulinate dehydratase is rich in sulfh y d r y l groups and is very sensitive to Pb 2÷. When hemolysates of whole h u m a n blood are assayed for this enzyme in the presence and absence of Pb 2÷, 50% inhibition is produced by 2 ~M Pb 2* [89]. At 100/~M Cd 2÷ or 250 ~M Zn 2÷ the inhibition by 2 ~M Pb 2* is completely reversed. When the response of the enzyme to Cd 2* or Zn 2÷ is measured in the absence of Pb 2*, stimulation up to 130% results at 3 ~M Cd 2÷, whereas in the case of Zn 2* the m a x i m u m stimulation of 160% occurs when the Zn 2+ concentration is 200 ~M. As the concentration of Zn 2÷ is increased, the stimulation is lost and inhibition is observed; 50% inhibition is caused by 40 ~M Cd 2+ and by ~ 2000 ~M Zn 2÷. Although Cd 2* is often considered a sulfh y d r y l reagent, as is Pb 2÷, in this case Cd 2+ reverses the inhibition by Pb 2+ before it too becomes inhibitory. Zn 2÷ also reverses the Pb 2÷ effects, but even higher concentrations of Cd 2÷ are needed. These very interesting relationships o f the 3 metals should provide considerable insight for understanding h o w specific metal ions can be distinguished by a protein. The studies were performed on crude hemolysates and are vulnerable to the criticisms t h a t the observed effect are n o t all representative of properties of 5-aminc~ levulinate dehydratase but m a y be, in part, due to complications resulting from the presence o f one or more other substances contributed by the blood. Indeed, in a short note describing 5-aminolevulinate dehydratase t h a t was purified 165-fold from h u m a n blood, the effects of Cd 2÷ were examined over the same concentration range as used above and no stimulation was observed [90]. However, a point of similarity between the 2 29

studies was that the inhibition caused by Hg 2÷ at 80 pM could be reversed completely by 80 pM Cd 2÷. Further study of these interesting relationships must include purification o f the enzyme. Adenosine 3',5'-phosphate (cyclic AMP) is involved in the mechanism of action of several hormones. Cyclic AMP formation results from adenylate cyclase reaction with ATP in the presence of Mg 2÷, and its hydrolysis is caused by cyclic nucleotide phosphodiesterase which also requires Mg 2÷. Eleven metal ions were examined as inhibitors of these enzymes that were obtained from rat cerebellum [91]. Cd 2÷ was the most inhibitory metal ion on the cyclase (LDs0 = 1 #M) and the second most effective on the diesterase. Pb 2÷ was nearly as effective as Cd2*; it also blocked the activation of the cyclase by norepinephrine (Cd 2÷ was n o t examined for this effect). Further studies on these enzymes should explore h o w one metal ion affects the action of another as was shown for a-aminolevulinate dehydratase [89], but again the enzymes should be isolated; the above studies were performed with crude homogenates or particulate preparations. I. Nucleic acids - - synthesis, transcription, and replication

Zinc is involved in regulating the formation and enzymatic activity of thymidine kinase [92--94]; the synthesis of DNA is defective during a dietary deficiency of Zn 2÷ [95--98]. It is of interest that both Zn 2+ deficiency and Cd 2÷ administration cause teratogenic effects in animals and the effects o f both are reversed b y Zn 2÷ [98--100]. Whether thymidine kinase is responsible for these effects is not known. Indeed, tightly b o u n d zinc is quite prevalent among enzymes involving nucleic acid synthesis and function: E. coil DNA-dependent R N A polymerase [101]; phage T7 R N A polymerase [102]; yeast RNA polymerases I [103], B [104], and III [105]; DNA polymerase from E. coli and sea urchin [106]; and RNAdependent DNA polymerase [107,108]. Methionyl-tRNA synthetase also has zinc as a c o m p o n e n t [109]. A number of metal ions that are carcinogenic and mutagenic have been tested to determine h o w they perturb the replication and transcription of DNA. DNA polymerase synthesizes deoxyribonucleotide polymers from deoxynucleoside triphosphates using DNA or RNA as a template, whereas R N A polymerase utilizes ribonucleoside triphosphates to form R N A from a DNA template. The incorporation of a nucleotide that does n o t form a Watson-Crick base pair with any deoxynucleotide in the template is a measure of erroneous incorporation or "infidelity". Among 31 metal salts examined b y Sirover and Loeb [110] there was a correlation between the mutagenic/carcinogenic properties of the metal ion and the ability to cause erroneous incorporation during DNA synthesis at a rate of 130% of the control. Cd 2÷, Ag +, and Cu 2÷ were the most p o t e n t of the cations tested. Certain problems in this sort of study are considered below in comparable studies of RNA polymerase and of the replication of DNA. RNA polymerase activity can be divided into 2 separate steps: initiation and elongation. These 2 steps were examined in another study [111 ] with 30

10 monovalent and divalent metal ions. For divalent ions elongation was inhibited in the order Pb 2+ > Cd 2+ > Co 2÷ > Zn 2+ > Cu 2+ > Mg 2+. On the other hand, at metal ion concentrations t h a t caused moderate inhibition of elongation ratio, initiation was stimulated by certain mutagenic and carcinogenic metal ions, e.g., up to 3-fold by Co 2+, whereas metals n o t in this category inhibited initiation. The relative order for the effect of metal ions on initiation was quite different from t h a t for their effect on elongation: Co 2+ ~ Cu 2+ ~ Mn 2+ ~ Cd 2+ ~ Pb 2÷, whereas Zn 2+ and Mg2+ were inhibitory. The opportunities for metal ions to cause effects in replication and transcription o f DNA can involve the enzyme, the substrate, the DNA template, and the RNA product. Moreover, artifacts can also occur in enzyme studies. These studies, as well as those of Sirover and Loeb on DNA polymerase, were conducted at pH 8 and in the presence of mercaptoethanol or dithiothreitol. At this pH and in the presence of sulfhydryl compounds, Niyogi observed t h a t m a n y of these metals form precipitates (unpublished). How these precipitates may affect either polymerase is of concern, and an uncertainty t h e n exists as to the cause of the inhibition or stimulation. Accordingly, Niyogi has undertaken a reexamination of the effects of various metal ions on the initiation and elongation reactions carried, o u t by E. coli RNA polymerase at pH 7.4 in the absence of mercaptoethanoL These results indicate t h a t the previous results of H o f f m a n and Niyogi are still obtained, but the effective metal ion concentration can be as much as an order o f magnitude lower. For example, Niyogi n o w finds t h a t Cd 2÷ strongly inhibits transcription, as tested with 3 different templates phage T4 DNA, calf t h y m u s DNA, and poly(dA-dT) ( m a n u script in preparation). The concentrations o f Cd 2÷ needed for 50% inhibition are 30, 45, and 250 pM, respectively, for the 3 templates; these values are much lower t h a n reported previously [111]. Furthermore, under these conditions the stimulatory effect of Cd 2÷ on initiation (at Cd 2÷ concentrations t h a t inhibit elongation) can still be o b t a i n e ~ Thus RNA polymerase is even more sensitive to certain carcinogenic metal ions than had been f o u n d previously. These studies were extended to examine the effect of metal ions on misincorporation of nucleotides into the RNA p r o d u c t (Niyogi, manuscript in preparation). There is a concentration-dependent increase in misincorporation (> 50-fold) of CMP ( i n place of UMP), as tested with poly(dA-dT) template. The misincorporation, particularly that at low concentrations of Cd 2÷, can be substantially overcome by higher concentrations of Mg 2÷, the obligatory metal ion needed for proper transcription. Mn :÷ also causes misincorporation, but only up to 5 times the background value; this effect could n o t be overcome by elevating the Mg2÷ concentration. These results suggest a possible mechanism for mutagenesis and carcinogenesis and indicate t h a t a careful evaluation of the concentration of metal ions in vivo should be made. The studies on RNA polymerase described above demonstrate that the studies on infidelity of DNA synthesis by Sirover and Loeb [110] could -

-

31

bear reexamination, since t h e y too were made under conditions t h a t caused metal ions to precipitate. A n o t h e r factor in determining the error rate caused by metal ions is the background, or error rate of the enzyme under standard assay conditions. The background error rate for RNA-dependent DNA polymerase of avian myeloblastosis virus used by Sirover and Loeb [110] was 1 error in 700 nucleotides incorporated, and a 30% increase in error rate was considered significant. S. Mitra and R. Bergen (Oak Ridge National Laboratory) have been pursuing similar studies using purified DNA-dependent DNA polymerase of rat liver, which has an error rate of ~ 1 0 -4 (manuscript in preparation). The error rate varies somewhat depending on the combination of template and "erroneous" nucleoside triphosphate employed. Using poly(dA-dT) as template-primer and measuring the misincorporation o f dCMP into the product, t h e y examined a number of metal ions at concentrations t h a t cause 50% inhibition o f normal synthesis. Hg 2÷ caused the most errors and the alkali metal ions the least. Cd 2÷ and Zn 2+ (at 0.1 mM) were intermediate and caused the error rate to increase 20-fold in both cases. These studies were conducted at pH 7.5 and in the absence of mercaptoethanol. In contrast to the remits of Sirover and Loeb, the carcinogenic and mutagenic metal ions were not distinguishable from those that are non~arcinogenic or non-mutagenic. These variabilities indicate a need to reconsider the possible correlation between the carcinogenic/mutagenic effect of a metal ion and the error induction on DNA polymerase. Especially important in such a reevaluation would be the background error production of the DNA polymerase; the rate o f 1/700 is unusually high and seriously interferes with the detection o f additional errors. J. C o m p e t i t i o n o f rig 2÷ and Pb 2÷, but n o t Cd 2÷, with K* and Na ÷ Cations t h a t are required for an enzyme's function can sometimes protect against inhibition by toxic metals. Adenosine triphosphate p h o s p h o h y d r ~ lase t h a t is dependent o n Na ÷ and K ÷ (Na ÷, K÷-ATPase) is associated with cellular ion transport and was examined for its inhibition by Cd 2÷, Hg 2÷, and Pb 2+ [112]. With microsomal preparations and homogenates as the enzyme sources, Hg 2+ was the most potent inhibitor (50% inhibition at 6 × 10 -6 M) as compared with Pb 2+ (8 × 10 -s M) and Cd 2÷ (1.4 × 10 -4 M). Inhibition by Hg 2+ was complicated by other components in the enzyme preparations. As Na ÷ was increased from 20 to 200 raM, the inhibition by Pb 2÷ and Hg 2+ decreased appreciably but t h a t by Cd 2÷ remained unaffected. As K ÷ was increased from 4 to 20 mM the inhibition by Pb 2+ increased while the Cd 2÷ and I-Ig2+ effects were unchanged. Comparison o f Na ÷, K+-ATPase from several mammals demonstrated similarities in their susceptibilities to divalent metal ions. The divalent ions Pb 2+ and Hg 2+ may prove to be useful probes for exploring the Na + and K ÷ sites and Cd 2÷ for exploring some other critical site of this enzyme.

32

VII. CONCLUSIONS

In this review we have focused atrention on Cd 2+ and its interactions with nucleic acids and proteins. Data for other metal ions, especially Zn 2+, Hg 2+, and Ca 2+, have also been presented for comparison. The discussions have included a survey of basic physical and chemical characterizations of metal ions with a view toward recognizing and understanding any general aspects that might emerge about the behavior of cadmium in biological systems. In this context, the most useful of the physicochemical concepts listed in Table II are those developed in the theory of hard and soft acids and bases. Use of a numerical softness parameter, e.g. Op (Section III), combined with the principle that hard/soft ions seek hard/soft ligands helps explain a n u m b e r o f diverse phenomen& When the soft ion Cd 2+ reacts with nucleic acids, the interaction with the purines and pyrimidines is greater than with the phosphates, as is characteristic o f a soft ion. Compared with Cd 2÷ the softer ion Hg 2+ has a higher affinity for nucleotides and for DNA, whereas the harder ion Zn 2+ has a somewhat lower affinity. The relative effects of a n u m b e r of cations on the stability o f DNA can be explained by considering the softness o f the metal ions. Among the amino acids f o u n d in proteins, Cd 2+ has the highest affinity for cysteine and histidine. Zn 2+ has a similar selectivity, b u t its affinity for cysteine greatly exceeds t h a t of Cd2+; this fact is n o t consistent with predictions based on softness alone, indicating t h a t other factors, e.g. stereochemistry, come into play. The affinity of Hg 2+ for cysteine is also higher than t h a t o f Cd 2+, and both o f these metal ions have proven useful as sulfh y d r y l reagents. The binding of Cd 2÷ to proteins is n o t parallel to the binding of Hg 2+, however, since some enzymes are inhibited by one or the other o f these cations but n o t both. Sulfhydryl groups are clearly sites for Cd 2+ binding in some cases, but other amino acid components are also active binding sites. When the amino acids are in polypeptide structure the assocation with Cd 2÷ is diminished, primarily because the free carboxyl groups are n o t present. A t e n d e n c y toward enhanced binding o f Cd 2+ is seen in peptides t h a t contain several cysteine residues. When Cd 2÷ is added t o an enzyme, stimulation or inhibition of the catalytic activity m a y occur. In some cases Zn 2+ or Ca 2÷ can be replaced by Cd 2+. Aside from the effect on catalytic properties, the presence of 113Cd in a protein can serve as a sensitive probe since 113Cd given an NMR signal; as the structure of the protein in the neighborhood of the H3Cd is altered, the NMR signal can change. The affinity of Cd 2+ for nucleic acids and their components is less than t h a t for proteins and amino acids. Since proteins are also more a b u n d a n t t h a n nucleic acids, the bulk of bound Cd 2+ in a living system should be

33

found in the former, b u t not to the exclusion of Cd 2÷ occurring in nucleic acids. Whereas many effects of Cd 2÷ on enzymes have been described, the mechanism b y which Cd 2÷ disrupts normal functions of both enzymes and nucleic acids has y e t to be elucidated. ACKNOWLEDGEMENTS

We express appreciation to our colleagues, Drs. J.D. Hoeschele, S. Mitra, S.K. Niyogi, R. Triolo, M.W. Williams, and H. Witschi for critical comments during the preparation of this report. We thank Drs. C.C. Travis and B.L. Whitfield and Ms. E.D. Copenhaver for assistance in obtaining information from the literature. This study was sponsored b y the Office of Health and Environmental Research, U.S. Department of Energy, under contract W7405-eng-26 with the Union Carbide Corporation. REFERENCES 1 T. Kjellstr~m, B. Lind, L. Linnman and C-G. Elinder, Arch. Environ. Health, 30 (1975) 321. 2 C-G. Elinder and T. Kjellstr~m, Ambio, 6 (1977) 270. 3 W. Fulkerson and H.E. Goeller (Eds.), Cadmium the Dissipated Element, Oak Ridge National Laboratory Report ORNL NSF-EP-21, May 1972. 4 A.S. Hammans, J.E. Huff, H.M. Braunstein, J.S. Drury, C.R. Shriner, E.B. Lewis, B.L. Whitfield, and L.E. Towill, Reviews of the environmental effects of pollutants: IV Cadmium, National Technical Info. Service, ORNL/EIS-106, EPA-600/1-78-026, June 1978. 5 L. Friberg, M. Piscator, G. Nordberg and T. Kjellstr~m, Cd in the environment, Vol. I: CRC Press (1971); Cleveland, APTD-0681, PB-199-795; VoL II: Environmental Protection Agency, Washington, D.C., EPA-R2-73-190, February 1973; Vol. III: Environmental Protection Agency, Washington, D.C., EPA-650/2-75-049, June 1975. 6 Cadmium 77, 1st Intern. Cadmium Conf., San Francisco, 1977, published by Metal Bulletin, London, 1978. 7 C.F. Baes, in W. Fulkerson and H.E. Goeller (Eds.), Cadmium the Dissipated Element, Oak Ridge National Laboratory Report ORNL NSF-EP-21, May 1972, p. 35. 8 A.O. Summers and S. Silver, Annu. Rev. Microbiol., 32 (1978) 637. 9 F.A. Cotton and G. Wilkinson, Advanced Inorganic Chemistry, 3rd edn., WileyInterscience, New York, 1972. 10 Handbook of Chemistry and Physics, 55th edn., CRC Press, West Palm Beach, Florida, 1976, p. F-213. 11 W.C. Fernelius, in R.E. Burk and O. Grummitt (Eds.), Structure of coordination compounds, Chap. III in Chemical Architecture, Interscienee, New York, 1948. 12 M.N. Hughes, The Inorganic Chemistry of Biological Processes, Wiley-Interscience, New York, 1972. 13 R. Gabler, Electrical Interactions in Molecular Biophysics, Academic Press, New York, 1978. 14 S. Ahrland, J. Chart and N.R. Davies, Q. Rev. (Lond.), 12 (1958) 265. 15 R.G. Pearson, J. Am. Chem. Soc., 85 (1963) 3533. 16 S. Ahrland, Structure and Bonding, Vol. 1, Springer-Verlag, 1966, p. 207. 17 S. Ahrland, Structure and Bonding, Vol. 5, Springer-Verlag, 1968, p. 118. 18 C.K. J ~ e n s e n , Structure and Bonding, Vol. 1, Springer-Verlag, 1966, p. 234. 19 C.K. J~gensen, Structure and Bonding, VoL 3, Springer-Verlag, 1967, p. 106. 20 S. Ahrland, Structure and Bonding, VoL 15, Springer-Verlag, 1973, p. 167.

34

21 R.G. Pearson and R.J. Mawby, in V. G u t m a n n (Ed.), Halogen Chemistry, Vol. 3, Academic Press, N e w York, 1967, p. 55. 22 R.J.P. Williams and J.D. Hale, Structure and Bonding, Vol. 1, Springer-Verlag, 1966, p. 249. 23 R.J.P. Williams, Proc. Chem. Soc., (1960) 20. 24 R.J.P. Williams, Ind. Chem. Belg., 4 (1963) 389. 24a. E. Nieboer and D.H.S. Richardson, Environ. Pollut. (Set. B), 1 (1980) 3. 25 G. Zubay and P. Doty, Biochim. Biophys. Acta, 29 (1958) 47. 26 G.L. Eichhorn, in G.L. Eichhorn (Ed.), Inorganic Biochemistry, Chap. 34, Elsevier, Amsterdam, 1973. 27 G.L. Eichhorn and Y.A. Shin, J. A m . Chem. Soc., 90 (1968) 7323. 28 R.M. Izatt,J.J. Christensen and J.H. Rytting, Chem. Rev., 71 (1971) 439. 29 U. Weser, Structure and Bonding, Voh 5, Springer-Verlag, 1968, p. 41. 30 I. Sisso~ff,J. Grisvard and E. Guill~, Prog. Biophys. Mol. Biol., ~! (1976) 165. 31 S.L. Davydova, in H. Segal (Ed.), Metal Ions in Biological Systems, Vol. 8, Marcel Decker, N e w York, 1979, p. 183. 32 R.W. Gellert and R. Bau, in H. Segel (Ed.), Metal Ions in Biological Systems, Vol. 8, Marcel Decker, N e w York, 1979, p. 1. 33 K. Gonda, S. Koga, M. Sakaguchi, Y. Miyata, H. Ogura and T. Okamoto, Yakugaku Zasshi, 98 (1978) 708. 34 R.B. Martin and Y.H. Mariam, in H. Segal (Ed.), Metal Ions in Biological Systems, Vol. 8, Marcel Decker, N e w York, 1979, pp. 57--124. 35 D.M.L. Goodgame, I. Jeeves, C.D. Reynolds and A.C. Skapski, Biochem. J., 151 (1975) 467. 36 D.M.L. Goodgame, I. Jeeves, C.D. Reynolds and A.C. Skapski, Nucleic Acids Res., 2 (1975) 1375. 37 P. deMeester, D.M.L. Goodgame, T.J. Jones and A.C. Skapski, Biochem. J., 139 (1974) 791. 37a B.A. Cartwright, D.M.L. Goodgame, I. Jeeves and A.C. Skapski, Biochim. Biophys. Acta 477 (1977) 195. 38 C.H. Wei and K.B. Jacobson, Abst. in Am. Crystallographic Assn. Meeting, March 1980, Lakeport Convention Center, Alabama. 39 V.S. Hiatt and K.B. Jacobson, Toxicoh Appl. Pharmacol., submitted. 40 M.A. Wosnick and B.N. White, Biochim. Biophya Acta, 561 (1979) 194. 41 A. Jack, J.E. Ladner, D. Rhodes, R.S. Brown and A. Klug, J. Mol. Biol., 111 (1977) 315. 42 S. Kominami, V.T. Wee and P. Riesz, Radiat. Res., 62 (1975) 422. 43 R.S. Mitra and I.A. Bernstein, J. Bacteriol., 133 (1978) 75. 44 R. Thomson, T.A. Kilroe-Smith and I. Webster, Environ. Res., 15 (1978) 309. 45 L.G. Silldn and A.E. Martell, Stability Constants of Metal Ion Complexes, Special Publication No. 25, The Chemical Society, 1971. 46 M. Webb, in S.S. Brown (Ed.), Clinical Chemistry and Chemical Toxicology of Metals, Elsevier North Holland Press, Amsterdam, 1977, p. 51. 47 R.B. Martin in H. Segal (Ed.), Metal Ions in Biological Systems, Voh 9, Marcel Decker, New York, 1979, p. 1. 48 H. Waldmann-Meyer, J. Biol. Chem., 235 (1960) 3337. 49 C. Tanford, J. Am. Chem. Soc., 7 4 ( 1 9 5 2 ) 211. 50 M.S.N. Rao and H. Lal, J. Am. Chem. Soc., 80 (1958) 3222. 51 W.L. Hughes, J. Am. Chem. Soc., 69 (1947) 1836. 52 J.L. Sudmeier and J.J. Pesek, Anal. Biochem., 41 (1971) 39. 53 Y. Hashimoto, Hyogo-ken Eisei Kenkyusho Kenkyu Hokoku, 12 (1977) 22. 54 Y. Hashimoto and I.i~ Watanabe, Hyogo-ken Eisei Kenkyusho Kenkyu Hokaku, 11 (1976) 27. 55 M.M. Goil, Z. Parasitenkd, 55 (1978) 59. 56 T.-Y. Liu, in H. Neurath and R.L. Hill (Eds.), The Proteins, Vol. III, Academic Press, New York, 1977, p. 239. 35

57 M. Webb, Biochim. Biophya Acta, 65 (1962) 47. 58 D.R. Sanadi, J.W. Littlefield and R.M. Bock, J. Biol. Chem., 197 (1952) 851. 59 D.R. Sanadi, M. Langley and F. White, J. BioL Chem., 234 (1959) 183. 60 M. Webh, Biochim. Biophys. Acta, 89 (1964) 431. 61 M.D. Enger, L.B. Rall and C.E. Hildebrand, Nucleic Acids Res., 7 (1979) 271. 62 J.H.R. K~igiand B.L. Vallee, J.Biol. Chem., 236 (1961) 2435. 63 A. Yoshida, B.E. Kaplan and M. Kimura, Proc. Natl. Acad. Sci. USA, 76 (1979) 486. 63a J.D. Otvas and I.M. Armitage, J. Am. Chem. Soc. 101 (1979) 7734. 64 D.D. Ulme~ in J. Maniloff, J.R. Coleman and M.W. Miller (Eds.), Effects of Metals on Cells, Suhcellular Elements and Macromolecules, C.C. Thomas, Springfield, Illinois, 1970, p. 11. 65 B.L. Vallee and D.D. Ulmer, Annu. Rev. Biochem., 41 (1972) 91. 66 O. Wada, S. Nakazawa, T. Ono, N. Yamaguchi and K. Toyokawa, Ind. Health, 11, (1973) 32. 67 E.M. Diamond, A. Jedeikin and J.E. Kench, Biochem. Biophys. Res. Commun., 52 (1973) 679. 68 N. Despaux, C. Bohuon, E. Comoy and C. Boudene, Biomedicine, 27 (1977) 358. 69 P. Mazliak, A.M. Decotte and J.C. Kader, Chem.-BioL Interact., 16 (1977) 115. 70 H. Theorell, A.P. Hygaard and R.K. Bonnichsen, Acta Chem. Scand., 9 (1955) 1148. 71 J.H.R. Kiigi and B.L. Vallee, J. BioL Chem., 235 (1960) 3188. 71a H. Eklund, B. NordstrSm, E. Zepperzauer, G. SSderlund, I. Ohlsson, T. Boiwe and C. Br'~inden. FEBS Lett., 44 (1976) 200. 72 D.E. Drum and B.L. Vallee, Biochem. Biophys. Res. Commun., 41 (1970) 33. 73 A.J. Sytkowski and B.L. Vallee, Biochemistry, 17 (1978) 2850. 74 A.J. Sytkowski, Arch. Biochem. Biophys., 184 (1977) 505. 75 J.F. Chlebowski and S. Mabrey, J. Biol. Chem., 252 (1977) 7042. 76 J.F. Chlebowski, I.M. Armitage and J.F. Coleman, J. Biol. Chem., 252 (1977) 7053. 76a J.D. Otvas, J.R. Alger, J.E. Coleman and I.M. Armitage, J. Biol. Chem.,254 (1979) 1778. 77 T. Drakenberg, B. Lindman, A. Cav~ and J. Parello, FEBS Lett., 92 (1978) 346. 78 S. Fors~n, E. Thulin and H. Lilja, FEBS Lett., 104 (1979) 123. 79 N. Haugaard, N.H. Lee, R. Kostrzewa, R.S. Horn and E.S. Haugaard, Biochim. Biophys. Acta, 172 (1969) 198. 80 E.E. Jacobs, M. Jacob, D.R. Sanadi and L.B. Bradley, J. BioL Chem., 223 (1956) 147. 81 A.L. Fluharty and D.R. Sanadi, Proc. Natl. Acad. Sci. USA, 46 (1960) 608. 82 A.L. Fluharty and D.R. Sanadi, Biochemistry, 1 (1962) 272. 83 A.L. Fluharty and D.R. Sanadi, Biochemistry, 2 (1963) 519. 84 E.M. Diamond and J.E. Kench, Environ. Physiol. Biocher~, 4 (1974) 280. 85 N. Revis and C. Horton, Toxicol. AppL Pharmacol., 43 (1978) 439. 86 A. Aitio, M. Ahotupa and M.G. Parkii, Biochem. Biophys. Rea Commun., 83 (1978) 850. 87 M. Sagai, F. Shiraishi and K. Kubota, Nippon Eiseigaku Zasshi, 32 (1977) 463. 88 B. Riba~Ozonas, M. Dean-Guelbenzu and A. Santos-Ruiz, in C.F. Mills (Ed.), Trace Element Metabolism in Animals, E. and S. Livingstone, Edinburgh, 1970, p. 173. 89 J.R. Davis and M.J. Avram, Toxicoi- Appl. Pharmacol., 44 (1978) 181. 90 O. Wade, Y. Yano, T. Ono and K. Toyokawa, Ind. Health, 10 (1972) 59. 91 J.A. Nathanson and F.E. Bloom, Mol. Pharmacol., 12 (1976) 390. 92 I. Lieberman, R. Abrams, N. Hunt and P. Ove, J. Biol. Chem., 238 (1963) 3955. 93 J.E. Dreosti and L.S. Hurley, Proc. Soc. Exp. Biol. Med. 150 (1975) 161. 94 A.S. Prasad, D. Oberleas and D. Koniuch, J. Lab. Clin. Med., 83 (1974) 634. 95 M. Fujioka and I. Lieberman, J. Biol. Chem., 239 (1964) 1164. 96 R.B. Williams, C.F. Mills, J. Quarterman and A.C Dalgarno, Biochem. J. 95 (1965) 29P. 97 H.H. Sandstead and R.A. Rinaldi, J. Cell. Physiol., 73 (1969) 81.

36

98 99 100 101 102 103 104 105 106 107 108 109 110 111 112

H. Swenerton, R. Shrader and L.S. Hurley, Science, 166 (1969) 1014. V.H. Ferrn and S.J. Carpenter, Nature, 216 (1967) 1123. R. Semba, K. Ohta and H. Yanamura, Teratology, 10 (1974) 96. M.C. Sc~utton, C.W. W u and D.A. Goldthwait, Proc. Natl. Acad. Sci. USA, 68 (1971) 2497. J.E. Coleman, Biochem. Biophys. Res. CommurL, 60 (1974) 641. D.S. Auld, I. Atsuya, C. Campino and P. Vanenzuela, Biochem. Biophya Rea CommurL, 69 (1976) 548. H. Lattke and U. Weser, FEBS Lett., 65 (1976) 288. T.M. Wandzilak and R.W. Benson, Biochemistry, 17 (1978) 426. J.P. Slater, A.S. Mildvan and L.A. Loeb, Biochem. Biophya Res. Commun., 44 (1971) 37. B.J. Poiesz, G. Seal and L.A. Loeb, Proc. Natl. Acad. Sci. USA, 71 (1974) 4892. D.S. Auld, H. Kawaguchi, D.M. Livingston and B.L. Vallee, Proc. Natl. Acad. Sci. USA, 71 (1974) 2091. L.H. Posorske, M. Cohn, N. Yanagisawa and D.S. Auld, Biochim. Biophys. Acta, 576 (1978) 128. M.A. Sirover and L.A. Loeb, Science, 194 (1976) 1434. D.J. Hoffman and S.K. Niyogi, Science, 198 (1977) 513. B.R. Nechay and J.P. Saunder~ J. Environ. Pathol. ToxicoL, 2 (1978) 283.

37