Electrostatic Theories of Salting-In and Salting-Out in Biochemistry, Manuais, Projetos, Pesquisas de Engenharia de Materiais

Baixe Electrostatic Theories of Salting-In and Salting-Out in Biochemistry e outras Manuais, Projetos, Pesquisas em PDF para Engenharia de Materiais, somente na Docsity! Critical Appraisal of Salting-Out and Its Implications for Chemical and Biological Sciences Phulwinder K. Grover* and Rosemary L. Ryall Division of Urology, Department of Surgery, Flinders University School of Medicine, Flinders Medical Centre, Bedford Park, South Australia 5042, Australia Received August 19, 2004 (Revised Manuscript Received October 25, 2004) Contents 1. Introduction 1 2. Definition 2 3. Mechanisms of the Salting Effect 3 3.1. Hydration Theories 3 3.2. Water Dipole Theories 4 3.3. Electrostatic Theories 5 3.4. Internal Pressure Theories 6 3.5. Theories Based on van der Waals Forces 6 4. General Significance of Salting-Out 7 5. Particular Significance: Kidney Stones 8 6. Summary 8 7. References 8 1. Introduction In simple terms, salting-out describes the precipi- tation of a less soluble material from a solution in which it is mixed with other substances. For at least 2 centuries, preparative chemists have known and described the process and have employed it for the isolation and purification of chemicals.1-3 However, the application of the concept in the biological and health sciences is a relatively modern phenomenon (see section “General Significance of Salting-Out”). We have recently demonstrated that salting-out is responsible for the observation that dissolved urate promotes calcium oxalate (CaOx) crystallization in undiluted human urine in vitro,4 a finding that has enormous implications for pathogenesis of urinary stones. A higher than normal excretion of urate in the urine (hyperuricosuria) has long been proposed as a predisposing factor in the development of CaOx urolithiasis5-7 but has lacked a firm scientific foun- dation for two principal reasons. First, the two theories most commonly cited to explain the associa- tion between urate excretion and urolithiasis, namely, epitaxy8,9 and depletion of glycosaminoglycan inhibi- tors of CaOx crystallization,10 are steeped in contro- versy and are not physiologically pertinent.11-16 Second, there has been no unequivocal demonstration that hyperuricosuria is a common, reproducible find- ing in CaOx stone formers.17-19 Nonetheless, these must be balanced against consistent reports that administration of allopurinol, a drug that decreases the synthesis and hence the urinary excretion of urate, reduces recurrence of calcium stones.20-27 Given such empirical but persuasive evidence, it would be unreasonable to deny the existence of some connection between the level of urate and the pre- cipitation of CaOx crystals in urine. Thus, we have demonstrated, as was originally suggested by Kal- listratos et al.,28 that the connection rests on the principle of salting-out.4 Further, because we have also shown that the ability of urate to provoke CaOx crystal formation depends on the prevailing urinary concentrations of calcium and oxalate,4 the credibility of our proposal does not depend on the need to explain the success of allopurinol by invoking a requirement for hyperuricosuria, which, as stated above, is not a well-documented feature of CaOx stone disease.7,20-21,29-44 More importantly, it accom- modates the possibility that urate could promote CaOx stone formation in patients with hyperurico- suria, as well as in those with normal levels of urate excretion who have relatively high urinary concen- trations of calcium and oxalate. This suggests that urate may, in fact, be a bigger culprit of CaOx stone pathogenesis than has been previously thought and reported by Yu and Gutman5,45 and others.46-52 To * To whom correspondence should be addressed: Urology Unit, Department of Surgery, Flinders Medical Centre, Bedford Park, South Australia 5042, Australia. Telephone: 61-8-8204-4870. Fax: 61-8-8204-5966. E-mail: pk.grover@flinders.edu.au. Volume 105, Number 1 10.1021/cr030454p CCC: $53.50 © 2005 American Chemical Society Published on Web 12/07/2004 the best of our knowledge, stone formation caused by promotion of CaOx crystallization by dissolved urate is the only known pathological example of salting-out in humans. Although our interest in salting-out stemmed from our interest in pathogenesis of urinary calculi, the phenomenon has much wider applications ranging from fundamental to applied science (see section “General Significance of Salting-Out”). A full com- prehension of salting-out necessitates a sound knowl- edge of the hydration and thermodynamic properties of ions, which, in turn, requires at least a basic understanding of physical chemistry, electrochemis- try, and mathematics. Because application of the principle of salting-out in industry and in the chemi- cal and biological sciences is rising dramatically, interest in understanding the phenomenon, particu- larly the basic mechanisms involved, is at its highest. Although there have been excellent reviews on the subject,53-57 they were all written either before or in the early 1960s. Furthermore, they were aimed at chemical engineers and/or physical chemists and thus are beyond the easy comprehension of most investi- gators in the biological and health sciences. There- fore, our aims were to provide a critical and updated review of the phenomenon of salting-out for chemists and biologists, particularly those working in the health sciences, and to highlight the ramifications of its use in chemistry, biochemistry, and molecular biology. 2. Definition The change in solubility of a nonelectrolyte in an aqueous solution, which results from the addition of an electrolyte, is known as the salting effect. Thus, there can either be an increase or a decrease in solubility of a nonelectrolyte with increasing concen- trations of added electrolyte. They are known as salting-out and salting-in, respectively.56,57 For the purpose of the definition, electrolytes and nonelec- trolytes are salts that have high and low solubilities, respectively. Mathematically, the influence of an electrolyte on the aqueous solubility of a nonelectro- lyte can be expressed by the physical equation for gases, commonly known as the Setschenow equa- tion,56 given below where s0 and s are the solubilities of the nonelectro- lyte in water and electrolyte in solution, respectively, cs is the concentration of the electrolyte in moles per liter, fc is the activity coefficient of the nonelectrolyte (expressed in concentration units), and k is the salting constant. A positive value for this constant indicates salting-out, and a negative value indicates Phulwinder K. Grover was born in India. He completed his B.S. (medical) and M.S. (biochemistry) from Panjab University in Chandigarh and Punjab Agricultural University in Ludhiana, respectively. Then, he moved to Adelaide and received his Ph.D. in Biochemistry from the Flinders University of South Australia under the supervision of Professors Rosemary L. Ryall and Villis R. Marshall. After a postdoctoral position with Professor Martin I. Resnick at the Department of Urology, at Case Western Reserve University in Cleveland, OH, he joined Professor Ryall’s research group at the School of Medicine, Flinders University of South Australia. Currently, he is a Senior Hospital Scientist in the Urology Unit of the Department of Surgery at the Flinders Medical Centre and the Flinders University of South Australia. He has published extensively on various aspects of urolithiasis and has given many presentations at national and international meetings. He was invited as a visiting lecturer to the University of the Witwatersrand in Johannesburg, South Africa, in 1994. He has been awarded prizes for scientific presentations, locally (at the 27th Annual Scientific Meeting of the Australian Society for Medical Research held in Canberra in December 1988), nationally (at the 42nd Annual Scientific Meeting of the Urological Society of Australasia held in Melbourne in March 1989), and internationally: for his contributions to urolithiasis research, he was awarded the Young Investigator Award at the eighth International Symposium on Urolithiasis and Related Clinical Research held in Dallas, TX, in 1996. His major research interests are exploring various aspects of urolithiasis especially the involvement of hyperuricosuria and proteins in the development of kidney stones. Rosemary Lyons Ryall was born in the United States when her father was working at the Australian Embassy in Washington, DC. She accompanied her parents back to Australia at the age of 2. Rose began her scientific career at the Australia National University in Canberra where she first completed an honors degree and then a Ph.D. in biochemistry. She moved to Adelaide in 1976 and was appointed to the Urology Unit at the Flinders Medical Centre, where she began her longstanding interest in kidney stone formation. Her research is presently concerned principally with exploring how proteins are involved in the development of kidney stones and the crystals from which they are built. As a result, her interests have widened to include other biominerals, especially those in higher plants such as spinach and oak trees, which use proteins to make the very same crystals but in a more salubrious and ordered fashion. She is fascinated with biomimetics. She has published numerous reviews, scientific papers, and book chapters and has given many presentations throughout the world. In 1998, her extensive contributions to kidney stone research gained her a Doctorate of Science from the Australian National University. Rose is currently Professor of Urological Research and Chief Medical Scientist in the Urology Unit of the Department of Surgery at the Flinders Medical Centre and the Flinders University of South Australia and chair of the Steering Committee of the emerging International Urolithiasis Society. log s0/s ) log fc ) kcs (1) 2 Chemical Reviews, 2005, Vol. 105, No. 1 Grover and Ryall 3.3. Electrostatic Theories To explain the observed variations in salting effects of different nonpolar solutes, Debye and McAulay81 and Debye82 suggested an electrostatic explanation of the salting effect. Their theory was further devel- oped by various workers to give qualitative and quantitative dimensions.83-86 The tenet of the theo- ries is that the amount of work necessary to dis- charge the ions in pure solvent is different from that required in a solution containing a solute. They therefore related both salting-out and salting-in to the influence of the solute on the dielectric constant of the solvent. On that basis, if the saturated solution of solute has a dielectric constant less than water, then salting-out occurs, and if the saturated solution of solute has a dielectric constant more than water, then salting-in occurs. This model is shown in Figure 3. The major advantages of the electrostatic theories are that they provide an explanation for and the correct order of magnitude of the salting effects of ordinary neutral electrolytes such as sodium and potassium chlorides.56 They also predict reasonably Figure 2. Diagrammatic representation of the water dipole theories. These theories suggest that favorable orientation of water molecules around polar solutes causes salting-in and vice versa. Figure 3. Diagrammatic representation of the electro- static theories. The tenet of these theories is that if the saturated solution of a solute has a higher dielectric constant compared with that of water, it causes salting-in and vice versa. Salting-Out Chemical Reviews, 2005, Vol. 105, No. 1 5 well the dependence of the salting constant, k, on the molecular size of the nonelectrolyte;57 that is, the degree of salting-in of nonpolar solutes increases with ionic size, although there are some notable exceptions to this generalization.56 These theories can also be used to estimate the volumes of hydrated ions from their hydration numbers.57 However, they cannot reasonably account for marked discrepancies in the ranking sequences of similar electrolytes, and they are completely unable to account for observed shifts from salting-out to salting-in with particular non- electrolytes.56 For example, while the theories predict salting-out by salts of smaller ions (such as sodium and potassium chlorides), they provide no explana- tion of salting-in caused by salts of large ions (such as tetramethylammonium, naphthalenesulfonate, and long-chain fatty acids). These drawbacks are not unexpected because they take into account only the primary electrical effect and not “displacement” and “structural” effects.56 3.4. Internal Pressure Theories In 1899, Euler87 made an empirical observation that the aqueous dissolution of ethyl acetate caused shrinkage in the volume of water. He also noted that the increasing order of these volume contractions upon the dissolution of different salts was related, in the same sequence, to an increase in salting-out. Later, Geffcken88 and Tammann89,90 showed a similar correlation between salting effects and the relative effects of salts in decreasing the compressibility of the solution, which prompted Tammann90 to suggest a theory commonly known as the “internal pressure” concept of salting-out. Later, McDevit and Long91 proposed an explicit model of the theory to study qualitative and quantitative aspects of the salting effect. According to that model, neutral solute mol- ecules in solution merely occupy volume. Their pres- ence exerts internal pressure on solvent molecules, which, in turn, modifies the ion-solvent interactions and causes precipitation of the nonelectrolyte. Thus, according to these theories, the degree of salting-out or salting-in of a nonpolar solute is determined by the extent to which the solvent medium is com- pressed or expanded when the ions are present. Generally, as the compressibilities of the solutions increase, the salting-out effect diminishes and vice versa.56 These theories, which are schematically represented in Figure 4, are supported by the fact that predicted and observed salting effects for non- polar nonelectrolytes correlate quite well with the corresponding volume changes that occur when the salts are dissolved in water. However, they cannot reasonably account for marked variations in the order of ranking of the salting effects of similar electrolytes and vice versa. For instance, according to these theories, the predicted order of salting-out for various salts is essentially the same for such disparate species as hydrogen, nitrous oxide, and benzene, which, in fact, is not true.56 Their major drawback, therefore, is that they hold well strictly for nonpolar nonelectrolytes91 but give no explanation for the effects of polar nonelectrolytes. 3.5. Theories Based on van der Waals Forces The tenet of these theories is that short-range electrostatic interactions, other than those mentioned in section 3.1, occur between ions and neutral mol- ecules. Such intermolecular interactions, which are collectively known as van der Waals forces, can be of two types: attractive and dispersive. Of these, the latter have been suggested to play an appreciable role in salting effects of ions.56 From time to time, various workers have proposed explicit models for the quan- tification of these dispersive forces.92 A diagrammatic representation of the theories of van der Waals forces is presented in Figure 5. The concept of van der Waals forces is supported by the fact that predicted salting-in effects of large Figure 4. Diagrammatic representation of the internal pressure theories. According to them, if the presence of a nonpolar solute decreases the internal pressure on solvent molecules, it causes salting-in and vice versa. 6 Chemical Reviews, 2005, Vol. 105, No. 1 Grover and Ryall ions (also known as hydrotropism), such as quater- nary ammonium ions and long-chain fatty acids, are observed experimentally. To further explain salting- in, Desnoyer et al.93 demonstrated that the dissolu- tion of salts of large ions increases the structure of water, which decreases the entropy of the system, increases the solubility, and hence causes salting-in. The theories of van der Waals forces explain only salting-in, which is not the primary focus of this paper but is discussed in detail elsewhere.56,69 Despite the success of the theories based on van der Waals forces in explaining some observed solubil- ity data, their quantitative application in the calcula- tion of dispersion potential of complex molecules is questionable.94 The theories also predict much less specificity in the effects of different ions than is actually observed,56 and they fail to account for the anomalously low salting-out effects generally caused by lithium and hydrogen ions, as well as some nonelectrolytes.56 On the basis of these shortcomings, along with the theoretical limitations of the concept, it has been argued56 that van der Waals forces may play only a secondary role, if any, in determining the relative effects of a series of ions. Hence, the concept must remain speculative until more exhaustive, convincing evidence becomes available. 4. General Significance of Salting-Out The phenomenon of salting-out is of both funda- mental and applied interest. Its study can provide a wealth of information of theoretical importance to understand the complex nature of interactions be- tween ions and solvent molecules, which, in particu- lar, allows an appreciation of the unique nature of water as a solvent.56 Data obtained from experimen- tal investigations can also have direct implications for studying kinetic salt effects,56 elucidating mech- anisms of reactions,56,95 determining protein micro- heterogeneity,96,97 and estimating the relative surface hydrophobicity of proteins98 and bacterial cells.99 From a practical perspective, it has already proved useful to differentiate virulent strains of Yersinia pseudotuberculosis100 and inhibit proteolytic en- zymes.101 Salting-out is especially useful for quantify- ing proteins102 and active metabolites of drugs and toxins in blood and other body fluids,103-107 because it improves recovery. Consequently, it is not surpris- ing that the concept is used in broad-spectrum drug screening.108 Data obtained from salting-out experiments are also invaluable for the colorimetric assays of free fatty acids109 and for high-grade purification of chemicals, pharmaceuticals, proteins,110-115 and DNA from fresh116-119 and stored samples,120,121 which is particularly important in forensic science, because most specimens are generally available in very limited amounts. Macromolecules required for re- search and/or molecular (health and forensic) diag- nostic purposes122-131 are commonly isolated using the salting-out principle, which is also invaluable for the routine synthesis of membrane vesicles of differ- ent compositions.132 Such vesicles, which are repre- sentative of different pathological conditions, can then be used as models for studying transport systems across biomembranes. Because salting-out increases recovery, it has quite a lot of industrial applications as well. For instance, it is used for large-scale purification of chemicals (synthetic and semisynthetic) and pharma- ceuticals.133-137 Also, it is used for large-scale puri- fication of petroleum-based products and en- zymes.138,139 The latter have applications in industry (e.g., amylase for starch processing used in the sugar industry, cellulase and pectinase for wood processing used in the pulp and paper industry, and zymase for fermentation used in brewing beer, wine making, and the baking industry), diagnostics, and biochemical research.140-141 It is remarkable that salting-out is an entirely physical phenomenon and does not affect properties of molecules or macromolecules (polyelectrolyte, neu- tral, or hydrophobic in nature) including RNA, DNA, and proteins. This is evidenced, as mentioned above, by the fact that pharmaceuticals, nucleic acids, and proteins purified by salting-out are used routinely in research, molecular health diagnostics, and, more importantly, in molecular forensic diagnostics. Figure 5. Diagrammatic representation of the theories of van der Waals forces. These theories suggest that increased van der Waals forces between ions and neutral molecules cause salting-in. Salting-Out Chemical Reviews, 2005, Vol. 105, No. 1 7