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Complexing behaviour of bishydroxycoumarin with macromolecules Cho, Moo Jung 1970

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THE COMPLEXING BEHAVIOUR OF BISHYDROXYCOUMARIN WITH MACROMOLECULES  by  MOO JUNG CHO B.S.P., SEOUL NATIONAL UNIVERSITY SEOUL, KOREA, 1966  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN PHARMACY i n the D i v i s i o n of Pharmaceutical Chemistry of the Faculty of Pharmaceutical Sciences  We accept t h i s thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA MAY, 1970  In p r e s e n t i n g t h i s  thesis  in p a r t i a l  f u l f i l m e n t o f the requirements f o r  an advanced degree at the U n i v e r s i t y of B r i t i s h the L i b r a r y s h a l l I f u r t h e r agree for scholarly  make i t f r e e l y  tha  permission  this  written  thesis  for financial  Apr*/.  >X,  I  gain shall  thesis  not be allowed without my  sC/€>iCg5>  <  Columbia  7 <?  f o r r e f e r e n c e and study.  i s understood that copying o r p u b l i c a t i o n  'phaM^hCcuii'Ca.I  The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada  that  by the Head o f my Department o r  permission.  Department o f  Date  It  I agree  f o r e x t e n s i v e copying o f t h i s  purposes may be granted  by h i s r e p r e s e n t a t i v e s . of  available  Columbia,  This thesis, in the opinion of the examiners, exceeds the usual standards considered necessary for research at the M. Sc. l e v e l .  It i s , in many respects, equal  to investigations carried out at the Ph. D. l e v e l and r e f l e c t s the student's a b i l i t y to carry out research at these more advanced levels.  - i = ABSTRACT  The strong binding of bishydroxycoumarin  to serum albumin  was f i r s t reported about 20 years ago. However, the mechanism of binding has not been studied. In t h i s investigation,  attempts  have been made to reveal the mechanism. The work was extended to some other synthetic macromolecules including polyvinylpyrrolidone. The l i t e r a t u r e survey covers the physicochemical properties and the complexing behaviours i n aqueous solution of the individual substances examined. The theory of multiple e q u i l i b r i a , which i s fundamental to an understanding of the binding process, has been summarized. Spectrophotometric, s o l u b i l i t y , dynamic and equilibrium dialyses, and viscometric methods were used and their t h e o r e t i c a l back-ground has been reviewed and discussed. Some physicochemical properties of BHC, necessary for the interpretation of binding data, were estimated. Maximum binding c a p a c i t i e s of macromolecules and association constant of each binding s i t e were obtained from the binding data. The nature of the s i t e and intermolecular forces were characterized from thermodynamic analysis. This abstract represents the true contents of the thesis submitted.  Supervisor  - i iTABLE OF CONTENTS  Page I. INTRODUCTION  1  I I . THEORY  3  I I I . METHODOLOGY  22  1. Spectrophotometry  23  2. Phase S o l u b i l i t y  26  3. Equilibrium D i a l y s i s  28  4. Viscometry  33  IV. THE CHEMICAL AND BIOLOGICAL CHARACTERISTICS OF THE SUBSTANCES USED IN THIS INVESTIGATION  38  1. Bishydroxycoumarin  38  (BHC)  2. Human Serum Albumin  (HSA)  3. Polyvinylpyrrolidone (PVP) 4. Dextran, Starch, and Hydroxyethyl Starch (HES)  41 44 .... 51  V. EXPERIMENTAL  53  1. Apparatus  53  2. Chemicals and Reagents  53  3. Determination of Apparent pKa Values f o r BHC  55  4. S o l u b i l i t y Measurements on BHC 5. The S o l u b i l i t y of BHC as a Function of Macromolecule Concentration  57 58  6. Spectrophotometrie Analysis  60  7. Equilibrium D i a l y s i s  61  8. Viscometric Analysis  66  - iii Page VI. RESULTS AND  DISCUSSION  1. Intramolecular  68  Hydrogen Bonding i n BHC  68  2. Apparent pKa Values of BHC  71  3. S o l u b i l i t y  85  (a) E f f e c t s of pH, Ionic Strength and Components (b) E f f e c t of Macromolecule i ) Starch Sol and HES i i ) HSA and PVP  Buffer  4. Spectrophotometry (a) Absorptivity Values of BHC (b) Absorbance Contribution of HSA and PVP (c) Depression of BHC Absorbance i n the Presence of HSA and PVP (d) Spectrophotometry Analysis of Complex Formation  85 91 91 94 96 96 96 98 98  5. Dynamic D i a l y s i s  106  6. Equilibrium D i a l y s i s  112  (a) (b) (c) (d)  Binding of BHC to Cellophane Membrane Permeability of Membrane to PVP Donnan E f f e c t Free Drug Concentration and Volume Ratio  112 112 114 116  7. Interpretation of Binding Data  118  (a) Langmuir-Type Plot (b) Scatchard Plot (c) Double Reciprocal Plot  118 123 123  8. Thermodynamic Analysis and Mechanism of Interaction  126  (a) HSA-BHC Interaction 126 i ) Enthalpy, Entropy, and Free Energy Changes .. 126 i i ) P o s s i b i l i t y of Ionic Interaction 128 i i i ) P o s s i b i l i t y of Pre-Existing Binding Site 129 iv) Nature of Binding Site and E f f e c t of Binding on Water Structure 131 (b) PVP-BHC Interaction 132 i ) Enthalpy, Entropy, and Free Energy Changes .. 132 i i ) Analysis of Enthalpy of Binding 135 i i i ) P o s s i b i l i t y of Hydrophobic Bonding 138 iv) Nature of the Intermolecular Forces and of the Binding Site 139  - ivPage 9. Viscometry 10. Comparison of Methods Used to Evaluate Binding VII. SUMMARY AND CONCLUSION  142 149 155  VIII. REFERENCES  157  BIOGRAPHICAL INFORMATION  174  - vLIST OF TABLES  Table 1.  Page The Complexing of Drugs  Behaviour of PVP with Various Types 47  2.  Absorptivity Values for BHC i n a pH - 7.4 Buffer  3.  Estimate of Molar Absorptivity Ratio of Bound BHC to Free BHC  104  4.  Analysis of Spectrophotometric Data for PVP-BHC Interaction i n 0.02$ PVP (5 umole/L.)  104  5.  Calculated Data for the Dynamic D i a l y s i s of BHC i n the Presence of PVP  109  6.  C a l c u l a t i o n Procedures for (Df) and r Value brium D i a l y s i s for HSA-BHC Interaction)  (Equili-  117  7.  C a l c u l a t i o n Procedures for (Df) and r Value brium D i a l y s i s for PVP-BHC Interaction)  (Equili-  8.  E f f e c t of Changes i n Temperature on the Binding of BHC to HSA  9.  10. 11.  12.  .... 96  117 128  Thermodynamic Data for the Binding of D-Phenyl(p-azobenzoylamino)-acetate by P u r i f i e d Antibody Specific for the Compound, i n 0.02M Phosphate Buffer of pH 7.4 Containing 0.15M NaCl (from Karush, 1956) .. 130 Thermodynamic Functions for the Binding of One Mole of BHC by One Mole of Vacant Binding Site on PVP  135  C a l c u l a t i o n Procedure for Estimating Specific and Reduced V i s c o s i t i e s of PVP Solution at 10°C i n the Presence and Absence of BHC  142  Influence of BHC Binding on the Rheological Properties of PVP at Various Temperatures  144  - vi LIST OF FIGURES  Figure  Page  1. Schematic Diagram of a Macromolecule with n Sites for the Attachment of a Simple Molecule  7  2. Schematic Diagram of a Macromolecule with Two Sets of Binding Sites  14  3. Hypothetical Binding Curve for a Macromolecule with Two Groups of Binding Sites  15  4. Schematic Diagram of Changes i n Water Structure Accompanied by an Interaction Between M and D  21  5. Hypothetical Curves for Changes i n Absorbance (of Small Molecule) as a Function of M Concentration  24  6. Calculated Values of R as a Function of Salt Concentrat i o n i n the M-Free Compartment (from B u l l , 1964a)  30  7. Chemical Structure of BHC  38  8. Chemical Structure of Monomer of PVP, N-Vinylpyrrolidone  44  9. Diagram of a Plexglas Block and an Assembled D i a l y s i s Cell  62  10. A Set-Up for Dynamic D i a l y s i s Method  63  11. A Water Bath with a Tumbler Used i n S o l u b i l i t y and Equilibrium D i a l y s i s Studies  65  12. Infrared Spectrum of BHC i n KBr  69  13. Chemical Structure of BHC showing Two Intramolecular Eight-Membered Chelations  70  14. Three Dimensional Structure of BHC  70  15. Resonance Structure of BHC after the F i r s t Ionization ..  72  16. T i t r a t i o n Curves for BHC i n 40$ v/v DMF i n Water  74  17. U l t r a v i o l e t Spectra Showing the Second D i s s o c i a t i o n of BHC 18. Absorbance-pH Curves for BHC  76 78  - viiFigure  Page  19. U l t r a v i o l e t Spectra Showing the Second Dissociation of BHC  i n 20$ v/v DMF  80  20. Absorbance-pH Curves for BHC at 276.5 and 315 mu  82  21. Apparent pKa Values of BHC as a Function of Per Cent DMF  83  22. Tautomerism of a BHC  Molecule  84  23. Effect of pH on the Apparent S o l u b i l i t y of BHC at 30°C ..  86  24. Effect of Ionic Strength (Chloride Ion) on the Apparent S o l u b i l i t y of BHC at 30°C 25. Buffer Value of T r i s Buffer Solutions 26. Two Dimensional Structure of T r i s Showing Intramolecular Hydrogen Bonds  87 90 91  27. Effect of Various Concentrations of HES and Potato Starch Sol on the Apparent S o l u b i l i t y of BHC at 30°C i n T r i s Buffer  92  28. Chemical  Structure and Molecular Configuration of HES  ..  93  29. Effect of HSA and PVP Concentrations on the Apparent S o l u b i l i t y of BHC i n T r i s Buffer at 20°C  95  30. Absrobance Measurements at 304 mu for HSA and Solutions i n T r i s Buffer  97  31. Absorption Spectra for BHC Absence of 0.1$ HSA  i n the Presence  32. Absroption Spectra for BHC Absence of 0.4$ PVP  i n the Presence  PVP  and 99 and 100  33. Predominating Chromophore ( oi,/9 -Unsaturated Lactone) i n the BHC Molecule  101  34. Absorbance Depression of BHC as a Function of HSA Concentration  102  35. Absorbance Depression of BHC as a Function of PVP Concentration  103  36. Absorbance Differences for BHC Solutions i n the Absence and Presence of Constant Amount of Macromolecules as a Function of Total BHC Concentration  105  37. Loss of Free BHC from Inside a D i a l y s i s Bag i n the Absence and Presence of 0.4$ HES and Dextran i n T r i s Buffer at 30°C  107  - viii Figure  Page  38. Loss of Free BHC from Inside a D i a l y s i s Bag i n the Absence and Presence of 0.4 and 0.2$ PVP i n T r i s Buffer at 30°C  108  39. Estimate of the Extent of Adsorption of BHC to Cellophane Membrane at Three Different Temperatures  .... 113  40. Colorimetric Determination of PVP 41. Plot of r Values versus the Concentration of Free BHC for HSA-BHC Binding at Two Temperatures 42. Plot of r Values versus the Concentration of Free BHC for PVP-BHC Binding at Four Temperatures 43. Binding Curves at 20°C for HSA-BHC and PVP-BHC Interactions i n T r i s Buffer  115 119 120 122  44. Scatchard Plot for HSA-BHC Interaction at 20 and 40°C ... 124 45. Scatchard Plot for PVP-BHC Interaction at 10, 20, 30, and 40°C  125  46. Double Reciprocal Plot for PVP-BHC Interaction  127  47. Van't Hoff Plot for the PVP-BHC Interaction  133  48. Schematic I l l u s t r a t i o n of the Binding Processes Between BHC and PVP Molecules 49. Molecular Model of PVP Chain Segment with Eight  136  Monomer Units  140  50. Proposed Configuration of PVP-BHC Complex  141  51. Densities of PVP Solutions 52. Reduced V i s c o s i t y of PVP as a Function of PVP Concent r a t i o n at Various Temperatures  143 145  53. E f f e c t of Temperature on the I n t r i n s i c V i s c o s i t i e s of PVP i n the Presence and Absence of BHC  148  54. Comparison of Binding Data Obtained from Spectrophotometric Analysis with Those from the Equilibrium D i a l y s i s Method for the HSA-BHC Interaction at 20°C  150  55. Comparison of Binding Data Obtained from Spectrophotom e t r y Analysis with Those from the Equilibrium D i a l y s i s Method for the PVP-BHC Interaction at 20°C  152  - ix ACKNOWLEDGEMENTS  The author would l i k e to thank his supervisor, Dr. M. Pernarowski, for h i s guidance and encouragement during the course of t h i s investigation. He would also l i k e to express his gratitude to Dr. A.G. Mitchell for h i s advice and professional understanding of the problems associated with t h i s study. The author i s grateful to the many professors and friends i n the Faculty of Pharmaceutical Sciences, University of B r i t i s h Columbia,  for t h e i r assistances at the various  stages of t h i s investigation. In p a r t i c u l a r , he would l i k e to thank Dr. B. Roufogalis for h i s help i n interpreting thermodynamic data, Dr. C.T. Rhodes for h i s guidance i n general physical chemistry, Dr. F.S. Abbott and Mr. J . Coates for t h e i r counselling i n organic chemistry, and Dr. J.O. Runikis for assigning one of h i s summer students (Miss K.G. Tom) to help with the viscometric experiments. The author would also l i k e to express h i s gratitude to Mr. A.J. Leathern for h i s cooperation and help with many of the technical aspects involved i n t h i s investigation. This study was financed, at least i n part, by funds made available to the author by the Faculty of Pharmaceutical Sciences. The author would, therefore, l i k e to thank the Dean of the Faculty, Dr. B.E. Reidel, not only for these funds but also for confidence i n the author's c a p a b i l i t i e s to embark on a M. Sc. program at t h i s u n i v e r s i t y .  I. INTRODUCTION  Many investigators have studied the interactions between drugs and a wide variety of organic and inorganic molecules which are associated with a therapeutically active substance i n either an jLn v i t r o or In vivo system. It i s known that drugs form 'complexes' with plasma proteins, enzymes, other drugs and many of the adjuvants which are added to dosage forms. The physicochemical properties of these complexes d i f f e r s i g n i f i c a n t l y , i n many instances, from those observed for the interacting drug. Although these properties can be determined, they are not based, i n general, on the complex i t s e l f but on studies i n which the complex, the drug, and the interacting molecule are associated with each other i n some In v i t r o system. A complex i s a co-ordination compound which arises from a Lewis acid-base reaction. This c l a s s i c a l d e f i n i t i o n includes those complexes which are formed by reacting the drug with a metallic ion or an organic molecule. For the purposes of t h i s thesis, the l a t t e r d e f i n i t i o n i s broader than necessary. The word 'complex', as used herein, i s defined as that substance formed by a reversible chemical reaction i n which equilibrium rates are much higher than any of the rates associated with the measuring process. Chemical (covalent) bonds are not formed and the long range forces which hold the interacting molecules together are much weaker than those found i n most chemical compounds.  Bishydroxycoumarin (BHC) i s strongly bound to plasma proteins. I t has been suggested that the interaction leads to e r r a t i c therapeutic r e s u l t s and that binding of drugs to macromolecules may a f f e c t the i n vivo a c t i v i t y of the substance. The object of t h i s study i s , therefore, to investigate the mechanism of i n t e r a c t i o n between t h i s drug and human serum albumin (HSA), starch s o l , polyvinylpyrrolidone (PVP), dextran, and hydroxyethyl starch (HES). The l a t t e r three substances have been used as plasma expanders. Complex formation may be studied by u t i l i z i n g a wide variety of methods. However, only the equilibrium and dynamic dialyses, s o l u b i l i t y , spectrophotometric,  and viscometric methods  w i l l be used i n t h i s i n v e s t i g a t i o n . Quantitative information on the interactions may be obtained from equations which are based on the law of mass a c t i o n . The nature of the intermolecular forces between the molecules i s derived from thermodynamic data obtained during the i n v e s t i g a t i o n . Although studies of t h i s type have been c a r r i e d out by many investigators, the s i g n i f i c a n c e of their observations has not always been evident. The bulk of the papers on the subject appeared i n the l i t e r a t u r e during the 1950's but, i n recent years, many investigators have again begun to study complex formation. This i s due, i n part, to the p o s s i b i l i t y that both the s t a b i l i t y of the drug and i t s in vivo a c t i v i t y may be affected by other drugs or adjuvants i n the dosage form. The r e s u l t s i n t h i s t h e s i s are not d i r e c t l y related to the l a t t e r problem but do o f f e r a d d i t i o n a l proof that therapeutically s i g n i f i c a n t drugs bind e a s i l y to macromolecules.  I I . THEORY  A general discussion of the p r i n c i p l e s and concepts fundamental to the binding capacity of proteins with various substances may be found i n the papers by  Scatchard (1949;  and others, 1954), Klotz (1946a; 1949a; 1953a), E d s a l l and Wyman (1958c), Foster (1960), Tanford (1965), and Weber (1965). The mathematical theory associated with such studies i s discussed i n d e t a i l by Kruger-Thiemer, et a l . ( 1 9 6 4 ) , Hart (1965), Sandberg, et a l .  (1966), and Rosenthal (1967).  Scatchard (1949; and others, 1954)  stated that four  questions should be answered at the conclusion of any study on the interaction between a protein and a small molecule. These are: "How many molecules are bound to the protein?" "How t i g h t l y are the molecules bound to the protein?" "Where does the binding occur?" "Why does the binding occur?" The answers to these questions deal, therefore, with the number of binding s i t e , the equilibrium constant at the s i t e , and the type of interaction between the molecule and the functional group or groups on the protein. If the functional groups on the large molecule act independently, the law of mass action may be used to explain the i n t e r a c t i o n with a small molecule and the binding strength can be expressed as a constant. I f the protein (M) combines with a molecule (D) to form a single complex (MD), then  M  +  D =  MD  (Eq. 1)  - 4 -  The association constant (K) i s defined by the  following  equation. (D ) b  K =  (M )  (Eq.  (D )  f  2)  f  The quantities i n parentheses represent the concentrations of the respective species and the subscripts  (b and f) indicate  bound or complexed and free or unbound species,  respectively.  However, (M ) t  (M^),  = (D ) + b  (M )  (Eq.  f  i n Eq. 3, indicates the t o t a l concentration  of macro-  molecule. Rearrange Eq. 2 and substitute (M^) - (D ) b  (D ) = K(D ) b  f  £(M )  -  t  (D ) b  3)  for  (Eq.  4)  Divide both sides of Eq. 4 by K(D )(D ) and rearrange. f  (D )  1  b  (M+)  b  1 +  1  (Eq.  5)  K(D ) f  The molar r a t i o of bound drug to t o t a l macromolecule i s equal to r . This r a t i o indicates the extent of  binding.  (D ) b  r r=  2—  (M ) +  Substitute the above into Eq. 5 and rearrange.  (Eq.  6)  (M ). f  - 5 -  K (D ) f  r =  (Eq. 7)  1 + k (D ) f  Interactions between macro and simple molecules are usually more complex than that indicated above. If there are n binding s i t e s , i f each s i t e i s not influenced by i t s neighbor, and i f each has the same i n t r i n s i c a f f i n i t y for D, then the successive interactions and their corresponding constants may be represented i n the following way.  M •+ D  (MD)  = MD  f  k  l ^  (M)(D ) f  MD + D  MD  2  -f-  =  f  D  f  (MD ) 2  MD  2  =  (MD)(Dj) (MD ) 3  MD  3  2  MD _ i  1  + D  f  (Eq. 8)  (MD )(D )  = MD^  f  (MD ) ±  k, -  (MD|_ j ) ( D ) f  MD n-1 + D_p = MD  (MD ) n  n  (MD _!)(D ) n  f  - 6 Furthermore,  the step a s s o c i a t i o n c o n s t a n t s are not  independent of each o t h e r . However, the r e l a t i o n s h i p s between c o n s t a n t s can be s t a t e d mathematically by a p p l y i n g the r u l e s of combination and permutations. A schematic diagram of a macromolecule i s shown i n F i g u r e 1. The e q u i l i b r i u m constant f o r the a s s o c i a t i o n between D and s i t e 1 on M i s the same as that  f o r the r e a c t i o n between D and any other p o s i t i o n on  the molecule. T h e r e f o r e :  M + D  M + D  K =  =  f  =  f  gMD  MD  ( MD) n  2  (M)(D ) f  (M)(D )  (M)(D )  (Eq.  10)  f  f  Therefore:  (Eq. 9)  ±  ( MD)  (jMD)  y  k  1  = nK  (Eq. 11)  since (MD)  = (,MD) i  + ( MD)+ d  + ( MD) n  0  (Eq. 12)  Similarly, k =( 2  n-1  J  K  (Eq. 13)  0 Figure 1. Schematic diagram of a macromolecule with n s i t e s for the attachment of a simple molecule (D).  r n- i+1  and  i  (Eq. 14)  K  This, then, i s the general r e l a t i o n s h i p between the step association constant  ( k ) for the formation of the MD i  i  and the i n t r i n s i c association constant  complex  (K). The c o e f f i c i e n t of  K i n Eq. 14 i s frequently c a l l e d the ' s t a t i s t i c a l factor' since t h i s may also be derived on the basis of s t a t i s t i c a l considerations. The value of r may now re-defined. (MD) + 2(MD )-r3(MD )^ 2  r =  3  ... *i(MDi)+  (M)+- (MD)+ (MD )+ ... +(MD )+ 2  i  ... +n(MD ) n  ... 4 (MD ) n  (Eq. 15)  The  (MD) terms i n Eq. 15 may  now be appropriately expressed  by K, (M) and (D^) terms i n Eq. 8. (D )  The f and f  f  f  r =  (Eq. 16)  f  represent the denominator of Eq. 15 and i t s f i r s t  derivative with respect to (D^). Substitute the appropriate terms of the l e f t side of Eq. 14 into the corresponding step association constant terms involved i n Eq. 16 and rearrange on the basis of the binomial theorem.*  Thus r can be expressed  without using step association constant terms.  r=  n K 1 + K  (D ) f  (Eq. 17)  (D ) f  Therefore, i f there are n independent binding s i t e s , the extent of binding i s n times that for a single s i t e and the i n t r i n s i c association constant i s the same as that i n Eq. 7. Rearrange Eq. 17.  1/r = 1/n + l/nK(D )  (Eq. 18)  f  Therefore, a plot of 1/r versus l / ( D ) i s a straight l i n e with f  a slope value of 1/nK  and an intercept value of 1/n. Both the  binding constant and the number of binding s i t e s can, therefore, be determined. Enzymologists r e f e r to t h i s method of presenting *  Eq. 16 and 17 are not derived herein. The equations leading to these may be found i n the papers published by Klotz (1946a; 1953a) or by E d s a l l and Wyman (1958c).  - 9data graphically as the Lineweaver - Burk plot (Lineweaver and Burk, 1943). The main problem with t h i s double r e c i p r o c a l plot i s that a few low solute concentrations w i l l outweigh many high solute concentrations. Small extrapolating errors at high (D ) f  w i l l r e s u l t i n large errors i n the n value (Goldstein, 1949; Dowd and Riggs, 1965). Klotz, Walker, and Pivan (1946b) used t h i s method of p l o t t i n g i n t h e i r investigations into the binding of azosulfonic acids with bovine serum albumin. Scatchard (1949), on the other hand, plotted h i s data i n a d i f f e r e n t manner. The 'Scatchard equation', which may be derived from Eq. 17, i s  r/(D ) = Kn - Kr f  A plot of r/(Dj) versus r i s , therefore, a straight  (Eq. 19)  line.  The intercept value on the abscissa y i e l d s n; the intercept value on the ordinate i s equal to Kn. This equation lays less stress on the values of r at very low (Df) values than does the double r e c i p r o c a l plot (Eq. 18). In addition, i t gives a more even r e l a t i v e weight to the d i f f e r e n t points on the curve. Eq. 17 i s similar to the equation that was derived by Langmuir (1918) to describe c e r t a i n adsorption isotherms. The equation i s m=  bkC 1 +kC  (Eq. 20)  where m i s the number of grams of solute adsorbed by one gram of adsorbent; C i s the t o t a l concentration of solute  -10in s o l u t i o n . The constants i n t h i s equation  (b and k) a r i s e s  from the mathematical derivation of the isotherm. Although Eq. 17 and 20 are similar, i t i s not correct to assume that binding and adsorption are i d e n t i c a l processes. The equations are similar because both have been derived from the law of mass action. The subject i s discussed i n d e t a i l by Goldstein (1949) and Klotz (1953a). The Freundlich isotherm i s one of the f i r s t  equations  proposed to explain adsorption phenomena.  m s k C  1 / n  (Eq. 21)  This isotherm can not be used i f the concentration of adsorbate with respect to adsorbent i s too high. The isotherm i t s e l f i s empirical. Patel and Foss (1965) described the binding of benzoic acids by polysorbate 80 and cetomacrogol 1000 i n terms of a Freundlich-type adsorption r e l a t i o n s h i p . Many binding systems cannot be described mathematically by using the simple mass action equation based on the assumption that there i s but one i n t r i n s i c association constant. Attempts have been made to correct for e l e c t r o s t a t i c interactions between the binding s i t e s on M. I f charged ions are bound to M, the f i r s t ion  tends to reduce the a f f i n i t y of M for the second oncoming ion  because of e l e c t r o s t a t i c repulsion between species of l i k e charge. Eq. 17 i s , therefore, no longer v a l i d even i f the i n t r i n s i c a f f i n i t y of each s i t e on M i s the same f o r the small i o n .  - 11 If e l e c t r o s t a t i c forces are s i g n i f i c a n t , there i s a r e l a t i o n s h i p between two successive binding constants and  . The correction procedure described by Klotz, Walker,  and Pivan (1946b) i s similar to that used by Kirkwood and Westheimer (1938) i n t h e i r study of the f i r s t and second ionization constants of a dibasic a c i d . The equation for the free energy changes for the reaction  MD _ + 2 MD. i  2  = 2 M  D  ^  _  (  E  q  .  22)*  consists of two terms. It takes into consideration the free energy change of the interaction i n the absence of e l e c t r o s t a t i c e f f e c t and the e l e c t r o s t a t i c free energy change ( AG ^) e  which  can be estimated from the Born and Debye-Hiickel Theory. K  LG  n-(i-2)i  i-1  = RT In  = RT In k  i  N  n- ( i - l ) i - l  - &G i e  (Eq. 23)  In order to calculate any constant k^, k-^ must f i r s t be obtained from a suitable extrapolation of experimental data. The kg value may be calculated from Eq. 23. By using a similar procedure, k o  may be calculated from the k  g  value. Other constants are obtained  i n a similar manner. If successive binding constants are known, r values can be estimated by using Eq. 16. * Eq. 22 i s the summation of two successive reactions to produce the complexes MD^_j and MD^.  - 12 Scatchard (1949) corrected f o r the e l e c t r o s t a t i c e f f e c t by using the following equation.  e  2 w r  =  Kn - Kr  (Eq. 24)  The w term may be calculated from theory or an approximate value may be determined  empirically. The Debye-Huckel equation  for a charge spread uniformly over the surface of a sphere of radius b which excludes small ions to a radius a i s given below. Ik w=  2Dk T  1/b  B  (Eq. 25)  1 + Ika  D i s the d i e l e c t r i c constant of the medium, kg i s the Boltzmann constant, T i s the absolute temperature,  £ i s the electronic  charge, z i s the valence of the small molecule, and Ik i s defined by the Debye-Huckel Theory.  Tanford, Swanson, and  Shore (1955a) reported that, i n the hydrogen ion t i t r a t i o n of bovine serum albumin, empirical values of w are independent of pH between pH values of 4.3 and 10.5 but change d r a s t i c a l l y when pH values are outside the l a t t e r l i m i t s . Karush and Sonenberg (1949) found that the binding of bovine serum albumin with three a l k y l sulfates could not be described mathematically by using the mass action equation even a correction i s made for e l e c t r o s t a t i c i n t e r a c t i o n . They assumed that the free energies of binding at the various s i t e s obeyed a Gaussian d i s t r i b u t i o n and from t h i s deduced a t h e o r e t i c a l expression which adequately described their data.  - 13 In a second study on the interaction between bovine serum albumin and an anionic azo dye, Karush (1950) found that the data f a i l e d to f i t the Gaussian d i s t r i b u t i o n hypothesis. Experimental r e s u l t s could, on the other hand, be explained by assuming the existence of two d i f f e r e n t groups of binding s i t e s .  Inter-  actions between macro and simple molecules are probably more complex than that indicated i n Eq. 17. Most macromolecules probably contain several sets or groups of s i t e s with d i f f e r e n t a f f i n i t i e s for the simple molecule. If the macromolecule contains m d i f f e r e n t sets of s i t e s , the f i r s t set with n^ equivalent and independent binding s i t e s , each with i n t r i n s i c association constant K^,. the second set with n^ such s i t e s , each with an i n t r i n s i c association constant Kg, and so forth, then the mean number of s i t e s occupied by D i s r  =  ™ > , i . 1  n^Df) (Eq. 26) 1  + K (D ) ±  f  The KJL i n t h i s equation i s an i n t r i n s i c association constant and i s different from the step equilibrium constant (designated as k^) i n Eq. 8. The K values have the following order; K^^ Kg>  •••^ '  The t o t a l number of binding s i t e s are defined by Eq. 27. m n  =  >  \ n i = 1  ±  (Eq. 27)  In order to further i l l u s t r a t e the e f f e c t of more than one set of binding s i t e s , a schematic diagram of a macromolecule with two sets of s i t e s i s shown i n Figure 2.  K  m  - 14 -  Figure 2. Schematic diagram of a macromolecule with two sets of binding s i t e s . The molecule contains four s i t e s with an association constant equal to and eight s i t e s with an association constant equal to Kg.  Eq. 26 may  now be re-written on the basis of two  of binding s i t e s i l l u s t r a t e d i n Figure 4^ 1 + K  (D )  8K  f  1  (D ) f  sets  2. (D )  2  1 + K  f  0  (D.)  (Eq.  28)  The m term, for the example c i t e d , i s equal to two. There are twelve s i t e s on the macromolecule.  - 15 -  r Figure 3. Hypothetical binding curve for a macromolecule with two groups of binding s i t e s . n = 4 , . K s 4000; n = 8 , K = 1000. 1  1  2  2  Sandberg, et a l . (1966) and Rosenthal  (1967) showed how  an experimental curve obtained from a Scatchard equation  can  be resolved into two or more straight l i n e s , each of which represents a different  set of binding s i t e s . This graphical  approach to the treatment of binding data i s i l l u s t r a t e d i n Figure 3. The K-^ and K  2  values for t h i s hypothetical system  are 4000 and 1000 respectively. If l i n e 1 represents the f i r s t binding system and l i n e 2 the second system, then curve 1 i l l u s t r a t e s experimental  data. Any point P on curve 1 i s the  sum of the binding coordinates of system 1 at point Pj and of system 2 at point P . 2  Points Pj, P , 2  and P are so chosen  - 16 that they l i e on a straight l i n e which passes through the o r i g i n . Consequently, the geometric relationship  OP =  0P  1  + 0P  (Eq. 29)  2  i s obtained. Experimental data i s resolved by drawing straight l i n e s under the curve so as to s a t i s f y the r e l a t i o n s h i p shown i n Eq. 29. For systems with more than two sets of binding s i t e s , the mathematical procedures for c a l c u l a t i n g K have been worked out by Hart (1965). Scatchard et a l . (1950), i n their study on the i n t e r a c t i o n between the thiocyanate i o n and human serum albumin (HSA), obtained a binding curve which when resolved indicated the presence of two sets of binding s i t e s with n values of 10 and 30 and K values of 1000 and 250. Karush (1950), i n h i s study on the interaction between bovine serum albumin (BSA) and an anionic azo dye, used the mathematical approach described by Scatchard et a l . (1950) but d i d not correct f o r e l e c t r o s t a t i c e f f e c t s between binding s i t e s . Binding studies may also be c a r r i e d out i n the presence of two or more substances, both of which can attach themselves to the same s i t e on M. I f the two reacting species C and D have corresponding association constants KQ and K , then the binding D  of D i n the presence of C i s explained mathematically by the following equation. n K r  °  =  (D )  D  f  1 + K (D ) + K ^ C j ) D  f  (Eq. 30)  If the n s i t e s are equivalent and independent, then  r  n K ' (D„) D  = .  D  K ' D  (Eq. 3 1 )  i  + Kp' (D )  1  f  i s defined by Eq. 3 2 .  K '  (Eq. 3 2 )  =  +  1  (C )  K  The above equations imply that, i n the presence of a constant concentration of C, the binding of D to M follows the same pattern as that indicated i n Eq. 1 7 , except that the K ^ ' value w i l l be lower than that observed for  and i s a function of (C).  If KJJ, or K i n Eq. 1 7 , i s known, and K  D  ' i s determined for a  known value of (C), then K^. can be calculated from an equation which i s derived from Eq. 3 2 .  Kp =  1  (C ) \ f  Klotz et a l .  (1948)  K  /  D  V  -  1  (Eq.  33)  *  studied the e f f e c t s of s a l i c y l a t e ,  dodecylsulfate, and other anions on the binding of methyl orange by serum albumin and found that t h e i r r e s u l t s could be explained by using Eq.  33.  Cogin and Davis  (1951)  studied, by use of Eq.  the competition i n the binding of long chain fatty acids and methyl orange to BSA.  33,  - 18 It has also been observed that, i n many instances, the binding of anions by albumin does not decrease over the pH range of 6 to 9 to the extent that would be expected from the increased negative charge on the protein. Karush (1951) observed an increase i n binding a b i l i t y of albumin for methyl orange when the pH was increased from 6.4 to 7.6. The net charge changes, under these conditions, from -8 to -16. These discrepancies have been a t t r i buted to a f a i l u r e of the Debye-Huckel Theory when applied to complex protein molecules or to the possible configurational changes which may occur i n the albumin molecule over the 6 to 9 pH range. Such configurational rearrangements could change n and K values i n a d i r e c t i o n which could compensate for the repulsive e l e c t r o s t a t i c e f f e c t s of an increasing pH. Binding studies at more than one temperature have resulted i n a thermodynamic  evaluation of complexes. Enthalpy of binding,  A H ° , can be computed from the temperature depencence of association constant. (Eq. 34)  The standard free energy of binding,  AG° , at equilibrium can  be estimated from a knowledge of binding constants at various temperatures. &G° = - RT In K  (Eq. 35)  For isothermal changes i n a system, the v a r i a t i o n of free energy with temperature i s expressed by the Gibbs-Helmholtz equation.  - 19 -  AG°- AH  0  (Eq.  T  36)  Since association constants are dependent on the composition of the buffer, the standard  state includes the buffer employed  i n the experiment. Thermodynamic parameters are, therefore, subj e c t to possible error which would arise i f the buffer ion binds s i g n i f i c a n t l y to M and varies considerably with temperature. Thermodynamic data helps to explain the nature of the intermolecular forces responsible for binding  (Karush,  1950;  Klotz and others, 1949a; 1949b; 1953b). Temperature changes do not appear to a f f e c t greatly the extent of binding of ions with serum albumin. However, Klotz and Ayers (1952) have shown that there i s a marked temperature-dependent binding between p-aminoazobenzene and bovine serum albumin. For any equilibrium reaction which i s not affected s i g n i f i c a n t l y by temperature, the heat of reaction i s small. It follows from Eq. 36 that, i f AH°is small, the magnitude of  AG at any fixed temperature i s determined primarily by the 0  value of  AS°  the entropy change i n the reaction. The  free energies of binding  ( i . e . , negative  favorable  AG°value) which have  been observed for many ion-albumin complexes seem, therefore, to be a r e s u l t of a favorable entropy change ( i . e . , positive AS°value) during binding rather than to be any favorable heat effect  ( i . e . , negative  AH°value).  - 20 The positive and r e l a t i v e l y high values of  AS°are i n  themselves unique because the reactions as written i n Eq. 8 and 9 are association reactions for which one would expect unfavorable entropy changes. One explanation for t h i s phenomenon i s given below. Although an anion i s usually written as D~,  i t has been claimed that t h i s ion has several polarized  water molecules 'frozen' to i t i n aqueous solution. The subject has been extensively discussed by various investigators: Frank and Evans (1945), Claussen and Polglase (1952), Masterton (1954), Buswell and Rhodebush (1956), Feates and Ives (1956), Frank and Wen  (1957), Klotz (1958), Nemethy and Scheraga  1962b), Nemethy, Steinberg, and Scheraga  (1962a;  (1963), Mohammad (1965),  and Bernal (1965). Similarly, the protein molecule i s highly hydrated.*  This probably occurs around the charged l o c i of  the cationic nitrogen atoms which seem to be d i r e c t l y involved i n the binding process. Consequently, the formation of a bond between these two oppositely charged species would release some of the 'frozen' water molecules. The system, therefore, becomes more randomized and i t becomes reasonable to expect an increase i n the entropy of the system. This implies that, at the molecular l e v e l , there would be an increase i n the number of molecule species upon formation of the anion-protein complex rather than a decrease as indicated i n the M + D » MD equation. A schematic diagram of the changes i n water structure around M and D i n the M + D = MD reaction i s shown i n Figure 4. *  It i s well known that, i n aqueous solution, macro and small molecules are hydrated. However, Frank and Evans (1945) were probably the f i r s t researchers to emphasize the importance of 'iceberg' around solute molecules i n water.  - 21 -  i c e - l i k e ' water  melted' water  Figure 4. Schematic diagram of changes i n water structure accompanied by an interaction between M and D.  Karush (1950), i n h i s study on the interaction between an anionic azo dye and BSA, reported  AS° values of 8.7 e.u.  and 3.3 e.u. for the f i r s t and second groups of binding s i t e s , respectively. He attributed the differences i n AS* values to the s t r u c t u r a l differences between the two binding groups. It was suggested that the cationic group 1 s i t e s are not bonded intramolecularly. Group 2 s i t e s , on the other hand, are linked to nearby anionic carboxyl groups. Therefore, the binding of the anionic dye by group 2 s i t e s would require the breaking of these bonds and would be accompanied by the release of an equal number of carboxyl groups. Binding on group 1 s i t e s would involve a net neutralization of charge and t h i s would r e s u l t i n a positive  AS° value because water molecules are liberated from  the ions. Such an entropy increase would not be observed at group 2 s i t e s .  I I I . METHODOLOGY  Goldstein (1949), i n h i s paper on the interaction between drugs and plasma proteins, has reviewed the methodology associated with binding studies. Similar papers have been published by Klotz (1953a). More recently, Meyer and Guttman (1968a) have reviewed those methods (e.g., k i n e t i c , or dynamic, d i a l y s i s , p a r t i t i o n i n g , gel f i l t r a t i o n , u t i l i z a t i o n of isotopes, nuclear magnetic resonance, and fluorescence quenching techniques) which have been developed during the past several years. These methods f a l l into one of two categories. The f i r s t group depends upon the properties of the interacting molecule; the second, on the behavior of the macromolecule. Quantitative investigations must be based, therefore, on a method which w i l l y i e l d numerical values for two of the three unknowns,  (D^),  (D ), and (D ) and for (M ). These symbols are an inherent part b  t  t  of Eq. 17. Of the many methods described i n the l i t e r a t u r e , only those based on spectrophotometry, s o l u b i l i t y analysis, equilibrium and dynamic d i a l y s i s methods, and viscometry w i l l be discussed here. Except viscometry, these methods measure changes i n the properties of D, the interacting molecule.  1. Spectrophotometry  The spectrum of D i s frequently changed by the macromolecule, M. These spectral changes have been used by many investigators to determine the extent of binding of a wide variety of substances with macromolecules (Job, 1926; Robinson and Hogden, 1941; Klotz, 1946c; 1947; Benesi and Hilderbrand, 1949; Oster and Immergut, 1954; Worley and Klotz, 1966; Connors and Mollica, 1966). If the concentration of M i s low, the t o t a l absorbance at a s p e c i f i e d wavelength of free and bound D i s defined by Eq. 37. A  -  £ b(D ) + f  f  £ b(D ) b  (Eq. 37)  b  A i s the absorbance; b i s the c e l l length; and  £ i s the  molar absorptivity of the s p e c i f i e d forms of D. The term  oL i s  defined by Eq. 38. (Eq. 38)  The f r a c t i o n of D^, F , i s expressed by the following equations. f  * £ (D ) - A f  F f  € (D ) F  T  t  \  *€ (D ) F  T  > or  app  (Eq. 39)  - 24 The apparent molar a b s o r p t i v i t y i n Eq. 39 i s defined by Eq. 40. A  = Gapp  ( D  t>  ( E <  1-  4 0  >  Molar a b s o r p t i v i t y values for the bound drug may be determined by extrapolating absorbance values for D i n the presence of increasing quantities of M. At high M concentrations, i t i s assumed that D i s completely bound to M. The hypothetical curves i n Figure 5 i l l u s t r a t e absorbance changes as a function of M concentration.  0  m  b  CONCENTRATION OF MACROMOLECULE Figure 5. Hypothetical curves for changes i n absorbance as a function of (M). £ values can be estimated from asymptotic values. At (M) = 0, m, b; F = 1, jt, 0, respectively. b  f  - 25 -  The precision of t h i s method depends on the magnitude of the difference i n absorption produced by the presence of M. Binding data obtained i n t h i s way must be complimented by data obtained i n other ways because D concentrations are r e s t r i c t e d by Beer's Law  ( i . e . , they are too low and too narrow). Further-  more, M should not absorb energy at the wavelength  at which the  absorbance change for D due to the presence of M i s maximum. In spite of these disadvantages, t h i s method i s important because small quantities of D can be determined with accuracy. Moreover, i t i s not necessary to separate a r t i f i c i a l l y  and  as i n the  equilibrium d i a l y s i s technique. Klotz (1946c), i n h i s paper on the i n t e r a c t i o n between an azo dye and BSA, reported good agreement between the spectrophotometric method and the equilibrium d i a l y s i s technique i n the region i n which the two methods overlap. Oster and Immergut (1954) found that the absorbance of iodine at 290 nyu  changed d r a s t i c a l l y  i n the presence of polyvinylpyrrolidone (PVP). This increase i n absorbance  i n the presence of increasing PVP concentrations resulted  i n a sigmoidal curve similar to that i n the upper part of Figure 5. However, at lower PVP concentrations, the curves changed slowly. This appears to indicate that the f i r s t few molecules of iodine are bound to PVP with d i f f i c u l t y but that further molecules are more e a s i l y taken up by the polymer.  2. Phase S o l u b i l i t y  This technique has been discussed i n d e t a i l by  Higuchi  and Connors (1965). It involves the addition of an equal quantity  ( i n excess of i t s normal s o l u b i l i t y ) of D into each  of several solutions containing successively increasing amount of Jfl. The solutions are brought to equilibrium at a constant temperature and then analyzed for D^.. A phase diagram i s constructed by p l o t t i n g the amount of D i n solution versus (Hj.) . If there i s no i n t e r a c t i o n between D and M, there w i l l be  no  changes i n (D) i n the presence of M. If a soluble complex i s formed, (Dj.) w i l l increase as (M^.) increases within a range of concentrations  which i s a c h a r a c t e r i s t i c of both the small  and  macro molecules. Increased quantities of D i n the presence of M represent D  since (D )  fe  f  i s a fixed constant under s p e c i f i e d  conditions and i s i n equilibrium with (D ) b  throughout the  (M)  range. When the theory of multiple e q u i l i b r i a i s applied to the data, the method becomes a 'spot' analysis because only  one  value for (D ), or the s o l u b i l i t y of D i n the absence of M, i s f  used throughout the experiment. Furthermore, t h i s method i s suitable only for substances of r e l a t i v e l y low  solubility.  Certain aspects of the binding process can, however, be e a s i l y studied by u t i l i z i n g phase s o l u b i l i t y analysis.  For example,  the method has been used to investigate the e f f e c t s of various solvents, pH,  ionic strength, and temperature on the extent of  binding. The value of r (see Eq. 6) i s a constant under controlled  - 27 conditions and therefore a comparison of r values, under d i f f e r e n t conditions, gives information about the binding mechanism. The thermodynamic parameters obtained from s o l u b i l i t y analysis need not be corrected for d i s o r i e n t a t i o n entropy, because the interacting molecules possess no r o t a t i o n a l freedom i n the c r y s t a l l i n e or bound state (Sahyun, 1964). In some methods (e.g., d i a l y s i s method), however, binding occurs i n an unsaturated system, which implies that the bound molecules possess fewer degrees of freedom (Tanford, 1950; McMenamy and Seder, 1963). Therefore comparison of thermodynamic data from d i f f e r e n t methods should be c a r r i e d out after corrections have been made for differences i n the standard state. Many investigators and i n p a r t i c u l a r Higuchi and h i s coworkers have used t h i s technique to study a wide variety of intermolecular reactions. The papers covering these interactions are not s p e c i f i c a l l y referenced here but are l i s t e d i n d e t a i l i n the 'REFERENCES' section of t h i s thesis (Higuchi and others, 1953a; 1953b; 1954a; 1954d; 1954e; 1954f; 1959; 1961; 1964;1965; Mader, 1954; Kostenbauder and Higuchi, 1956; Poole and Higuchi, 1959; D i t t e r t and others, 1961; Breuninger and Goettsch, 1965; Wadke and Guttman, 1965; Wolfson and Banker, 1965; Singh and others, 1967). Most of these papers have no d i r e c t bearing on t h i s study. However, several w i l l be b r i e f l y reviewed i n order to i l l u s t r a t e the a p p l i c a b i l i t y of the method. Higuchi and Lach (1954d) reported that an insoluble complex was formed between phenobarbital and polyethylene g l y c o l . Their r e s u l t s indicated that a 2:1 complex was formed ( i . e . , two  - 28 ethylene oxide u n i t s reacted with one phenobarbital  molecule).  Mansour and Guth (1968) studied the complexing behaviour of starch and starch fractions with benzoic acid, some of i t s derivatives, sorbic acid, and other selected molecules. Breuning and Goettsch (1965) studied the interactions between p-chlorometaxylenol and various synthetic polymers. The method has not been used extensively to study the i n t e r a c t i o n between macromolecules. Laurent (1963) reported a r e l a t i v e decrease i n the s o l u b i l i t y of human serum albumin, y - g l o b u l i n , and fibrinogen i n the presence of various types of dextran. He studied the e f f e c t of ionic strength and pH  and  showed that the s o l u b i l i t y of proteins i n the presence of dextran increased with an increase i n the size of the protein.  3. Equilibrium D i a l y s i s  Interactions between small and macro molecules may  be  studied q u a n t i t a t i v e l y by u t i l i z i n g the equilibrium d i a l y s i s technique.*  A container i s divided into two compartments by  a semi-permeable membrane. A macromolecule solution i s placed i n one compartment; a solution containing the small molecule i s placed i n the second compartment. The small molecule passes through the membrane but the macromolecule i s retained i n i t s own compartment. At equilibrium, the t o t a l number of small *  The k i n e t i c or dynamic d i a l y s i s technique (Andreoli and others, 1965, Stein, 1965; Agran and Elofsson, 1967; Reuning and Levy, 1968; Meyer and Guttman, 1968b; 1970a; 1970b) w i l l be b r i e f l y discussed i n the 'RESULTS AND DISCUSSION' section of t h i s t h e s i s .  - 29 molecules i n the M compartment w i l l exceed that i n the M-free compartment. The difference between these two i s a measure of (D^). Two  concentrations  possible sources of error, the Donnan  e f f e c t , and membrane binding of small molecules must, however, be taken into consideration before applying t h i s technique. When a charged macromolecule i s retained i n one of the two compartments, at equilibrium, the concentration of d i f f u s i b l e ions i s no longer i d e n t i c a l across the membrane. This phenomenon has been described as the Donnan equilibrium (Gverbeek, 1956). The ion r a t i o characterizing the d i s t r i b u t i o n of d i f f u s i b l e ions across the membrane (R) i s expressed by Eq. 41 when both anion and cation are univalent.  R .  (Eq. (C )  41)  +  F  The subscripts, F and M, represent the M-free and M compartments, and the parentheses represent the concentrations  of the specified  ion species. Values of R may  be expressed as a function of the  concentra-  t i o n of the neutral s a l t i n the M-free compartment, ( C ) , the +  F  valence on the macromolecule, Z , M  and  (M) i n molality (Bull, 1964a).  (Eq.  Figure 6 i l l u s t r a t e s the r e l a t i o n s h i p between R and  (C ) 4  F  42)  when  the M-compartment contains 10 grams of the macromolecule per grams of solvent. The molecular weight of M i s 40,000; the  1000  valence,  - 30 2.5  2.0 R 1.5  1.0 0  0.2  0.4 (c )  0.6  +  F  Figure 6. Calculated values of R as a function of s a l t concentration i n the M-free compartment. See B u l l (1964a)  i n one s e r i e s of experiments, i s -10 and i n the other i s -40. In a d i l u t e solution of M, the Donnan e f f e c t can be neglected only i f the concentration of the d i f f u s i b l e ion i s reasonably high and the valence of M i s f a i r l y low. In a solvent system of high ionic strength and pH at which the macromolecule has a small valence charge, the abnormal d i s t r i b u t i o n of small molecules across the membrane due to the Donnan equilibrium can be neglected. The d i a l y s i s membrane may act as a binding s i t e for the small molecule and a correction must be made for t h i s i n t e r a c t i o n . Most corrections are made by using a control i n which no macromolecule i s present i n the apparatus.  I t i s then possible to  - 31 measure the 'loss' of small molecule from the s o l u t i o n . It has been either observed or assumed that the extent of membrane binding i s proportional to the amount of small molecule added to the system. Osborne (1906), at the turn of the century, studied the i n t e r a c t i o n between s a l t and proteins by u t i l i z i n g the d i a l y s i s method. The procedure was refined by Klotz, Walker, and Pivan (1946b) and, of approximately 400 papers on protein binding reviewed by Meyer and Guttman (1968a), more than 130 papers reported the use of d i a l y s i s thechnique. Its main advantage i s that an interaction can be studied through a range of small molecule concentrations. Nearly complete saturation of the macromolecule with a given D can often be achieved. I t i s one of the few methods which are conducive to quantitative work and i s thermodynamically sound. By using t h i s method, i t i s possible to cover a wider range of r value (see Eq. 6) and thus obtain more information about the i n t e r a c t i o n . Karush and Sonenberg  (1949) covered an r range from 0 to 10 i n  a study of the i n t e r a c t i o n between a l k y l sulfates and bovine serum albumin. In a similar study, Pollansch and Briggs (1954) studied r values up to 40. They used volume r a t i o s of protein compartment to protein-free compartment of 1 to 79 and 1 to 9. By using the former r a t i o , they were able to u t i l i z e a large quantity of detergent i n t h e i r study. The maximum amount of detergent which could be used was limited only by the s o l u b i l i t y of the detergent i n the protein-free compartment.  - 32 Equilibrium d i a l y s i s would be c a r r i e d out by e q u i l i b r a t i n g the macromolecule solution i n a cellophane  bag with an external  solution containing the small molecule. However, the  concentra-  t i o n of the small molecule i s frequently limited by i t s solubil i t y . Consequently, a large volume of solution containing a small quantity of the small molecule i s required to cover the whole range of i n t e r a c t i o n . Under these conditions, a long period of time i s required to e q u i l i b r a t e the system. Yang and Foster (1953) used a 1 to 100 volume r a t i o of protein compartment to proteinfree compartment i n t h e i r study on the i n t e r a c t i o n between dodecylbenzenesulfonate (SDBG) and bovine plasma albumin. However, even a f t e r a one-month time i n t e r v a l , d i a l y s i s equilibrium  was  not attained. They, therefore, modified t h e i r procedure by storing the mixed solutions for at least two days at 1 to 3° C and then d i a l y z i n g against an equal volume of the buffer used for an additional two days. The amount of free SDBG was  then  determined i n the dialyzate. Patel and Foss (1964) used a d i a l y s i s c e l l consisting of two plexiglas blocks separated Eide and Speiser  by a semi-permeable membrane.  (1967a) used a s i m i l a r apparatus but  stirred  the solutions i n the chambers with a magnetic s t i r r e r . By using these approaches, a better control of membrane binding  was  attained because the surface area of the membrane i s more or less constant *  throughout the study.*  Goldstein (1949) reported that loss of small molecules due to membrane binding i s not only large (for example, to 20 per cent of t o t a l methylene blue i n h i s study) but also variable from bag to bag.  - 33 A d i a l y s i s membrane i s a very t h i n layer of a s p e c i f i e d substance or mixture of substances. Various types of membranes are described by Craig (1965). Kostenbauder et a l . (1969) discussed the use of nylon membranes. Nylon reacts with phenolic compounds, however, and M i t c h e l l and Brown (1966) used rubber latex membranes i n a study of the i n t e r a c t i o n between p-chlorometaxylenol and a non-ionic however, use cellophane  surfactant. Most investigators,  membranes. These membranes are usually  marketed i n r o l l s and are stored i n p l a s t i c bags to prevent drying. They contain g l y c e r i n and small amounts of other  solutes  but these can be e a s i l y removed by washing. When stored i n a r e f r i g e r a t o r , t h e i r porosity remains f a i r l y constant  over long  periods of time. The main advantage of t h i s type of membrane i s that i t i s r e l a t i v e l y free of fixed charges which would be ion s e l e c t i v e (Craig, 1965).  4. Viscometry  A d i l u t e s o l u t i o n of concentration C w i l l have a s l i g h t l y higher v i s c o s i t y (^)  than the solvent i t s e l f C 7 ) . The 0  v i s c o s i t y i s defined by Eq.  °?rel  -  relative  43.  °7 /  (Eq-  «)  The r e l a t i v e v i s c o s i t y i s , therefore, s l i g h t l y more than one and includes the e f f e c t of solvent (unity) and solute. S p e c i f i c v i s c o s i t y , which i s o l a t e s the solute e f f e c t , i s defined by Eq.  44.  - 34 -  ^sp = ^ r e l "  < 1-  1  E(  44  >  Specific v i s c o s i t y depends on concentration, i s small number, and i s r e l a t e d the reduced v i s c o s i t y .  Vred  =  ^sp  /  (Bq.  c  45  >  The reduced v i s c o s i t y i s a large number, does not change much with concentration i n d i l u t e solutions, and measures the increase i n v i s c o s i t y per unit concentration i n a slotuion of concentration C. If the above value i s determined at several low concent r a t i o n s , an extrapolation to zero concentration w i l l y i e l d a value which i s due to the solute at i n f i n i t e d i l u t i o n per unit concentration. This value i s c a l l e d the i n t r i n s i c v i s c o s i t y and i s the value which i s q u a n t i t a t i v e l y important  i n solute-  solvent i n t e r a c t i o n s .  C°?3= c ^ O  Isp / C  (Eq. 46)  Huggins (1942) related concentration to reduced v i s c o s i t y by using Eq. 47.  ^ s  P  / c = (*}]••  VTO  2 C  (EQ  -  4 7 )  - 35 KJJ i s a constant, i s known as Huggins parameter, and i s determined experimentally.* I n t r i n s i c v i s c o s i t y depends on hydration and molecular shape. The r e l a t i o n s h i p between p a r t i c l e assymetry and v i s c o s i t y are complex but Mysels (1959), Yang (1961), and Flory (1953) have discussed the changes i n i n t r i n s i c  viscosity  due to s t r u c t u r a l changes i n macromolecules. M i l l e r and Hamm (1953) studied the properties of polyvinylpyrrolidone (PVP) by measuring v i s c o s i t y , sedimentation v e l o c i t y , and d i f f u s i o n . Configurational changes i n bovine serum albumin have been investigated by Yang and Foster (1954) by measuring i n t r i n s i c v i s c o s i t y and s p e c i f i c r o t a t i o n over a 1.3 to 7.0 pH range. They concluded that v i s c o s i t y changes are due to swelling rather than coulombic repulsion and suggested that the expansion reaction i s an all-or-none rather than a stepwise phenomenon which i s fast and completely r e v e r s i b l e . A similar study was c a r r i e d out by Tanford and Buzzell (1956). They concluded that the expansion process was much more complicated than that suggested by Yang and Foster (1954). Doty et a l . (1957) measured the  i n t r i n s i c v i s c o s i t y of poly-L-glutamic acid as a function of  pH i n 0.2 M sodium chloride-dioxane (2:1). They concluded that the  polypeptide e x i s t s i n an  - h e l i x below a pH of 5.5 and as  a random c o i l at pH values i n excess of 6.5. *  Kraemer (1938) proposed a similar equation; ln°? l/C • £ . " 1 ) - K ' C H C (Eq. 48). K and K' are related by each other i n the following way; K + K' = 0.5 (Eq. 49). Both Eq. 47 and 48 indicate that a plot of ^gp/C and ln°?rel/C versus C should y i e l d the same intercept, C ? 3 ,and the l i m i t i n g slopes at C = 0 should s a t i s f y the Eq. 49 r e l a t i o n s h i p . By using both equations, i n t r i n s i c v i s c o s i t y can be determined with some confidence. re  2  H  H  1  - 36 Changes i n v i s c o s i t y have been used to detect interactions between small and macro molecules. Frank et a l . (1957) measured the reduced v i s c o s i t y of PVP i n the presence of dye. In the absence of s a l t , the reduced v i s c o s i t y was  increased by the dyes  and a sharp v i s c o s i t y maximum was observed. The reduced v i s c o s i t y increased with a decrease i n PVP concentration. This appears to be t y p i c a l p o l y e l e c t r o l y t e s because e l e c t r o s t a t i c repulsion of i d e n t i c a l charges w i l l lead to an unfolding of the polymer molecule. As the PVP concentration was decreased  i n the presence of  a constant quantity of dye, the concentration of unbound 'gegenions' (counterions) increased. This increase w i l l tend to suppress the eleetroviscous e f f e c t . A v i s c o s i t y increase i n the presence of s a l t was explained by assuming a c r o s s - l i n k i n g e f f e c t due to aggregation of dye ions. A similar study was c a r r i e d out by Molyneux et a l . (1961b). Their r e s u l t s indicated that, i n general, the polymer expands i n the presence of anionic cosolutes. With non-ionic cosolutes, the polymer contracts and,  i n the presence of c a t i o n i c cosolutes,  no appreciable v i s c o s i t y e f f e c t s were observed. The influence of buffers and complexing substances on the r h e o l o g i c a l properties of PVP has been studied by Eide and Speiser (1967b). The properties of PVP,  i n water and i n s a l t solution, were  determined by Goldfarb and Rodriguez (1968) by measuring heat capacities, s p e c i f i c volumes, and reduced v i s c o s i t i e s . They concluded  that the decrease i n i n t r i n s i c v i s c o s i t y of aqueous  PVP with increased temperature was due to the progressive c o i l ing of the molecule. Interactions between dodecyl s u l f a t e  - 37 anions and BSA at high pH have been studied by Lovrien (1963). He maintained pH at a value at which the protein has a large negative charge. Under such conditions, an increase i n hydrodynamic volume would be expected. However his viscometric data indicated that the detergent reduces expansion. The hydrocarbon portion of the molecule appears to induce conformational changes which counter unfavorable e l e c t r o s t a t i c energy changes. Complexation  has been studied by using c a p i l l a r y viscometers.  The operating c h a r a c t e r i s t i c s of these viscometers have been described by Van Wazer et a l . (1963). Specifications may be found i n documents issued by the American Society for Testing and Mate r i a l s (A.S.T.M., 1966a; 1966b). The v i s c o s i t y equation applicable to c a p i l l a r y viscometers i s based on Poiseuille's Law. ^  / f = Ct - B/t  (Eq. 50)  f> i s the density of the l i q u i d ; t i s the flow time i n seconds; the constant C and B have been characterized by Cannon et a l . (1960); the quantity B/t i s c a l l e d the kinetic energy correction factor. In a well designed viscometer, B/t i s usually a small per cent of the Ct term. When the correction factor can be neglected, the c a l i b r a t i o n constant C i s determined by measuring the flow time of a standard l i q u i d of known kinematic v i s c o s i t y . I t may also be determined by comparing the flow time of a l i q u i d i n an uncalibrated viscometer with that observed i n a master viscometer with a known C value.  IV. THE CHEMICAL AND BIOLOGICAL CHARACTERISTICS OF THE SUBSTANCES USED IN THIS INVESTIGATION  1. Bishydroxycouraarin  Bishydroxycoumarin (BHC), was f i r s t synthesized by Link (1943-1944),  and i s o f f i c i a l i n the U.S.P. It i s also described  as dicoumarin or dicumarol. I t s chemical name i s 3,3'-methylenebis-(4-hydroxycoumarin) or 3,3'-methylene-bis-(4-hydroxy-l,2benzopyrone).  OH  OH  Figure 7. Chemical structure of BHC.  BHC i s a white c r y s t a l l i n e or amorphous powder p r a c t i c a l l y insoluble i n water. It i s a weak dibasic acid and soluble i n a l k a l i n e solutions. Burns, Wexler, and Brodie (1953) reported a pKa value of 5.7. This value was obtained by t i t r a t i n g 10 ml. of a 90$ ethanolic solution (containing 20 mg. of BHC) with 0.025N sodium hydroxide solution. Nagashima, Levy and Nelson (1968a) obtained a value of 6.5 by using a p a r t i t i o n i n g technique. In a l k a l i n e solution, BHC absorbs a maximum of radiant energy at 314 mu.. The pH of the solution a f f e c t s the spectrum and isosbestic points are observed at 254 and 286 mu  (Findlay  - 39 and others, 1965). The molecular weight of BHC Melting points of 287-293°C (Merck Index, 1968)  i s 336.29. and 288-290°C  (Nagashima, Levy, and Nelson, 1968a) have been reported. French and Wehrli (1965) published the infrared spectra of some of the coumarin anticoagulants, including BHC. The f i r s t c l i n i c a l t r i a l s u t i l i z i n g BHC were c a r r i e d out i n the e a r l y 1940•s. Ingram (1961) and Douglas (1962) reviewed the use of BHC  i n the treatment  of myocardial i n f a r c t i o n , angina  pectoris, rheumatic heart disease, cerebrovascular disease, venous thrombosis,  and pulmonary embolism. The  pharmacological  properties of coumarin anticoagulants have been reviewed by Levine (1967). Owren (1963a; 1963b) discussed the use of a n t i coagulant  medication.  The importance of d i s s o l u t i o n rate on c l i n i c a l e f f e c t  was  f i r s t reported by Lozinski (1960). Findlay et a l . (1965) reported that the p a r t i c l e size d i s t r i b u t i o n i s a major factor governing d i s s o l u t i o n r a t e . O'Reilly, Aggeler, and Leong (1964) observed that the absorption of BHC  from solution (or when administered  as a powder) was rapid but that absorption was slow when whole tablets were administered to the patient. Weiner et a l . (1950) studied the physiological d i s p o s i t i o n of BHC  i n man.  They reported a strong i n t e r a c t i o n between the  drug and plasma albumin. A similar study was i n i t i a t e d by Lee et a l . (1950) but the test animals i n t h i s instance were mice and r a b b i t s . O'Reilly et a l . (1964) c a r r i e d out a pharmacodynamic study of BHC  and warfarin i n man.  Other investigations u t i l i z i n g  d i f f e r e n t animal species were c a r r i e d out by Jaques et a l . (1957),  - 40 Christensen (1964), Solomon et a l . (1967), and Nagashima et a l . (1968b; 1968c; 1968d). The metabolism of BHC was  investi-  gated by Christensen (1966). The in v i t r o binding of warfarin to albumin was extens i v e l y studied by O'Reilly et a l . (1966; 1967;  1968;  1969).  In addition to equilibrium d i a l y s i s , they used a heat burst micro-calorimeter to measure the heat evolved i n the interaction (O'Reilly and others, 1968). The exothermic and nonionic nature of the i n t e r a c t i o n was observed. The introduction of a polar hydroxy group on the coumarin ring during metabolism reduced i t s hydrophobic binding surface and thus decreases albumin binding (O'Reilly and others, 1969). During t h e i r investigation of the analysis of BHC i n b i o l o g i c a l f l u i d s , Nagashima et a l . (1968a) observed a decrease i n plasma albumin binding at pH 4. I t i s at t h i s pH that the plasma albumin undergoes a largely reversible s t r u c t u r a l a l t e r a t i o n from compact to expanded form. The configurational expansion r e s u l t s i n a disruption of non-polar c l u s t e r s located i n the i n t e r i o r of the albumin molecule and thus causes a decrease i n binding strength.  2. Human Serum Albumin  Human serum albumin (HSA) i s characterized by i t s s o l u b i l i t y i n water or by i t s electrophoretic behaviour. Its physicochemical properties may be explained i n terms of an elongated e l l i p s o i d with a molecular weight of 69,000, a length of 150 A, and a diameter of 38 A. Certain investigators report a molecular weight of 65,000. The arguments for or against these values are given by Putnam (1965). U t i l i z i n g i n t r i n s i c v i s c o s i t y measurements, Tanford and Buzzell (1956) obtained an a x i a l r a t i o of about 3 to 1. HSA  appears to be made up of amino acid residues but t h e i r  primary sequence has not been established. The residues are joined together by a single long peptide chain which i s i n t e r n a l l y cross-linked by 17-18 d i s u l f i d e bridges. These d i s u l f i d e  linkages  contribute greatly to the s t a b i l i t y of the configuration. The importance of such c r o s s - l i n k s i n protein structures has been discussed by E d s a l l and Wyman (1958a). A f r a c t i o n a t i o n procedure for the i s o l a t i o n of albumin has been worked out by Cohn et a l . (1950). This 'Conn Fraction V i s mainly albumin and i s prepared by p r e c i p i t a t i o n at pH of 4.8 and an ethanol concentration of 40$. Spectrophotometric and t u r b i d i metric  (Layne, 1956), Kjeldahl and isotopic techniques (Hauro-  witz, 1963) have been used to analyze proteins. Although serum albumin i s available i n c r y s t a l l i n e form, the substance i t s e l f i s micro-heterogeneous. This heterogeneity  may be established  by examining the substance e l e c t r o p h o r e t i c a l l y at low ionic strength, chromatographically,  i n t e r f e r o m e t r i c a l l y , or serolo-  g i c a l l y . Foster (1968) has written a review on t h i s subject.  - 42 Because of i t s a v a i l a b i l i t y , HSA  i s one of the most widely  investigated proteins. I t s ion binding behaviour, i t s amphoteric heterogeneity at low pH, and i t s denaturation have been i n v e s t i gated. Foster (1960) reviewed these c h a r a c t e r i s t i c s . T i t r a t i o n curves have been evaluated by Tanford et a l . (1950; 1955a; 1955b). They estimated the number of dissociable groups and calculated t h e i r i n t r i n s i c d i s s o c i a t i o n constants.  A s a t i s f a c t o r y agreement  was obtained with regard to the amino acid contents. They and Foster et a l . (1956), Aoki et a l . (1957) and Clark et a l . (1962) observed that both HSA  and bovine serum albumin exhibited an  anomalous t i t r a t i o n behaviour below the i s o e l e c t r i c point, 4.7, and beginning at about a pH of 4. Yang and Foster (1954), on the basis of a study of the e f f e c t of pH on o p t i c a l r o t a t i o n and v i s c o s i t y , concluded that an isotropic expansion of the albumin molecule ocurred i n acid s o l u t i o n . They attributed the molecular expansion to a mutual repulsion of the p o s i t i v e l y charged ammonium groups. This phenomenon was confirmed by Tanford et a l . (1955b; 1956). They proposed that the expansion takes place through an intermediate expandable form - the so-called 'F form'. Foster and Clark (1962) presented evidence that the native form (the ' N form') has a large number of carboxylate groups that are masked and that isomerization to the F form leads to the normalization of a l l carboxylate s i t e s . Because i t has a strong a f f i n i t y for ions and other  substances,  albumin has been used to elucidate protein-ion interactions. Klotz (1953a) was the f i r s t investigator to review t h i s subject. Karush (1950) attributed the high r e a c t i v i t y of albumin with a  - 43 variety of anions tb the p a r t i c u l a r configurational adaptability of the molecule. Serum albumin interacts r e a d i l y with detergents ions such as dodecyl s u l f a t e . Interaction of t h i s type have been investigated i n various laboratories (Putnam, 1945; Karush,  1949;  Pollansch and Briggs, 1954; Lovrien, 1963) and reviews on the subject have been published by Foster (1960) and Ray  (1968).  The c l a s s i c a l review on drug-albumin interactions was published by Goldstein (1949). More recently, Meyer and Guttman (1968a) have re-reviewed t h i s p a r t i c u l a r aspect of serum albumin binding. The p h y s i l o g i c a l consequences of ion binding and the a f f i n i t y of serum albumin for dyes, drugs, and similar molecules have been emphasized by Bennhold  (1961). The e f f e c t of drug-albumin  interactions on the absorption, d i s t r i b u t i o n , and excretion of the drug has been discussed by Martin (1965). Wishnia and Pinder (1964) observed that the extent of binding of alkanes (Cg to Cg) to the F form of bovine serum albumin i s much lower than that obserbed for the N form. They concluded that the large hydrophobic c l u s t e r s i n the i n t e r i o r of the protein are responsible for the i n t e r a c t i o n . Nagashima et a l . (1968a) observed a similar decrease i n binding capacity of BHC to HSA i n the acid range. The r o l e of hydrophobic bonding i n r e l a t i o n to physicochemical behaviour of protein solutions, has been discussed by Kauzmann (1959), Klotz (1958; Scheraga  1960),  (1961; 1963), Nemethy et a l . (1962c; 1963), and C e c i l  (1967). In t h i s context, studies on water structure have been made by various investigators (see p. 20). Solvent e f f e c t s on the binding or organic ions by proteins have been discussed by Klotz and Luborsky (1959).  3. Polyvinylpyrrolidone  Polyvinylpyrrolidone  (PVP) i s a water soluble, high  molecular weight polymer. I t may be represented s t r u c t u r a l l y i n the following way. HC 2  0  HC 2  I CH  CHg-  N Figure 8. Chemical structure of monomer of PVP, N-vinylpyrrolidone (C6H9ON - 111.14).  Because of i t s chemical and physical properties, the polymer has been used as a plasma extender. The molecular weight of PVP i s defined i n terms of the v i s c o s i t y of a d i l u t e solution. For t h i s purpose, the Fikentscher  K  F  value i s commonly used  (G.A.F., 1957). The average molecular weight of the PVP which i s used as a plasma extender i s approximately 40,000 (K = 30). F  This value i s derived from u l t r a c e n t r i f u g a t i o n and osmosis measurements. I t s physicochemical properties were investigated by May et a l . (1954). An aqueous solution of PVP i s s l i g h t l y a c i d i c (pH of 4) but has no buffering action. Using l i g h t scattering measurements and assuming that the PVP molecule i s a random c o i l , Hengstenberg et a l . (1952) reported a mean square molecular diameter of 360 A corresponding  - 45 to a molecular weight of 249,000. A similar study was c a r r i e d out by M i l l e r and Hamm (1953). Assuming an elongated e l l i p s o i d  shaped  model, they obtained an a x i a l r a t i o of 22 to 1 and a root mean square distance of 169 A for PVP of molecular weight of 41,500. At a lower temperature, PVP was believed to be more t i g h t l y c o i l e d . * Drugs, dyes, and toxins bind with PVP. References on t h i s p a r t i c u l a r subject and to i t s physicochemical and physiological properties are available from the manufacturer of the polymer (G.A.F., 1967). Oster and Immergut (1954) studied the iodine-PVP complex and t h i s complexation process has been used quantitatively to determine PVP (Campbell, 1953). Scholtan (1953) expressed the binding process of PVP with dyes by use of Langmuir adsorption isotherm. His thermodynamic data indicated that i n one case the a f f i n i t y of the binding process was determined preferably by the entropy, i n the other (meta-benzopurpurin  4B) preferably by the  heat of reaction. In solutions containing PVP, albumin and dyes simultaneously, a complexation balance was formed. Theoretical r e l a t i o n s were given between these three components. The calculated and experimental values showed a close agreement. Spitzer and McDonald (1956) studied the interactions between bovine serum albumin and PVP with  bromophenol blue (BPB).  There was no evidence of an e l e c t r o s t a t i c factor with respect to the exothermic PVP-BPB binding. Frank et a l . (1957) studied the i n t e r a c t i o n between PVP and azo dyes. Orange II and benzopurpurin 4B appear to be bound to the chain segment of seven *  This statement i s i n contradiction to that appeared on p. 36 (Goldfarb and Rodriguez, 1968). See 'RESULTS AND DISCUSSION' section of t h i s thesis for more d e t a i l .  - 46 or ten monomer u n i t s . Molyneux and Frank (1961a; 1961b) studied the interaction of PVP with a large number of aromatic compounds. They observed entropy gains i n almost a l l systems and concluded that hydrophobic bonding ocurred. Infrared spectra of dry PVP films containing varying concentrations of cosolutes indicated the presence of polymer-cosolute hydrogen bonds (1961a). Using l i g h t scattering and viscometric methods, they concluded that anionic cosolutes expand the polymer, nonionic cosolutes contract the molecule, and cationic cosolutes had no e f f e c t on molecular size  (1961b). Worley and Klotz (1966) studied the e f f e c t of PVP on the  near infrared spectra of H 0 - DgO solutions. They suggested 2  that PVP e x h i b i t s a structure making character. Goldfarb and Rodriguez  (1968), however, found no evidence for the existence  of more 'structural water' i n the v i c i n i t y of the PVP molecule. PVP has several properties which makes i t of p a r t i c u l a r interest to the pharmaceutical s c i e n t i s t . PVP forms water soluble or water dispersible complexes with a wide variety of water insoluble drugs (G.A.F., 1967). References covering these  com-  plexation studies are given i n Table 1. Binding depends not only on the nature of the simple molecule but also on the concent r a t i o n of the macromolecule (Edsall and Wyman, 1958c). In most of the investigations c i t e d i n Table 1, at a fixed t o t a l drug concentration, concentration r a t i o of t o t a l to free (or vice versa) i s a l i n e a r function of macromolecule concentration. The r a t i o can, therefore, be an approximation of the extent of binding. Ratios at 1$ PVP concentration are shown i n Table 1.  - 47 -  Table 1. The Complexing Behaviour of PVP with Various Types of Drugs.  Drug  Benzyl Penicilin Mandelic acid Caffeine Theophylline Cortisone  C i t r i c Acid Aminopyrine Phenol  Chlorobutanol  Benzyl Alcohol Phenylethyl Alcohol  0  Water  0 0  Water Water  0 30 30  Water Water Water  0 0  Water Water  (Dt)xlO $PVP mole/L. 3  (D ) Methodology (D ) and Reference t  f  1.0  1.42  1.0 1.0  1.1 1.00  1.547 1.547 3.095  1.0 1.0 1.0  1.05 1.05 1.05  2.68 6.619  1.0 1.0  1.00 1.08  .393 12.49 5.56  No Evidence of Complex Formation  0  Water  5.74  1.0  1.13  0 0 0 0 0  Water Water Water Water Water  3.62 1.81 7.25 1.82 7.25  1.0 1.0 1.0 1.0 1.0  1.22 1.43 1.47 1.40 1.40  0 Water ? 1.0 1.23 30 Water 2.58 1.0 1.19 30 Water 4.31 1.0 1.19 No Evidence of Complex Formation 0 0  Water Water  200.0 160.0  15 30 30 45  Water Water Water Water  28.5 28.7 57.7 28.8  .37 1.19 .12 1.23 1.0 1.0 1.0 1.0  1.06 1.08 1.08 1.11  No Evidence of Complex Formation  Equilibrium Dialysis and Solubility, Higuchi and Kuramoto (1954c), Guttman and Higuchi (1956).  Benzoic Acid Salicylic Acid m-Hydroxy Benzoic Acid p-Hydroxy Benzoic Acid p- Ami no Benzoic Acid Phenobarbital  Solvent System  c  Equilibrium Dialysis, Higuchi and Kuramoto (1954b).  Sulfathiazole Sodium Salicylate Procaine HC1 Chloramphenicol  o  Equilibrium Dialysis, Bahal and Kostenbauder (1964).  (Continued on next page.)  - 48 (Table 1. Continued) Drug  °C  Solvent System  (D )xl0 $PVP mole/L. 5  t  p-Chlorometaxylenol *a  30 30 30 30 30 30  Water Water Water Water Water Water  Methylparaben Propylparaben Hexylresorsinol  27 30  Water Water  23  Water  Tannic Acid *b  Soluble Complex Formed  Benzoic Acid  Phenol Aniline  Nitrobenzene p-Nitrophenol p-Nitrobenzoi c Acid p-Hydroxy Benzoic Acid  p-Amino Benzoic Acid  .113 .140 .197 .225 .283 .401 6.12 1.23 .858  (D ) (D ) t  f  2.0 2.0 2.0 2.0 2.0 2.0  1 .13 1 .17 1 .19 1 .18 1 .18 1 .15  1.0 1.0  1 .15 1 .30  .05 3 .00  Equilibrium D i a l y s i s and Solubility, Breuninger et a l . (1965). Eqm. D i a l y s i s , Miyawaki et a l . (1959), P o l l i et a l . (1969). Kabadi et a l . (1966).  22 Ionized 22 pKa 22 pKa 22 Union. pKa 22 22 pKa 22 Union. 22 Ionized 22 pKa 22 pKa 22 Union. 22 Union. 22 Union. 22 Ionized 22 pKa 22 pKa 22 Union.  33.0 13.7 36.3 10.1 22.5 45.1 32.4 35.8 38.5 74.3 33.9 3.52 13.3 4.49 1.32 3.82 1.02  1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1*0 1.0 1.0 1.0 1.0 1.0. 1.0  1 .02 1 .03 1 .03 1 .11 1 .02 1 .05 1 .07 1 .00 1 .02 1 .02 1 ,04 1 .03 1 ,11 1 .02 1 .02 1 .03 1 .06  22 Ionized 22 rpKa of 22 tcocr 22 Union.  13.5 12.5 33.7 1.51  1.0 1.0 1.0 1.0  1 .08 1 .11 1 .13 1 .25  22 -cocr 22 fpKa of 22 ICOO" 22 rpKa of t-NH +  12.1 12.3 21.5  1.0 1.0 1.0  1 .08 1 .09 1 .11  11.9  1.0  1 .11  3  Methodology and Reference  W  M  0- C h-  1  P  3  H*  cr  09 P  w(6 g  C HO> HO (D  P  .—, u) co co cnP  (Continued on next page.)  - 49 (Table 1. Continued) °C  Solvent System  Bezocaine  22  pKa of -NH Union. pKa pKa Union. Union. pKa pKa Union. 3  Methylparaben 22 22 22 Butylparaben 22 Propylparaben 22 22 22 Methyl22 be nzoate Ethylbanzoate 22 Procaine HC1 22  Union. Union. Ionized  (Dt)xlO $PVP mole/L. 3  1.09 2.25 1.13 .74 2.66 1.32 .517 1.06 1.59 .875 ?  4.1 8.16  (Dt.) (Df)  1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0  1.11 1.12 1.13 1.09 1.11 1.10 1.20 1.13 1.15 1.18  1.0 1.0 1.0  1.02 1.03 1.02  Methodology and Reference  Equilibi•ium Dialysis, Eide et al. (1967a).  Drug  *a - Insoluble complex i s formed up to PVP concentrations of 0.4$, after which s o l u b i l i t y increases l i n e a r l y with regard to PVP concentration. *b - The presence of dextrose s l i g h t l y enhances the complexation.  Some investigators presented t h e i r r e s u l t s i n graphical form and i t was necessary, therefore, to make approximations d i r e c t l y from these graphs. Similarly, the d i f f e r e n t concent r a t i o n terms i n the various publications necessitated a conversion to an i d e n t i c a l concentration term. Several of the factors a f f e c t i n g binding are i l l u s t r a t e d by the r e s u l t s i n Table 1. Bezoic acid, for example, i s more weakly bound to PVP than i t s derivatives. Orthohydroxy benzoic acid  (Sali-  c y l i c acid) has a lower binding tendency than does the meta or para forms. The former substance has a higher internal coordination or chelation between -OH and -COOH groups.  - 50 The lesser complexing tendency of the corresponding  para-  amino compound i s probably due to the weaker e l e c t r o p h i l i c nature of amino hydrogen as compared with the  hydroxyl  hydrogen. This r e s u l t s i n weaker hydrogen bond formation (Higuchi and Kuramoto, 1954c). The e f f e c t of temperature i s i l l u s t r a t e d by the studies of the PVP-chlorobutanol  complex. Binding strength increases  with temperature. This indicates an endothermic reaction. Bahal et a l . (1964) concluded  that the large p o s i t i v e entropy  change accompanying the binding was due to the formation of hydrophobic bonds. I f the PVP concentration i s fixed, the extent of i n t e r a c t i o n i s often enhanced by increasing t o t a l drug concentration (Breuninger and Goettsch,  1965;  Eide and Spei-  ser, 1967a). This concentration dependency i s not, however, observed i n most i n v e s t i g a t i o n s . The r e s u l t s obtained by Eide and Speiser (1967a) strongly suggest that the nonionic species react more r e a d i l y with  PVP.  The low i n t e r a c t i o n tendency of the ionic compounds i s most l i k e l y due to the hydrophobic nature of the substances.  The  increased complexing tendency of ionic compounds containing -OH,  -NHg,  or -COOH groups indicates that hydrogen bonding  also plays a s i g n i f i c a n t role i n reactions of t h i s type. Buffer substances (Eide and Speiser, 1967b) and t h i r d components i n the solvent system (Kabadi and Hammarlund,  1966)  w i l l also  a l t e r PVP-drug binding. Simonelli et a l . (1969) reported that the apparent s o l u b i l i t y and rate of solution of s u l f a t h i a z o l e  - 51 from compressed tablets containing PVP i s greatly increased i f the drug i s f i r s t coprecipitated with the polymer. These investigators developed methods for the preparation of these coprecipitates i n water and i n 95$ alcoholic solution.  4. Dextran, Starch, and Hydroxyethyl Starch  Dextrans are polysaccharides and consist of a variety of oC-polyglucosans produced by Leuconostoc mesenteroides  and  c l o s e l y related bacteria under suitable environmental conditions. Synthetic procedures have been developed by Ruckel and  Schuerch  (1966). The concentration of dextran i n solution may be determined by heating the carbohydrate with anthrone i n s u l f u r i c acid (Scott and Melvin, 1953). Arond and Frank (1954) studied the molecular weight d i s t r i b u t i o n of native dextran by u t i l i z i n g l i g h t scattering techniques and the i n t r i n s i c v i s c o s i t y i n aqueous solution. Granath  (1958) studied the properties of  branched dextran i n solution. By measuring v i s c o s i t y , l i g h t scattering properties, and sedimentation rates, Granath  was  able to obtain a more complete picture of the hydrodynamic behaviour of dextran molecule. Gronwall (1957) discussed the use of dextran solutions as plasma extenders. It i s generally recognized that as the molecular weight of dextran increases i t s interaction with proteins increases. In t h i s context, Ricketts (1966) published data on the molecular composition of dextran solutions which are currently used as plasma extenders. Reese et a l . (1966) compared the extent of  - 52 branching of the synthetic dextran with that of the natural product. Enzymatic analysis confirmed the basic s i m i l a r i t y of the two  forms.  More recently, Laurent and Granath (1967) fractionated dextran by using Sephadex G-200 packed i n t o chromatographic columns. Greenwood (1956) reviewed the physicochemical properties of starch. BeMiller (1965) b r i e f l y discussed complexation of carbohydrates with organic substances. A n a l y t i c a l procedures for determining starch s o l have been described by Launer (1963) and  Whistler  et a l . (1965). Saito (1957) reported that anionic surfactants are r e a d i l y adsorbed on various nonionic polymers including starch and PVP,  e s p e c i a l l y above the c r i t i c a l micelle concentration. They  suggested that the polar part of the anion i s probably adsorbed on the oxygen atom i n the polymer. However, l i t t l e attention has been given to t h i s complexation phenomenon. Gray and Schoch (1962) studied the influence of various f a t t y adjuvants on the swelling behavior of several starches. Goudah et a l . (1965) and Mansour et a l . (1968) studied the s o l u b i l i t y c h a r a c t e r i s t i c s of benzoic a c i d derivatives i n the presence of various starch s o l s . They concluded that amylose i s the main complexing component i n starch. Hydroxyethyl starch (HES) has been hydroxyethylated  i s a waxy or branched starch which  to retard intravascular hydrolysis.  The substance i s being promoted as a plasma substitute. However, not much i s known about the physicochemical properties of  HES  solutions. Most publications deal with the in vivo behaviour i n animals and have been c o l l e c t e d by the National Academy of Sciences  (N.A.S. - N.R.C., 1965).  V. EXPERIMENTAL  1. Apparatus  (a) Spectrophotometers Beckman DU Beckman DU-2 Bausch & Lomb Spectronic 505 Beckman IR-10 (b) Fisher Accumet 310 pH Meter (c) International Equipment Company HN Centrifuge (d) American Laboratory S t e r i l i z e r (e) Westphal Balance (f) Blue M E l e c t r i c Company Refrigerated Bath (g) Cannon-Fensk Viscometer (Size 50) (h) Haake Thermoregulator (Type FE) (i) B-D Cornwall Continuous Pipetting Outfit with Swinny F i l t e r Adopter. MF-Millipore (WP) F i l t e r Paper (pore size, 0.65 ± 0.30 ji) was used with the apparatus.  2. Chemicals and Reagents  (a) Bishydroxycoumarin, U.S.P., (BHC). The melting point of the substance was 287-288 °C. The drug was obtained from Abbott Laboratories Limited, Montreal, Quebec and was  identified  by infrared spectrophotometry. (b) Polyvinylpyrrolidone (PVP). Plasdon C was purchased from the General A n i l i n e Corporation, New York, N.Y. The Kj. value range i s 28 to 32 (upper 15$ not higher than Kp, 41; lower 25$ not less than Kp, 16) and the molecular weight i s 40,000. To remove monomer, the PVP was extracted with  - 54 anhydrous ether i n a soxhlet apparatus for 24 hours. The PVP was then dried i n an oven, under vacuum and at a temperature of 35-40°C for 12 hours. May (1954) reported that the monomer could be extracted with methylene c h l o r i d e . It was found, however, that t h i s solvent dissolved the polymer and for t h i s reason, anhydrous ether was used to extract the monomer (see Higuchi et a l . , 1954b). The water content of PVP was determined from the loss i n weight after drying i n an oven at 110-115°C for 24 hours. Five such determinations were c a r r i e d out. The average value was 3.79$ (range, 0.28$). (c) Dextran-75. The sample was obtained from Abbott Lab., Montreal, Quebec. I t s molecular weight was 75,000 ± 15,000. I t s water content was determined i n the manner indicated above. The average value was 2.24$ (range 0.6$). (d) Human Serum Albumin (Cohn Fraction V, HSA). The albumin was obtained from Pentex Inc., Kankakee, 111. No loss i n weight on drying was detected. (e) Hydroxyethyl Starch (HES). The starch was obtained from McGaw Lab., Glendale, C a l i f . The company also supplied a 6$ solution containing 0.9$ sodium c h l o r i d e . (f) Potato Starch. The starch was purchased from Baker and Adamson Products, New York,  N.Y.  (g) Tris(hydroxymethyl)aminomethane ( T r i s ) . Reagent Grade. (h) 0.1N I  2  T.S., IN HC1, IN NaOH, U.S.P.  (i) Buffer Components. Reagent Grade. (j) Dimethylformamide(DMF). Reagent Grade.  3. Determination of Apparent pKa Values for BHC (a) Potentiometric T i t r a t i o n . Dissolve an accurately weighed sample of BHC (approximately 140 mg.) i n 200 ml. of DMF. To a 10.0 ml. aliquot of t h i s solution, add 2.5 ml. of 0.01N HC1, 30.0 ml. of DMF, and s u f f i cient water to make 100.0 ml. of s o l u t i o n . T i t r a t e the solution with 0.01N NaOH, using a glass-calomel combination electrode to follow pH changes. Perform a blank t i t r a t i o n . (b) Spectrophotometric Determination. Add 400 ml. of 0.01N NaOH to an accurately weighed sample of BHC (approximately 50 mg.). Shake u n t i l dissolved (approximately 2 hours) and d i l u t e to 500.0 ml. with 0.01N NaOH. D i l u t e t h i s solution to give a f i n a l concent r a t i o n of approximately 10 mg./L. A series of buffer solutions were prepared (pH range of 2.5 to 10.5) by u t i l i z i n g Perrin's buffer tables (Perrin,  1963).  The ionic strength of these solutions was 0.0i. The pH difference between buffer solutions was 0.5 u n i t s except i n the case of the buffers i n the 4.0 to 7.0 range. For these solutions, the pH i n t e r v a l between solutions was of the order of 0.2 u n i t s . Chloroacetic acid - KOH gave a 2.5 to 3.0 range; formic acid KOH, a 3.2 to 4.2 range; acetic acid - KOH, a 4.4 to 6.2 range; phosphates, a 6.4 to 7.6 range; T r i s - HC1, an 8.0 to 8.8 range; borates, a 9.0 to 9.7 range; and carbonates, a 10.0 to 10.5 range. A 2.5 ml. aliquot of the BHC stock solution (BHC concent r a t i o n of approximately 10 mg./L.) was diluted to 50.0 ml. with buffer. Visual observation indicated the BHC remained i n solution over the entire pH range. Actual pH values were determined by using a pH meter.  - 56 Absorbance values were determined by the trace analysis technique (Reilley and Crawford, 1955; Pernarowski, 1969) at 276.5 and 315 mu. For convenience, a BHC solution of 0.01N NaOH (BHC concentration of approximately 3 mg./L.) was used to adjust zero per cent transmittance (0$ T) at both wavelengths.  Absorbance  values were determined i n the following manner. Select wavelength and adjust instrument to read 0$ T. Place reference solution i n the l i g h t beam. Turn the selector switch on the Beckman DU spectrophotometer to the one position and zero the instrument using the dark current control knob. Return selector switch to the check p o s i t i o n . Place buffer solution i n the beam and set the instrument to 100$ T. Determine the absorbance of a solution containing the drug. Absorbance was plotted versus pH. The pH values at the i n f l e c t i o n points corresponds to the apparent pKa values. The mathematical basis f o r the c a l c u l a t i o n i s given i n Eq. 51.  pKa = pH - log  (ionized form) (unionized form)  (Eq. 51)  The s o l u b i l i t y of BHC i n buffer solutions of low pH i s the l i m i t i n g factor i n the determination of pKa^ value. However, the second pKa value can be more e a s i l y determined since the drug i s more soluble at higher pH values. In determining pKa.^ value, BHC concentration of approximately 0.5 mg./L. was used to a pH of 7. The absorbance values of such solution were determined at 276.5 mjx by the trace analysis method. Two more runs were made i n buffer-DMF systems which containing 1.0 and 0.8 mg. drug per l i t e r of solution containing 2.5 and 10.0$ v/v DMF, respectively.  - 57 The pKag value was determined by using solutions containing 10 mg. drug per l i t e r . The  spectrum-wavelength  curves of these solutions (pH range of 6.0 to 10.5) were recorded by using a B & L Spectronic 505 spectrophotometer. The procedure was repeated with solutions containing 5 and 20$ v/v DMF.  4. S o l u b i l i t y Measurements on BHC  (a) E f f e c t of pH on the S o l u b i l i t y of BHC. Buffer solutions (pH range of 6.4 to 8.0) were prepared by using T r i s and hydroc h l o r i c a c i d . The buffers were prepared i n the following manner. D i l u t e 50.0 ml. of IN HC1 with water. Insert glass-calomel electrodes into the solution and t i t r a t e with 0.5M T r i s solution to the desired pH. Dilute with water to make 250 ml. and redetermine the pH. The ionic strength of these buffers was 0.2. S o l u b i l i t y measurements were c a r r i e d out i n the following manner. Transfer 50 mg. of BHC to a 125-ml. glass b o t t l e and add 100 ml. of buffer. Tumble i n a water bath at 30°G at a r o t a t i o n a l speed of approximately 30 rpm. After 24 and 48 hours, withdraw aliquots f i l t e r e d through a Swinny f i l t e r adaptor and determine the absorbance at 286 mu, the isosbestic point for BHC. The solutions must be d i l u t e d prior to measurement, the extent of d i l u t i o n depending on the pH of the buffer. The f i l t e r i n g device must be maintained at 30°C i n order to prevent drug from p r e c i p i t a t i n g from s o l u t i o n . The pH values of the solutions were determined both before and after s o l u b i l i z a t i o n .  - 58 (b) The E f f e c t of Ionic Strength on the S o l u b i l i t y of Hydrochloric  acid solutions were prepared from IN HC1.  each of these solutions was or 0.5M  adjusted  BHC.  The pH of  to 7.2 with either  0.1  T r i s s o l u t i o n . The ionic strength of each of the  solutions was calculated from the amount of hydrochloric acid used to prepare the buffer. The s o l u b i l i t y of BHC buffer solutions was  i n these  determined i n the manner described i n the  previous section.  5. The  S o l u b i l i t y of BHC as a Function Macromolecule Concentration  (a) Starch Sol. A starch s o l was  of  prepared by a procedure  similar to that described by Goudah and Guth (1965). Prepare a s l u r r y , i n a 100-ml. beaker, containing 25 Gm. of potato starch with T r i s buffer (pH of 7.2; ionic strength, 0.2). Slowly add the s l u r r y to 350 ml. of hot buffer at 90-95°C. Agitate continuously for f i v e minutes i n a b o i l i n g water-bath. Transfer the starch s o l to an autoclave and heat for three hours at approximately 125°C. Remove the s o l from the autoclave, cool, and adjust the pH, i f necessary, to 7.2 with 0.1M T r i s . Make volume to 500 ml. with T r i s buffer. Calculate the starch concentration on the basis of the amount of starch weighed i n i t i a l l y . S o l u b i l i t y measurements were c a r r i e d out i n the following manner. Prepare starch solutions by d i l u t i n g the s o l with T r i s buffer. To each solution, add excess BHC. Tumble these preparations i n closed containers i n a 30°C water-bath for 40 hours. F i l t e r the solutions through a coarse sintered glass f i l t e r . Maintain the f i l t e r i n g apparatus at 30°C p r i o r to use. D i l u t e the samples, i f necessary, and determine the absorbance of the solution at 286 mu. Place a starch solution of the same concentration as that i n the t e s t s o l i n the reference beam of the B & L Spectronic 505 spectrophotometer.  - 59 (b) HES.  S o l u b i l i t y measurements were c a r r i e d out i n the  following manner. Prepare a 3$ HES solution by d i l u t i n g a 6$ solution containing 0.9$ sodium chloride with T r i s buffer. Add hydrochloric acid solution to obtain an ionic strength of 0.2 and a pH of 7.2. T y p i c a l quantities are l i s t e d below. 6$ HES i n 0.9$ IN HC1 IM T r i s Water  NaCl q.s. to  500.0 123.0 134.6 1000.0  ml. ml. ml. ml.  Calculate the ionic strength from the quantity of hydrochloric acid and sodium chloride i n the solution. Prepare a series of HES solutions containing various concentrations of HES by d i l u t i n g with T r i s buffer. Add an excess of BHC to each solut i o n and determine the s o l u b i l i t y i n the manner described i n the previous section. (c) PVP.  S o l u b i l i t y measurements were c a r r i e d out i n the  following manner. Prepare a 4$ PVP solution by d i s s o l v i n g the substance i n T r i s buffer (pH of 7.4; ionic strength, 0.15). D i l u t e t h i s stock solution with T r i s buffer to prepare solutions containing various concentrations of PVP. Add excess BHC to 25.0 ml. of each of these solutions. Transfer the sealed 30-ml. cent r i f u g e tubes to a 20°C water-bath and tumble for 40 hours. Transfer aliquots to a second series of centrifuge tubes. Centrifuge for 20 minutes at 2,500 rpm. Withdraw aliquots, d i l u t e with T r i s buffer and determine the absorbance at 304 mu using Beckman DU-2 spectrophotometer. Use T r i s buffer as the blank s o l u t i o n . Correct measured absorbance for PVP content and absorbance depression due to complex formation (a t y p i c a l c a l i b r a t i o n curve w i l l be shown l a t e r ) . (d) HSA.  The procedures for determining s o l u b i l i t y were  the same as that given above (a t y p i c a l c a l i b r a t i o n curve w i l l be shown l a t e r ) .  6. Spectrophotometric Analysis (a) Infrared Spectrum of BHC. Prepare a KBr p e l l e t and record the spectrum on a Beckman IR-10 spectrophotometer. (b) The Absorptivity Value of BHC. Weigh accurately 100.0 mg. of BHC and dissolve i n 500.0 ml. of 0.01N NaOH. D i l u t e aliquots with 0.01N NaOH to give f i n a l concentrations of 2 to 18 mg./L. Prepare f i v e such solutions from three d i f f e r e n t stock solutions. Record spectrum on a B & L Spectronic 505 spectrophotometer and calculate absorptivity values at 286 mu. Repeat the procedure but d i l u t e the stock solution with T r i s buffer of pH 7.4. Prepare ten solutions from each of four d i f f e r e n t stock solutions i n such a way that the f i n a l concentration varies from 4 to 12 mg./L. Check the pH of the solutions both before and after reading the absorbance on a Beckman DU and DU-2 spectrophotometers set at 304 mu. Calculate the absorptivity value at 304 mu. (c) Absorbance Values of HSA and PVP Solutions. Prepare 4$ stock solutions using T r i s buffer. Prepare a s e r i e s of solutions by d i l u t i n g the stock solution with T r i s buffer. Determine the absorbance on a Beckman DU-2 spectrophotometer set at 304 mu. Prepare c a l i b r a t i o n curves by p l o t t i n g absorbance versus the per cent concentration of macromolecule i n s o l u t i o n . (d) Determination of Changes i n Absorbance at a Fixed BHC Concentration as a Function of Macromolecule Concentration. Shake an excess of BHC with T r i s buffer for one hour. F i l t e r through a Milipore f i l t e r paper. To each of a s e r i e s of 50-ml. volumetric flasks, add d i f f e r e n t amounts of 4$ macromolecule solut i o n and a constant amount of the BHC stock solution. To two flasks, add only BHC stock solution. Dilute to volume with T r i s buffer.  - 61 (e) Determination of the Changes i n Absorbance at a Fixed Macromolecule Concentration as a Function of BHC Concentration. Add varying q u a n t i t i e s of the BHC stock solution (see previous section) to fixed amounts of the macromolecule* Dilute and record absorbance as indicated above. Calculate the t h e o r e t i c a l absorbance value of each solution from the amount of BHC i n s o l u t i o n . Subtract the observed value from the t h e o r e t i c a l value and plot the difference against the observed value. Prepare solutions to contain 0.02$ PVP and use d i f f e r e n t BHC concentrations. Data from (d) above and that obtained here i s used to prepare the c a l i b r a t i o n curves.  7. Equilibrium D i a l y s i s  (a) D i a l y s i s C e l l . A diagram of a d i a l y s i s c e l l i s shown i n i n Figure 9. C e l l s of t h i s type are described i n d e t a i l by Patel and Foss (1964). Total volumes i n the d i a l y s i s c e l l may  be  changed by adding additional spacers on either side of the membrane. (b) Preparation of the Cellophane Membrane. Immerse dialyzer tubing ( f l a t width i s equal to 1.735 inches) i n d i s t i l l e d water for several minutes. Unfold, cut to size, and transfer to 1.5 l i t e r s of water. Shake the container for approximately ten hours and, at hourly i n t e r v a l s , replace the water with fresh water. Store i n a r e f r i g e r a t o r at 0-5°C and use within two weeks. Prior to use, immerse the membrane i n T r i s buffer. Remove the membrane from the buffer, drain but do not allow drying to occur, and attach the membrane to the c e l l .  - 62 -  Rubber 'O' Ring  Cellophane Membrane  Figure 9. Diagram of a plexiglas block and an assembled d i a l y s i s c e l l . (c) D i a l y s i s Equilibrium and Membrane Binding of  BHC.  Prepare three solutions containing varying concent r a t i o n s of BHC. Transfer a portion of the solution to one side of. the d i a l y s i s c e l l and dialyze against T r i s buffer i n the other compartment. Remove aliquots and analyze for BHC. Continue the process u n t i l the BHC concentration i n both compartments i s the same for at least two sampling periods. On the basis of the data obtained, determine the time required for e q u i l i b r a t i o n i n the c e l l . Membrane binding of BHC  was determined i n the following  manner. Transfer a s o l u t i o n containing a known quantity of BHC to the c e l l . Dialyze u n t i l both compartments contain the same amount of drug. Calculate the t o t a l amount of drug i n s o l u t i o n . Subtract t h i s value from the amount of drug added to the c e l l . Calculate the per cent recovery and estimate membrane binding. In experiments involving macromolecules, a control c e l l containing no macromolecule was maintained under the conditions s p e c i f i e d for that study. Thus additional information on membrane binding was obtained throughout the i n v e s t i g a t i o n .  - 63 (d) Dynamic D i a l y s i s . A diagram of the apparatus used i s shown i n Figure 10. The procedure has been used by Meyer and Guttman (1968b) to study protein binding with drugs. Add excess of BHC to a solution containing a macromolecule. Shake for four to f i v e hours. F i l t e r the solution through M i l l i p o r e f i l t e r paper. Analyze for BHC i n a s i m i l a r manner described previously (see p. 59). To the cellophane bag, transfer 50.0 ml. of the macromolecule solution nearly saturated with BHC. Add 500.0 ml. of T r i s buffer to the apparatus. S t i r t h i s external solution with a magnetic s t i r r e r . S t i r the macromolecule solut i o n within the bag i n the manner indicated i n Figure 10. At s p e c i f i e d times, remove 50.0 ml. of solution from the main part of the apparatus. Immediately replace t h i s s o l u t i o n with the same volume of T r i s buffer. Calculate the amount of drug i n the cellophane bag by subtracting the amount of drug removed from i n i t i a l amount added to the system.  Motor Holes for Introduction and Removal of Solution Rubber Stopper To a Constant-Temperature Water Bath BHC-Macromolecule Stock Solution t  Cellophane Bag T r i s Buffer Magnetic S t i r r i n g Device  Figure 10. A set-up for dynamic d i a l y s i s method.  - 64 (e) Permeability of PVP through the Cellophane Membrane. Prepare PVP solutions i n T r i s buffer (0.1 to 0.4$). Transfer aliquots to one of the compartments i n the d i a l y s i s c e l l and dialyze against T r i s buffer for 40 hours. Analyze the T r i s buffer compartment for PVP, following the method of Campbell et a l . (1953). To a 10.0 ml. of sample of the solution, add 0.1 ml. of 0.1N iodine i n 0.1M potassium iodide s o l u t i o n . After 12 to 15 minutes, read the absorbance of the solution on a Beckman DU-2 spectrophotometer set at 500 mu. D i l u t e the iodine t e s t solution to 10.0 ml. of T r i s buffer and use as the blank s o l u t i o n . Calculate concentration from a c a l i b r a t i o n curve based on solutions containing known amount of PVP. (f) .Equilibrium D i a l y s i s Studies. Prepare a series of BHC and BHC-macromolecule solutions (see p. 63). Determine BHC concentration i n these solutions (see p. 59). Transfer 20.0 ml. (or 40.0 ml., depending on compartment volume) of the BHC solution to the d i a l y s i s c e l l and dialyze versus the BHC-macromolecule solution or macromolecule solution containing no BHC. Also dialyze BHC-macromolecule solution against T r i s buffer. In one run of experiment, use s i x to twelve c e l l s depending on the experiment and the number of control c e l l s required. Attach the c e l l s to the shaft of the water bath and tumble at 30 rpm for 40 hours (a diagram of the apparatus i s shown i n Figure 11). Remove aliquots from the BHC compartment, store i n a water bath at 20°C, and analyze for BHC content. D i l u t e the aliquots, i f necessary ( d i l u t i o n factors of from 1:1 to 1:10), with T r i s buffer. Carry out at least two analyses on each aliquot taken from the BHC compartment. PVP studies were c a r r i e d out at 10, 20, 30, and 40°C and at PVP concentrations  of 0.1,  0.2,  and 0.4$.  Similar HSA  concen-  t r a t i o n s were used but studies were c a r r i e d out at 20 and 40°C only. HES of 0.5$  studies were c a r r i e d out at 30°C and at a  HES.  Since HES  concentration  does not s i g n i f i c a n t l y increase  s o l u b i l i t y , BHC-HES stock solutions were not used i n the  BHC  -  6 5  -  Figure 11. A water bath with a tumbler used i n s o l u b i l i t y and equilibrium d i a l y s i s studies.  i n v e s t i g a t i o n . Dialyses were based on BHC and HES  solutions  only. Temperature fluctuations during d i a l y s i s were less than 0.1°C.  8. Viscometric Analysis  (a) C a l i b r a t i o n of the Viscometer. Calibrate seven Cannon-Fenske Viscometers i n the manner described i n an A.S.T.M. b u l l e t i n (1966b). Use freshly d i s t i l l e d water as the reference standard. V i s c o s i t y and density values for water at various temperatures are given i n the l i t e r a t u r e (Bull, 1964b; Weast, 1969). With respect to the v i s c o s i t y values for water, the values from B u l l ' s textbook were used i n the c a l c u l a t i o n . The viscometers were f i l l e d and allowed to stand i n a water bath for at least two hours. Flow time was determined to 0.1 seconds. At least f i v e determinations were made for each viscometer. The average flow time for each viscometer was substituted into Eq. 50 along with the two water constants and the instrument constant at the s p e c i f i e d temperature was c a l c u l a t e d . Temperature v a r i a t i o n i n any experiment was less than 0.1°C. (b) Density Measurement. Calibrate a Westphal balance at each temperature with d i s t i l l e d water. Maintain the s p e c i f i e d temperature by immersing the glass cylinder of the balance i n a water bath (800 ml.) set to the desired temperature. Make c a l i b r a t i o n both before and after measurement of PVP solutions. The reprod u c i b i l i t y was 0.0002 density u n i t s . Prepare a series of PVP solutions and determine density. Note that the apparatus measures apparent s p e c i f i c density but, for the purposes of the experiment, the two terms were considered to be synonymous. (c) V i s c o s i t y Measurements of PVP Solutions i n the Presence of BHC. Dissolve PVP i n a BHC stock solution to y i e l d a f i n a l PVP concentration of 4$. D i l u t e with BHC stock solution to y i e l d solutions which contain the same quantity of BHC and from 0.2 to 3.0$ PVP.  -  67 -  Prepare seven such solutions. Determine flowtimes i n the manner described i n (a) above. Substitute the mean flow-time, the density value, and the instrument constant into Eq. 50 and c a l culate, the v i s c o s i t y . Calculate r e l a t i v e v i s c o s i t y by dividing each v i s c o s i t y value by that of the BHC solution without PVP.* Calculate reduced and s p e c i f i c v i s c o s i t i e s by using Eq. 44 and 45. Determine i n t r i n s i c v i s c o s i t y and Huggins parameter from the intercept and slope of the graph obtained by p l o t t i n g reduced v i s c o s i t y versus PVP concentration.  *  This implies that the r e l a t i v e v i s c o s i t y of the BHC solution was considered to be unity (see Eq. 43).  V I . RESULTS AND DISCUSSION  1. Intramolecular Hydrogen Bonding i n BHC  The infrared spectrum of BHC  i s shown i n Figure 12 and i s  similar to that published by French and Wehrli  (1965).  The v i b r a t i o n a l frequency corresponding to an -OH occurs at approximately 3500 Cm. * -  usually observed at 7000 Cm.  stretch  The f i r s t overtone i s  I f chelation occurs with the  -1  carbonyl group i n phenols through the formation of an i n t r a molecular hydrogen bond, the former frequency value becomes less but the extent of change depends on the strength of the hydrogen bond. The band i s broad and, at times, very weak (Nakanish,  1964;  Dyer, 1965). With BHC a broad absorption band occurs at approximately 3100 Cm.~*  and t h i s may  indicate an intramolecular  hydrogen bond. The molecular model for BHC  suggests the possible formation  of two intramolecular hydrogen bonds between the carbonyl group at Cg and the -OH group at C^t (or vice versa). S o l u b i l i t y data (see l a t e r ) appear to support t h i s conclusion. These two internal eight-membered chelations (see Figure 13) appear to r e s t r i c t the r o t a t i o n of the one-sided moiety of the methylene bridge and f i x i t s configuration. The most probable three dimensional structure of BHC i s shown i n Figure 14.  - 70 -  F i g u r e 13. Chemical s t r u c t u r e o f BHC showing two i n t r a m o l e c u l a r eight-membered c h e l a t i o n s .  F i g u r e 14. Three dimensional s t r u c t u r e o f BHC Black, red, and white bars r e p r e s e n t carbon, oxygen, and hydrogen atoms, r e s p e c t i v e l y . The b l a c k bar below the model has a l e n g t h of 4 A  2. Apparent pKa Values of BHC  If a dibasic acid i s symmetrical with respect to the two ionizable hydrogen atoms and i f the negative charge on the f i r s t ionizable group i s s u f f i c i e n t l y removed from the remaining hydrogen atom, then the e f f e c t on the second group i s negligible and the f i r s t i o n i z a t i o n constant i s approximately four times as large as the second constant (Robinson and Stokes, 1968). In general, however, the e f f e c t of the negative charge does make i t more d i f f i c u l t for the second hydrogen to i o n i z e . For example, for  oxalic acid, the f i r s t constant i s approximately 1000 larger  than the second. On the other hand, there i s only a s i x f o l d difference for azelaic acid (C00H(CH2)7COOH). Kirkwood and Westheimer (1938), under reasonable assumptions with respect to the size and configuration of the molecules, discussed the e f f e c t of e l e c t r o s t a t i c i n t e r a c t i o n between ionizable groups on the cosecutive i o n i z a t i o n constants.* Although pKa values have been reported for BHC (Burns and others, 1953; Nagashima and others, 1968a), the uncertainties associated with the determination and the absence of values for the t o t a l i o n i z a t i o n process made i t necessary to study the i o n i z a t i o n behaviour of t h i s drug. The molecule has two ionizable hydrogen atoms and both are involved i n the i n t e r n a l hydrogen bond. As shown i n Figure 15, the negative charge on the oxygen *  This study formed the basis for the theory of multiple e q u i l i b r i a i n the complexation process i n the presence of e l e c t r o s t a t i c interactions between binding s i t e s (see p. 11).  - 72 -  Figure 15. Resonance structure of BHC after the f i r s t i o n i z a t i o n .  atom produced by the f i r s t i o n i z a t i o n i s expected to make the oxygen atom i n the carbonyl group more negative by an e l e c t r o n i c s h i f t through the conjugated chain. This increase i n electronegativity apparently increases i t s a b i l i t y to form hydrogen bond with the remaining strengthened  hydrogen atom (Pauling, 1967). The  hydrogen bond w i l l , therefore, make i t more d i f f i -  c u l t f o r the second hydrogen atom to ionize. For t h i s reason, and the fact that the s t a t i s t i c a l considerations of two successive i o n i z a t i o n constants would appear to be sound when applied to long and t h i n molecules but not to shorter and more spherical molecules (Robinson and Stokes, 1968) l i k e BHC, the r a t i o of the f i r s t to the second i o n i z a t i o n constants f o r BHC should be at  least 1000:1. I t i s rather surprising, therefore, that only  single i o n i z a t i o n constants for BHC have been reported i n the literature. The pK values of acids and bases may be determined i n many d i f f e r e n t ways (Albert and Serjeant, 1962). BHC i s , however, very insoluble i n a c i d i c solution and the basic t i t r i m e t r i c approach  - 73 i n aqueous media cannot be u t i l i z e d . However, s o l u b i l i t y can be increased by u t i l i z i n g an organic solvent - water mixture and potentiometric and spectrophotometric t i t r a t i o n s can be c a r r i e d out i n such systems. Alcohol, dioxane, and DMF have been recommended i n such systems (Parke and Davis, 1954). Attempts were made, therefore, to measure the apparent pKa values of BHC i n DMF-water systems containing varying quantities of DMF. These values may be extrapolated to zero per cent DMF i n order to determine the value i n water alone. Garrett (1963) studied the v a r i a t i o n of pKa values of t e t r a c y c l i n e s i n a DMF-water system i n a similar manner. A solution containing 706.0 mg. BHC per l i t e r of DMF was prepared. A 10.0 ml. aliquot of t h i s solution was transferred to a 100 ml. volumetric f l a s k . When 2.5 ml. of 0.01N HC1 were added to the flask, the solution became turbid but the t u r b i d i t y disappeared when 30.0 ml. of DMF was added. Addition of water to 100.0 ml. produced no further t u r b i d i t y (see p. 55). I t became necessary to consider the following factors when preparing BHC sample solutions f o r t i t r a t i o n ; pH of the solution before t i t r a t i o n , strength of t i t r a n t , concentration of BHC, size of sample, and per cent DMF. The r e s u l t s i n Figure 16 were obtained when a 100.0 ml. sample of BHC (70.6 mg. per l i t e r of a 40$ solution of DMF i n water) solution containing 2.5 ml. of 0.01N HC1 was t i t r a t e d with 0.01N NaOH. The blank t i t r a t i o n curve was obtained by t i t r a t i n g 100 ml. of 40$ DMF solution containing 2.5 ml. of 0.01N HC1 with 0.01N NaOH. The blank t i t r a t i o n curve was subtracted  u X)—-•  4  <  •  1  •  1  5  6  7  8  9  PH  10  - 75 volumewise from the BHC curve and the difference converted to equivalents of OH ion per mole of BHC  and plotted as a function  of pH. The pH range covered i n these t i t r a t i o n s was Because BHC  from 3.5 to 10.  i s insoluble i n acidic media, t i t r a t i o n s could not  be c a r r i e d out below pH 3.5 and no potentiometric break i s observed. One endpont region (pH 5 to 8) i s present; the second region f a i l s to appear on the curve. It i s impossible, therefore, to report quantitative pKa values for BHC  i n t h i s system. However,  the difference curve does show the presence of two pKa values, the one below pH 4, the other, above pH 9. No further attempts were made to determine pKa values i n t h i s way because i t would have been necessary to increase DMF  concentrations and hence  operate much further from the zero per cent DMF The low s o l u b i l i t y of BHC  point.  i n aqueous media caused similar  d i f f i c u l t i e s during the spectrophotometric values. The s o l u b i l i t y of the unionized BHC  determination of pKa (AHg form) i s approxi-  mately 0.5 mg./L. (1.5 x 10"6 mole/L.). At these concentrations, the spectral c h a r a c t e r i s t i c s of the unionized species cannot be obtained. However, the change from the AH be followed. These spectrophotometric  -  to the A  form can  curves are shown i n  Figure 17. Each s o l u t i o n contained 10.0 mg. BHC  per l i t e r of  the s p e c i f i e d buffer. As the pH decreases, an absorption maximum appears at 276.5 mu; at 315  mu.  with increasing pH, a peak i s observed  330  320  310  300  290 Wavelength, mu  280  270  260  250  If a similar spectral change occurs when AH  2  i s converted  to AH~ and H~, the most suitable wavelengths for analysis would appear to be 276.5 or 315 mu. A s e r i e s of solutions were prepared to contain 0.5 mg. BHC per l i t e r of buffer. To prevent spectral d i s t o r t i o n , the ionic strength of each solution was kept as low as possible (Sager and others, 1945). Perrin (1963) describes buffers of t h i s type. They have a low ionic strength (0.01), cover the 2.2 to 11.6 pH range, and absorb very l i t t l e  energy  i n the u l t r a v i o l e t region of the electromagnetic spectrum. The absorbance of a solution containing small quantities of drug can be determined  i n one of two ways. F i r s t , a long  spectrophotometrie c e l l may be used i n the determination and, secondly, precision spectrophotometry  (trace analysis) may  used to analyze the solutions. Sager et a l . (1945) used a  be 5-Cm.  c e l l to determine the pKa values of some esters of p-hydroxybenzoic a c i d . The s o l u b i l i t i e s of these esters ranged from to 10"^ molar. Precision spectrophotometry ford, 1955;  Pernarowski,  10"  4  (Reilley and Craw-  1969) has not been used to determine  such values. By using the p r i n c i p l e s inherent i n precision spectrophotometry, the absorbance values of a series of BHC  solutions were  determined at 276.5 and 315 mu and plotted versus pH values. These plots are shown i n Figure 18. Absorbance values did not exceed 0.1 absorbance unit even though the absorbance scale  was  expanded to a maximum by appropriate use of reference standards. A s l i g h t t u r b i d i t y was observed i n those BHC  solutions i n  0.01N  HC1. No t u r b i d i t y was observed i n the solutions with higher pH  .09  .08 -  Figure 18. Absorbance-pH curves for BHC. C i r c l e s and t r i a n g l e s indicate readings at 276.5 and 315 mu, respectively. Closed and open symbols were obtained from separate experiments.  .07 "  .06  A .05  A  .04 -  .03  A  "  .02 .  O .01 •  - 79 values but undetected over-saturation may  be the cause of the  scattered values below pH 4 i n Figure 18. Absorbance changes show c l e a r l y the two i o n i z a t i o n steps. The calculated values from the curves i n Figure 18 are 4.6 and 4.2  for pKa^ and  7.75  and 7.9 for pKag. The former values were obtained from absorbance readings at 276.5 mu; Average values are 4.44  the l a t t e r , from readings at 315  mu.  and 7.83.*  Figure 18 also shows that the spectral changes due to the f i r s t i o n i z a t i o n i s apparently d i f f e r e n t from those associated with the second i o n i z a t i o n . At both 276.5 and 315 mu,  absorbance  increases as pH increases from 3 to 5 (compare with Figure 17). This implies that no isosbestic point occurs between these two wavelengths on the f i r s t i o n i z a t i o n process. The pKa value may 2  be determined d i r e c t l y by measuring  absorbance values of buffered solutions of BHC, has a reasonable  because  BHC  s o l u b i l i t y at that pH range (see l a t e r ) .  Solutions containing 10.0 mg. of drug per l i t e r of buffer, 5$ i n buffer, and 20$ DMF  i n buffer were prepared.  DMF  Absorbance-  wavelength curves were recorded on B & L Spectronic 505  spectro-  photometer. Figure 19 shows spectral changes occurred i n the presence of 20$  DMF.  However, at low pH values, drug s o l u b i l i t y must be increased by the addition of DMF.  This solvent does not a f f e c t the pH of  the buffer and absorbs l i t t l e energy at the two a n a l y t i c a l wavelengths. Solutions containing 0.5 mg. of drug per l i t e r of buffer, 1.0 mg. *  of drug per l i t e r of 2.5$ DMF  i n buffer, and 0.8  mg.  The mean of 4.2 and 4.4 i s 4.44 because a log scale i s being used. Antilog values for the pair are added. An average value i s the log of the value obtained divided by two. S i m i l a r l y the average of 7.75 and 7.9 i s 7.83.  F i g u r e 19. U l t r a v i o l e t s p e c t r a showing the second d i s s o c i a t i o n of BHC i n 20$ v/v DMF. S o l u t i o n pH v a l u e s a r e : 1, 11.30; 2, 10.75;  .80 .75 t-70 .65 .60  1.55 > |.50 w o  cr P  .45 g © .40  • .35 " .30 .25 4.20  340  330  320  310  300  290 Wavelength, mu  280  270  260  250  - 81 of drug per l i t e r of 10$ DMF  i n buffer were used i n determining  pKa^ value. Absorbance values of these solutions were measured at 276.5 mu by means of precision spectrophotometry  (see p. 56).  Results are shown i n Figure 20. The r e l a t i o n s h i p between pKa values and per cent DMF  i s shown i n Figure 21. Values obtained  from t h i s data are 4.4 for pKa^ and 8.2 for pKa . The 2  constant for DMF  i s reported to be 36.7  dielectric  (Leader, 1951). As the  solvent becomes less polar by addition of DMF  the f i r s t  ionization  occurs more e a s i l y and the weak acid behaves as a stronger acid i n t h i s system. In aqueous media, the BHC molecule i s believed to e x i s t i n the enol form which i s more acidic than keto form (see Figure 22).*  The enol-keto r a t i o would be greater when the  p o l a r i t y of the environment decreases (Gould, 1963). The increase i n the mole f r a c t i o n of enol form may be partly responsible for the increased a c i d i t y of BHC  i n the DMF  solvation around the monoionized  system. The extent of  BHC molecule would be expected  to decrease as the p o l a r i t y of the solvent decreases ( i . e . , removal of charges become more d i f f i c u l t ) . As a r e s u l t , the second i o n i z a t i o n constant increases s l i g h t l y when DMF content i s i n creased. These r e s u l t s and those obtained by potentiometric t i t r a t i o n (see p. 75) indicate pKa values of the same magnitude. *  After t h i s work had been completed, a study on the structure of BHC and a related compounds appeared i n the l i t e r a t u r e . Hutchinson and Tomlinson (1969) proposed a hydrogen-bonded structure for BHC, i d e n t i c a l to ours (Figure 13), from a study of nuclear magnetic resonance and infrared spectra. They also made the conclusion that i n tautomeric structures similar to Figure 22 4-hydroxycoumarin i s preponderantly i n the enolic form both i n the s o l i d state and i n solution i n polar solvent. We had further inferred that enol:keto r a t i o increases with decrease i n p o l a r i t y of solvent. We are g r a t e f u l to Mr. J . Coates for bringing t h i s paper to our attention.  Absorbance at 276.5 mu Measured by Precision Spectrophotometry  ro ,  CM  •a  cn ..  cn  Absorbance at 315 mu CM  —»-  —  cn  cn —t—  03 —»-  3  \ cn  o  01 CD  CD  P rt3  a  ro ->i  a cn f t . . P 3 ro • cn cn a  CD  o  CD 3  S3 .  0) ><!  IO I—  1  01  o  8  3 •  rt 01 O • H»  co  cd  CD f  p P 3 TO P M 3 CD CO  a  F i g u r e 21. Apparent pKa v a l u e s o f BHC as a f u n c t i o n of per cent DMF. V a l u e s were o b t a i n e d s p e c t r o p h o t o m e t r i c a l l y .  - 84 -  O  O Keto Form  Figure 22. Tautomerism of a BHC molecule.  From a l l of the data obtained herein, i t i s reasonable to assign a pKaj value of 4.4 and a pKa value of 8.0 to BHC. By 2  means of non-logarithmic linear t i t r a t i o n curves, Levy (1969) obtained values of 4.6 and 7.7. However, i n order to obtain these values, he assumed a s o l u b i l i t y of 0.2 mg./L. for the unionized BHC.  3. S o l u b i l i t y  (a) E f f e c t s of pH,  Ionic Strength and Buffer Components.  The e f f e c t of pH on the apparent s o l u b i l i t y of BHC  at 30°C i s  shown i n Figure 23. A Tris-HCl buffer system (ionic strength, was used i n the determinations.  0.2)  S o l u b i l i t y increases r a p i d l y with  increase i n pH from 7.6 to 8.0. BHC  appears to be unstable i n  0.1N  NaOH solutions, since they turn yellow a f t e r standing at room temperature for three days. Low aqueous s o l u b i l i t y strongly indicates that the groups at C^ and C4«  hydroxyl  do not contribute to s o l u b i l i t y by forming  hydrogen bonds with water. A p a r a l l e l s i t u a t i o n e x i s t s with c y l i c a c i d i n which the -OH  sali-  group i s involved i n an intramolecular  hydrogen bond (Martin, 1968b). S o l u b i l i t y i s expected to increase a f t e r the f i r s t i o n i z a t i o n since an intramolecular hydrogen bond, in which the ionized hydroxyl group has been involved, i s no longer present. However, a marked change i n s o l u b i l i t y (such as observed around pH 8) i s not expected, because the molecule i s s t i l l more or l e s s r i g i d l y fixed by one remaining internal hydrogen bond to allow l i t t l e interaction-with water. After complete i o n i z a t i o n , a s i g n i f i c a n t ion-dipole i n t e r a c t i o n between BHC  and water can  be expected and t h i s w i l l r e s u l t i n a marked increase i n s o l u b i l i t y . The e f f e c t of ionic strength on the apparent s o l u b i l i t y of BHC at  i s shown i n Figure 24. The pH of each s o l u t i o n was maintained 7.2.  If neutral s a l t s are used to a l t e r ionic strength, organic  molecules w i l l , i n general, show a decreased water s o l u b i l i t y  - 86 -  Figure 23. Effect of pH on the apparent s o l u b i l i t y of BHC at 30°C (ionic strength, 0.2). S o l u b i l i t y was determined after 24 ( O ) and 48 (•) hours.  — i —  7.4  pH  7.6  7.8  8.0  - 87 -  Figure 24. Effect of ionic strength (chloride ion) on the apparent s o l u b i l i t y of BHC at 30 C (pH, 7.2), after 24 ( O ) , 48 ( • ) , and 72 ( A ) hours. 8  - 88 because of the 'salting out' e f f e c t . *  The r e s u l t s for BHC do  not follow t h i s pattern. However, the ionic strength of these solutions was governed by HC1 content and the study may, therefore, be nothing more than the s p e c i f i c e f f e c t of chloride ion on the s o l u b i l i t y of BHC. According  to Frank and Wen (1957),  halide ions except f l u o r i d e ion are water structure  breakers.  Fewer 'icebergs' would be expected around the chloride ion and t h i s should enhance BHC-water i n t e r a c t i o n , i . e . , the number of unbound water molecules i s greater i n the presence of chloride ion and t h i s provides more c a v i t i e s for the solution of the hydrocarbon (Nemethy and Scheraga, 1962b; Mohammad, 1965). A l l drug-macromolecule interactions were studied i n buffer systems. Buffer composition i s , therefore, an important part of the i n v e s t i g a t i o n . For example, the ionic strength of the buffer a r i s e s from the hydrochloric acid added to the buffer. This implies that buffer with a low ionic strength contains l e s s buffer component. Consequently, the buffer capacity of the T r i s buffer ( f3) i s lower at low ionic  ' Ka Ka C C (H+) (IT)  d(b) B '  =  « 2.303 d(pH)  strength.  (Ka + ( H * ) ]  2  + (H*-)  +  (OH")  (Eq. 52)  * Hydrocyanic acid and glycine, which are a l i k e i n that t h e i r aqueous s o l u t i o n have higher d i e l e c t r i c constants than pure water, become more soluble i n the presence of s a l t s . The data herein may r e f l e c t such a 'salting i n ' e f f e c t (Edsall and Wyman, 1958b), but evidence for t h i s i s lacking because the d i e l e c t r i c constant of the BHC solution was not determined.  - 89 In Eq. 52, Ka and C are the i o n i z a t i o n constant of the buffer component and i t s concentration, respectively, and b i s the number of gram equivalents of a l k a l i added to one l i t e r of buffer solution (Bates, 1961). As shown i n Eq. 52, buffer capacity i s a function of pH and the concentration of the buffer component. These r e l a t i o n s h i p s between buffer capacity and pH for T r i s buffer i s shown i n Figure 25. As expected, maximum buffer capacity i s obtained at a pH equal to the pKa of T r i s . The value reported i n the l i t e r a t u r e i s 8.075 (Bates, 1961). The T r i s buffer used i n t h i s i n v e s t i g a t i o n i s 0.063M with respect to T r i s and 0.15N  with  respect to hydrochloric a c i d . The buffer capacity i s 0.021. Buffer components may  i n t e r a c t with macromolecules. T r i s ,  however, has an intramolecular hydrogen bond as shown i n Figure (Benesch and others, 1955)  26  and should not react s i g n i f i c a n t l y  with macromolecules. For example, Higuchi and Kuramoto (1954c) claimed that complexation of s a l i c y l i c acid with PVP i s less than observed between p-hydroxy-benzoic acid because a strong i n t e r n a l hydrogen bond e x i s t s i n the former drug (see pp. 49-50). Chloride ions, on the other hand, may  also i n t e r f e r e with BHC-macromolecule  i n t e r a c t i o n . Klotz (1953a) b r i e f l y reviewed the binding of chloride ion to serum albumin. However, these e f f e c t s were neglected i n t h i s investigation because competitive  i n t e r a c t i o n of chloride ion  with the macromolecule would be far l e s s than that usually observed drug-macromolecule i n t e r a c t i o n s . correction for competitive  A t y p i c a l example of  e f f e c t of buffer components may  be  found i n the paper published by Klotz and Urquhart (1949c). Using Eq. 33, they corrected for the contribution of phosphate to the i n t e r a c t i o n between methyl orange and albumin.  - 90 -  Figure 25. Buffer value of T r i s buffer solutions i n the pH range from pKa - 1.3 to pKa + 1.1. Total T r i s concentrations are 0.2M (a), 0.1M (b), and 0.05M ( c ) . See Bates (1961). Open c i r c l e represents the buffer capacity of T r i s buffer used i n t h i s investigation.  - 91 -  Figure 26. Two dimensional structure of T r i s showing intramolecular hydrogen bonds. See Benesch and Benesch (1955).  (b) Effect of Macromolecule - i ) Starch Sol and HES. The binding tendency of BHC to potato starch s o l and HES i n T r i s buffer (pH, 7.2; ionic strength, 0.2) i s i l l u s t r a t e d i n Figure 27. A l l solutions were e u i l i b r a t e d f o r at least 40 hours at 30°C. Concentration r a t i o of t o t a l to free BHC i s plotted against macromolecule concentration. When linear r e l a t i o n s h i p i s observed the slope (r value i n Eq. 6) can be an approximation of binding capacity (see p. 4 and 46). I f the drug and macromolecule do not interact, a straight l i n e with zero slope passing through the unity should be obtained.  This type of presenting s o l u b i l i t y  and equilibrium d i a l y s i s data has been frequently used i n the l i t e r a t u r e (see, for example, Higuchi and Kuramoto, 1954b). Results i n Figure 27 show that approximately  60 and 72$ of the  t o t a l BHC e x i s t ;in the; complexed form when the drug i s e q u i l i brated with 3$ starch s o l and HES solutions, respectively.  - 92 -  Figure 27. E f f e c t of various concentrations of HES ( A ) and potato starch s o l ( • ) on the apparent s o l u b i l i t y of BHC at 30°C i n T r i s buffer (pH, 7.2; ionic strength, 0.2)  4.5  4.0  3.5  3.0  2.5  2.0 J  1.5  1.0  % Macromolecule  - 93 -  Figure 28. Chemical structure (a) and molecular configuration (b) of HES.  The chemical structure of HES i s shown i n Figure 28 (a). Its molecular configuration, i l l u s t r a t e d i n Figure 28 (b), i s believed to be similar to that of amylopectin i n that both molecules have many d i l a t e d branches. The average number of hydroxyethyl groups per glucose unit has been reported to be 0.7 to 0.9 (N.A.S. - N.R.C., 1965). Potato starch consists of 20$ amylose and 80$ amylopectin (Greenwood, 1956) whereas HES has no amylose-type configuration. It has been reported that amylose i s more l i k e l y to undergo a side-by-side association with organic molecules than amylopectin (BeMiller, 1965; Goudah and Guth, 1965; Mansour and Guth, 1968). The starch s o l should, therefore, have a s l i g h t l y higher a f f i n i t y  - 94 for BHC than HES and t h i s i s confirmed by the r e s u l t s shown i n Figure 27. Unfortunately, the limited r e s u l t s obtained do not lend themselves to an interpretation of mechanisms. Mansour and Guth (1968) assumed that benzoic acid, derivatives of benzoic acid, and sorbic acid form complexes with starches i n a manner similar to that observed for the starch-iodine  and starch-  n-butanol systems. There appears to be an entrapment of the 'guest' molecule i n the <*-helical structure of amylose with a supplimentary s t a b i l i z a t i o n of dipole-dipole  interactions  (Stein, 1948). A s i m i l a r mechanism might explain BHC-starch and BHC-HES interactions. (b) E f f e c t of Macromolecule - i i ) HSA and PVP. The apparent s o l u b i l i t y of BHC i n T r i s buffer  (pH, 7.4; ionic strength, 0.15)  at 20°C as a function of either HSA or PVP concentration i s i l l u s trated i n Figure 29. Solutions were analyzed for BHC spectrophotometrically. due  Corrections  were made for depression of BHC absorbance  to complex formation i n a manner described  elsewhere i n t h i s  thesis (see pp. 59-60 and 104-105). In 1.0$ HSA or PVP solution, apparent s o l u b i l i t y i s enhanced by approximately 5.4 and 21.5 times, respectively. In these solutions, approximately 84.4 and 95.6$ of t o t a l BHC e x i s t i n the complexed form. In low macromolecule concentration range (up to 1.0$ for HSA and 0.4$ for PVP),  linear r e l a t i o n s h i p i s  observed between change i n s o l u b i l i t y and macromolecule concentration.  When BHC-PVP binding  a f f i n i t y i s compared with that  for other drugs (see Table 1), t h i s i n t e r a c t i o n i s unusually strong. Interaction mechanisms w i l l be extensively  discussed l a t e r .  - 95 55  F i g u r e 29. E f f e c t o f HSA ( c i r c l e s ) and PVP ( A ) c o n c e n t r a t i o n s on the apparent s o l u b i l i t y o f BHC i n T r i s b u f f e r (pH, 7.4; i o n i c s t r e n g t h , 0.15) at 20°C. Analyses f o r BHC c o n c e n t r a t i o n were c a r r i e d out a f t e r 40-hour s o l u b i l i z a t i o n . Each t r i a n g l e i s average of t h r e e d i f f e r e n t e x p e r i ments. Open and c l o s e d c i r c l e s r e p r e s e n t separate experiments.  50  45  40  35  30  25 P 4->  S  20  15 -  10  0  0  .5  1.0  1.5  2.0  $ Macromolecule  2.5  3.0  3.5  4.0  4. Spectrophotometry  (a) Absorptivity Values of BHC. BHC concentrations were determined spectrophotometrically. Absorbance readings were c a r r i e d out at either 304 mu, the wavelength at which BHC exhibits maximum absorption i n pH 7.4 buffer, or at 286 mu, an isosbestic point. Absorptivity values ( a ) are reported i n Table 2. Molar g  absorptivity values may be obtained by multiplying a  g  values  by 336.29, the molecular weight of BHC.  N S s  \^  Wavelength  Instrument  286 mu  B & L 505  47.35 * 0.26  Beckman DU  48.38 * 0.48  Beckman DU-2  304 mu  55.5 ± 0.57  Table 2. Absorptivity Values f o r BHC i n a pH-7.4 Buffer.  (b) Absorbance Contribution of HSA and PVP. HSA and PVP absorb some radiant energy at the above wavelengths. I t i s necessary to correct f o r t h i s absorption by using appropriate c a l i b r a t i o n curves, before c a l c u l a t i n g BHC concentrations i n the presence of these macromolecules. Such curve i s shown i n Figure 30. Both HSA and PVP obey Beer's Law over the concent r a t i o n range investigated. HSA appears to absorb 10 time more energy than PVP, when both macromolecule  solutions are expressed  on a w/v percentage basis. Under experimental conditions,  - 97 -  Figure 30. Absorbance measurements at 304 mu for HSA ( • ) and PVP ( O ) solutions i n T r i s buffer (pH, 7.4). Each point represents the average of at least two determinations on Beckman DU-2.  ,  1  _  ,  2  *  3  ==^-»—  4  »  5  TF  6  % Macromolecule, xlO PVP and x l O  r,  •  7 2  HSA  8  r  9  :  r  10  - 98 absorbance values corresponding to the macromolecule  concen-  t r a t i o n i n the solution, were determined from t h i s curve and subtracted from the values obtained for the BHC-macromolecule solution. (c) Depression of BHC Absorbance  i n the Presence of HSA  and PVP. The spectrophotometrie c h a r a c t e r i s t i c s of bound BHC d i f f e r from those observed for the free drug. Spectral changes i n the presence of HSA and PVP are i l l u s t r a t e d i n Figures 31 and 32. The reference solution, i n both instances, contained the same quantity of macromolecule  as the BHC s o l u t i o n . The spectra  are, therefore, due to BHC i n the s o l u t i o n . For comparison, spectra of BHC i n the absence of macromolecule  are also shown  i n Figures 31 and 32. Because the absorption maximum at 304 mu i s depressed, i t i s probable that the functional group producing t h i s absorption i s involved i n the complexation process. I t i s d i f f i c u l t to r e l a t e u l t r a v i o l e t maximum to configuration. However, the chromophore i n BHC i s probably the d,-unsaturated  lactone structure  (see Figure 33). As the pH of the solvent changes, the -OH group i n the /3-carbon position has a tendency to ionize and cause the type of electronic s h i f t shown i n Figure 15. This implies that the energy repuired for the e l c t r o n i c t r a n s i t i o n w i l l be altered r e s u l t i n g i n a s h i f t of the spectrum. (d) Spectrophotometrie Analysis of Complex Formation. Since absorbance i s depressed, attempts were made to evaluate these spectral changes q u a n t i t a t i v e l y . The procedures used are s i m i l a r to those described by Klotz (1946c) and Oster and Immergut (1954). A series of BHC solutions containing i d e n t i c a l  Figure 32. Absorption spectra for BHC i n the presence ( ) and absence ( ) of 0.4$ PVP.  —i  330  >  .  320  1  1  310  1  1  300  .  i  290  Wavelength, mu  .6  1  1  280  1  1  270  ,  1—  260  - 101 -  Figure 33. Predominating chromophore ( <A,(3 unsaturated lactone) i n the BHC molecule.  quantities of BHC but varying amount of HSA and absorbance values were determined  and PVP were prepared  at 304 mu. The  depressions  i n absorbance values, plotted as a function of macromolecule concentration, are i l l u s t r a t e d i n Figures 34 and  35.  Beyond a c e r t a i n macromolecule concentration, there i s no further depression, which implies that a l l BHC molecules are i n the bound form. Molar absorptivity values of bound BHC were estimated from the known per cent depression of absorbance readings. The r e s u l t s are summarized i n Table 3. The  £  f  and  €  b  values represent molar a b s o r p t i v i t i e s for the free and bound species, respectively. The per cent depression i s equal to oL value i n Eq. 38. In Figure 36, absorbance depression at a constant macromolecule concentration was plotted as a function of BHC concent r a t i o n . Most of the r e s u l t s i l l u s t r a t e d are from the experiments used to produce the plots i n Figures 34 and 35. A linear r e l a t i o n ship was observed between the difference i n absorbance i n the absence and presence of macromolecule and the observed absorbance. From t h i s , i t i s possible to obtain information which can be used  — 102 — Figure 34. Absorbance depression of BHC as a function of HSA concentration. BHC concentrations are 26.7 (a), 23.7 (b), 15.4 (c), and 9.1 (d) x l O " mole/L. 6  .06  .08  .10  % HSA  A  - 103 -  F i g u r e 35. Absorbance d e p r e s s i o n o f BHC as a f u n c t i o n of PVP c o n c e n t r a t i o n . BHC c o n c e n t r a t i o n s are 26.5 (a) and 16.9 (b) x l O " mole/L. 6  (a)  (b)  .2  •3 %  .4 PVP  -+—  -5  .6  .7  .8  - 104 i n the law o f mass a c t i o n . From Eq. 37 t o 40, ( D ) and r v a l u e s f  can be e s t i m a t e d . The c a l c u l a t i o n procedure  i s illustrated i n  T a b l e 4, when PVP c o n c e n t r a t i o n i s equal t o 0.02$ ( i . e . ,  5xl0  - 6  mole/L.).  € (D )  Experiment  f  HSA-BHC (a)  (b)  (O  (d)  PVP-BHC (a)  (b)  t  $ Depression  *b<V  Average  .499 .442 .288 .170  .443 .402 .256 .155  88.8 90.0 88.9 91.1  89.9  .493 .315  .400 .252  81.1 80.0  80.5  T a b l e 3. Estimate o f Molar A b s o r p t i v i t y R a t i o o f Bound BHC t o Free BHC.  € f  (D ) t  .0985 .197 .246 .315 .394 .493 .616  3 0 4 irai Observed .0895 .180 .224 .284 .355 .445 .553  A  AA .009 .017 .022 .031 .039 .048 .063  (D )  (D )  2.47 4.67 6.04 8.52 10.72 13.19 17.31  2.81 5.89 7.14 8.36 10.39 13.22 15.69  b  5.28 10.56 13.18 16.88 21.11 26.41 33.00  f  r 0.494 0.934 1.21 1.70 2.14 2.64 3.46  T a b l e 4. A n a l y s i s o f Spectrophotometrie Data f o r PVP-BHC I n t e r a c t i o n i n 0.02$ PVP (5 umole/L.). Absorbance observed i s average o f a p a i r o f separate measurements on sample s o l u t i o n s s e p a r a t e l y prepared. The c o n c e n t r a t i o n terms a r e i n jimole/L. C^, = 0.805 € f = 0.805 x 18,660 (see T a b l e 3 ) .  F i g u r e 36 was a l s o used t o c o r r e c t f o r the presence o f macromolecule i n BHC a n a l y s e s . F o r example, i f a BHC s o l u t i o n i n 0.02$ PVP has an absorbance v a l u e o f 0.400, the c o r r e c t i o n factor  (0.043, r e a d d i r e c t l y from the o r d i n a t e i n F i g u r e 36) i s  added, and the new v a l u e i s d i v i d e d by c o n c e n t r a t i o n o f BHC i n the s o l u t i o n .  6^ t o y i e l d the molar  - 105 Figure 36. Absorbance differences for BHC solutions i n the absence and presence of constant amounts of macromolecules as a function of t o t a l BHC concentration. Closed symbols: HSA-BHC; open symbols: PVP-BHC. Macromolecule concentrations are: squares, 0.004$; triangles, 0.01$; c i r c l e s , 0.02$.  Absorbance Observed  5. Dynamic D i a l y s i s  The r e s u l t s of the dynamic d i a l y s i s experiments are i l l u s t r a t e d i n Figures 37 and 38. On the basis of molecular weights of 75,000 and 450,000, the systems contained 53.3 and 8.89 micromoles dextran and HES per l i t e r of T r i s buffer, respectively. In studies of the PVP-BHC interaction, two d i f f e r e n t PVP concentrations (50 and 100 jimole/L.) were used. In the absence of macromolecule, d i a l y s i s of BHC follows Fick's law of f i r s t order k i n e t i c s . This i s indicated by the straight l i n e obtained when log concentration i s plotted versus time. The interactions of BHC with dextran and HES appear to be weak. On the other hand, the d i a l y s i s rate of BHC i n the presence of PVP i s s i g n i f i c a n t l y d i f f e r e n t from that observed i n the absence of the macromolecule. Quantitative analysis of t h i s data can be c a r r i e d out by u t i l i z i n g Eq. 21 and Eq. 53 given below. D i a l y s i s rate i s proportional to the concentration of free BHC inside the d i a l y s i s bag. -  d(D ) t  dt  =  k  d  (D.)  (Eq. 53)  1  The concentration of unbound ( i . e . , free) BHC i n the macromolecule compartment at any t o t a l BHC concentration can be calculated from Eq. 53. The k^ value i s obtained from the slope of the semilog plot of (D.J.) versus time i n the absence of macromolecule. The instantaneous rate at a value of (D^) i n the presence of macromolecule can be estimated from a plot of (D ) versus time on t  l i n e a r graph  paper.  - 107 -  Figure 37. Loss of free BHC from inside a d i a l y s i s bag i n the absence ( O ) and presence of 0.4$ HES ( • ) and dextran ( A ) i n T r i s buffer (pH, 7.4; ionic strength, 0.15) at 30°C. j  150.-  •*  0  +-—  — • —  1  »  1  2  1  3 Hours  1  1  4  1  (c)  Figure 38. Loss of free BHC from inside a d i a l y s i s bag i n the absence (d) and presence of 0.4$ (a and c) and 0.2$ (b) PVP i n T r i s buffer (pH, 7.4; ionic strength, 0.15) at 30°C.  Hours  t-—*—t—t—t—r  10  11  - 109 The f i r s t - o r d e r rate constant  ( k ) , which characterizes d  the d i f f u s i o n process and incorporates the area and thickness of the membrane, was  found to be 5.71  x 10"3 Cm. /min. 2  Calculations of (Df) and r values for the experiment (b) i n Figure 38 are i l l u s t r a t e d i n Table 5. The i n i t i a l BHC concent r a t i o n was  828x10" mole/L. and PVP concentration was 6  ( i . e . , 50xl0~  6  0.2$  mole/L.). The second colume shows BHC concen-  t r a t i o n s at a given time i n the PVP-free compartment which contains 500 ml. of T r i s buffer. These values were converted to amount terms and subtracted from the t o t a l amount of drug i n the PVP compartment which contains 50 ml. of PVP-BHC solution. The amount of BHC remaining  inside the bag was re-converted to  a concentration term and t h i s value i s shown i n column three. The fourth column i s the slope of a tangent l i n e at a given time obtained from a plot of (D ) versus time. +  Time (Hour) 0 1 2 3 4 5 6 7 8 9 10  (C) 0 4.7 8.8 11.5 13.6 15.5 16.6 17.4 18.2 18.4 18.8  (Dt) 828 781 735 700 667 635 608 583 558 537 515  k (D ) d  f  (D )  (Db>  r  132 111 101 97 83 • 2 80.5 72.5 67.8 63.5 61.3  649 624 599 570 552 527 510 490 473 454  13.0 12.5 12.0 11.4 11.0 10.5 10.2 9.8 9.5 9.1  f  —.  .756 .631 .578 .554 .475 .460 .413 .387 .363 .350  Table 5. Calculated Data for the Dynamic D i a l y s i s of BHC i n the Presence of PVP. The concentration terms are i n jimole/L. PVP concentration was 50 umole/L. See the text for more d e t a i l .  - 110 A single dynamic d i s l y s i s experiment would f a i l to cover a wide range of r values or (D ) (especially when the binding f  a f f i n i t y i s remarkably high as i n the case of PVP-BHC i n t e r a c t i o n ) . It i s , therefore, necessary to repeat the procedure several times with d i f f e r e n t experimental conditions (e.g., use of much lower macromolecule concentration) i n order to obtain data covering a wider range of r values or ( D ) . f  For  example, the r e s u l t s i l l u s t r a t e d i n curves (a) to (c)  i n Figure 38 cover a (D ) range from 36.8 to 68.9, from 61.3 f  to  132.0, and from 6.3 to 14.7 umole/L., respectively. In terms  of  r value, they are from 7.4 to 8.8, from 9.1 to 13.0 (see  Table 5), and from 1.19 to 1.22. These ranges could be extended by dialyzing f o r longer periods of time but a n a l y t i c a l errors w i l l increasev' with time and r e s u l t i n unreliable data. These d i f f i c u l t i e s are mentioned by Stein (1965) but not by Meyer and Guttman (1968b; 1970a; 1970b). For  t h i s reason, the experimental design ( i . e . , volume  r a t i o of the two compartments,  concentration of drug and macro-  molecule, size of sample, duration of d i a l y s i s , etc.) i s c r i t i c a l when t h i s technique i s used to study completely unknown drug-macromolecule interactions. However, t h i s method can be e f f i c i e n t l y used for a study of well known binding systems such as albumin-dye complexations (Meyer and Guttman, 1970b). Data obtained for HES-BHC and dextran-BHC  interactions f a i l e d  to cover a wide range enough (Df) range to permit an evaluation of  the mechanism of the i n t e r a c t i o n . Because of t h i s , the data  obtained for these macromolecules i s not reported here.  - Ill Dynamic d i a l y s i s experiments confirm the r e v e r s i b i l i t y of binding of BHC with the macromolecules investigated. Continuous d i a l y s i s appeared to remove a portion of the BHC which had been bound to the macromolecule within the d i a l y s i s bag. For example, i n 0.2$  PVP solution, s o l u b i l i t y data indicates that approxi-  mately 84$ of the t o t a l BHC  e x i s t s i n the bound form (see Figure  29). This value i s not necessarily equal to that i n the i n i t i a l stages of the dynamic d i a l y s i s experiment (b) i n Figure 38 because extent of binding depends on BHC concentration. However, i f i t i s assumed that they are approximately 133  equal,*  then  ( i . e . , 828-828x0.84) umole/L. e x i s t s i n the unbound form  at time zero. After ten hours of continuous d i a l y s i s , the  BHC  concentration inside the bag was reduced to 515 umole/L. (see Table 5). This means that 313 umole/L. have been removed from the bag i n t h i s period of time. This, of course, i s much greater than the value of 133 umole/L. and indicates d i s s o c i a t i o n of the PVP-BHC complex during the experiment.  *  This assumption seems to be v a l i d , since the PVP s o l u t i o n for the dynamic d i a l y s i s experiment (b) was nearly saturated with BHC (see p. 63).  6. Equilibrium D i a l y s i s  (a) Binding of BHC  to Cellophane  Membrane. One of the main  sources of error i n the d i a l y s i s method i s the irregular adsorption of small molecules by the membrane (see pp. 30-32). necessary, basis.*  It was  therefore, to correct for t h i s binding on an  Extent of binding of BHC  experimental  to membrane (and also possibly  to the p l e x i g l a s c e l l ) was studied at three d i f f e r e n t temperatures. Results are shown i n Figure 39. No s i g n i f i c a n t differences i n binding were observed at the three temperatures. Although the same procedure for membrane preparation was used throughout the study, the extent of binding varied. However, on the basis of a l l the data, a correction factor of four per cent of the t o t a l  BHC  was used throughout t h i s i n v e s t i g a t i o n . (b) Permeability of Membrane to PVP. Hengstenberg and Schucht (1952) reported that the permeability of PVP molecule through the membrane can be neglected only i f i t s molecular weight i s greater than 10,000. However, Spitzer and McDonald (1956) found that some PVP molecules cross the membrane even i f the molecular weight i s higher than 40,000. By using d i f f e r e n t i a l t i t r a t i o n , they confirmed  that the dialyzable PVP i s t i t r a t a b l e  species which i s either a sub-fraction of the PVP or an impurity (or impurities) arisen during synthesis of the polymer.  *  When the concentration of small molecules i s analyzed on both compartments, the c o r r e c t i o n for membrane binding i s unnecessary. However i n t h i s work only macromolecule-free compartment was analyzed for BHC concentration.  - 113 -  Figure 39. Estimate of the extent of adsorption of BHC to the cellophane membrane at three d i f f e r e n t temperatures: 10 ( A ) , 20 ( • ) , and 40 (• ) C.  - 114 Although the PVP used i n t h i s i n v e s t i g a t i o n was p u r i f i e d by extraction with anhydrous ether, experiments were c a r r i e d out to determine i f the polymer passed through the membrane. Analysis of the PVP-free compartment was c a r r i e d out spectrophotometrically using the iodine-PVP reaction. A c a l i b r a t i o n curve i s shown i n Figure 40. When 20 ml. of a 0.4$  PVP solution was added to one of  the compartments and dialyzed for 40 hours against T r i s buffer, approximately  10 mg./L. PVP was detected i n the PVP-free compart-  ment. This implies that approximately  0.25$ of the t o t a l amount  of PVP used can pass through the membrane. This value i s so small that i t can be  neglected.  (c) Donnan E f f e c t . In equilibrium d i a l y s i s , an appropriate ionic strngth must be maintained  i n order to prevent Donnan  equilibrium across the membrane (see p. 30). The a l t e r n a t i v e i s to use low macromolecule concentrations. HSA charge of 18 at pH 7.4  has a net  negative  (White and others, 1968). The ionic strength  i n the T r i s buffer used i s 0.15 used i n the i n v e s t i g a t i o n was  and the highest HSA  0.4$  concentration  (5.8 x 10~5 mole/L.). When  these values are substituted into Eq. 42, the d i s t r i b u t i o n r a t i o of univalent anions across the membrane (R value i n Eq. 42) i s equal to 1.0035.  It i s possible, therefore, to neglect the abnormal  d i s t r i b u t i o n of BHC  anion due to the Donnan e f f e c t . PVP has no  e l e c t r i c a l charge at pH 7.4 e f f e c t can, therefore, be *  (May and others, 1954). The Donnan  neglected.*  PVP w i l l also behave as a p o l y e l e c t r o l y t e after binding with the BHC anion. However t h i s c h a r a c t e r i s t i c w i l l not be remarkable i n a solution of high ionic strength (see Frank and others, 1957, for example).  - 115 Figure 40. Colorimetric determination of  mg. PVP / L.  PVP.  - 116 (d) Free Drug Concentration and Volume Ratio. In order to study binding mechanisms, i t i s necessary to cover a wide range of (Df) or r values. Pollansch and Briggs (1954) claimed that maximum e f f i c i e n c y i s obtained by assigning appropriate volume r a t i o s to the macromolecule to macromolecule-free compartments. Furthermore, the t o t a l amount of BHC  that can be handled under  given conditions can be enhanced by dialyzing BHC solutions against macromolecule solutions which have been nearly saturated with BHC.  Yang and Foster (1953) used t h i s approach i n t h e i r  binding studies (see pp. 31-32). T y p i c a l design and r e s u l t s of a d i a l y s i s experiment are shown i n Tables 6 and 7. Calculations can be c a r r i e d out on the basis of either concentration or amount term. If 1:1 volume r a t i o i s used, i t i s more convenient  to use concentration term. However,  i n t h i s investigation at least one of the d i a l y s i s c e l l s had a 2:1 volume r a t i o . It i s , therefore, more convenient amount term.*  The corrected t o t a l amount of BHC  (D^. i n the  column of the tables) i s obtained by multiplying D^, corrected i n i t i a l amount of BHC, for  by 0.96,  to use fifth  the un-  the c o r r e c t i o n factor  membrane binding. The BHC content of the macromolecule-free  compartment i s determined spectrophotometrieally and converted to the amount present i n either 20 or 40 ml. The i n i t i a l BHC concent r a t i o n i n the macromolecule compartment was determined from the c o r r e c t i o n curves i l l u s t r a t e d i n Figure 36. *  Attention must be paid to term used i n the c a l c u l a t i o n procedure. In the l i t e r a t u r e , some investigators f a i l e d to convert concentration term to amount term, although they used volume r a t i o rather than 1:1. For example, O'Reilly and Kowitz (1967) showed a t y p i c a l miscalculation of r values. It was pointed out by Meyer and Guttman (1970b).  - 117 Results  Designing No. ml.of Stock Solutions IA 40(BHC/1) IB 20(H-B) 2A 20(BHC/1) 2B 20(H-B) 3A 20(BHC/2) 3B 20(HSA) 4A 20(BHC/4) 4B 20(HSA) 5A 20(BHC/5) 5B 20(HSA) 6A 40(BHC/10) 6B 20(HSA)  D.F,  DH  7.65 4.41 3.83 4.14 1.91 0 0.96 0 0.765 0 0.765 0  304  (D )  D,  D»  l  f  11.58  10  .241  129.1  7.75  3.83  6.60  7.91  5  .427  114.4  4.58  3.33  5.74  1.85  1  .198  10.6  0.42  1.42  2.45  0.92  1  .039  2.1  0.084  0.834 1.44  0.734  1  .031  1.67 0.064  0.67  1.16  0.734  1  .047  2.52 0.15  0.58  1.01  Table 6. C a l c u l a t i o n Procedures for (Df); aridrr Value. Compartment B contains HSA. The BHC concentration of the stock solution (BHC/1) was 191.3 umole/L. Denominator represents the d i l u t i o n factor (D.F.) of the stock s o l u t i o n . HSA stock solution nearly saturated with BHC i s designated by H-B, for which the BHC concentration i s 220.7 umole/L. HSA concentration i s 0.2$ (28.98 umole/L. = 0.58 umole/20 ml.). Temperature was maintained at 20°C. The concentration terms are i n umole/L. The amount terms are i n jumole.  No. IA IB 2A 2B 3A 3B 4A 4B 5A 5B 6A 6B  Designing ml.of Stock D± Solutions 40(BHC/1) 7.87 20(P-B) 9.60 40(3BHC/4) 5.90 20(P-B) 9.60 20(3BHC/4) 2.95 20(P-B) 9.60 20(BHC/2) 1.97 20(P-B) 9.60 20(Tris) 0 20(P-B) 9.60 20(3BHC/4) 2.95 20(PVP) 0  Results D  t  D.F.  A  304  (D ) f  D  f  D  b  r  16.77  10  .243  130.0  7.80  8.97  17.94  14.88  10  .216  115.5  6.93  7.95  15.9  12.05  10  .206  110.4  4.42  7.64  15.27  11.10  .189 .370 .300  100.2  4.01  7.10  14.19  9.22  10 5 5  80.4  3.22  6.00  12.00  2.83  1  .429  23.0  0.92  1.91  3.82  Table 7. C a l c u l a t i o n Procedures for (Df) and r Value. Compartment B contains PVP. The BHC concentration of the stock solution (BHC/1) was 196.6 umole/L. The fract i o n a l expression gives the d i l u t i o n factor f o r the stock s o l u t i o n . PVP stock solution nearly saturated with BHC i s designated by P-B, for which the BHC concentrat i o n i s 480.1 umole/L. PVP concentration i s 0.1$ (25 jimole/L. = 0.5 umole/20 ml.). Temperature was maintained at 20°C.  7. Interpretation of Binding Data  (a) Langmuir-Type P l o t . The simplest way to handle binding data i s to plot r value versus  (D^). The curve obtained from  Eq. 17, which i s applicable only to a binding system containing a single set of binding s i t e s , i s a segment of a rectangular hyperbola  passing through the o r i g i n . I f (D ) i n Eq. 17 becomes f  i n f i n i t e , the r value approaches n as a l i m i t ,  r  and a t r  s  n/2,  In Eq. 55, K  - 1  =  (D ) - K  n  (  q  -  5  4  )  (Eq. 55)  - 1  f  represnts the i n t r i n s i c  E  'dissociation' constant.  These two equations indicates the importance of a wide range of ( D f ) values. For example, Dowd and Riggs (1965) pointed out that many investigators chose unsuitable substrate concentration ranges i n studies of enzyme-substrate complexation. They discussed the s i g n i f i c a n c e of the Michaelis-Menton equation which i s similar to Eq. 17 or 20. Binding data for BHC to HSA and PVP i s i l l u s t r a t e d i n Figures 41 and 42. For the ( D f ) range studied, both macromolecules f a i l e d to show saturation of binding s i t e s .  As i l l u s t r a t e d i n  Figure 41, at lower BHC concentrations the drug i s more e a s i l y bound to HSA than at high concentrations. However, with PVP the curve does not change as ( D f ) increases. In other words, the ( D f ) range investigated i s too narrow to permit an evaluation  - 119 -  Figure 41. Plot of r values versus the concentration of free BHC for HSA-BHC binding at two temperatures; 20°C (closed symbols) and 40°C (open symbols). Values were obtained from three d i f f e r e n t HSA concentrations; 0.1$ (triangles),0.2$ ( c i r c l e s ) , and 0.4$ (squares).  7  -  I  0  1  , 20  \  »  40  1  1  1 80  1  60  1  (D ) x 10 f  1  1  100 b  mole/L.  1  120  1  1  140  <  >—  160  - 120 10  (D ) f  x  10  b  mole/L.  - 121 of binding mechanisms. It was  impossible, from equilibrium  d i a l y s i s method, to obtain data at a higher (Df) range, even though attempts were made to saturate binding s i t e s with  BHC  prior to d i a l y s i s experiments. However, s o l u b i l i t y method can give information at high (Df). The r values calculated from, s o l u b i l i t y data at 20°C are 6.8 for HSA-BHC and 20.2  for PVP-BHC i n t e r a c t i o n s . * For PVP-BHC interaction,  a further equilibrium d i a l y s i s experiment was c a r r i e d out i n which the PVP concentration was reduced from 0.4$  to 0.1$  to get addi-  t i o n a l data at a high (Df) range. These r e s u l t s at 20°C and r e s u l t s from s o l u b i l i t y data are shown i n Figure 43. For HSA-BHC interaction, complete saturation of the binding s i t e s was reached. The curve shows that about s i x or seven binding s i t e s occur on the HSA  molecule (see Eq. 54). The i n t r i n s i c association constant  i s approximately  3.5 x 10  4  L./mole (see Eq. 55). However these  r e s u l t s are v a l i d only i f there i s a single set of binding s i t e s for BHC  (for further discussion, see p.123). In case of PVP-BHC  interaction, even the data obtained from s o l u b i l i t y method f a i l e d to show complete saturation of binding s i t e s . It was,  therefore,  impossible to interprete the binding curve q u a n t i t a t i v e l y .  *  From Figure 29, i t can be seen that up to 1$ HSA and 0.4$ PVP, the apparent s o l u b i l i t y of BHC changes l i n e a r l y with respect to macromolecule concentration. Therefore i n t h i s range of macromolecule concentrations, the r value (slope of a l i n e ) i s a constant and independent of macromolecule concentration. The r values reported was obtained i n t h i s range of macromolecule concentrations. The fact also explains why data obtained from d i a l y s i s studies i n which 0.1, 0.2, and 0.4$ HSA were used, produced i d e n t i c a l binding curves as shown i n Figure 41.  20 j Figure 43. Binding curves at 20°C for HSA-BHC (closed symbols) and PVP-BHC (open symbols) interactions i n T r i s buffer. Data obtained from s o l u b i l i t y analysis (large open c i r c l e s ) are included on the curves. Data for 0.1, 0.2 and 0.4$ macromolecule are represented as triangles, c i r c l e s , and squares, 16 t respectively. 'A  12 -  -4-  20  -»40  -4-  60  -4  80  100 (Df) x 10  120 6  mole/L<  - 123 (b) Scatchard P l o t . The binding curves for HSA-BHC i n t e r a c t i o n i l l u s t r a t e d i n Figures 41 and 43 are replotted on the basis of Eq. 21 i n Figure 44. At both 40 and 20°C, the binding data follows a c u r v i l i n e a r course that bends sharply near the abscissa. This indicates that more than one set of binding s i t e s i s present i n the HSA  molecule. The values of n, the average number of the  f i r s t set of binding s i t e s , 2.8 at 40°C and 3.0 at 20°C, was rounded o f f to the nearest whole number. This rounding-off procedure has been used by Scatchard et a l . (1957). The intercepts of the curves on the ordinate, nK, are 51 x 10^ and 105 x 10^, which y i e l d i n t r i n s i c association constants of 17 x 10^ and 35 x 10^ (L./mole) at 40 and 20°C, r e s p e c t i v e l y . The association constant at 20°C i s nearly twice that at 40°C. The second set of binding s i t e s w i l l not be considered i n t h i s discussion because the i n t r i n s i c a f f i n i t y of the BHC  molecule for these s i t e s i s far  smaller than that for the f i r s t set. Binding data for the PVP-BHC i n t e r a c t i o n at various temperatures plotted according to the Scatchard equation  (Eq. 21) i s  i l l u s t r a t e d i n Figure 45. Although the data i s scattered, d i f f e r ences i n binding strength at various temperatures can be seen. Straight l i n e s were drawn on the assumption that there are 50 i d e n t i c a l binding s i t e s on the PVP molecule irrespective of temperature (for further discussion, see p.126). The point marked with an arrow i s the value obtained from the s o l u b i l i t y a n a l y s i s . (c) Double Reciprocal P l o t . Because of error inherent i n the double r e c i p r o c a l plot, i t i s preferable to use the  Scatchard  plot for quantitative analysis of binding whenever possible. However i n the case of PVP-BHC i n t e r a c t i o n i t was  d i f f i c u l t to  n o •-  ioo  90 -  80 "  70  !o  60  D  50  40 -  3 0 ••  20  10 ••  r  s  (D )/(HSA) b  Figure 45. Scatchard plot for PVP-BHC interaction at 10 ( • ) , 20 ( O ) , 30 ( A ) , and 40 ( A ) *C. Data obtained from solub i l i t y i s indicated by an arrow.  21 20 19 18 17 16 15 "  11  1  10 9 8  8  9 r =  10  11  12  (D )/(PVP) b  13  W  15  16  17  18  19  20  126 derive accurate binding parameters from the Scatchard p l o t . Hence a double r e c i p r o c a l plot (see Eq. 20) was used, Figure 46. These curves pass through approximately the same point on the ordinate ( i . e . , 0.02). The r e c i p r o c a l of the intercept ( i . e . ,  50)  was taken as the average number of binding s i t e s on the PVP molecule and used i n the extrapolation of the Scatchard plot (see Figure 45). I n t r i n s i c association constant at various temperatures was calculated from the slopes of the curves.  8. Thermodynamic Analysis and Mechanism of Interaction  (a) HSA-BHC Interaction. i ) Enthalpy, Entropy, and Free Energy Changes. Assuming that there i s no s i g n i f i c a n t temperature change within the temperature  dependence of enthalpy  range i n which the interaction was  c a r r i e d out, i t i s possible to estimate the standard enthalpy change ( AH°) for the association of one mole of BHC to one mole of the binding s i t e s of the f i r s t set on the HSA molecule. The c a l c u l a t i o n i s shown below.  (Eq. 56)  Standard free energy  ( AG")  and entropy ( A S ) 0  changes were  calculated using Eq. 35 and 36, and are reported i n Table 8. Because of possible competitive r o l e of the buffer ions during binding, the K values must be regarded as being dependent on buffer composition. With respect to the thermodynamic quantities, t h i s implies that the standard state includes the T r i s buffer  - 128 K x 10~ (L./mole)  5  °c  AH AG° (Kcal/mole) 0  AS (e.u.) 0  20  3.5  - 6.58  - 7.43  42.90  40  1.7  - 6.58  - 7.49  + 2.91  Table 8. E f f e c t of Changes i n Temperature on the Binding of BHC to HSA.  used i n the investigation. This approach to c a l c u l a t i o n i s that used by Karush (1950). The decrease i n binding strength of HSA for BHC with increasing temperature i s c h a r a c t e r i s t i c of exothermic reaction. Similar decreases have been observed for many protein interactions with a wide variety of substances (see, for example, O'Reilly and Kowitz, 1967). The negative sign for AG° means that the binding process i s spontaneous. The A S ° value, the disorder factor of thermodynamic changes, i s p o s i t i v e . This i s i n agreement with observations for most of albumin-anion interactions. However, the magnitude of A S ° i s small compared with that observed for the i n t e r a c t i o n between albumin and azo dye anions. i i ) P o s s i b i l i t y of Ionic Interaction. At pH 7.4, the HSA molecule has a net negative charge of 18 (White and others, 1968). On the basis of the determined pKa values (see p. 84), 80 and 20$ of t o t a l BHC e x i s t s as the monoionized and di-ionized species, r e s p e c t i v e l y . This does not mean that the p o s s i b i l i t y of ionic i n t e r a c t i o n between these substances can be ruled out. A net negative charge on the protein molecule merely implies an excess of negative charges over positive residues. There i s evidence i n the l i t e r a t u r e to substantiate the view that cationic centers on the protein at a pH 7 are intimately involved i n the binding  - 129 with anionic molecules (Klotz, 1949a). Thermodynamic changes for the HSA-BHC i n t e r a c t i o n indicate large contribution to the - AG°value by the - A H °  an unusually  value. In general, anion-protein  interactions have shown l i t t l e  temperature dependence (Klotz and others, 1949b). A small temperature c o e f f i c i e n t i s a c h a r a c t e r i s t i c of an i n t e r a c t i o n between oppositely charged species  (Klotz and others, 1952).  I f the  nature of the HSA-BHC i n t e r a c t i o n were largely e l e c t r o s t a t i c , i . e . , i f the ionic part of the BHC molecule combined with the c a t i o n i c o parts of the HSA molecule, the main source of the - A G value would be a large p o s i t i v e  AS° value with l i t t l e contribution  o  from the A H factor. I t i s very u n l i k e l y , therefore, that the HSA-BHC i n t e r a c t i o n i s ionic i n nature. O'Reilly et a l . (1967;1969) proposed similar suggestions for the i n t e r a c t i o n between HSA and warfarin. i i i ) P o s s i b i l i t y of Pre-Existing Binding  S i t e . In the  absence of information about attendant conformational  changes,  most interpretations of binding processes make the assumption that pre-existing binding s i t e s are involved (Lovrien, 1963). On the other hand, Karush (1950) postulated that serum albumin possesses 'configurational a d a p t a b i l i t y ' for a variety of small molecules. I t i s c l e a r from Karush's discussion that there i s a strong p o s s i b i l i t y that some proteins might form binding during the binding process. Using t h i s hypothesis, 1957)  sites  Karush (1956;  s a t i s f a c t o r i l y interpreted the differences i n thermodynamic  parameters usually observed i n non-specific anion-albumin and s p e c i f i c hapten-antibody i n t e r a c t i o n s .  - 130 The positive  AS  0  associated with many reaction involving  proteins are usually attributed to disorientation and unfolding of the protein molecule. This does not appear to be a s a t i s factory explanation for the HSA-BHC i n t e r a c t i o n because the enthalpy changes observed are very negative whereas a process of unfolding presumably requires the breaking of several bonds and should, therefore, r e s u l t i n an endothermic  reaction of appreciable  magnitude (Klotz and others, 1949b). It i s , therefore, preferable to postulate that the HSA molecule may have some kind of pree x i s t i n g s i t e for the BHC molecule. This s i t e may be more or less r i g i d as the binding s i t e s of antibody for haptens.  Thermodynamic  data obtained for the HSA-BHC i n t e r a c t i o n i s compared, i n Table 9, with that obtained by Karush (1956) for antibody-anionic hapten binding. K x 10~ (L./mole) 4.4 - 6.7  AH* AG° (Kcal/mole) -(7.1 - 7.3) -(7.24 - 7.48)  AS° (e ,u.) + (0.3 - 0 .7)  2.1 - 3.1  -(7.1 - 7.3)  + (0.3 - 0 .7)  5  °c  7.1  25  -(7.25 - 7.50)  Table 9. Thermodynamic data for the binding of D-phenyl-(p-azobenzoylamino)-acetate by p u r i f i e d antibody s p e c i f i c for the compound, i n 0.02M phosphate buffer of pH 7.4 containing 0.15M NaCl (from Karush, 1956).  Free energy changes, for both interactions, are of the same magnitude. In the s p e c i f i c binding between antibody and haptens, the free energy change i s due almost e n t i r e l y to the enthalpy. The contribution of the entropy term i s n e g l i g i b l e .  - 131 iv) Nature of Binding Site and E f f e c t of Binding on Water Structure. The BHC combining region on the HSA molecule probably consists of an i n t e r - h e l i c a l cavity whose van der Waals contour i s c l o s e l y complementary to, and therefore selective for the BHC molecule. In t h i s respect, the interaction may be considered s p e c i f i c , and i s explained by a large contribution of enthalpy to free energy changes and by a r e l a t i v e l y sharp change i n slope of the binding curves near the abscissa (see Figure 44). The change i n slope i s similar at both 20 and 40°C which implies that there i s no s i g n i f i c a n t change i n the average number of binding s i t e s at the two temperatures. Presumably the cavity i s not completely r i g i d , as i n case of an antibody for haptens, and cannot r e s i s t the disruptive tendencies of i n t r a molecular e l e c t r o s t a t i c repulsions to which the protein i s subjected at extreme values of pH, e.g., a pH value of 3. Therefore, as Nagashima and others (1968a) have observed, the HSA-BHC i n t e r a c t i o n would be much less at pH ranges where N to F conversion of the protein takes place (see p. 42). The cavity can, therefore, be e a s i l y disrupted by expansion of the albumin molecule. The HSA-BHC binding involves a transfer of a hydrophobic molecule from an aqueous environment  to a region with a lower  d i e l e c t r i c constant. After binding occurs, a hypothetical hole, previously occupied by the BHC molecule, remains and t h i s w i l l then be f i l l e d with an equal volume of hydrogen-bonded water molecules (Karush, 1956). The number of hydrogen bonds formed i n t h i s way w i l l exceed the number of hydrogen bonds which  - 132 the free BHC molecule previously formed with i t s neighboring water molecule.*  On the basis of t h i s explanation, complex  formation would be exothermic and should be associated with a substantial decrease i n the enthalpy of the system, which w i l l exceed the endothermic nature of the heat of fusion of icebergs around the BHC and HSA molecules. The melting of iceberg-structured water around BHC  and  HSA molecules w i l l r e s u l t i n an increase i n randomness ( i . e . , positive  A S ) . The BHC molecule w i l l lose r o t a t i o n a l and 0  t r a n s i t i o n a l degrees of freedom a f t e r binding to y i e l d a higher ordering ( i . e . , negative  A S ° ) . Formation of a bonded-water  c l u s t e r at the hypothetical hole l e f t by the BHC molecule w i l l give an ordering e f f e c t (Nemethy and Scheraga, 1962a). The disordering e f f e c t s mentioned  two  f i r s t probably exceed the last  two ordering e f f e c t s and the r e s u l t i s a net entropy change of + 2.9.  In more s p e c i f i c interactions (such as antibody-hapten  interactions), the loss of degrees of freedom would be highly s i g n i f i c a n t and could r e s u l t i n a net negative change (Karush, 1957). (b) PVP-BHC Interaction. i ) Enthalpy, Entropy, and Free Energy Changes. I n t r i n s i c association constants are shown i n Figure 47, a van't Hoff type plot, as a function of temperature. The enthalpy change appears to be temperature dependent. A s i m i l a r temperature dependence has been observed i n other studies (see, for example, Hymes and others, 1969). From the slope of a tangent l i n e at a given temperature the standard enthalpy change was estimated. Thermodynamic parameters were calculated by u t i l i z i n g Eq. 35 and 36. *  From low aqueous s o l u b i l i t y of BHC, i t was assumed that BHC molecule has l i t t l e hydrogen bonds with neighboring water molecules (see p. 85).  - 133 -  Figure 47. Van't Hoff plot (log K versus for the PVP-BHC i n t e r a c t i o n .  1/T)  - 134 Results are l i s t e d i n Table 10. If the concentration used i n c a l c u l a t i n g the association constant are expressed per l i t e r ,  A S  0  i n moles  i s the entropy change when one mole of BHC  and  one mole of a binding s i t e on a PVP molecule, each at a concent r a t i o n of one mole per l i t e r , react to give one mole of the complex, again at concentration of one mole per l i t e r . If d i f f e r ent concentration units are used,  A S  0  w i l l have a d i f f e r e n t  value. It i s desirable to eliminate t h i s rather arbitrary factor before trying to interpret the molecular  the magnitude of  AS  0  i n terms of  structures present i n the s o l u t i o n . If we assume  that the A S ° values l i s t e d i n Table 10 are determined from measurements at s u f f i c i e n t l y high d i l u t i o n , the so-called 'unitary (or contact)' entropy change ( /_u)  for the reaction  can be calculated from Eq. 57 (Gurney, 1953;  Kauzmann, 1959).  ASu »  _S° + 7.98  (Eq. 57)  The unitary entropy change depends, therefore, only on those factors which involve the i n t e r a c t i o n of the BHC  molecule  and the binding s i t e on a PVP molecule with the solvent and with each other and not on the contribution due to randomness of the mixing with the solvent. The  ' c r a t i c ' term (7.98  originates from the expression, - R In (1/55.6), where  e.u.) 55.6  (mole/L.) i s the concentration of water i n a highly diluted aqueous s o l u t i o n . This term takes into account the reduction i n number of independent solute species by one on the combinat i o n of a BHC molecule with a PVP molecule (Molyneux and Frank, 1961a). In order to compare t h i s data with that for the inter-  - 135 actions between PVP and a variety of organic substances (Molyneux and Frank, 1961a), the unitary values for PVP-BHC interaction are also l i s t e d i n Table 10. K x 10" °c (L./mole) 2  10  3.64  AG° (Kca 1/mole) -2.74 -3.32  AS°  20  3.05  -2.96  -3.33  + 1.28  + 9.26  30  2.48  -4.69  -3.32  -4.53  + 3.45  40  1.72  -8.57  -3.20  -17.14  - 9.16  ASu (e .u .) + 2.05 +10.03  Table 10. Thermodynamic functions for the binding of one mole of BHC by one mole of vacant binding s i t e on PVP.  i i ) Analysis of Enthalpy of Binding. Emphasizing the 'iceberg' concept of water structure (Frank and Evans, 1945) around the BHC and PVP molecules, i t i s possible to divide the binding enthalpy into the following f i v e contributions (Molyneux and Frank, 1961a). The corresponding binding processes are schematically i l l u s t r a t e d  i n Figure 48. (1) Heat i s needed to  break (or bend) hydrogen bonds i n the icebergs neighboring the polymer and BHC molecules. (2) Enthalpy w i l l also have to be provided to overcome any s p e c i f i c  interactions (e.g., true  hydration) between the water and PVP and between the water and BHC. (3) The actual binding process between the 'dehydrated' e n t i t i e s w i l l be exothermic.  (4) The interaction between the  complex formed and the neighboring water ( i . e . , reformation of true hydration) w i l l be exothermic.  (5) Enthalpy w i l l , f i n a l l y ,  also be gained by the reforming of hydrogen bonds i n the icebergs associated with the complex.  -  136  -  (1)  AH^  (+)  AHg (+)  (2)  V  (3)  AH  AH  4  (-)  3  (-)  (4)  (5)  AH  5  (-)  Figure 48. Schematic i l l u s t r a t i o n of the binding processes between BHC and PVP molecules. Small c i r c l e s represent a hydrogen bonded water cluster indicating the formation of a p a r t i a l cage around a solute.  - 137 The net enthalpy change associated with the purely hypothet i c a l binding processes i l l u s t r a t e d i n steps two to four i n Figure 48 was reported to be e s s e n t i a l l y a constant (-5 Kcal/mole) for  interactions of PVP with a variety of substances (Molyneux  and Frank, 1961a). If t h i s value i s applied to the data herein, the net enthalpy changes associated with the f i r s t and steps would be approximately +2.3, at  +2.0,  +0.3,  and -3.6  fifth Kcal/mole  10, 20, 30, and 40°C, respectively. Total changes are shown  i n Table 10. As temperature  increases, the enthalpy change  decreases to a negative value at 40 C. 8  The mole fractions of various water species i n the f i r s t  layer  around an aromatic hydrocarbon were estimated as a function of temperature  by Nemethy and Scheraga  (1962b) on the basis of  t h e i r theory of water structure (Nemethy and Scheraga,  1962a).  For example, the f r a c t i o n of unbroken hydrogen bonds changes from 59.3$ at 10°C to 49.0$ at 40°C. On t h i s basis, the decreasing tendency of the enthalpy changes, associated with the f i r s t and f i f t h binding processes, with increasing temperature can be e a s i l y explained. At a given temperature, of  the absolute value  the enthalpy change associated with the breaking hydrogen  bonds i n the icebergs around the solutes,  ^H-p  will  exceed  that for the formation of hydrogen bonds i n the icebergs around the complex,  ^Hg,  since the number of hydrogen bonds around the  solutes before binding would exceed the number of hydrogen bonds around the complex. However, the difference becomes less important as temperature  increases. The value approaches zero and  f i n a l l y becomes negative at 40°C.  - 138 i i i ) P o s s i b i l i t y of Hydrophobic Bonding. The so-called 'iceberg' concept (Frank and Evans, 1945) assumes that hydrocarbon groups, such as those present both i n the polymer and i n the BHC molecules, are surrounded i n aqueous solution with one or more layers of water molecules which are more highly ordered than the molecules i n ordinary l i q u i d water. Entropy gains i n protein (Klotz, 1958; Kauzmann, 1959; Nemethy and Scheraga, 1962c; Nemethy and others, 1963; C e c i l , 1967; Hymes and others, 1969) and (Molyneux and Frank, 1961a; Bahal and Kostenbauder,  PVP  1964; Eide  and Speiser, 1967a) interactions with a variety of small molecules or ions have been attributed to increased disorderness of the iceberg structure due to complex formation. The positive unitary entropy change observed over the room temperature range (see Table 10) can be explained i n a manner similar to that above. It i s postulated, therefore, that the PVP-BHC complex i s accompanied by either a less ordered iceberg or by an iceberg containing a smaller number of water molecules as compared with the iceberg of the two separate e n t i t i e s . The release of water molecules from the ordered structure should produce a proportional gain i n entropy. The free energy change associated with PVP-BHC i n t e r a c t i o n shows no s i g n i f i c a n t temperature dependence. The contribution to the free energy of entropy term becomes more important as temperature increases from 17.5$ at 10°C to 38.5$ at 40°C. A similar trend has been observed for the formation of a t y p i c a l hydrophobic bond between leucine and isoleucine (Nemethy and Scheraga, 1962c) although, i n the case, the magnitude of i t s contribution i s approximately  - 139 eighty per cent. At a high temperature (e.g., 40°C), hydrophobic bonding seems to play a lesser r o l e i n the PVP-BHC i n t e r a c t i o n . The exothermic reaction here i s enhanced by temperature whereas the formation of hydrophobic bonds i s e s s e n t i a l l y endothermic i n nature  (Nemethy and others, 1963). This inference i s also  supported by the decrease i n the favorable entropy change with increase i n temperature. iv) Nature of the Intermolecular Forces and of the Binding S i t e . The PVP molecule has no ionizable groups (May others, 1954)  and  and i t i s , therefore, u n l i k e l y that e l e c t r o s t a t i c  interactions would play an important r o l e i n i t s binding with BHC  anion. The high temperature dependence of binding strength  also r u l e s out the p o s s i b i l i t y of s i g n i f i c a n t e l e c t r o s t a t i c interactions (see p. 129). However, the lactam bond i n the pyrrolidone r i n g represents a dipole, which i s l i k e l y to undergo ion-dipole i n t e r a c t i o n with BHC of the anion i n such a way  anion. It w i l l aid the binding  as to supply the necessary  attraction  force to bring the two components into close contact (Frank and others, 1957). After the close contact of the two e n t i t i e s by ion-dipole force, which varies with the inverse f i f t h power of the distance, van der Waals forces*  w i l l s t a b i l i z e the complex.  The photograph i n Figure 49 shows a molecular model of a PVP chain segment with eight repeating units and i s similar to that proposed by Frank et a l . (1957). To avoid unnecessary complications, a l l hydrogen atoms are omitted. Pyrrolidone *  The term 'van der Waals forces' i s often used without explanation. In t h i s context, the dipolar forces of Keesom (dipole-dipole), Debye (dipole-induced dipole), and London (induced dipole-induced dipole) are c a l l e d van der Waals forces. The forces vary inversely with the seventh power of the distance (Martin, 1968a).  - 140 -  Figure 49. Molecular model of PVP chain segment with eight monomer u n i t s . Black, blue, and red colours represent carbon, nitrogen, and oxygen atoms, respectively.  rings appear to make a channel-type of cavity i n both sides of the p a r a f f i n backbone which i s apparently accessable for complexation with BHC. A PVP molecule of molecular weight, 40,000, has approximately 360 monomer units, since the molecular weight of the l a t t e r i s about 111. The binding data shown i n Figure 46 indicates an average number of binding s i t e s on a PVP molecule of approximately 50, irrespective of temperature. This implies that on the average, 7.2 repeating units provide a binding s i t e for BHC molecule. As shown i n Figure 50, a BHC molecule f i t s quite well on approximately eight pyrrolidone r i n g s . As pointed out e a r l i e r (p. 85), a monoionized BHC molecule i s expected to have less r o t a t i o n a l degrees of freedom around the methylene bridge than a f u l l y ionized molecule. A model  - 141 -  Figure 50. Proposed Configuration of PVP-BHC complex.  of the PVP-BHC complex indicated that a better f i t of monoionized BHC molecule may require a s l i g h t bending, or folding, of the PVP molecule toward the BHC anion. However, no such requirement i s necessary i n case of binding of the di-ionized BHC species, since the high degrees of freedom would allow the anion to have a suitable form for f i t t i n g to PVP. The p o s s i b i l i t y of binding of another BHC anion on the other side of the PVP molecule (below the plane of the paper i n Figure 50) can be ruled out by an expected repulsive forces between BHC anion bound to the polymer and an oncoming BHC anion.  9. Viscometry  Any changes i n hydrodynamic volume as a r e s u l t of c o n f i g u r a t i o n 1 changes i n the macromolecule during the binding process should a l t e r the reduced v i s c o s i t y . The reduced v i s c o s i t y was calculated using Eq. 43 to 45 i n the presence and absence of BHC. The BHC concentration was 185 mg./L. (550 x I O  - 6  mole/L.).  Results of the density measurements f o r PVP s o l u t i o n necessary for the c a l c u l a t i o n of reduced v i s c o s i t y are shown i n Figure 51. Table 11 shows the c a l c u l a t i o n procedure for s p e c i f i c and reduced v i s c o s i t i e s from measurements of flow time and density. In Figure 52, the reduced v i s c o s i t y was plotted against PVP concentration. Slopes and intercepts were calculated using the method of least squares. Table 12 shows slope and intercept ( i . e . , i n t r i n s i c v i s c o s i t y ) values f o r each l i n e . The c o r r e l a t i o n c o e f f i c i e n t , Rc, was calculated between the two variables, reduced v i s c o s i t y and PVP concentration. From the slope, the Huggins constant, K , was also estimated H  $PVP 4.0 3.0 2.0 1.5 1.0 0.4 0  t ? AxlO (Gm./ml.) (sec.) 2.72 1.0184 1274.6 2.71 1.0161 1034.7 847.9 2.64 1.0138 2.48 1.0127 804.6 2.47 1.0115 716.3 2.46 1.0102 620.8 2.42 1.0092 571.3 5  (Eq. 47).  °1  .03531 .02849 .02269 .02021 .01790 .01543 .01395  ^sp 1.5312 1.0423 .6265 .4487 .2832 .1061 0  ^sp/C 38.28 34.74 31.33 29.91 28.32 26.53  -  Table 11(a). Calculation procedure for estimating s p e c i f i c and reduced v i s c o s i t i e s of PVP s o l u t i o n at 10°C i n the absence of BHC. See pp. 33-37 for d e f i n i t i o n of terms used.  - 143 -  1.020  1.002  1.000  - 144 $PVP 4.0 2.0 1.5 1.0 0.4 0  AxlO  ?  5  2.72 2.71 2.64 2.48 2.47 2.42  (Gm./ml.) 1.0184 1.0183 1.0127 1.0115 1.0102 1.0092 .  t (sec.) 1260.9 820.9 747.9 705.5 611.3 566.9  .03493 .02255 .02000 .01770 .01525 .01385  °7 sp *?s /C 1.5220 38.05 .6282 31.41 .4440 29.60 .2780 27.80 .1011 25.28 0 P  Table 11(b). Calculation procedure for estimating s p e c i f i c and reduced v i s c o s i t i e s of PVP solution at 10°C i n the presence of 550 umole/L. BHC. See pp. 33-37 f o r d e f i n i t i o n of terms used. In the Absence of BHC  °c  Slope 339.0 305.1 271.2 248.6  10 20 30 40  H .55 .56 .54 .55 K  24.81 23.35 22.38 21.22  Rc 1.048 1.004 1.003 .991  In the Presence of BHC (550 umole/L.) Slope 343.5 330.3 303.8 290.6  H .58 .65 .67 .73 K  24.32 22.54 21.28 20.02  Rc .980 1.001 .995 1.015  V .98 .97 .95 .94  Table 12. Influence of BHC binding on the rheological properties of PVP at various temperatures. I n t r i n s i c v i s c o s i t y ( t°73 ) units are ml./Gm. Rc i s the correlat i o n c o e f f i c i e n t . V i s the i n t r i n s i c v i s c o s i t y r a t i o (Eq. 58). See pp. 33-37 for d e f i n i t i o n of other terms.  In the absence of BHC, the i n t r i n s i c v i s c o s i t y of PVP decreases with increase i n temperature (Table 12). The same temperature e f f e c t was reported by Goldfarb and Rodriguez (1968). This finding may be interpreted i n terms of a progressive c o i l i n g of the polymer with an increase i n temperature.*  I t may be  postulated that the van der Waals contour of the binding s i t e i s changed by the configurational change i n an unfavorable way for BHC binding which r e s u l t s i n lower binding a f f i n i t y at higher temperature. *  M i l l e r and Hamm (1953) found that the d i f f u s i o n c o e f f i c i e n t of PVP i s higher at 5°C than at 21.4°C. From t h i s they postulated that the PVP molecule i s more t i g h t l y c o i l e d at the lower temperature. In t h i s respect, our data and that obtained by Goldfarb et a l . (1968) contradict that of M i l l e r et a l . The Fikentscher's Kp- value was 30 i n each case.  - 145 39 38 37  Figure 52. Reduced v i s c o s i t y of PVP as a function of PVP concentration at various temperatures; 10 ( c i r c l e s ) , 20 (squares), 30 (triangles), and 40 (hexagons) °C. Solid and dotted l i n e s represent the v i s c o s i t i e s i n the presence and absence of BHC (550 umole/L.), respectively.  36 35 34 33 32 31 30 29 28 27 26 25 24 23 22 21 20 19 18 .5  1.0  1.5  2.0  % PVP  2.5  3.0  3.5  4.0  - 146 At a l l temperatures studied, binding of BHC i s accompanied by a s l i g h t but s i g n i f i c a n t decrease i n i n t r i n s i c v i s c o s i t y . The shrinking of the polymer c o i l , indicated by the reduction i n i n t r i n s i c v i s c o s i t y , supports the postulate previously made (see p. 141). Molyneux and Frank (1961b) employed the ' i n t r i n s i c v i s c o s i t y r a t i o ' (V), defined i n Eq.58, as a d i r e c t i n d i c a t i o n of the e f f e c t of cosolute on the size of the polymer molecule i n s o l u t i o n . C?) i n the Presence of Cosolute 1  V =  (*]) i n the Absence of Cosolute  (Eq. 58)  The r a t i o for BHC-PVP binding at 30°C (see Table 12) i s i n the same order as that for the interactions of sodium hydrogen phthalate and sodium-p-hydroxy benzoate with PVP studied by Molyneux and Frank (1961b).  In solutions of high ionic  strength,  expansion due to the coulombic repulsion between the BHC anions bound to the polymer c o i l i s believed to be greatly reduced by the 'screening' e f f e c t of the free counterions,  T r i s of RH^  4-  form, within the T r i s buffer solution encompassed by the polymer molecule. In other words, van der Waals forces s t a b i l i z i n g the PVP-BHC complex probably exceed repulsive forces and hence a s l i g h t degree of c o i l i n g can be maintained. Molyneux and Frank (1961b) also observed shrinking of the polymer c o i l i n the presence of nonionic cosolutes such as phthalic acid and benzoic a c i d . The bulky nonionic part of BHC molecule may also have such an e f f e c t and cooperate to the shrinking of PVP.  - 147 Changes i n i n t r i n s i c v i s c o s i t y caused by the cosolute are generally not a linear function of the concentration of the l a t t e r (Eliassaf and others, 1960). In the present study, measurements were made at only one BHC concentration, a concentration which i s near the s o l u b i l i t y l i m i t . Therefore, the changes measured only serve to ascertain the existence of an e f f e c t at that concentration and the general nature of t h i s e f f e c t . As shown i n the l a s t column i n Table 12, the extent of c o i l ing  of PVP due to complexation  with BHC  becomes greater as the  temperature increases. The v a r i a t i o n of i n t r i n s i c v i s c o s i t y with temperature i s more c l e a r l y demonstrated i n Figure 53, i n which Eq. 59 analogous to the Arrhenius equation of k i n e t i c s (Martin, 1968c) i s used to i l l u s t r a t e the data. «9 - A  E  v  /  R  T  e  (Eq. 59)  A i s a constant and depends on the solution being studied. Ev i s the 'activation energy' required to i n i t i a t e flow of the solution. As shown i n Figure 53, the data obtained f i t s the equation w e l l . From the slope, the a c t i v a t i o n energy was c a l c u l a t e d to be and 1.13  0.92  Kcal/mole i n the absence and presence of BHC, r e s p e c t i v e l y .  The Huggins constant i s some function of the solute-solvent i n t e r a c t i o n (Huggins, 1942;  Yang, 1961). As the temperature increa-  ses, the parameter for the polymer i n the presence of BHC  anion  shows a gradual but s i g n i f i c a n t deviation from the average value of  0.55  for the polymer i n the absence of BHC.  No attempt has  been made to interpret these findings, since the deviations are, i n general too i r r e g u l a r for them to be correlated with any d e f i n i t e molecular e f f e c t (Molyneux and Frank, 1961b).  - 148 -  26  3.1  3.2  3.3  3.4  3.6  3  1  - ^ r  3-5  x io  5  F i g u r e 53. E f f e c t o f temperature on the i n t r i n s i c v i s c o s i t i e s o f PVP i n the presence and absence o f BHC. Symbols have same meanings as those i n F i g u r e 52.  10. Comparison of Methods Used to Evaluate Binding  In the present work, f i v e d i f f e r e n t methods have been used to investigate the mechanism of binding of BHC  to various macro-  molecules; spectrophotometric, dynamic and equilibrium dialyses, s o l u b i l i t y , and viscometric methods. The f i r s t four methods are based on the analysis of changes i n properties of BHC  due to  complex formation. Viscometry measures changes i n properties occurring i n the macromolecule. The data from the spectrophotometric method i s , i n general, of doubtful value. The main d i f f i c u l t y i s estimation of absorptiv i t y value of bound drug (Klotz, 1953a). As shown i n Table 3, the per cent depression of BHC absorbance i s reproducible i n the presence of excess amount of macromolecules. However, i t can be expected that bound BHC w i l l have d i f f e r e n t molar absorptivity values i f binding s i t e s on a macromolecule shows heterogeneity (e.g., HSA)  because the actual binding environment w i l l be  d i f f e r e n t from one set of s i t e s to another set of s i t e s . Figure 54 compares the spectrophotometric data with that obtained from the equilibrium d i a l y s i s method for the HSA-BHC i n t e r a c t i o n . A l l points are from the absorbance depression measured i n 0.04$ HSA  (see Figure 34 for data and Table 4 for  c a l c u l a t i o n procedure). Different values are assigned for molar absorptivity of bound drug. It i s surprising to note that the largest deviations occur when the experimentally  determined  absorptivity value (closed c i r c l e s i n Figure 54) i s used.  - 150 •  '  3.2 •  ~  •  «  3.0 • 2.8 • 2.6 •  0  2  4  6  8  10  12  14  16 18 20  22  24  26  28  (Df) x 10 mole/L. Figure 54. Comparison of binding data obtained from spectrophotometric analysis (closed symbols) with those from the equilibrium d i a l y s i s method (open c i r c l e s ) for the HSA-BHC i n t e r a c t i o n at 20°C. Various values were given to ot i n Eq. 38; 0.899 (• , experimentally determined), 0.880 ( A , a r b i t r a r i l y chosen), and 0.805 (•, value for PVP-BHC i n t e r a c t i o n ) . See Table 3 for more d e t a i l . 6  - 151 Figure 55 shows the comparison for the PVP-BHC i n t e r a c t i o n . At extremely low concentration range of free drug, data from both methods agree. The agreement may  be due to the fact that the  molecule has only one set of binding s i t e s for BHC.  PVP  However, the  precision of the spectrophotometric method f a l l s o f f rapidly at higher concentrations  of free drug and the data cannot be used by  i t s e l f . Klotz (1946c) arrived at a s i m i l a r conclusion. The dynamic d i a l y s i s method i s based on the fact that  non-  d i f f u s i b l e macromolecule-drug complexes are 'reversibly' formed i n the macromolecule compartment and that the rate of loss of drug molecule from that compartment i s d i r e c t l y proportional to the free drug concentration, provided that care i s taken to ensure that 'sink' conditions are maintained for the d i f f u s i n g species. The main advantage of t h i s method i s that i t i s less tedious  and  time-consuming and requires fewer i n d i v i d u a l measurements to define binding behaviour. However, i n order to prevent back d i f f u s i o n of drug molecule into the macromolecule compartment, large amounts of buffer are required. In addition, i t i s d i f f i c u l t to obtain the experimental conditions for an unkown binding system over a wide range of free drug concentration. Numerical comparison of the data from t h i s method under a s p e c i f i e d condition (see p. 63) been made already  (see p.  has  110).  Equilibrium d i a l y s i s i s one of most suitable methods for binding studies. The volume r a t i o of the two compartments must be appropriately assigned drug concentration  i n order to cover a wide range of free  (or to improve precision of a n a l y s i s ) . A d i a l y s i s  c e l l which has a fixed volume (e.g., p l e x i g l a s block used by Patel and Foss, 1964)  i s not completely s a t i s f a c t o r y for the study of  very insoluble compounds.  - 152 -  Figure 55. Comparison of binding data obtained from spectrophotometric analysis (closed symbols) with those obtained from the equilibrium d i a l y s i s method (open c i r c l e s ) f o r the PVP-BHC i n t e r a c t i o n at 20°C. Data are from absorbance depression measured i n 0.02$ (•) and 0.1$ ( A ) PVP.  - 153 S o l u b i l i t y analysis has been widely used to study molecular interaction i n solution. If there i s a sharp break i n the s o l u b i l i t y curve, the o v e r a l l step s t a b i l i t y constant can be estimated chi and Connors, 1965; Connors and Mollica, 1966).*  (Higu-  However, i n  the present work, the macromolecules studied have appreciable aqueous s o l u b i l i t y and formed soluble complexes with BHC;  the  s o l u b i l i t y curves were non-linear and f a i l e d to show a break. The r value obtained from the linear portion i s a constant (see p. 121). Hence the method gives only one point on any type of binding curve ( i . e . , Langmuir, Scatchard, and double r e c i p r o c a l plots). For the PVP-BHC interaction, the r value was approximately  20  compared with a value of 50 obtained from equilibrium d i a l y s i s (see p. 121 and p. 126). The molecular model of the PVP-BHC complex supports the value of 50 i d e n t i c a l binding s i t e s per PVP  molecule  (see pp. 140-141). It i s apparent, therefore, that a l l the binding s i t e s are not accessible to BHC molecules and that uptake of BHC *  The o v e r a l l step s t a b i l i t y constant (k y) i s defined by 0  (MD ) n  x>v for the reaction,  (M)(D)  nD + M  =  (Eq. 60)  n  MD  n  (Eq. 61)  The right-hand side of Eq. 60 can be obtained by multiplying Eq. 14 by i t s e l f from i = l to n. Eq. 62 then shows the r e l a tionship between o v e r a l l step s t a b i l i t y constant ( k ) , step s t a b i l i t y constant (k_), and i n t r i n s i c association constant (K) o v  k  o v  s  k_.k2«kg.••-k = K n  n.^).^)....^)  (Eq. 62)  - 154 i n t erferes with the i n t e r a c t i on at other s i t e s . Interference due to repulsion of bound BHC  anion for unbound BHC  anion i s  u n l i k e l y because of the screening e f f e c t of high ionic strength (see p.  146).  Viscometry does not provide quantitative information about binding and i n the present study i t s a p p l i c a b i l i t y was the low s o l u b i l i t y of BHC.  limited by  However, a configurational change i n  PVP was detected from measurements made at one BHC  concentration.  C o i l i n g of the polymer, as a r e s u l t of binding (see p. 146), i s a possible explanation of the i n a c c e s s i b i l i t y of a l l binding s i t e s to BHC  molecules.  V I I . SUMMARY AND CONCLUSION  1. Physicochemical  Potentiometric  P r o p e r t i e s o f BHC i n Aqueous S o l u t i o n s  and spectrophotometric  methods were used t o  determine the apparent pKa v a l u e s o f the weak d i b a s i c a c i d , BHC. BHC i s very i n s o l u b l e i n a c i d i c s o l u t i o n s and i t was to modify the u s u a l t i t r a t i o n approach t o pKa  necessary  determinations.  Values obtained were 4.4 and 8.0 f o r pKa^^ and pKag, r e s p e c t i v e l y . The e f f e c t o f i o n i c s t r e n g t h  ( c h l o r i d e i o n ) and pH on the  apparent s o l u b i l i t y o f BHC was i n v e s t i g a t e d . Increase  i n solu-  b i l i t y o f BHC with i n c r e a s e i n c h l o r i d e i o n c o n c e n t r a t i o n was e x p l a i n e d i n terms o f water s t r u c t u r e . The e f f e c t o f pH on the BHC s o l u b i l i t y was i n t e r p r e t e d on the b a s i s o f i n t r a m o l e c u l a r hydrogen bonding. T h i s view was supported  by the i n f r a r e d spectrum  of the drug i n KBr d i s c .  2. B i n d i n g  Studies  Data obtained by u t i l i z i n g the s o l u b i l i t y a n a l y s i s and dynamic d i a l y s i s methods i n d i c a t e d t h a t b i n d i n g s t r e n g t h o f BHC i n c r e a s e s i n the order dextran,  HES and potato s t a r c h s o l .  HSA and PVP possess much g r e a t e r a f f i n i t y  f o r BHC than the  o t h e r s and f o r these two macromolecules the mechanism o f i n t e r a c t i o n with BHC was i n v e s t i g a t e d i n d e t a i l . The f o l l o w i n g c o n c l u s i o n s were reached.  (a) i)  With respect  Both  under  to  interactions  the  lactone  structure  (b)  exothermic  conditions,  i n BHC i s  With respect  -  b o t h HSA-BHC a n d PVP-BHC  are  experimental  156  to  and occur ii)  The  HSA-BHC  binding sites  was o b s e r v e d .  of  the  was a p p r o x i m a t e l y t h r e e ,  association  set  constant  is  equal  at  40  and 20°C,  to  be  inter-helical cavities  mates, iv)  and i s  The  large  selectivity  is  (c)  2  ii)  the  AH  to  binding sites  The  the  AG  i i i )  role  i n the data  a significant indicates  binding  that  process.  0  seven  c o i l i n g of  sites  L./mole  are  believed  Waals contour  approxi-  the  BHC m o l e c u l e ,  was s u p p o r t e d by  value,  or  eight  constant  and van der  i n the  10^  the  v)  The  main b i n d i n g  i)  A PVP m o l e c u l e  provides approximately  binding process,  part  binding  intrinsic  a n d 35 x  for,  BHC m o l e c u l e s .  indicates  The  interaction:  association  Ion-dipole  heterogeneity  sources.  40,000  of  A  The b i n d i n g s i t e s  selective  to  for  ii)  10^  binding sites 0  i)  number o f  whose v a n d e r  PVP-BHC  consists  intrinsic  thermodynamic  plays data  With respect  L./mole.  important the  the  average  17 x  d e r i v e d from non-ionic  binding site  PVP. 10  of  to i i i )  sense  a molecular weight of  identical one  in a  c o n t r i b u t i o n of  energy  with  respectively,  thus  The  -unsaturated  complexations.  interaction:  of  first  spontaneously  ol,/3  i n v o l v e d i n the  interactions:  that  implies  that  monomer u n i t s  of  is  i n the  order  Waals  forces  play  iv)  At low  v)  Intrinsic  polymer takes  of an  temperatures,  h y d r o p h o b i c bond  process, the  This  50  place  formation viscosity during  VIII. 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