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Separation of antimicrobial protein fractions from animal resources for potential use in infant feeding Al-Mashikhi, Shalan Alwan Edan 1987

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SEPARATION OF ANTIMICROBIAL PROTEIN FRACTIONS FROM ANIMAL RESOURCES FOR POTENTIAL USE IN INFANT FEEDING  by SHALAN ALWAN EDAN AL-MASHIKHI B . S c , University of Baghdad M . S c , University of Baghdad A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Food Science)  We accept this thesis as conforming to the required  standard  THE UNIVERSITY OF BRITISH COLUMBIA August,  1987  (c) Shalan Alwan Edan Al-Mashikhi,  1987  In presenting  this thesis in partial fulfilment  of the  requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department  or  by  his  or  her  representatives.  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  DE-6(3/81)  ii  ABSTRACT In  the  first  part  of  this  study,  a  non-ferric  method  for  selective  elimination of p - l a c t o g l o b u l i n from cheese whey was i n v e s t i g a t e d .  A new method  was developed based on hexametaphosphate treatment of cheese whey.  When Cheddar  cheese whey was treated under the optimized c o n d i t i o n s , i . e . ,  1.33 mg/mL sodium  hexametaphosphate at 22°C and pH 4.07 for 1 h r , more than 80% of B-lactoglobulin was removed by p r e c i p i t a t i o n .  Almost a l l  of the  immunoglobulins and the major  portion of a-lactalbumin were retained in the supernatant as indicated by sodium dodecyl s u l f a t e polyacrylamide gel electrophoresis (SDS-PAGE) and immunochemical assays.  By d i a l y s i s against d i s t i l l e d water 72.2% of the phosphorus was removed  from the  supernatant.  In  the  second and the  were used f o r  i s o l a t i o n of  By using gel  filtration  active cheese  immunoglobulin whey,  at  pH  on Sephacryl  G were  with  1,4-Butanediol  obtained,  the  copper first  ion  the  thesis,  and  for  99,  chromatographic methods from whey p r o t e i n s .  and 92.1% whey,  selectively  diglycidyl  for  yellowish peak was  the rich  and Cheddar  adsorbed  to  the  containing  0.5M  ether-iminodiacetic chromatography  same purpose. in  biologically  acid  with 5 mM Veronal-HCl  chelate-interaction  used  peak was r i c h in immunoglobulins.  83.3  colostral  Lactoferrin,  Sepharose, was eluted  7.2.  of  S-300,  obtained  Sepharose 6B, or s o - c a l l e d metal loaded  part  immunoglobulins and l a c t o f e r r i n  respectively.  heparin-attached NaCl,  third  Of  lactoferrin,  the  while  binding  protein-metal  to  bacterial  interaction  on  (MCIC), was two  peaks  the second  Some of the physical and chemical properties  of the proteins in these peaks, including immunochemical p r o p e r t i e s , points,  acid  1ipopolysaccharides,  via histidine modification,  and  the  and the  isoelectric  mechanism capacity  of  of the  i ii  method  were  lactoferrin In for to  studied.  possibility  of  isolating  immunoglobulins  and  from e l e c t r o d i a l y z e d whey was also i n v e s t i g a t e d .  the f o u r t h ,  i s o l a t i o n of isolate  The  f i f t h and sixth parts  the  immunoglobulins and l a c t o f e r r i n  these b i o l o g i c a l l y important  and egg white.  of  thesis,  the method developed  from whey protein was applied  proteins d i r e c t l y  from skimmilk,  blood  The casein in skimmilk was found to compete with immunoglobulins  for binding to copper ion in MCIC column when skimmilk was loaded in presence of 0.05 was  M T r i s - a c e t a t e buffer solved by changing  containing 0.5  the  equilibrating  containing 0.5  M NaCl, pH 7.0.  the  biologically  yield  of  M NaCl, pH 8.2; buffer  to  however, t h i s problem  0.02  M phosphate  buffer  When blood was d i r e c t l y applied to MCIC column,  active  IgG  was  more  than  95%.  Ovotransferrin,  strongly adsorbed to the MCIC column, was eluted with two-step e l u t i o n protocols which  suggests  immunoglobulins,  it  exists  caseins,  in  two  transferrin  forms. and  The  histidine  ovotransferrin  were  involved in the mechanism of the i n t e r a c t i o n with the MCIC column.  residues  in  found  be  to  iv TABLE OF CONTENTS Page ABSTRACT  ii  TABLE OF CONTENTS  iv  LIST OF TABLES  vii  LIST OF FIGURES  viii  APPENDIX  xvii  ACKNOWLEDGEMENTS  .  xviii  INTRODUCTION  1  LITERATURE REIVEW A. History B. Human milk v s . cow's milk C. Humanizing infant formula D. A l l e r g e n c i t y of whey proteins E. Antimicrobial system in human milk and milk substitutes 1. Immunoglobulins 2. L a c t o f e r r i n 3. Lactoperoxidase and lysozyme 4. B i f i d o b a c t e r i a 5. Ovotransferrin F. I s o l a t i o n of bioactive proteins 1. General methods 2. Metal chelate interaction chromatography  4 4 5 9 10 13 13 18 19 20 22 23 23 24  MATERIALS AND METHODS A. Materials B. Acid whey preparation C. Sodium dodecyl s u l f a t e polyacrylamide gel electrophoresis 1. Discontinous SDS-PAGE 2. Gradient SDS-PAGE D. Immunochemical analysis 1. Immunoelectrophoresis and immunodiffusion 2. Enzyme linked immunosorbent assay for anti-1ipopolysacchardies a c t i v i t y determination 3. Sandwish enzyme linked immunosorbent assay for IgG assays E. Sodium hexametaphosphate treatment of cheese whey F. Optimization procedure 1. Mapping super simplex 2. Centroid mapping optimization and simultaneous f a c t o r s h i f t . . . . G. Evaluation of separation e f f i c i e n c y (response value) H. Surface plot I. Phosphorus determination  26 26 26 27 27 28 28 28 29 30 30 32 32 32 33 34 34  V  Page J.  K.  L. M. N. 0. P. Q. R. S. T.  Fractionation procedures of bioactive components 1. Gel f i l t r a t i o n chromatography 2. S i l i c a adsorption chromatography 3. Heparin-Sepharose chromatography 4. Metal c h e l a t e - i n t e r a c t i o n chromatography Determination of capacity of MCIC 1. Immunoglobulins 2. Ovotransferrin 3. T r a n s f e r r i n Preparation of egg white Preparation of apo, d i f e r r i c and d i c u p r i c ovotransferrin H i s t i d i n e modification of proteins I s e l e c t r i c focusing Preparation of a n t i s e r a Measurement of b a c t e r i o s t a t i c a c t i v i t y Extraction of 1ipopolysaccharides Lactoperoxidase assay Separation of heavy and l i g h t chains of immunoglobulins  RESULTS AND DISCUSSION PART I. A. B. C. D. PART II. A. B. C. D. E.  Reduction of B - l a c t o g l o b u l i n content of cheese whey by using sodium hexametaphosphate Optimum conditions for separation of immunoglobulins and B-lactoglobulin Three dimensional i l l u s t r a t i o n of e f f e c t s of pH and hexametaphosphate on separation e f f i c i e n c y Elimination of phosphorus Proposal of new infant formula Separation of bovine immunoglobulins and l a c t o f e r r i n from whey proteins by gel f i l t r a t i o n techniques Gel f i l t r a t i o n of Sephacryl S-300 Gel f i l t r a t i o n on TSK HW-5S I s o l a t i o n of immunoglobulins from whey proteins I s o l a t i o n of l a c t o f e r r i n from whey proteins Anti-1ipopolysacchardies a c t i v i t y of i s o l a t e d immunoglobulins  Separation of immunoglobulins and l a c t o f e r r i n from cheese whey by adsorption and chelating chromatography techniques A. Adsorption chromatography methods B. Metal c h e l a t e - i n t e r a c t i o n chromatography 1. Acid whey 2. Cheddar cheese whey 3. E l e c t r o d i a l y z e d and sweet whey powders a . IgG a c t i v i t y and protein content b. E f f e c t of pH adjustment c . I s o l a t i o n of immunoglobulins by c o n t r o l l e d pore glass d. I s o l a t i o n of immunoglobulins by metal c h e l a t e - i n t e r a c t i o n chromatography C. Binding capacity and recovery of immunoglobulins from metal c h e l a t e - i n t e r a c t i o n chromatography column  34 34 35 35 35 36 36 39 40 40 40 41 41 41 42 42 43 44 45  45 46 48 53 55 57 58 61 67 67 75  PART III.  79 80 83 83 86 90 90 93 97 99 104  vi Page D. Anti-1ipopolysaccharides a c t i v i t y of immunoglobulins r i c h fraction E. Lactoperoxidase content of l a c t o f e r r i n r i c h f r a c t i o n F. I d e n t i f i c a t i o n of glycoproteins in l a c t o f e r r i n r i c h f r a c t i o n G. I s o e l e c t r i c points of l a c t o f e r r i n and immunoglobulins r i c h fractions H. H i s t i d i n e modification and metal c h e l a t e - i n t e r a c t i o n chromatography properties I. Separation of heavy and l i g h t chains of immunoglobulins J . Separation of l a c t o f e r r i n and lactoperoxidase in l a c t o f e r r i n rich fraction 1. Gel f i l t r a t i o n method 2. Stepwise pH e l u t i o n 3. Imidazole gradient e l u t i o n PART IV. Metal c h e l a t e - i n t e r a c t i o n chromatography of skimmilk A. MCIC with T r i s - a c e t a t e buffer B. MCIC with phosphate buffer C. Mechanism of casein-metal i n t e r a c t i o n . . 1. a-casein 2. a j and 8-casein 3. K - c a s e i n s  PART V. Separation of immunoglobulins and t r a n s f e r r i n from blood serum and plasma by metal c h e l a t e - i n t e r a c t i o n chromatography A. Metal c h e l a t e - i n t e r a c t i o n chromatography 1. Blood serum on Cu-loaded MCIC 2. Blood serum on MCIC columns loaded with other metal ions 3. Blood plasma on MCIC column B. Immunochemical assays C. B a c t e r i o s t a t i c a c t i v i t y of blood immunoglobulins and transferrin D. Anti-1ipopolysaccharides a c t i v i t y of blood immunoglobulins E. Capacity of MCIC column for t r a n s f e r r i n F. Mechanism of protein-metal i n t e r a c t i o n Separation of ovotransferrin from egg white by metal c h e l a t e - i n t e r a c t i o n chromatography A. Metal c h e l a t e - i n t e r a c t i o n chromatography of egg white B. Capacity of MCIC column for ovotransferrin C. Mechanism of ovotransferrin separation by MCIC  106 108 108 110 110 112 117 117 117 121 124 125 129 132 133 133 137  141 142 142 142 147 149 149 149 152 155  PART VI.  157 158 162 164  CONCLUSIONS AND RECOMMENDATIONS  168  REFERENCES CITED  170  APPENDIX  184  vii LIST OF TABLES Page Table 1.  Gross Composition of Human and Cow's Milk (Grams per 100 Grams of F l u i d Product)  6  3  Table 2.  Protein Composition of Human and Cow's M i l k  Table 3.  Concentration of bovine immunoglobulins in serum and secretions (mg/mL)  8  a  15  a  Table 4.  Biochemical c h a r a c t e r i s t i c s of bovine immunoglobuins  Table 5.  Phosphorus d i s t r i b u t i o n in supernatant and p r e c i p i t a t e obtained by SHMP treatment. P, phosphorus Protein composition of human and cow's milks and wheybased and proposed B - l a c t o g l o b u l i n (B-Lg)-free infant formula  Table 6.  Table 7.  Table 8. Table 9.  17  a  54  56  Immunoglobulin G contents* of f r a c t i o n s , obtained from gel f i l t r a t i o n on Sephacryl S-300 and crude Ig prepared by ammonium s u l f a t e treatment  62  IgG a c t i v i t y of peak and unbound f r a c t i o n s from MCIC treatment of whey  87  Whey proteins d i s t r i b u t i o n * in acid whey, f r a c t i o n s obtained from MCIC of bovine acid whey, and the unbound materials to MCIC column  88  Table 10. IgG and protein contents of reconstituted ED and sweet whey powders compared to l i q u i d cheese whey  92  Table 11. IgG a c t i v i t y of pH 4.5 and pH 8.2 supernatant (S) and p r e c i p i t a t e (P) f r a c t i o n s from sweet whey and ED whey  96  a  Table 12. IgG content of d i f f e r e n t stages of the i s o l a t i o n of IgG from skimmilk on MCIC column  128  Table 13. Binding of casein f r a c t i o n s to MCIC column before and a f t e r modification of h i s t i d i n e groups  135  Table 14. IgG contents* of blood serum or plasma f r a c t i o n s obtained from MCIC column loaded with d i f f e r e n t metal ions  150  LIST OF FIGURES  Flow diagram of the procedure for elimination B - l a c t o g l o b u l i n from Cheddar cheese whey with SHMP Preparation of metal  of  chelate agarose  Flow chart of MCIC process of i s o l a t i o n of Ig  cheese whey treatment  for ,  Approximate response surface patterns f o r (A) pH and (B) SHMP concentration obtained by mapping accumulated data from simplex optimization (Vertices 1-9) and centroid optimization (Vertices 10-15). T, target values of pH and SHMP SDS-PAGE of supernatant (S) and p r e c i p i t a t e (P) obtained a f t e r treatment with 1.33 mg/mL SHMP at pH 4.07. CCW, Cheddar cheese whey; a-La, a-lactalbumin; B-Lg, B-lactoglobulin; IgG-HC, immunoglobulin G heavy chain; IgG-LC, immunoglobulin G l i g h t chain; BSA, bovine serum albumin; OVA, ovalbumin Immunoelectrophoretic pattern of Cheddar cheese whey. S, supernatant;CCW, Cheddar cheese whey; P, p r e c i p i t a t e ; IgG, immunoglobulin G; B-Lg, B - l a c t o g l o b u l i n ; abwp, antibovine whey proteins Contour (A) and 3-dimensional (B) surface plots of r e l a t i o n s h i p between pH, SHMP and Separation effeciency (SE) of cheese whey treatment. ("about" angle=60 and "above" angle=35 for 3-dimensional plot) Gel f i l t r a t i o n of bovine c o l o s t r a l whey on Sephacryl S-300 Superfine column (94 x 2.5 cm) eluted with 0.1 M T r i s - H C l buffer pH 8.0 containing 0.5 M NaCl. Flow r a t e , 12 mL/hr. 1 and 3 are f r a c t i o n s 1 and 3, respectively Gel f i l t r a t i o n of crude Ig obtained from ammonium s u l f a t e treatment on Sephacryl S-300 Superfine column (94 x 2.5 cm), eluted with 0.1 M T r i s - H C l buffer pH 8.0 containing 0.5 M NaCl, flow rate 12 mL/hr. 1 and 3 are f r a c t i o n s 1 and 3, respectively SDS-PAGE of f r a c t i o n s obtained from gel f i l t r a t i o n on Sephacryl S-300. F l , f r a c t i o n 1; F3, f r a c t i o n 3 (Figure 8); Ig, crude immunoglobulin; L F , l a c t o f e r r i n ; CW, untreated c o l o s t r a l whey; HC, LC, immunoglobulin heavy and l i g h t chains, respectively  ix Page Figure 11.  Figure 12.  Figure 13.  Figure 14,  Figure 15.  Figure 16.  Figure 17.  Figure 18,  Figure 19.  Immunoelectrophoretic analysis against anti-whole bovine antiserum of f r a c t i o n s obtained from Figure 8. F3, f r a c t i o n 3; F l , f r a c t i o n 1; BSA, bovine serum albumin; T F , transferrin Gel f i l t r a t i o n pattern of c o l o s t r a l whey on TSK column (40 x 2.6 cm) eluted with 0.07 M imidazole-0.05 M KC1 b u f f e r , pH 6.5; flow r a t e , 50 mL/hr. 1 and 2 are f r a c t i o n s 1 and 2, r e s p e c t i v e l y  64  65  SDS-PAGE of f r a c t i o n s (Fl and F2) obtained from Figure 12 as compared to standards. Lane 1, a-lactalbumin; Lane 2, B - l a c t o g l o b u l i n ; Lane 3, bovine serum albumin; Lane 4, transferrin; M, standard mixture; AW, acid whey, CW, c o l o s t r a l whey; HC and LC, immunoglobulin heavy and l i g h t chains, respectively  66  Immunoelectrophoresis of f r a c t i o n s obtained by Sephacryl S-300 and Fractogel TSK column against anti-whole bovine serum antiserum. ( F l - S and F3-S, f r a c t i o n 1 and 3 of Figure 8, respectively) ( F l - T and F2-T f r a c t i o n 1 and 2 of Figure 12, respectively) IgG, immunoglobulin G  68  Gel f i l t r a t i o n of acid whey on Sephacryl S-300 Superfine column (94 x 2.5 cm), eluted with 0.1 M T r i s - H C l b u f f e r , pH 8.0 containing 0.5 M NaCl. Flow rate 18 mL/hr. 1, 2, 3 and 4 are the f r a c t i o n s obtained  69  Gel f i l t r a t i o n of Cheddar cheese whey on SepharylS-300 Superfine column (94 x 2.5 cm), eluted with 0.1 M T r i s - H C l b u f f e r , pH 8.0 containing 0.5 M NaCl. Flow rate 18 mL/hr. 1, 2, 3 and 4 are the f r a c t i o n s obtained ,  70  SDS-PAGE of f r a c t i o n s (1, 2, 3, 4) obtained from Figure 15. AW, acid whey; IgG, immunoglobulin G; HC and LC, heavy and l i g h t chains of immunoglobulins, respectively  71  SDS-PAGE of f r a c t i o n s (1, 2, 3, 4) obtained from Figure 16. IgG, immunoglobulin G; CCW, Cheddar cheese whey; HC and LC, heavy and l i g h t chains of immunoglobulins, respectively  72  Immunoelectrophoresis of fractions obtained from gel filtration of whey protein against anti-whey protein antiserum. AW, acid whey; F l - H , f r a c t i o n 1 from Figure 20; F2-A, f r a c t i o n 2 from Figure 15; F2-C, f r a c t i o n 2 from Figure 16; IgG, immunoglobulin G  73  X  Page Figure 20.  Figure 21.  Figure 22.  Figure 23.  Heparin-Sepharose chromatography of Cheddar cheese whey. Cheese whey (400 mL) dialyzed against 0.05 M NaCl in 5 mM v e r o n a l - H C l , pH 7.4 was applied to the column (10 mL settled gel). The column was washed with the same buffer and then eluted with a l i n e a r gradient of NaCl ( . . . . ) as indicated. The flow rate was 50 mL/hr. UB, unbound p r o t e i n s ; 1, f r a c t i o n 1  74  SDS-PAGE of f r a c t i o n s obtained from Figure 20, CCW, Cheddar cheese whey; UB; unbound whey proteins to Heparin-Sepharose column; F l , l a c t o f e r r i n r i c h f r a c t i o n ; L F , l a c t o f e r r i n  76  Anti-1ipopolysaccharide a c t i v i t y of c o l o s t r a l IgG i s o l a t e d by gel f i l t r a t i o n on Sephacryl S-300. U-U , E. c o l i LPS; D - n , S_;_ typhi murium LPS; O - O , parapertussis LPS  77  E l u t i o n p r o f i l e s of adsorbed proteins from s i l i c a (S), c o n t r o l l e d pore glass (C) and alumina (A) chromatographic treatment of Cheddar cheese whey. One l i t r e of Cheddar cheese whey in 0.005 M Na HP0 , pH 8.2 was passed through 1.3 x 7.0 cm column of (S), (C) or (A) e q u i l i b r a t e d with 0.005 M phosphate buffer at pH 8.2. A f t e r washing with 30 mL of e q u i l i b r a t i n g b u f f e r , the adsorbed proteins were eluted with El (50 mL 0.1 M a c e t i c acid pH 2.77 containing 0.5 M NaCl), then E2 (60 mL 0.1 M T r i s - H C l pH 9.0 containing 0.5 M NaCl). The flow rate was 1 mL/min  81  SDS-PAGE p r o f i l e s of cheese whey and f r a c t i o n s obtained from Figure 23.Lane 1, untreated Cheddar cheese whey; Lane 2, a c e t i c acid f r a c t i o n from s i l i c a sand; Lane 3, a c e t i c acid f r a c t i o n from controlled pore g l a s s ; Lane 4, a c e t i c acid f r a c t i o n from alumina; Lane 5, T r i s - H C l f r a c t i o n from s i l i c a sand; Lane 6, T r i s - H C l f r a c t i o n from c o n t r o l l e d pore g l a s s ; Lane 7, T r i s - H C l f r a c t i o n from alumina; Lane 8, unbound f r a c t i o n from s i l i c a ; Lane 9, unbound f r a c t i o n from controlled pore g l a s s ; Lane 10, unbound fraction from alumina; Lane 11, untreated Cheddar cheese whey; Lane 12, a c e t i c acid f r a c t i o n from alumina, L F , l a c t o f e r r i n ; HC and LC, immunoglobulin heavy and l i g h t chains, respectively  82  Elution profiles of adsorbed proteins from MCIC on Sepharose 6B treatment of 1 L Cheddar cheese whey (CCW) and 1 L acid whey (AW) (obtained from raw m i l k ) , using l i n e a r gradient elution of 0.05 M T r i s - a c e t a t e containing 0.5 M NaCl, pH 8.0 to 2.8. Flow rate was 0.8 mL/min. CCW, Cheddar cheese whey; AW, acid whey  84  2  Figure 24.  Figure 25.  4  xi Page Figure 26.  Figure 27.  Figure 28.  Figure 29.  Figure 30.  Figure 31.  Figure 32.  SDS-PAGE p r o f i l e s of acid whey and f r a c t i o n s obtained by MCIC and gel f i l t r a t i o n . Lanes 1 and 2, are f r a c t i o n s obtained by MCIC; Lane 3, unbound material to MCIC column; Lanes 4 and 5, peak 1 and 2 obtained by gel f i l t r a t i o n on Sephacryl column; Lane 6, mixture of standard proteins ( t r a n s f e r r i n , bovine serum albumin, B - l a c t o g l o b u l i n s and a-lactalbumin); Lane 7, acid whey, L F , l a c t o f e r r i n ; HC and LC, heavy and l i g h t chains of immunoglobulins, respectively . . . .  85  Immunoelectrophoretic a n a l y s i s of f r a c t i o n s obtained by MCIC of acid whey. IgG, immunoglobulin G; P2 and PI are Peak 2 and 1, respectively of Figure 25; T F , t r a n s f e r r i n ; BSA, bovine serum albumin  89  SDS-PAGE of Cheddar cheese whey and f r a c t i o n s obtained by MCIC on Sepharose 6B treatment. Lane 1, control cheese whey; Lanes 2, 3 and 4, areunbound f r a c t i o n ; Lanes 5 and 7, f i r s t eluted peak; Lanes 6 and 8, second eluted peak; LF, lactoferrin; HC and LC heavy and light chains of immunoglobulin, respectively  91  SDS-PAGE p r o f i l e of l i q u i d cheese whey a f t e r pH adjustment and c e n t r i f u g a t i o n . Lanes 1, 3, 5, 7, 9 and 11 are the p r e c i p i t a t e and Lanes 2, 4, 6, 8, 10 and 12 are the supernatant of samples treated at pH 8 . 5 , 8.0, 7.0, 6.0, 5.0 and 4 . 5 , r e s p e c t i v e l y , L F , l a c t o f e r r i n ; BSA, bovine serum albumin; C, casein  94  SDS-PAGE p r o f i l e s of f r a c t i o n s from ED whey a f t e r pH adjustment and centriguation. Lanes 1 and 3, are p r e c i p i t a t e at pH 8.2 and 4.5 r e s p e c t i v e l y ; Lanes 2 and 4, are supernatant at pH 8.2 and 4.5 r e s p e c t i v e l y ; Lane 5, e l e c t r o d i a l y z e d whey; L F , l a c t o f e r r i n , HC and LC, heavy and l i g h t chains of immunoglobulins, respectively  95  E l u t i o n p r o f i l e of adsorbed proteins from CPG (10 mL) treatment of 250 mL of e l e c t r o d i a l y z e d whey (EDI), 250 mL of sweet whey (SW), and 1 L of ED whey (ED2). Arrows indicate s t a r t of e l u t i o n with E l (0.1 N a c e t i c acid pH 2.8 containing 0.5 M NaCl) and E2 (0.1 M T r i s - H C l , pH 9.0 containing 0.5 M NaCl) buffers  98  Elution profile of adsorbed proteins from MCIC on Sepharose 6B treatment of 960 mL e l e c t r o d i a l y z e d whey (EDW) and 720 mL sweet whey (SW)powders reconstituted in water, using l i n e a r gradient elution of 0.05 M Tris-acetate containing 0.5 M NaCl, pH 8.2 to 2.8. Flow rate was 0.8 mL/min. 1 and 2 are f r a c t i o n s obtained  100  xi i Page Figure 33.  Figure 34.  Figure 35.  Figure^36.  Figure 37.  Figure 38.  Figure 39.  SDS-PAGE p r o f i l e s of ED whey and f r a c t i o n s obtained by MCIC on Sepharose 6B treatment. Lane 1, control untreated whey; Lanes 2, 3 and 4, are unbound f r a c t i o n s ; Lane 5, wash f r a c t i o n ; Lane 6, f i r s t eluted peak; Lane 7, second eluted peak; BSA, bovine serum albumin; HC, heavy chain of immunoglobulins  101  SDS-PAGE p r o f i l e s of sweet whey and f r a c t i o n s obtained by MCIC on Sepharose 6B treatment. Lanes 1, 12, control untreated whey; Lane 2, p r e c i p i t a t e ; Lanes 3, 4 and 5; unbound f r a c t i o n s ; Lanes 6, 7, 8 and 9, wash f r a c t i o n s ; Lane 10, f i r s t eluted peak; Lane 11, second (shoulder) eluted peak; BSA, bovine serum albumin  102  Saturation point for adsorption of crude Ig (prepared from colostrum by ammonium s u l f a t e method) on Cu-loaded IDA-BGE Sepharose 6B (SROSE) and Sephacryl S-300 (SACRYL). 0.3% crude Ig was passed through a 10 mL column (7.0 x 1.4 cm) e q u i l i b r a t e d with 0.05 M T r i s - a c e t a t e 0.5 M NaCl, pH 8.2. W, washing with the s t a r t i n g b u f f e r s ; E, e l u t i o n with 0.05 M T r i s - a c e t a t e / 0 . 5 M NaCl, pH 4 . 0 . The flow rate was 20 mL/hr  105  Anti-1ipopolysaccharide a c t i v i t y of Ig i s o l a t e d from cheese whey by MCIC method. • - • , E^ col i LPS; o - a S^ typhi murium LPS; O - O . i L parapertussis LPS  107  SDS-PAGE of whey proteins (1), l a c t o f e r r i n r i c h f r a c t i o n (2), l a c t o f e r r i n (3) and lactoperoxidase (4). (A) stained with Commassie B r i l l a n t Blue and (B) stained with periodic acid S c h i f f (PAS). HC and LC heavy and l i g h t chains respectively  109  E l u t i o n p r o f i l e s of control (Ig) and diethyl pyrocarbonate treated immunoglobulins (DEP Ig). Samples (30 mg/5 mL 0.05 M T r i s - a c e t a t e containing 0.5 M NaCl, pH 8.2) were applied to the column (1.4 x 7.0 cm) and washed (W) with the s t a r t i n g buffer then eluted (E) with 0.01 M imidazole. Flow rate was 30 mL/hr  Ill  E l u t i o n p r o f i l e s of Fl-MCIC f r a c t i o n before (Fl) and a f t e r diethyl pyrocarbonate treatment (DEP F l ) . Samples (30 mg/5 mL 0.05 M T r i s - a c e t a t e containing 0.5 M NaCl, pH 8.2) were applied to the column (1.4 x 7.0 cm) and washed (W) with the s t a r t i n g buffer then eluted (E) with 0.01 M imidazole. Flow rate was 30 mL/hr  113  xi i i Page Figure 40.  Figure 41.  Figure 42.  Figure 43,  Figure 44.  Figure 45.  Figure 46.  Figure 47.  Figure 48.  Elution profiles of reduced and alkylated heavy and l i g h t chains of immunoglobulin on Sephadex G-75 eluted with 1 M propionic a c i d . 1 and 2 are f r a c t i o n s obtained  114  SDS-PAGE profiles of heavy and light chains of immunoglobulins i s o l a t e d by gel f i l t r a t i o n . Lanes 1 and 2, crude immunoglobulins; Lane 3 and 4, immunoglobulin l i g h t and heavy chains, respectively obtained from Figure 40  115  E l u t i o n p r o f i l e of reduced and alkylated heavy and l i g h t chains of Ig on Ultrogel ACA 54 eluted with 0.1 M T r i s - H C l buffer containing 4 M Guanidine-HCl and 1 mM iodoacetamide, pH 8.2. 1 and 2 are f r a c t i o n s obtained  116  Sephacryl S-300 column chromatography of l a c t o f e r r i n r i c h f r a c t i o n obtained by MCIC of acid whey. 100 mg sample was applied to Sephacryl column (83 x 2.5 cm) and eluted with 0.05 M potassium phosphate b u f f e r , pH 7.4 containing 0.01 M NaCl. 1 and 2, are f r a c t i o n s obtained. The flow rate was 30 mL/hr  118  Stepwise e l u t i o n p r o f i l e of acid whey on MCIC eluted by decreasing pH values. Arrows indicate pHs 7, 6, 5 and 4 of 0.05 M T r i s - a c e t a t e containing 0.5 M NaCl. 1 and 2 are f r a c t i o n s obtained  119  Elution p r o f i l e of bound proteins of acid whey on MCIC column, eluted (E) by using pH gradient (5-2.8) of 0.05 M T r i s - a c e t a t e containing 0.5 M NaCl. 1 and 2 are f r a c t i o n s obtained  120  Elution p r o f i l e of l a c t o f e r r i n rich fraction on MCIC column. E l , e l u t i o n with l i n e a r gradient of 0-10 mMimidazole solution (....); E2, e l u t i o n with 0.05 M T r i s - a c e t a t e containing 0.5 M NaCl, pH 2.8. 1, 2 and 3 are f r a c t i o n s obtained  122  SDS-PAGE p r o f i l e s of f r a c t i o n s obtained from Figure 46. Lane 1, whey p r o t e i n s ; Lane 2, control Fl-MCIC; Lane 3, unbound f r a c t i o n ; Lanes 4, 5 and 6 are peak 1, 2 and 3 of Figure 46; L F , l a c t o f e r r i n , LP, lactoperoxidase  123  Elution p r o f i l e of skimmilk on MCIC column. 100 mL skimmilk undiluted (SM) or 50% d i l u t e d (DSM) with 0.05 M Tris-acetate/0.5 M NaCl) was passed through Cu-loaded Sepharose 6B (1.4 x 7.0 cm), and washed (W) with same buffer. E l , elution with the same buffer at pH 4.0; E2, e l u t i o n with 0.01 M imidazole s o l u t i o n . 1 and 2 are eluted f r a c t i o n s . The flow rate was 21 mL/hr  126  xiv Page Figure 49.  Figure 50  Figure 51  Figure 52.  Figure 53.  Figure 54.  Figure 55.  Figure 56.  SDS-PAGE of f r a c t i o n s obtained in Figure 48. Lanes 1 and 2, skimmilk; Lane 3, unbound skimmilk to MCIC column; Lane 4, washing f r a c t i o n ; Lanes 5 and 6, are peak 1 and 2, r e s p e c t i v e l y ; Lane 7, standard IgG; Lane 8, a-casein  127  Elution p r o f i l e s of skimmilk (SM), Ig and LF mixture on MCIC column. 60 mg of Ig and LF was mixed with 1 mL skimmilk (SM+Ig+LF) and 30 mg Ig was mixed with 1 mL skimmilk (SM+Ig) and passed through MCIC column (1.4 x 7.0 cm). W, washing with 0.02 M phosphate buffer containing 0.5 M NaCl, pH 7.0; E l , e l u t i o n with 0.01 M imidazole; E2, e l u t i o n with T r i s - a c e t a t e containing 0.5 M NaCl, pH 3.0  130  SDS-PAGE of f r a c t i o n s obtained in Figure 50. Lane 1, skimmilk; Lane 2, skimmilk and Ig mixture; Lanes 3 and 4 are unbound and peak 1 of SM-Ig mixture a p p l i c a t i o n , r e s p e c t i v e l y ; Lane 5, SM-Ig-LF mixture; Lanes 6 and 7 are unbound and peak 1 of SM-Ig-LF mixture application, r e s p e c t i v e l y ; Lane 8, immunoglobulins; Lane 9, lactoferrin and Lane 10, a-casein  131  Metal chelate i n t e r a c t i o n chromatography of a - c a s e i n . 3 mL of protein (10 mg/mL) before (a-CAS) and after diethylpyrocarborate modification (DEP a-CAS) e q u i l i b r a t e d with 0.05 M T r i s - a c e t a t e / 0 . 5 M NaCl, pH 8.2 and applied to copper chelate Sepharose 6B (1.4 x 7.0 cm). W, washing with the same e q u i l i b r a t i n g b u f f e r ; E, e l u t i o n with 0.01 M imidazole. Flow rate was 30 mL/hr  134  Metal chelate interaction chromatography of a i-casein before (as-CAS) and a f t e r diethylpyrocarbonate modification (DEP as-DAS). See Figure 52 for conditions of separation  136  Metal chelate i n t e r a c t i o n chromatography of 8-casein before (8-CAS) and a f t e r diethylpyrocarbonate modification (DEP B-CAS). See Figure 52 for conditions of separation  138  Metal chelate i n t e r a c t i o n chromatography of (A) polymer K - c a s e i n ( K - C A S ) (B) monomer K - c a s e i n s (MK-CAS) before and a f t e r diethylpyrocarbonate modification (DEP K - C A S ) . See Figure 52 for separation conditions  139  Immobilized copper a f f i n i t y chromatography of blood serum. Blood serum (1 g in 10 mL 0.05 M T r i s - a c e t a t e / 0 . 5 M NaCl, pH 8.2) was applied to the column (1.4 x 7 cm). The column was washed with the s t a r t i n g buffer and then eluted with E l , 0.05 M T r i s - a c e t a t e 0.5 M NaCl, pH 4 . 0 , and with E2, 0.1 M imidazole. The flow rate was 30 mL/hr. F l and F2 are f r a c t i o n s obtained  143  s  XV  Page Figure 57.  Figure 58.  Figure 59.  Figure 60.  Figure 61.  Figure 62.  Figure 63.  Figure 64.  SDS-PAGE p r o f i l e s of blood f r a c t i o n s from MCIC on Sepharose 6B column. Sample i d e n t i f i c a t i o n : Lanes 1, 2 and 3 are F l of Figure 59 from Zn, Ni and Co loaded columns r e s p e c t i v e l y ; Lane 4, plasma protein eluted from Cu-loaded column with pH 4 b u f f e r ; Lanes 5, 6 and 7 are unbound, F l , and F2 in Figure 59, r e s p e c t i v e l y ; Lanes 8, 9 and 10 are standard t r a n s f e r r i n ( T F ) , bovine serum albumin (BSA), and immunoglobulins ( I g ) r e s p e c t i v e l y ; Lane 11, blood plasma  144  Immunoelectrophoresis of f r a c t i o n s obtained in Figure 56.P, blood plasma; F l and F2 f r a c t i o n s obtained in Figure 56; TF, t r a n s f e r r i n ; Ig, immunoglobulins, BSA, bovine serum albumin; abws, rabbit antibovine whole serum  145  Immobilized Z n - , N i - and Co- a f f i n i t y chromatography of blood serum. Blood serum (2 g in 20 mL 0.05 M T r i s - H C l / 0 . 1 5 M NaCl, pH 8.0) was applied to the column (2.8 x 8.5 cm). The column was washed with the s t a r t i n g buffer then eluted (E) with 0.1 M Na-acetate/0.8 M NaCl, pH 4.6. The flow rate was 30 mL/h. 1, i s f r a c t i o n obtained  146  Elution p r o f i l e s of adsorbed hemoglobin from MCIC columns (1.4 x 7.0 cm) loaded with Zn, N i , Co and Cu. 2 mL of hemoglobin (3 mg/mL in 0.05 M T r i s - H C l / 0 . 1 5 M NH«C1, pH 8.0) was applied to the column and washed (W) with 2-3 times bed volumes of the s t a r t i n g b u f f e r . E l , 0.1 M Na-acetate / 0 . 8 M NaCl, pH 4 . 5 ; E4, 50% ethanol  148  Bacteriostatic activity of i s o l a t e d immunoglobulins and t r a n s f e r r i n against E^ c o l i . C, c o n t r o l ; T F , t r a n s f e r r i n (10 mg/ml); M, mixture of TF (5 mg/ml) and Ig (5 mg/ml); Ig, immunoglobulins (10 mg/ml)  151  Figure 62. Anti-1ipopolysaccharide a c t i v i t y of blood IgG isolated by metal chelate interaction chromatography method. E^ c o l i LPS; o-a , typhimurium LPS; O - O » J L parapertussis LPS  153  Saturation point of adsorption of standard TF on Cu-loaded IDA-BGE Sepharose 6B. 0.2% TF was passed through 10 mL column (7 x 1.4 cm) e q u i l i b r a t e d with 0.05 M T r i s - a c e t a t e containing 0.5 M NaCl, pH 8.2, W, wash with starting b u f f e r , E l , e l u t i o n with 0.05 M T r i s - a c e t a t e containing 0.5M NaCl, pH 4 . 0 . Elution with 0.01 M imidazole  154  Elution p r o f i l e s of control (TF) and diethyl pyrocarbonate treated t r a n s f e r r i n (DEP-TF). Samples (30 mg/5 mL 0.05 M T r i s - a c e t a t e / 0 . 5 M N a c l , pH 8.2) were applied to the column (1.4 x 7.0 cm) and washed (W) with the s t a r t i n g buffer then eluted (E) with 0.01 M imidazole. The flow rate was 30 mL/hr  156  xvi Page Figure 65.  Figure 66.  Figure 67.  Figure 68.  Figure 69.  Figure 70  Metal c h e l a t e - i n t e r a c t i o n chromatography of egg white. 2 mL of undiluted blended egg white was passed through Cu-loaded Sepharose 6B MCIC column (7 x 1.4 cm). UB, unbound p r o t e i n s ; FW, f r a c t i o n eluted with washing step; E l , e l u t i o n with s t a r t i n g b u f f e r , pH 4.0; 1, fraction eluted with E l ; E2, elution with 0.01M imidazole; 2, f r a c t i o n eluted with E2  159  SDS-PAGE of f r a c t i o n s of egg white obtained by MCIC column shown in Figure 65. Lanes 1, 2 and 3, are unbound f r a c t i o n s ; Lane 4, washing f r a c t i o n ; Lane 5, peak 1; Lane 6, peak 2; Lanes 7 and 8, standard ovotransferrin and ovalbumin, r e s p e c t i v e l y ; Lanes 9 and 10, control egg white  160  Immunoelectrophoresis against anti whole egg white antiserum of ovotransferrin f r a c t i o n (F) prepared by the MCIC method as compared to commercial ovotransferrin (OVT), and egg white (EW)  162  Saturation profile of commercial ovotransferrin on Cu-loaded Sepharose 6B column. 0.2% ovotransferrin was passed through 3 mL of a Cu-loaded column (7 x 1.4 cm). E l , e l u t i o n with 0.05 M T r i s - a c e t a t e / 0 . 5 M NaCl, pH 4.0; E2, e l u t i o n with  163  Metal chelate i n t e r a c t i o n chromatography of apo-ovotransferrin (APO-OVT), Fe-ovotransferrin (Fe-OVT) and Cu-ovotransferrin (Cu-OVT). 3 mL (8 mg/mL) was applied to Cu-loaded Sepharose 6B (7 x 1.4 cm) a f t e r e q u i l i b r a t i o n with 0.05 M T r i s - a c e t a t e / 0 . 5 M NaCl, pH 8.2. W, Washing with the e q u i l i b r a t i n g b u f f e r ; E, elution with 0.01 M imidazole; flow rate was 30 mL/hr  165  Metal chelate interaction chromatography of control ovotransferrin (OVT) and diethyl pyrocarbonate treated ovotransferrin (DEP-OVT). 3 mL (8 mg/mL) was applied to Cu-loaded Sepharose 6B (7 x 1.4 cm) a f t e r e q u i l i b r a t i o n with 0.05 M T r i s - a c e t a t e / 0 . 5 M N a c l , pH 8.2. W, washing with the e q u i l i b r a t i n g b u f f e r ; E, e l u t i o n with 0.01 M imidazole; flow rate was 30 mL/hr  166  XVI1  APPENDIX Page N o n - f e r r i c methods for B - l a c t o g l o b u l i n removal from Cheddar cheese whey  184  \  xvi i i  ACKNOWLEDGEMENTS  I  would  like  to  express  supervisor Dr. Shuryo Nakai and  enthusiasm  preparation  of  throughout this  my  gratitude  and  appreciation  for his unlimited encouragement, the  thesis.  sincere  I  course  of  also would  this like  invaluable  investigation to  thank  the  and  to my advice in  members of  the the  committee, Drs. Brent Skura and William Powrie, Department of Food Science, and Dr.  Robert  Fitzsimmons,  suggestions and a s s i s t a n c e . Thanks throughout  are the  also years  Department  Animal  Science  for  their  valuable  Thanks also to Sherman Yee who eased the lab work.  extended of  of  study.  to  my A  parents special  for thanks  their to  constant my  wife  support for  her  encouragement and patience during the study. Finally,  I wish to express my deep appreciation and gratitude  to the Iraqi  Government f o r giving me the opportunity to study abroad and for the support provided to me throughout the study.  financial  1  INTRODUCTION In  developed s o c i e t i e s , many infants  human m i l k .  National  Academy of P e d i a t r i c s Pediatric  and  international  (Committee on N u t r i t i o n ,  (1981),  recommend breast  due to short supply of breast m i l k , nursing mothers, and the  continuing e f f o r t  infant  formula such  (ESPGAN,  1982)  of  and  infants  is d i f f i c u l t  Although, there are many d i f f e r e n t  1981).  Nutrition  dealing with  infant  formulae.  It  the  World  Health  possible.  some mothers having to work. and protective  of is  Thus, ability  1985).  infant formulas on the market today,  required  and prevention  American  i n s u f f i c i e n t n u t r i t i o n and health conditions  necessity of  reached the  the  than  for women to nurse infants  has been made to decrease n u t r i t i o n a l  them have not  as  whenever  differences between human milk and cows' milk (Kuwata et a l . ,  of  rather  1980), the European Society for  feeding  There are many cases, however, where i t  of  fed  organizations  Gastroenterology and Nutrition  Organization  are  standard,  i.e.  disease both believed  human milk  have to  that  the  (Wilkinson,  be considered when  premature  infant  benefit from breast milk to an even greater extent than can the f u l l - t e r m (Workshop P a r t i c i p a n t s , 1976). feeding  is,  protect  the  however, infant  the  all  can  infant  The most compelling argument in favor of breast  contention  that  breast  milk  contains  against both systemic and g a s t r o i n t e s t i n a l  factors  that  infections.  The  most dramatic testimonials in t h i s regard are provided by the r e s u l t s of nursery epidemics  where  otherwise  normal  infants  quickly  i n f e c t i o n s unless they are fed raw human milk The  protection  premature  accorded  infants  enterocolitis  can  Touloukian et a l . ,  where be  the  newborn  a relatively prevented  1967;  by  by  (Dortmann,  breast  milk  common f a t a l breast  Barlow et a l . ,  succumb  It  a  variety  of  1967; Gerrard, 1974). is  also  critical  syndrome c a l l e d  feeding  1974).  to  (Mizrawi  et  for  necrotizing al.,  1965;  also has been reported that  2  the incidence of sudden death syndrome in infants i s lower in breast fed than i t  i s in b o t t l e - f e d  The several  resistance review  Bezkorovainy, 1983;  infants  of  1977;  (Hanson  Rieter,  Lonnerdal, 1985;  human milk  to factors  (Mobbs, 1972).  breast-fed  articles  infants  against  and Winberg,  1978;  Rieter,  Gurr,  infection  1972;  1981;  immunoglobulin  bifidus factor the  factors  is  summarized  Goldman and Smith,  Packard,  1985a) which ascribe the  1982;  Friend  beneficial  A,  lactoferrin,  and i n t e r f e r o n . mentioned  above  carbohydrate, p r o t e i n ,  fat,  lysozyme,  lactoperoxidase,  Manufacturers and  mineral  not  of  just  al.,  effects  the most important  in  1974;  et  that are therein but are absent from bovine milk.  factors a r e , f o r the most p a r t , proteins in nature, are  infants  of  These  of which  leucocytes,  the  infant formulae must consider the  and vitamins.  nutrient Therefore,  content,  i.e.,  one possible way  to humanize cows' milk i s to enrich cow's milk with these bioactive components. Prevention of pathogenic i n f e c t i o n s i s only part of the problem.  One must  consider the prevention of immune response reaction in i n f a n t s .  The newborn is  particularly  even to whole  proteins. in  vulnerable.  For a few days the stomach i s porous,  The milk of any non-human species i s more l i k e l y to induce an a l l e r g y  sensitive  infants  than  mother's  milk.  allergenic  reactions due to infant formulae  (Packard,  1982).  antigenic  than  data  from  sensitivity  five to  Animal  either  experiments  In  of  cows'  p-lactoglobulin  milk in  suggest  protein 82%  the  occurrence  of  that found  B-lactoglobulin  is  in cow's m i l k .  Compiled  intolerance the  cases  in  infancy  (Wilkinson,  8-Lactoglobulin has been regarded as a major allergen  for b o t t l e - f e d  several  1981;  al.,  outstanding papers  1982;  Heppell  et  al.,  (Lebenthal, 1984;  1975;  Kurisaki  of  i s much more common than human milk  casein or a-Lactalbumin  studies  general,  et  Wharton, al.,  1985;  more  showed 1981).  infant  in  Moneret-Voutrin  et  Otani  et  al.,  1985;  3  Pahud et a l . ,  1985).  A l l e r g e n i c i t y of B - l a c t o g l o b u l i n may be a t t r i b u t e d  to the  fact that human m i l k , based on immunological r e a c t i o n s , contains only a trace of p-lactoglobulin Therefore, protein  (Brignon et a l . ,  1985).  the  of  elimination  composition of  cow's milk  B-lactoglobulin for  infant  is  one way to humanize  feeding.  Several  attempts  been made f o r separating B - l a c t o g l o b u l i n from bovine whey (Forsum, 1974; and Shahani, 1979;  Amundson and Watanawamchakorn,  ferric  method  chloride  3-lactoglobulin However,  recently  established  from bovine whey  because  lactoferrin  was  when  of  the  it  is  (Kaheko  potential saturated  et  loss with  1982).  for al.,  of iron,  Kuwata  Mathur a  precipitating et  al.,  antimicrobial  non-ferric  have  our laboratory,  selectively 1985;  the  In  the  1985).  activity  methods  have  of been  investigated.  The objectives of t h i s study were (1) for eliminating  p-lactoglobulin  chromatographic methods  for  from cheese whey;  the  from cheese whey;  (3)  for  immunoglobulins;  isolation  i s o l a t i o n of  of  to e s t a b l i s h a new n o n - f e r r i c method  isolation  to investigate  of  to  investigate  immunoglobulins and  (4)  to  utilize  i s o l a t i o n of ovotransferrin  the  lactoferrin directly  developed technique  from blood and from egg white.  (5)  to  apply  for the  The b i o l o g i c a l  a c t i v i t y and the p o s s i b i l i t y of using these proteins in f o r t i f i c a t i o n formula were i n v e s t i g a t e d .  different  the p o s s i b i l i t y of using skimmilk  immunoglobulins and t r a n s f e r r i n  same technique for  (2)  of  infant  4  LITERATURE REVIEW A. HISTORY Infant formula i s required for infants whose mothers do not breast feed for socioeconomic,  physical  revolution,  the  the  or  infant  milk). invented.  psychological  only a l t e r n a t i v e s the  However  or  services  by the  of  end of  Thus modern  reasons.  Before  a wet-nurse the  18th  technological  (directly  century,  a conical  developments  o r i g i n s in the mid-19th  supplied  for  the  first  in  the  nutritional  20th  needs  scientists  century, of  recognized  breast-feeding malnutrition  in  infants the  order  resulting  however,  to from  or  need  of  deal  with  the  adaptations  of  (1915).  On  a  product  fluid  homogenized mixture  basis  this  of both plant  of  by  early  H.J.  formula  1880's,  of  But  of  alternatives  childhood mothers  disease  to  Gerstenberger  fat),  consisted  to and  breast-feed  and of  the  medical  the United States market  formula  and animal  understanding  effective  many in  made  Switzerland  In the  requirements.  and  formulas  designed  little  problems  failure  Most present-day infant  was  safe  was  1983).  sanitary  for  adequately.  the  there  of  breast  time  The modern infant  century when Nestle  canned evaporated milk made i t s appearance ( M i l l e r ,  donor  baby bottle  and Borden in the U.S. began producing sweetened condensed m i l k .  Until  industrial  to mothers breast-feeding were starvation  possible a widespread s h i f t away from b r e a s t - f e e d i n g . industry found i t s  the  are  co-workers  4.6%  fat  (a  6.5% carbohydrate and 0.9%  protein and was given the name Synthetic Milk Adapted (SMA).  Over the years the  composition of infant formulas has been altered and adjusted, mostly in response to s c i e n t i f i c evidence of need (Packard, 1982).  5  B. HUMAN MILK VS. COW'S MILK M i l k , whether from the human or other mammals, i s an exceptionally complex mixture  of more than 200  fat-soluble  and water-soluble  components.  The milk  components may o r i g i n a t e from d i r e c t transfer from the blood, from biosynthesis from  blood  precursors  composition  of  f a c t o r s which  milk  or  is  from  a  affected  influence the  combination by  of  Consequently,  biochemical, physiological  composition of blood,  rate of transfer of nutrient  both.  the  and hormonal  factors which influence  the  from blood to milk and factors which influence the  rate of biosynthesis of compounds in the mammary gland (Blanc, 1981). Human and cow's milk  consist mainly  and minerals; however, t h i s gross  composition of  surprising mammalian  if  one  fat,  i s where the s i m i l a r i t i e s  human and  looks  of water,  at  species consists of  cow's milk  mammalian the  (Gurr,  animals  constituents  in  carbohydrate,  end.  1981).  Table  1 gives  This  is  general.  listed  Milk  above but  proportions of each vary as much as the species (Jenness, 1982). fats,  etc.  also vary  in t h e i r  protein  not  the very  from  the  all  relative  The p r o t e i n s ,  biochemical and physical properties  as well as  t h e i r d e t a i l e d composition. The f a t  concentration in human and bovine milk  and 3.7% respectively (Packard, 1982).  It  variable  absolute  constituent  Human milk  of  has a lower  milk level  both of  in  short  is not comparable i . e .  should be noted that fat quantity  chain f a t t y  and  adds  4.4  i s the most  in composition.  along with a higher  concentration of polyunsaturated f a t t y acids than cow's m i l k .  Another notable  difference  of  i s that human milk  unsaturated f a t t y acids while  has approximately a 50:50 r a t i o in bovine milk  that r a t i o  is 65:35.  vegetable o i l s may be used to adjust the r a t i o in cow's m i l k .  saturated  to  Unsaturated  6 Table 1.  Gross Composition of Human and Cow's Milk Grams of F l u i d P r o d u c t ) .  (Grams  a  Component  Human milk  Cow's mi 1k  Fat  4.4  3.7  Protein  1.0  3.3  6.9  4.7  Milk sugar (lactose) Mineral  matter (as ash)  Water Energy, KJ/100ml_  D  a  Packard, 1982..  b  Gurr,  1981.  0.2  0.7  87.5  87.6 273  290  per  100  7  The p r i n c i p a l this  secretion.  and  cow's  formula milk.  It  milk,  to  carbohydrate  in milk  contributes  about 40 and 29% of the total  respectively.  increase  the  i s l a c t o s e , a disaccharide s p e c i f i c to  A carbohydrate  concentration,  so that  is  generally  it  is  lactose  malabsorption  and  fermentative  carbohydrates should be added, i . e . , The major differences milk about  0.2%  primarily milk.  ash,  the  to  much  in the mineral  mineral  range  infant  of  human  Cow's milk 3.3  protein  vs.  and  K-caseins.  can  in  cow's  broken  Cow's milk  While  protein  a-lactalbumin,  of  over three  contains,  after  on the  incineration.  calcium and  milk  Cow's milk  and  down  times as much protein  human milk  into  three  (Gurr,  major  approximately  of t h i s component.  p-casein  fractions  p-lactoglobulin,  that  is due  in  cow's  cow's milk i s to be considered  The coagulation properties  acidified  of  average,  phosphorus  Table 2 gives a breakdown  to be more d i g e s t i b l e for the infant Other  Human milk  remaining  consists of  consists of very l i t t l e  different.  sources  human milk and cow's  as human milk  of  the  1981).  times as much casein as human m i l k .  be  resulting  1981).  respectively.  has mainly p - c a s e i n .  Other  content between  concentrations  contains well  contains over eight protein  matter  feeding (Wharton,  fractions  lactose,  diarrhea.  content should be reduced, i f  1.00,  amount of  The higher percentage of ash, or rather minerals,  higher  High mineral  for use in infant  milk  the  to  maltodextrin.  i s not so much in kind as amount.  averages about 0.70%.  i.e.  added  There are some problems in deciding which carbohydrate to add, since the  gut of some babies would be unable to handle t h i s in  in  energy of human  different  Cows'  milk  Casein i s a complex  fractions:  a -,  45% a - c a s e i n s  B-  s  while  and  human  Human milk on the other hand, of a - and p-casein are s  forms a soft  curd,  a -casein s  is  quite  thought  (Packard, 1982). differ  lactoferrin  between and  the  human  and  cow's  immunoglobulins  milk  are  fraction.  8  Table 2.  Protein Composition of Human and Cow's M i l k  Human Milk Protein  g/lOOmL  a  Cow's milk  Total, %  g/lOOmL  Total,%  0.88  100  3.30  100  Caseins  0.31  35  2.6  79  Total  0.57  65  0.7  17 —  0.12 0.30  21 3.5  B-lactoglobulin  0.15 tr  lactoferrin  0.15  17  tr  serum albumin  0.05  6  lysozyme  0.05  6  tr  1.0 —  immunoglobul ins  0.10  11  0.10  3.0  others  0.07  8  0.15  4.5  Total whey:  a-lactalbumin  a  Gurr,  tr=trace.  1981.  0.03  9.0 —  9  B-lactoglobulin milk while  makes up a high percentage of the protein in cow's  (B-Lg)  human milk contains very l i t t l e .  The concentration of B-Lg in human  milk  i s so low that f o r years i t  milk  (Packard, 1982; Brignon et a l . ,  milk  but only trace amounts are found in cow's m i l k .  tion  is relatively  milk  contains  similar  mostly  was thought 1985).  in concentration  IgA  while  in  that B-Lg was exclusive to cow's Lactoferrin  in the  cow's  (LF)  i s found in human  The immunoglobulin f r a c -  two milk  milk  IgG  sources but human  is  the  predominant  it  can be seen  immunoglobul i n . From the previous discussion on compositional d i f f e r e n c e s , that i t  i s important  cow's milk  to look beyond the gross make-up and investigate human and  on a more d e f i n i t i v e  basis.  The differences  the two milk sources cause currently a v a i l a b l e optimal  infant  in composition between  formulae to be less than  substitutes f o r human m i l k .  C. HUMANIZING INFANT FORMULA Due to many compositional differences  between  human and cow's m i l k ,  attempts have been made to humanize infant formula. ways in which cow's milk human m i l k :  and skimmilk (Gurr,  There have been three major  has been modified to bring  added carbohydrate,  substitute  fat  of  carbohydrates.  sucrose are  examples  of  two  per unit of energy  carbohydrates  been used in the infant formula industry (Wharton,  The purpose  carbohydrate content and to  the concentration of p r o t e i n , fat and mineral or  composition closer to  1981; Wharton, 1981).  for adding carbohydrates i s to increase the overall  Maltodextrin  its  and using a combination of whey  The simplest change involves the addition  dilute  many  that  intake.  have commonly  1981).  In some cases the fat from cow's milk has been removed and substituted with a mixture  of plant  and animal  from plant and animal  fats.  The purpose for  the  combination of  fats  sources i s to obtain a f a t t y acid composition s i m i l a r  to  10  that found in human m i l k . efficient  in  breast milk  the  Studies have indicated that absorption i s much more  substituted  (Wharton,  fat  formulas and becomes close to  that  found  1981).  Formulas based on demineralized whey have been used increasingly in years.  Demineralized whey,  minerals,  in  containing whey protein  i s used as the base and to t h i s  recent  and low concentrations  i s added a small amount of  of  skimmilk.  The range of casein/whey protein r a t i o s of human milk and cow's milk are 0.4 0.7 and 3.0 - 4 . 7 ,  respectively.  To humanize t h i s r a t i o ,  infant formulae are f o r t i f i e d with whey p r o t e i n s . with 4 volumes of whey changes the  ratio  some of the commercial  For instance, mixing skimmilk  to 0.75.  However  simple mixing  bovine whey with skimmilk does not minimize the compositional differences ween the two m i l k s , i . e . and immunogobulins (Ig)  higher contents of a-lactalbumin, in human milk than in cow's milk  lactoferrin,  human milk  (Liberatori  it  of  bet-  lysozyme  (Hambraeus, 1977).  While B - l a c t o g l o b u l i n i s the dominant whey protein in cow's milk mately 60% of the total whey p r o t e i n s ) ,  -  (approxi-  i s completely lacking or very low in  and Napolitano, 1980; Brignon et a l . ,  1985).  D. ALLERGENICITY OF WHEY PROTEINS In  adapted  bovine o r i g i n  formulas,  the mass balance of  can be corrected to a 40:60 r a t i o  (Anderson et a l . ,  1982;  Theuer,  1983).  casein versus whey proteins  in favor of the whey proteins  This adjustment  formula c l o s e r to. the protein composition of human m i l k . content well  as  of  some essential  cystine  in  amino acids  comparison with  (threonine, normal  of  cow's  addition of whey protein corresponds to n u t r i t i o n a l  adapts It  cow's milk  also increases the  tryptophan, milk;  the  thus,  and lysine)  as  adaptation  by  improvement (Forsum, 1974).  Presumably any of the individual proteins in cow's milk may induce s p e c i f i c antibody and provoke a l l e r g y in a susceptible c h i l d , but some proteins seem more antigenic than others (Wharton,  1981).  The newborn i s p a r t i c u l a r l y  vulnerable.  11  For a few days the stomach i s porous, even to whole p r o t e i n s . non-human species i s more l i k e l y mother's milk will  (Savilahti,  IgE.  The  s p e c i f i c antigen. base of Y.  1981).  simply be absorbed whole.  producing  the  The  IgE  is  are  in  The body reacts to the offending agent by simply  s p e c i a l l y designed to  to  instance  bind  to  to  mast  the  antigen.  cells  to  mast  cells  of  the  mast  cell.  command to  body tissue In  and at  essence,  the  the  fingers of  the  base,  same time  the  (Packard,  IgE i s produced.  the  It  issues  command c a l l s  this  the F c , or  The  or basophils  When an infant has been exposed to an a l l e r g e n , itself  recognize and bind to  and the Fab, the two protruding  designed  this  than  A p r o t e i n , or an antigenic fragment of protein  Y-shaped antibody,  binds  in s e n s i t i v e infants  There a r e , however, two ends to an antibody:  fingers  segment,  to induce an a l l e r g y  The milk of any  for  F  c  1982).  attaches  a chemical release  of  histamines which in turn cause the various disorders that accompany an a l l e r g i c reaction. Animal either et  experiments  suggest  that  B-lactoglobulin  casein or the small amount of a-lactalbumin  al.,  1958;  Goldman et  Compiled data  from f i v e  showed s e n s i t i v i t y  al.,  1963  studies  of  a,b;  in 82%,  more  antigenic  found in cow's milk  Moneret-Vautrin  cow's milk  to p - l a c t o g l o b u l i n  is  protein  In  view  casein in 43%, a-lactalbumin  denaturation  in  patients  of has  the to  above be  results,  considered  the when  well-known  allergencity  manufacturing  infant  enzymatic  hydrolysis  or  selective  elimination  of the protein was suggested by Ratner et a l .  (1979), McLaughlan et a l .  of  of  formula.  approaches have been considered to decrease P-Lg a l l e r g e n c i t y :  denaturation,  al.  infancy  1975).  p-lactoglobulin Different  1979).  in  41%, bovine serum globulin in 27%, bovine serum albumin in 18% of the (Lebenthal,  (Ratner  and G r i l l i a t ,  intolerance  than  P-Lg.  heat Heat  (1958), Anderson et  (1981), and more recently by Kilshaw et a l .  (1982)  12  and Heppell et a l . the  biological  (1984).  However, heat treatment of whey proteins may destroy  activity  immunoglobulins,  of  lactoferrin  bioactive and  proteins  present  lactoperoxidase.  in  Tryptic  hydrolysis of whey proteins suggested by Pahud et a l .  the and  whey  i.e.  chymotryptic  (1985) and A s s e l i n et a l .  (1986) are not recommended for the same reasons. S e l e c t i v e elimination of the a l l e r g i c compound may therefore  be the method  of choice to humanize the protein composition of cow's milk for infant feeding. Several attempts have been made f o r separating p-Lg from bovine whey.  Sephadex  G-75 gel f i l t r a t i o n was suggested as a method for whey protein f r a c t i o n a t i o n to humanize infant  formula  was p r e f e r e n t i a l l y by  (Forsum, 1974;  Mathur and Shahani, 1979).  p r e c i p i t a t e d at pH 4.65  ultrafiltration  and  from cheese whey a f t e r  demineralization  by  Watanawamchakorn, 1982; Slack et a l . , 1985). and pH treatment Cheddar  for  cheese whey.  separating Recently,  electrodialysis  p-Lg  concentration (Amundson  and  Pearce (1983) used heat treatment  p-Lg and an a-La a ferric  Also,  rich  fraction  from  bovine  chloride method was established for  s e l e c t i v e l y p r e c i p i t a t i n g P-Lg from bovine whey (Kaneko et a l . ,  1985; Kuwata et  al.,  l a c t o f e r r i n and  1985).  However,  the  abolish i t s antimicrobial  use of f e r r i c activity.  Antigenic r e a c t i v i t i e s al.  (1985).  tryptophan  P-Lg  was  of chemically modified p-Lg was studied by Otani  These researchers found that modification residues, or  reactivity.  chloride may saturate  sulfhydryl  groups had l i t t l e  However, a s i g n i f i c a n t decrease in the  acetylated,  succinylated or  coupled with glycine amide.  modified with  of  et  arginine  residues,  on the  antigenic  effect  reactivity diethyl  These results suggest that there  was noted when pyrocarbonate  or  is a p o s s i b i l i t y  that the amino group, h i s t i d i n e residue and carboxyl group may play an important role in the antigencity of bovine p-Lg.  13  E.  ANTIMICROBIAL  Several  1980).  I N HUMAN M I L K AND M I L K  substances in  particularly Cunningham,  SYSTEM  human milk  diseases of the 1979;  Fallot  Cunningham (1979)  provide  intestinal  et  al.,  noted  tract  1980; that  the  first  four  months  the  resistance  first  to  infant diseases,  (Cunningham, 1977;  France et  extent of formula feeding and was two-fold during  SUBSTITUTES  al.,  1980;  year m o r t a l i t y  Larsen, Pullan  was  16-fold.  et  al.,  increased with  higher in a r t i f i c i a l l y  difference  1978;  fed  the  infants;  Similarly,  breast  feeding reduced the incidence of i n f e c t i o n by Salmonella (France et a l . ,  1980),  respiratory syncytial virus (Pullan et a l . , 1980), and the incidence of hospital admissions f o r i n f e c t i o n in infants The factors infant's  in  increased  (Fallot  human milk  thought  resistance  to  et a l . ,  to  1980).  be responsible for  diseases  have  been  the  reviewed  breast  fed  extensively  (Gothefors and Winberg, 1975; Bezkorovainy, 1977; Reddy et a l . , 1977; McClelland et a l . ,  1978;  Reiter,  1978;  Pittard,  1979;  Welsch and May,  Gur.r, 1981; Hanson and Soderstrom, 1981; Packard, 1982). bial  factors are associated with c e l l u l a r  present  in  cow's milk  and other  substitutes  in  1.  The function of the antimicrobial  low  1981;  in human milk and  Other antimicrobial  lactoperoxidase i s present in larger quantities milk.  Blanc,  Some of the antimicro-  components present  thus are not present in human milk s u b s t i t u t e s .  1979;  or  trace  factors are  amounts.  Only  in cow's milk compared to human  factors w i l l be discussed.  Immunoglobulins Immunoglobulins or antibodies are a class of proteins that are comprised of  four polypeptide chains. heavy  chains  disulfide antibodies  and  bridges and  two  Each immunoglobulin unit i s formed from two identical  (Butler,  they  are  light  1983). named  for  chains. In  humans,  their  heavy  The  peptides  there  are  chains.  are  identical linked  by  five  classes  of  They  consist  of  14  immunoglobulin  G  (IgG),  immunoglobulin  M  (IgM),  immunoglobulin D ( I g D ) a n d immunoglobulin E (IgE). class  are  designated  respectively lambda  (Atassi  (A).  by  the  et a l . ,  Light  appropriate 1984).  The l i g h t  chains have one constant  Heavy chains consist of three constant are  also  hypervariable  Nisonoff,  of  four  immunoglobulin  polypeptide  daltons.  When the  into  heavy  two  proteolytic  al.,  chains  molecule  and  two  enzymes  (antigen binding) F(ab')2  regions  is  as i t  is  light  such  as  IgG,  and  has  (Fab-dimer)  IgA)  with  a,  S and  region.  region.  regions  e,  kappa (K) or  and one variable  variable  each  There  (Brock,  1979;  a  i s comprised of only one basic  low  From early  of  exist  have been  in  chains. papain,  and various  The to  molecule  give  investigations bovine  it  colostral  weight  may  types  it  also of  of  150,000  dissociates be  split  by  fragments:  Fb a  Proteolysis by pepsin at pH 4 y i e l d s an  80%  in  from fragment  Fc (Whitney  Ig  et  IgG  (Murphy  in colostrum i s  concentration  bovine  However,  of  et  al.,  (Butler,  high at  (Butler,  species  and  colostrum  IgG,  IgM  (Butler,  i s already known that d i f f e r e n t subclasses  with each successive postpartum milking various  immunoglobulins ( i . e . ,  both milk  by some groups of workers  Concentration of  secretions.  two  peptides derived  comprising about  identified  of  molecular  1983).  IgG  1983).  1972).  y,  treated with a reducing agent,  and Fc ( c r y s t a l l i n e ) .  1976; B u t l e r ,  IgG  y,  chains are either  In bovine colostrum, there are three major and  letter:  regions and one v a r i a b l e the  (IgA),  The heavy chains for  region  within  A  1982).  The simplest unit  Greek  immunoglobulin  1964).  IgG2 and  IgGi  1969;  Duncan et  al.,  parturition  1983).  Table 3 summarizes the  immunoglobulins  these values may provide a rough guide for  since there are d i f f e r e n t factors a f f e c t i n g  and decreases  in  serum  and  investigators,  the immunoglobulins concentration  in  15  Table 3.  Concentration  of  bovine  immunoglobulins  in  serum  and  secretions  (mg/mL) . a  IgA  igM  9.2  0.37  3.05  2.87  5.36  6.77  0.081  0.086  1.56  0.055 —  2.81  0.04  Saliva  0.034  0.016  0.34  0.006  Tears  0.32  0.01  2.72  .176  Urine  0.009  0.0013  tr  Bile  0.10  0.09  0.08  0.05  0.23  0.13  0.90  —  Body F l u i d Samples  IgGi  Serum  11.2  Colostrum (whey)  46.4 0.58  Milk Nasal  secretion  Vaginal  a  secretion  B u t l e r , 1983.  IgG  2  tr  16  milk  i.e.  age,  breed  differences,  differences  in  the  techniques  used  for  measurement and immunization. The  amino  acid  and  carbohydrate  composition  have been studied by several investigators al.,  1975).  The  physicochemical  carbohydrate  characteristics  bovine  IgGi  and  IgG2  (Groves and Gordon, 1967; Lisowski et  content,  of  of  bovine  sulfhydryl  Igs  are  content  shown in  and  Table  4  other (Butler,  1983). The human fetus  and infant  receives antibodies  absorbed through the placenta, and from human milk.  in  utero,  where IgG  is  Packard (1982) noted that  the milk antibodies function as s p e c i f i c host resistance factors by aggregating bacteria  in  bacterial  infants'  i n t e s t i n e , to  colonization  resistance viruses.  the  factors,  of  the  fixing  facilitate  their  intestinal  complement,  Thus they provide c r u c i a l  removal,  lining,  by  by  assisting  by n e u t r a l i z i n g  toxins  immunological protection  defence systems can be established  interfering  (Ogra and Ogra,  other  and by  until  host  killing  the  1978;  with  newborn  Hanson and  Soderstrom, 1981). Although classes of immunoglobulins ( i . e . , IgA,  IgG and IgM)  are present in  human milk throughout the period of l a c t a t i o n , the highest concentrations are in colostrum (Reddy et a l . , Ogra and Ogra,  1978;  1977; Hambraeus et a l . ,  Ones,  1979;  Goldman et  1978; McClelland et a l . ,  al.,  1982).  Small  colostrum IgG are absorbed from the i n t e s t i n e during the f i r s t birth  (Ogra  et  al.,  1977),  although  the  significance  Intact secretory IgG,  the major milk antibody,  of breast-fed infants  (Hambraeus et a l . ,  the  passive  penetration 1977;  transfer  of  specific  Hanson  and  Escherichia c o l i and  Soderstrom,  1981).  c o l i enterotoxin  amounts  to  mucosal  this  is  not  known.  intestine  appears to function surfaces  and antigens  (Walker  Antibodies  against  of  18-24 hours a f t e r  has been found in the  1978), where i t  immunity  by microorganisms, viruses  of  1978;  and  to  in  prevent  Isselbacher,  enteropathogenic  have been postulated to play a role  in  17  Table 4.  Biochemical c h a r a c t e r i s t i c s of bovine immunoglobulins . 3  Characteristics  IgGi  Heavy chain  IgG  2  IgA  IgM  igE  SC —  1  2  S20,w  6.9  6.9  10.9  19.5  —  4.1  EiP  13.5  12.3  —  11.8  —  —  2.8  2.6  8  11  —  5.9  2.6  —  Carbohydrate (%)  Total  Sulfhydryl groups Half-cysteine/100  residues 3.1  Total SH (mole/mole)  —  —  Free SH (mole/mole)  43.6 0.9  S-S linkages/mole  21  —  162K  152K  408K  1.030K  —  74K  Heavy chain (mol. wt.)  57K  54K  62K  76K  —  —  Light chain (mol. wt.)  25K  23K  23K  22.5K  —  —  Molecular weight  a  S  Butler,  1983.  20,W= Svedberg values  EH  8=  Absorbance of 1% protein solution at 278 nm.  18  preventing  infant  diarrhea  and Synge, 1978).  (Gindrat  et a l . ,  1972;  Stoliar  et a l . ,  1976; Rogers  IgM also provides protection against gram negative pathogens  (Packard, 1982). The  importance  of  oral  administration  and human infants i s well documented.  of  immunoglobulins in both  animals  In animals, supplementing a milk  replacer  with Ig separated from porcine blood maintained 86% survival of p i g l e t s compared to no survival 1978).  of the control  Similar  survival  rates  colostrum (McCallum et a l . , feeding  whey  containing  group fed with the milk are  1977).  Ig  has  obtained  replacer  by feeding  alone  piglets  (Elliot,  with  bovine  Improved survival and immunity of chicks by also  been  reported  from  USSR  (Kuznetsov  and  Rebrova, 1983). In  humans,  the  importance  demonstrated in c l i n i c a l test  of  Ig  in  infant  r e s u l t s from India  feeding  (Narayanan  has  et a l . ,  been  well  1983).  s i x t y - s i x low b i r t h weight infants s p l i t into two equal groups only 7 infants the group fed with human colostrum developed i n f e c t i o n infant  formula-alone  group,  which  is  significantly  S i g n i f i c a n t r e s u l t s were also shown by H i l p e r t fed  et a l .  colostrum from s e n s i t i z e d cows had an i n f e c t i o n  prevented  by o r a l l y  administering  different  18  rate  cow's colostrum  of  in  in the  (P<0.01).  (1974/1975) where  67.4% for the control group who were not fed colostrum. also  compared to  Of  infants  24% compared to  Rotavirus i n f e c t i o n was (Ebina  et  al.,  1985).  Diarrhea developed in only 1 of 6 infants given Rota colostrum, while 6 out of 7 infants given milk developed d i a r r h e a .  2.  Lactoferrin L a c t o f e r r i n , an iron-binding protein of breast m i l k ,  great al.,  importance for the breast-fed 1971;  Bullen et a l . ,  1972,  infant.  Reiter,  1983)  It  i s considered to be of  has been shown (Kirkpatrick  that l a c t o f e r r i n  et  can bind iron In  19  vitro  and  in  vivo,  microorganisms.  thereby  preventing  the  growth  of  L a c t o f e r r i n i s a s i n g l e - c h a i n glycoprotein with an approximate  molecular weight of 75,000-85,000 (Blackberg•and H e r n e l l , of  lactoferrin  consists of  own iron-binding s i t e .  two  largely  independent  1980).  The structure  domains, each carrying  its  This proposed structure has received strong support from  studies demonstrating that cleavage of l a c t o f e r r i n , under c e r t a i n  iron-requiring  usually by proteases, could  conditions y i e l d half-molecules capable of binding j u s t  a single  iron atom (Brock, 1985). Human milk lactoferrin  as  iron-binding  contains cow's milk  protein  1979;  (Arnold et a l . ,  3  to  (Packard,  transferrin.  enteropathogenic E. c o l i Honour,  from  Samson et  100  times  1982),  as much  and a  Lactoferrin  al.,  1979;  Samson et  trace  is  (Rogers and Snyge, 1978;  iron-binding amount  active  1980;  1977), Streptococcus mutans (Arnold et a l . ,  albicans (Kirkpatrick  et a l . ,  in  Spik et a l . ,  al.,  of  ),  protein  the  vitro 1978;  serum  against Dolby and  Vibrio  cholerae  1977), and Candida  1971), presumably by chelating iron and making  it  unavailable f o r microbial growth (Packard, 1982). Lactoferrin,  together  with  considerable b a c t e r i o s t a t i c  effect  coli  (Stephens  colostral  et  al.,  1980;  secretory  IgA  from  human  milk,  have  a  against human enteropathogenic strains of E.  Dolby and Stephens,  IgG, together with l a c t o f e r r i n ,  1983).  Similarly,  bovine  i s found to be active against a strain  of E i c o l i pathogenic to calves (Stephens et a l . , 1980).  Since the a n t i - E . col i  activity  digestion,  of  lactoferrin  i s not destroyed by p r o t e o l y t i c  may also play an a n t i b a c t e r i a l  role in vivo (Samson et a l . ,  lactoferrin  1980).  3. Lactoperoxidase and lysozyme Lactoperoxidase  (LP),  which catalyzes the oxidation  of the thiocyanate by  20 hydrogen peroxide to hypothiocyanate, serves as a major antimicrobial cow's  milk  (Reiter  Lactoperoxidase typhimurium  et  al.,  1976;  i s a c t i v e against  and  strains  of  1978;  coli,  Klebsiella  Bovine LP behaves d i f f e r e n t l y is  Reiter,  Pruitt  aeroqenes  (Pruitt  Human peroxidase  within  (Gothefors  is  highest  and Marklund,  in  average of  in bovine milk.  lactoperoxidase (Gothefors  is  and  not  inactivated  Marklund,  1985b).  4-5  is  important  (1978)  below the  to note  by the g a s t r i c j u i c e from an infant  1975),  whereas  pepsin  at  pH  2.5  days  rapidly  Bjorck  ug/mL human milk which i s f a r It  1985).  concentration  reach a peak at  Reiter,  of 0.5  ug/mL present  to  Its  colostrum and declines  1975;  reported a concentration l i m i t 10-30  1985).  and Tenovuo,  from other protective p r o t e i n s .  post-partum.  Tenovuo,  in  Pseudomonas f l u o r e s c e n s . Salmonella  low in bovine colostrum and increases rapidly  1 week  and  agent  that  (pH 5)  inactivates  lactoperoxidase (Paul and Ohlsson, 1985). Lysozyme positive activity  and  cleaves  gram  the  negative  cell  wall  microorganisms,  of IgA against E. c o l i  milk  Human milk (Chandan  et  and  ( A d i n o l f i et a l . ,  and with peroxide and with ascorbate to 1969).  peptoglycan  lyse g±  of  a  appears  to  number  of  potentiate  gram the  1966; H i l l and Porter, 1974), coli  and Salmonella  (Miller,  contains approximately 3000 times as much lysozyme as cow's al.,  1964;  Chandan et  al.,  1968).  Human  milk  possesses a molecular weight, amino acid composition, s p e c i f i c a c t i v i t y , s t a b i l i t y and a n t i g e n i c i t y which are quite d i f f e r e n t  lysozyme thermal  from the lysozyme in cow's  milk ( E i t e n m i l l e r et a l . , 1974; E i t e n m i l l e r et a l . , 1976).  4. B i f i d o b a c t e r i a The b i f i d o b a c t e r i a the  infant  against  (previously  disease  by  designated  producing  Lactobaci11 us b i f i d u s )  volatile  acids  which  protect  inhibit  the  21  proliferation  of  pathogenic microorganisms in  the  gut  (Friend  et  al.,  1983).  Within 3-4 days a f t e r b i r t h , the i n t e s t i n a l  t r a c t of breast fed infants contains  up to 99% Bifidobacterium bifidum type IV  (Gyllenberg and Raine, 1957; Haenel,  1970).  The f l o r a of formula fed infants does, not contain type  30-40% B_j_ b i f i d i u m Type II human  milk  have  bifidobacteria.  been  (Haenel, 1970). reported  to  IV,  but  rather  A number of substances or factors in  stimulate  the  proliferation  of  the  These factors include: buffer c a p a c i t y , l a c t u l o s e , l a c t o f e r r i n ,  pantothenic a c i d s , oligosaccharides and g l y c o p r o t e i n s . Infants fed e i t h e r human milk or a test formula with low buffering capacity have  a  relatively  low  fecal  pH  (5.1-5.4)  and  a  higher  proportion  of  b i f i d o b a c t e r i a than fecal coliforms or streptococci ( W i l l i s et a l . , 1973; Bullen et a l . ,  1977).  Those infants who were fed highly buffered  cow's milk  formula  had a s i g n i f i c a n t l y higher fecal pH (5.9-8.0) and a mixed fecal f l o r a ( W i l l i s et al.,  1973;  Bullen et a l . ,  that E_j. c o l i  1977).  It  and S^. faecium i n i t i a l l y  breast fed infants the pH of the  has been suggested (Bullen et  i n t e s t i n e drops and growth conditions  drop in pH and thus the p r o l i f e r a t i o n  of  the b i f i d o b a c t e r i a  infants are fed highly buffered formulas (Friend et a l . , Even though lactulose i s not present in human milk  during  of  sterilized  formula)  increases the  In  become  The crucial  is prevented when  1983). or cow's milk,  that supplementation of prepared formulas with lactulose  heating  1976)  colonize the gut and produce a c i d .  favorable for b i f i d o b a c t e r i a and unfavorable for other organisms.  been reported  al.,  proportion  of  it  has  (formed  intestinal  b i f i d o b a c t e r i a (Mendez and Olano, 1979; Shvedova, 1981). L a c t o f e r r i n may i n d i r e c t l y promote the growth of the b i f i d o b a c t e r i a by inhibiting  the  growth  acid d e r i v a t i v e s (Tamura et a l . ,  of  competing E± c o l i  have also been shown to 1972).  (Spik  stimulate  et  al.,  1978).  one s t r a i n  of  Pantothenic B^ i n f a n t i s  A nitrogen-containing oligosaccharide (Bezkorovainy and  22  Topouzian, Nichols,  1981) 1976)  and have  (Gyorgy, 1953).  glycoproteins been  The greatest  followed by human m i l k , and Monte,  shown  1983)  (Hirano  to  et  al.,  stimulate  the  stimulatory a c t i v i t y  1968; growth  that human milk  Penn. b i f i d u s stimulating  factors,  of  bifidobacteria  A recent report (Ashoor  contains two d i s t i n c t  the a c t i v i t y  and  i s found in human colostrum,  cow's colostrum and cow's m i l k .  noted  Bezkorovainy  B^ bifidum  of which varies  var.  from sample to  sample.  5.  Ovotransferrin Ovotransferrin  molecular  weight  phosphorus.  (OVT), about  also  called  76,000  and  conalbumin,  contains  no  is  a  free  glycoprotein  sulfhydryl  with a  groups  or  The protein moieties of ovotransferrin of egg white and t r a n s f e r r i n  of chicken blood serum are i d e n t i c a l , but the carbohydrate prosthetic groups are different  (Powrie and Nakai, 1986).  Schade and Caroline  (1944,  serum t r a n s f e r r i n  inhibited  This a n t i b a c t e r i a l  effect  1946)  first  reported  that  ovotransferrin  the growth of E. c o l i and other bacterial  and  species.  was destroyed by the addition of Fe which  saturated  +3  the iron binding s i t e s of the proteins (Brock, 1985). Valenti  et  ovotransferrin lactoferrin.  al.  (1983)  concluded that  was q u a n t i t a t i v e l y These  proteins  experimentally-induced  bacterial  the antimicrobial  and q u a l i t a t i v e l y  with  breast  (Packard, 1982).  than  those  of  to  of  human  that  similar  protective  infections  in  newborn  guinea  fed with  artificial  This has been a t t r i b u t e d ,  of a large quantity  hen's  a  resistance against enterobacterial  milk  of  demonstrated  observations have led to the concept of " n u t r i t i o n a l A greater  similar  activity  lactoferrin  immunity"  pigs.  (Weinberg,  on  These 1977).  i n f e c t i o n of human infants fed formula  to a great  in human milk  effect  is  well  documented  extent, to the presence  compared to cow's milk.  The  23  similarities  in  lactoferrin  justify  formula. infants  structure  In  and b i o l o g i c a l  the antimicrobial  addition,  effect  ovotransferrin  (Giacco-Del et a l . ,  activity  did  between  ovotransferrin  of ovotransferrin not  sensitize  added to  and  infant  ovotransferrin  fed  1985).  F. ISOLATION OF BIOACTIVE PROTEINS 1. General methods With a view  to the  i s o l a t i o n of  immunoglobulins as well  as of a l l  other  biopolymers, one must make use of those physicochemical properties or parameters that  are  peculiar  to  the  polymer  in  question,  different  from the  physicochemical properties  unwanted)  polymers (Van Oss, 1982-83).  of  and  that  the  other  There are f i v e  physicochemical parameters of biopolymers: s o l u b i l i t y , t e n s i o n , s i z e and shape, and ligand s p e c i f i c i t y . be  reviewed  in  isolation  strategies  ovotransferrin from d i f f e r e n t  of  are  quantitatively  accompanying  fundamentally electric  (but  different  change, surface  Some of these parameters w i l l  immunoglobulins,  lactoferrin  and  b i o l o g i c a l sources.  Methods currently a v a i l a b l e for the i s o l a t i o n of bovine immunoglobulins and their  subclasses  based on s o l u b i l i t y ,  batch processes, which are d i f f i c u l t  electric  charge and size and shape are  to mechanize (Butler and Maxwell, 1972; Fey  et a l . , 1976; Kanamaru et a l . , 1977; Butler et a l . , 1980; Kanamaru et a l . ,  1980;  Kanamaru et a l . , 1981; Kanamaru et a l . , 1982a; Kanamaru et a l . , 1982b; Shimazaki and Sukegawa, 1986).  1982;  Butler,  1983;  Brooks and Stevens,  Other methods based on a f f i n i t y  (Ey et a l . ,  1978; Martin,  1982)  1985;  Bokhout et  al.,  chromatography using protein A-Sepharose  or immuno-adsorbents (Bokhout et a l . , 1986)  are  quite expensive f o r large scale p u r i f i c a t i o n . L a c t o f e r r i n was f i r s t cow's milk  by Gordon et  isolated al.  (1962)  from human milk by  by Groves (1962) and from  using ammonium  sulfate  precipitation  24  and/or  ion  exchange  column.  Several  described but most of them are rather al.,  1971;  (1977)  Law, and R e i t e r ,  used metal  chelate  1977;  methods  salt  precipitation  Azari  intensive and d i f f i c u l t  Antonini,  little  proteins  more  using  Porath et a l .  than  Keen,  and  separation  1951;  al.  Hernell  Williams,  however,  a  decade  ago,  a  affinity  novel  purification  chromatography"  This technique was l a t e r c a l l e d metal  human  and  et  are  1962; labor-  Chromatography (MCIC)  Sulkowski, 1985). of  Lbnnerdal et  Blackberg  These methods,  Since i t s  has gained wide acceptance and was recently  1982;  been  isolation.  chromatography (MCIC) by Rassi and Horvath (1986). technique  However,  (Warner and Weber,  1977).  "immobilized metal  (1975).  have  to mechanize.  2. Metal Chelate-Interaction A  isolation  used i s o l a t i o n methods for ovotransferrin are based on  and ion exchange  and Baugh, 1967;  1984).  chromatography  (1980) used heparin-Sepharose for l a c t o f e r r i n The most frequently  its  laborious (Johansson, 1969; Querinjean  Kawakata,  affinity  for  serum  The a p p l i c a t i o n  proteins  (Porath  Ramadan and Porath, 1985), human l a c t o f e r r i n  of  et  was  introduced by  introduction,  reviewed  (Lonnerdal  MCIC has been al.,  1983;  for  chelate-interaction the and  reported  Andersson,  (Lonnerdal et a l . ,  (Torres et a l . , 1979), human f i b r o b l a s t i n t e r f e r r o n  technique  for 1984;  1977), lysozyme  (Edy et a l . , 1977)  and human  serum albumin (Hansson and Kagedal, 1981). As believed  explained to  histidine,  by  be the  Lonnerdal result  of  and the  Keen  (1982),  ability  of  the  binding  electron-rich  of  proteins  ligands,  is  such as  cysteine and tryptophan, to substitute weakly bonded l i g a n d , such as  water or buffer  ions,  in the complexes.  When a p r o t e i n ,  with surface exposed  amino acids having electron-donating c a p a c i t y , is exposed to a metal, a multipoint attachment  can r e s u l t .  strong  This binding is stable even in 1M NaCl ruling  25  out  the  possibility  binding.  It  is  of  ionic  important  interaction  to  realize  being that  the  principal  this 'kind  of  force  in  the  interaction  is  independent of whether the protein i s iron binding or not o r , in the case of an iron-binding apo-form. as  the  1982).  protein,  whether  the  Fe-saturated l a c t o f e r r i n  apo-form of  lactoferrin  protein  is  in  Fe-saturated  form  or  in  the  can bind to a copper-loaded gel as strongly  (Lonnerdal  et  al.,  1977;  Lonnerdal  and Keen,  26  MATERIALS AND METHODS A. MATERIALS Sodium hexametaphosphate Scientific  Company  immunoglobulins, alkaline  from  (Fairlawn,  rabbit  phosphotase  ovalbumin,  Chemical  NJ);  anti-bovine conjugated  ovotransferrin,  Sigma  (SHMP, p u r i f i e d  Company  Chemalog (South P l a i n f i e l d , NJ). of  Zittle  and  Custer  bovine  rabbit  (St.  was purchased from Fisher  8-lactoglobulin, IgG,  a-casein  grade)  a-lactalbumin,  serum  anti-bovine  and  diethyl  Louis,  MO).  albumin, IgG,  lactoferrin,  lactoperoxidase,  pyrocarbonate B-Casein  bovine  were purchased  was  obtained  from  K-Casein was prepared according to the method  (1963).  a i-Casein s  was  a  gift  from  Dr.  R.  Yada and  sulfhydryl blocked K - c a s e i n ( s s s - K - c a s e i n ) was a g i f t from Dr. S. Nakai. Materials f o r column chromatography were: s i l i c a (sand)  ( f i n e granular  type  No. S-150 from Fisher Laboratory Chemical, Fairlawn, NJ), c o n t r o l l e d pore glass 80-120 mesh, PG 1400-120 and fumed s i l i c a S-5055.(from Sigma Chemical Company, St.  Louis,  MO);  Laboratories, from  Mead  obtained  alumina  AG7, 100-200 mesh, No.  Mississauga, ON).  Johnson and from  (neutral  Company  Intercontinental  purchased from a local market. Foods (Burnaby, BC).  E l e c t r o d i a l y z e d and (Evansville, Packers  IN).  Ltd.,  132-1140 from BioRad  sweet  whey  Bovine  Vancouver,  powders were  blood  BC.  plasma  was  Skimmilk  was  Cheddar cheese whey was obtained from Dairyland  E s c h e r i c h i a c o l i Serotype 0142:K86(B):H6 (ATCC No. 23985),  Salmonella typhimurium  (ATCC No. 13311) and Bordetella parapertussis  15311) were supplied by American Type Culture C o l l e c t i o n ( R o c k v i l l e , other chemicals were of a n a l y t i c a l  (ATCC No. MD).  All  reagent grade.  B. ACID WHEY PREPARATION Raw milk Farm.  Acid  and colostrum were obtained whey  was  prepared  from  raw  from the milk  and  University colostrum.  centrifuged at 4,000 x g for 30 min at 5°C for cream separation.  Animal Science The milk  was  Acid whey was  27  prepared from the skimmilk by adding 50% a c e t i c acid solution to pH 4.6 at 25°C and c e n t r i f u g i n g at 10,000 x g for 15 min to remove casein p r e c i p i t a t e s .  C. SODIUM DODECYL SULFATE - POLYACRYLAMIDE GEL ELECTROPHORESIS The method of Laemmli gel  electrophoresis  presence of  0.2%  was performed with a slab type v e r t i c a l  gel  Electrophoresis unit  in the  (1970) was used a f t e r m o d i f i c a t i o n s .  Polyacrylamide  sodium dodecyl s u l f a t e  (SDS-PAGE)  system using the Atto SJ 1060 DSH  (Atto C o . , Tokyo, Japan).  1. Discontinous SDS-PAGE A whole gel was composed of separating gel long, and 13.5 cm wide, and stacking gel  (lower gel)  (upper g e l ) .  0.2 cm t h i c k ,  11 cm  Ten and 3% polyacrylamide  gels were used as the separating and stacking g e l s , r e s p e c t i v e l y , of which the ratio  of acrylamide to N.N'-methylene-bis-acrylamide  was 25.  Polymerization  of  both gels was catalyzed by 0.02% ammonium p e r s u l f a t e . One mL of whey solution (2-4 mg protein/mL) was treated with 5% SDS and 0.2 mM 2-mercaptoethanol  in b o i l i n g water for  1.5 min, followed by the addition of  200 mg sucrose and 50 jiL of 0.05% bromphenol blue tracking dye s o l u t i o n . five  Twenty  yL of the treated whey solution was applied to the sample s l o t a f t e r  the  sample s l o t s and upper electrode chamber were f i l l e d with T r i s - g l y c i n e electrode buffer  (3g T r i s + 14.4g glycine + lg SDS in 1 L, pH 8.3).  Electrophoresis was performed at of 90 v o l t s u n t i l in approximately floater  room temperature  4.5  hr.  (a gel supporter),  water,  voltage  the tracking dye marker migrated to 1 cm from the gel bottom, The gel  was then  removed, placed on a net  immersed in 0.25% Coomassie B r i l l i a n t  solution (Weber and Osborn, 1969), and stained for 1.5 with  with a constant  transferred  to  a  diffusion  destainer  hr.  plastic  Blue R-250 dye  The gel was rinsed  (model  172A,  Bio  Rad  28  Laboratories, circulation methanol)  Richmond, CA), and destained v e r t i c a l l y of  destaining  solution  through a cartridge  by using p e r i o d i c  (a  mixture  of activated  acid-Schiff  (PAS)  of  10%  carbon.  technique  for  18 to 20 hr with a  acetic  acid  and  7.5%  Glycoproteins were stained  described by Zacharius  et  al.  (1969).  2. Gradient SDS-PAGE Solutions solution of 0. 25  M  containing  3% and  20% acrylamide  were  60% acrylamide with 4% c r o s s l i n k i n g .  Tris-HCl  ethylenediamine  (pH  8.3)  (TEMED).  containing  0.2%  Ammonium persulfate  the solutions immediately  before mixing.  prepared  from  a  stock  The solutions were made SDS  for  and  0.125%  polymerization  in  tetramethyl was added to  Gradients were generated using a two  chamber device containing 20 ml of 20% acrylamide in the mixing chamber and 20 ml of 3% acrylamide in the reservoir chambers. a vertical  The mixture was then pumped into  slab mould of the Atto SJ 1060 SDH Electrophoresis Unit  Tokyo, Japan), at a flow rate of 2 mL/min. conditions,  staining  and destaining  were  (Atto C o . ,  Sample preparation, electrophoresis performed  as  described  in  Section  (C-l).  D. IMMUNOCHEMICAL ANALYSIS 1. Immunoelectrophoresis and immunodiffusion Immunoelectrophoresis  and  immunodiffusion  analysis  were  carried  according to the method of Williams and Chase (1971) with m o d i f i c a t i o n s . mL of  1% agarose in 0.05  over Gelbond f i l m Bioproducts,  (0.02  M Na-barbital  x 7.5  Rockland, MA).  sample well with a diameter room temperature  acetate  buffer,  pH 8.3  was  out Nine  gelatinized  x 10 cm, FMC Corporation Marine C o l l o i d Division Three yL of whey sample was applied to a punched  of 2 mm and immunoelectrophoresis was performed  for 45 min with a constant voltage of 60 v o l t s .  at  Sixty uL of  29  antibody  (Miles  Laboratories  Inc.)  was added to  performed overnight in a cold room. and  0.15  M NaCl  solutions  the  trough  After deproteinization  and then  in  water,  each  and d i f f u s i o n was  by shaking in 0.3 M  for  1 day,  the  gel  was  a i r - d r i e d , and stained with Amido Black 10B dye s o l u t i o n . Quantitative immunodiffusion  immunochemical  analysis  (R.I.D.) with a R.I.D.  of  kit  IgG  (Miles  was  carried  out  Laboratories  by  radial  Inc.).  Whey or  protein samples were dialyzed against 20 mM sodium phosphate buffer pH 7.0 for 2 days and f r e e z e - d r i e d . acetate for  buffer  Whey samples were then  pH 8.3  determination  dissolved in  0.05  M  barbital  to give a concentration within the range of the k i t  of  immunoglobulins.  After  deproteinization,  the  used  gel  was  a i r - d r i e d and then stained with Amido Black 10B dye s o l u t i o n .  2. Enzyme  linked  immunosorbent  assay  for  anti-1ipopolvsaccharide  activity  determination The method of Stephens (1984) was used with s l i g h t m o d i f i c a t i o n s . 2  flat-bottomed  microtitre  1ipopolysaccharides for 2 hr at bufferred  (LPS)  (PBS)  plates  incubated  for  2  coated (0.05  0.05%  Tween,  with  in 0.01  pH 7.2.  PBS/Tween, were dispensed in hr  at  100  uL  room temperature.  Serial  After  further  washing  in PBS/Tween) were added and incubated  diethanolamine  buffer  at  sodium azide) was added. 20  uL 5N NaOH and  the  pH 9.8 After  (p-nitrophenyl  containing 30 min the  absorbance  0.5  9.6)  100 uL volumes and  IgG  substrate  pH  dilutions  100 uL of a l k a l i n e phosphatase conjugated rabbit  were washed and 100 uL of  0.01%  M sodium phosphate  PBS/Tween (3 times), (1:750 d i l u t i o n  of  M sodium carbonate,  and washed three times  containing  immunoglobulins, made in  were  in coating buffer  room temperature  saline  plates  Immulon  for  phosphate,  the with  antibovine  2 hr.  Plates  1 mg/mL in  I'M  mM magnesium chloride and 0.2%  reaction was stopped by addition  change was  of  read  on an  ELISA plate  of  reader  30  (Titertek  Multiscan,  Flow  Laboratories,  Scotland)  Corrections were made for n o n - s p e c i f i c adsorption of  with  a  405  nm  filter.  Igs.  3. Sandwich enzyme linked immunosorbent assay for IgG assays The method of Troncone et a l . 2 flat-bottom m i c r o t i t r e (1:100 d i l u t i o n washed  three  (1986) was used with m o d i f i c a t i o n s .  plates were coated with 100 uL rabbit anti-bovine  in PBS/Tween) and incubated for times  in  Immulon  PBS  containing  0.05%  2 hr at Tween.  room temperature Serial  dilutions  IgG and of  immunoglobulin samples, made in PBS/Tween, were dispensed in 100 uL volumes and the plates  incubated for 2 hr at  room temperature.  A f t e r further washing, 100  uL of a l k a l i n e phosphatase conjugated rabbit anti-bovine IgG (1:750 d i l u t i o n PBS/0.05% Tween) were added and incubated f o r 2 hr. uL of substrate  (p-nitrophenyl  in  Plates were washed and 100  phosphate disodium 1 mg/mL in 1 M diethanolamine  buffer at pH 9.8 containing 0.5 mM magnesium chloride and 0.2% sodium azide) was added. and the  A f t e r 30 min the reaction was stopped by the addition of 20 uL 5 N NaOH absorbance was  read on an ELISA plate  reader with a 405  Corrections were made for n o n - s p e c i f i c adsorption of  nm f i l t e r .  Igs.  E. SODIUM HEXAMETAPHOSPHATE TREATMENT OF CHEESE WHEY An a l i q u o t of 10% sodium hexametaphosphate (SHMP) solution was added to 25 mL of pH adjusted cheese whey while maintaining the pH by dropwise addition of 3 N NaOH or  3  N HC1.  10,000 x g f o r 15 min. buffer,  pH 6.8,  The mixture  was  held  for  1 hr,  then  centrifuged  at  The p r e c i p i t a t e was dispersed in 5 mL of 0.5 M T r i s - H C l  and made up to 25 mL a f t e r  further  supernatant was neutralized to pH 6.8 with 3 N NaOH.  pH adjustment  to 6.8.  The  The samples were dialyzed  against d i s t i l l e d water for 48 hr and then freeze dried (Figure  1).  31  CHEDDAR CHEESE WHEY pH 4.0-4.5 SHMP 1.0-1.4 mg/mL at room temp, hold f o r l h r centrifuge at 10,000 X g PREC PITATE  SUPERNATANT  disperse in Tris-HCl b u f f e r , pH 6.8 dialyze f o r 48 hr centrifuge at 10,000 X g  SUPERNATANT freeze dry p-LG RICH POWDER  Figure 1.  PRECIPITATE (discard)  SUPERNATANT freeze dry  adjust, pH 6.8 dialyze f o r 48 hr centrifuge at 10,000 X g  PRECIPITATE (discard)  IG RICH POWDER  Flow diagram of the procedure f o r elimination of B-lactoglobulin Cheddar cheese whey with SHMP.  from  32  F. OPTIMIZATION PROCEDURE The mapping super simplex used to f i n d the most s u i t a b l e cheese whey which would give  optimization  (MSO) of Nakai  et a l .  conditions f o r the polyphosphate the maximum separation  efficiency  minimum amount of B - l a c t o g l o b u l i n in the supernatant.  treatment of of Igs and. a  An IBM PC computer was  used f o r computation f o r the MSO and centroid mapping optimization method of Aishima and Nakai  (1986).  The experimental  in MSO and CMO were within the following 1.0-1.4 mg/mL.  (1984) was  (CMO) by the  conditions (factors)  used  ranges: pH 4 . 0 - 4 . 5 , SHMP concentration  A l l experiments were carried out at room temperature  (22°C).  1. Mapping super simplex Mapping super simplex the  introduced by Nakai  et a l .  (1984)  and written f o r  IBM-PC was used in order to speed up the i t e r a t i v e optimization  and g r a p h i c a l l y i l l u s t r a t e experiments,  the level  divided  four  into  medium and small  the experimental  values  f o r each f a c t o r  groups based on t h e i r limits.  response s u r f a c e .  The large  used in  and f i n a l  concentration  and small  limits  of SHMP and pH).  average of both large and small l i m i t s . data.  Data points  factors were joined  After doing nine  the optimization  locations on the scale within  individual plots of response value (Separation e f f i c i e n c y ) (initial  procedure  were  were large,  determined  from  v s . each factor  level  The medium  limit  was an  These l i m i t s were used f o r grouping the  f o r one f a c t o r which belonged to the same groups of other together  thus giving an estimate  of the response surface.  The maps f o r a l l factors provided new level values f o r each f a c t o r .  2. Centroid mapping optimization and simultaneous factor Centroid mapping optimization to improve the optimization  (Aishima and Nakai,  shift 1986) was used in order  e f f i c i e n c y and to allow f o r a series of experiments  33  to be run simultaneously. f a c t o r was generated  A f t e r doing another six experiments, the map for each  in s i m i l a r manner as in  (F-l).  The maps provided target  values where the high separation e f f i c i e n c i e s were located. A Simultaneous Factor S h i f t IBM-PC was  used.  Target  Program (Nakai  values  determined from the graphs.  (estimated  et  best  al.,  1984)  separation  the present best value and the target value. resulting  from  investigated and t h e i r  G.  the  factor  Factor  Shift  an  were levels  the distance  The new experimental  Simultaneous  for  efficiency)  The program i s designed to s h i f t a l l  obtained from the mapped graphs simultaneously one f i f t h  (vertices)  written  between  conditions  Program  were  response values were c a l c u l a t e d .  EVALUATION OF S E P A R A T I O N E F F I C I E N C Y  (RESPONSE V A L U E )  Peak areas of whey proteins on the electrophoretograms were analysed using a Kontes f i b e r  optic  K-494800,  Kontes S c i e n t i f i c  Instruments,  Vineland, NJ) together with a Varicord variable  response recorder  (Model 42 B,  Photovolt Corp, NY). p-Lg  ratio"  scanner  (Model  Separation e f f i c i e n c y  calculated  from  peak  area  of  (SE) was expressed as the Igs  (PAi )  and  P-Lg  chain  peak  gs  "Igs  to  (PAp_|_ )  on  g  the densitometric patterns as: SE= P A PAi  was  g s  coefficient difficult  I g s  of  /  (PA  I g s  + PAp-Lg)  estimated 1.4  by  since  multiplying  the  the  determination  heavy of  light  due to overlapping with other minor p r o t e i n s .  chain  area  peak  area  by  a  was  The c o e f f i c i e n t 1.4 was  derived from analysis of IgG standards. For  quantitative  analysis,  the  variation  of  staining  and  conditions during electrophoresis was standardized using an internal ovalbumin.  Ten m i c r o l i t e r s  2-mercaptoethanol  (similar  of  to the  0.1%  ovalbumin  treatment of  solution sample)  destaining standard of  treated with SDS and  was added to each whey  34  sample solution and analyzed simultaneously. for  every  run  was  compared  ovalbumin standard. correction  with  the  The ovalbumin peak area measured  peak  areas  measured  for  a  series  of  The r a t i o of ovalbumin values, thus obtained, was used as a  factor.  H. SURFACE PLOT Contour and 3-dimensional  surface plots were obtained using the UBC Surface  V i s u a l i z a t i o n Routines program (Mair, 1982) 3-dimensional The  "about"  on an Amdahl 470 V/8 computer.  plot was rotated and t i l t e d f o r the best view of the surface: angle was the  angle  of  turn,  in  degrees,  z - a x i s , measured clockwise from the p o s i t i v e x - a x i s ; the angle of t i l t , xy plane.  I.  in degrees, of rotation  of  (b)  rotation  the  about  The (a) the  "above" angle was  about the y - a x i s , measured above the  In t h i s work, x=pH, y=SHMP and z=SE (Separation  efficiency).  PHOSPHORUS DETERMINATION The phosphorus d i s t r i b u t i o n of the f r a c t i o n s obtained by SHMP treatment was  determined according to the method of Morrison (1964).  J.  FRACTIONATION PROCEDURES OF BIOACTIVE COMPONENTS  1. Gel f i l t r a t i o n  chromatography  Immunoglobulins  were  isolated  from  colostral  whey,  acid whey and cheese  whey using Sephacryl S-300 (Pharmacia Fine Chemicals, Uppsala, Sweden) cm)  and Fractogel  TSK HW-55  (EM Science,  column of Sephacryl S-300 was e q u i l i b r a t e d containing Fractogel 6.5.  0.5  M NaCl  (Pharmacia  TSK was e q u i l i b r a t e d  Fine  with 0.07  Gibbstown, with 0.1  Chemicals,  NJ)  (40  x 2.6  M Tris-HCl 1978), while  M imidazole  -  0.05  (94 x 2.5 cm).  buffer, the  The  pH 8.0  column of  M KC1 b u f f e r ,  pH  35  2. S i l i c a adsorption Chromatography Chromatographic conditions were based on the process recommended by Spring and Peyrouset columns (1.3 pH 8.2,  x 7.5  then  washing o f f  (1982)  whey  for  silica,  controlled  pore  glass  and  alumina.  Small  cm) were e q u i l i b r a t e d with 0.005 M sodium phosphate s o l u t i o n , containing  0.005  the unbound m a t e r i a l s ,  a c e t i c acid containing 0.5  M phosphate  was  passed  the bound l a c t o f e r r i n  M NaCl, and the  through.  After  was eluted with 0.1 M  immunoglobulin  fraction  was  eluted  with 0.1 M T r i s - H C l buffer containing 0.5 M NaCl, pH 9.0.  3. Heparin-Sepharose Chromatography L a c t o f e r r i n was i s o l a t e d from cheese whey by using heparin-Sepharose column after  equilibration  with 0.005 M Veronal-HCl  (Blackberg and H e r n e l l ,  M NaCl,  pH  7.4  Chromatography  Sepharose 6B was activated  according to the method of Sundberg and Porath  One hundred grams of s u c t i o n - d r i e d Sepharose 6B was washed on a glass  filter-funnel ether  0.05  1980).  4. Metal C h e l a t e - i n t e r a c t i o n  (1974).  containing  with water and then mixed with 100 mL of 1,4-butanediol  (BGE) and 100 mL of  0.6  M sodium hydroxide  sodium borohydride per m i l l n i t r e . hr at 25°C and the reaction  solution  diglycidyl  containing  2 mg of  The suspension was mixed by rotation  stopped by washing the gel on a glass  for 8  filter-funnel  with large volumes of water. Epoxyactivated according  to  epoxyactivated  gel  obtained  the  method  gel  250  of  mL of  above  Porath  was  and  2 M Na2C03,  01 in 12.5  and 0.15 gram of sodium borohydrate were added. overnight with slow s t i r r i n g .  coupled  to  (1983). g of  iminodiacetic To  disodium  100  grams  acid of  iminodiacetate,  The suspension was kept at 60°C  The gel was washed thoroughly on a Blichner funnel  36  with  water,  with  diluted  washings were neutral Iminodiacetic  acetic  (Figure acid  acid  (5%)  and  again  with  water  until  2).  1,4-butanediol  diglycidyl  Sepharose  6B  (IDA-BGE  Sepharose Sepharose 6B) was packed into glass columns with d i s t i l l e d water. upper  one-half  copper  ions  as  to  two-thirds  indicated  of  the  by t h e i r  chelating  blue  color,  Sepharose was followed  d i s t i l l e d water and e q u i l i b r a t i o n with the s t a r t i n g buffer containing 0.5 M NaCl, pH 8.2). 0.5  M NaCl, pH 8.2  elution  (0.05  Liquid whey containing 0.05  elute  bound p r o t e i n s ;  with  M Tris-acetate  A f t e r washing linear  gradient  was formed  using equal volumes of s t a r t i n g buffer at pH 8.2 and a l i m i t buffer  at pH 2.8.  Alternatively,  pH 4.0  were eluted with the  the  with  pH gradient  bound proteins  the  off  The  M T r i s - a c e t a t e and  was passed through the copper loaded column.  was used to  saturated  by washing  o f f the unbound whey protein f r a c t i o n s with the s t a r t i n g b u f f e r , of  the  same buffer  at  and  0. 01 M imidazole as a two step e l u t i o n . The eluent was c o l l e c t e d in f r a c t i o n s and protein peaks were detected by UV absorbance  at  280  nm  using  Cary  210  Spectrophotometer  (Varian  Instrument  D i v i s i o n , CA). Following gradient  elution,  the chelating gel was regenerated with 0.05 M  Na EDTA solution to s t r i p off  the copper i o n s , followed by 6 M urea to remove  any remaining bound p r o t e i n s .  After washing with d i s t i l l e d water, the chelating  2  Sepharose was (Figure  ready  for  the  next  cycle of  copper loading and whey  treatment  3).  K. DETERMINATION OF CAPACITY OF MCIC 1.  Immunoglobulins Crude  Ig  was  isolated  form  bovine  colostral  p r e c i p i t a t i o n , according to the method of Fey et a l .  whey  by  (1976).  ammonium  sulfate  A solution of the  37  Agarose-OH + CH2CH-CH2-0-(CH2)4-0-CH2-CH-CH 0 V 1,4-butanediol d i g l y c i d y l ether (BGE) 2  Agarose-0-CH2-CH0H-CH -0-(CH )4-0-CH2-CH-CH2 2  2  V  BGE-Agarose ,CH -COOH 2  HN' CH -C00H iminodiacetic acid (IDA) v  2  ,CH -C00H Agarose-0-CH2-CH0H-CH2-0-(CH )4-0-CH2-CH0H-CH -N CH2-COOH IDA-BGE-Agarose 2  2  2  N  Cu2+  ,CH ?-Q Agarose-0CH2-CH0H-CH -0-(CH2)4-0-CH2-CH0H-CH2-N N Jpu CH C 2  2  S  2  Cu-IDA-BGE-Agarose  Figure 2.  Preparation of metal chelate agarose  38  •  IDA-BGE Sepharose 6B (1) (2) (3) (4)  Cu-IDA-BGE  H 0 wash Copper (0.05 M CuCl2) loading H2O wash starting buffer equilibration 2  3  Sepharose 6B (1) Whey (2) Starting buffer wash > Unbound Fraction ( P - L g ,  a-La)  T Protein bound Cu-IDA-BGE Sepharose 6B pH gradient e l u t i o n > Fraction 1 ( L f , Ig, BSA) > Fraction 2 (Ig) b  Cu-IDA-BGE Sepharose 6B (1) (2)  a b  EDTA wash (-> C u ) 6 M urea (-> remaining 2 +  proteins)  s t a r t i n g buffer = 0.05 T r i s acetate, pH 8.2, 0.5 M NaCl l i n e a r gradient: s t a r t i n g buffer as in Footnote a , l i m i t buffer M T r i s acetate, pH 2.8, 0.5 M NaCl  Figure 3.  Flow chart of i s o l a t i o n of Ig  MCIC  process  of  cheese  whey  = 0.05  treatment  for  39  crude Ig  (roughly  0.3% w/v)  a small column (1.4 6B e q u i l i b r a t e d While  the  eluted  Ig  buffer  (0.05  M Tris-acetate  solution was being applied  to  f r a c t i o n s were continually monitored with  of the column ( i . e . ,  no further  the  became equal  eluted  2.1 was passed through  x 7 cm) containing 2.2 mL copper-loaded chelating Sepharose  in s t a r t i n g  crude  with A28O of approximately  fractions  the  o  f  t  n  top of  0.5  column, the  respect to A280«  Saturation  was indicated when A28O of  original  e  crude Ig  The binding capacity of the copper-loaded gel was c a l c u l a t e d , at t h i s point,  M NaCl).  the  binding of protein) to A28O  pH 8.2,  solution. saturation  as the amount of applied crude Ig minus the amount of unbound Ig  amount of  (This  Ig was calculated from the A28O and volume of each f r a c t i o n ) .  washing o f f  unbound protein with the  eluted with 0.05 recovery of  M Tris-acetate  starting  buffer  at  buffer,  pH 4.0  After  the bound proteins were  containing 0.5  M NaCl.  Ig was calculated as the percentage of eluted protein  The  compared to  bound p r o t e i n .  2.  Ovotransferrin A solution  (0.2%  w/v)  of  was passed through a small  commercial  column (1.4  chelating Sepharose 6B e q u i l i b r a t e d  OVT with A28O ° f  approximately  1.94  x 7 cm) containing 3 mL copper-loaded  with  the  starting  buffer.  While  the OVT  solution was being applied to the top of the column, the eluted f r a c t i o n s were continually further became  monitored  binding equal  of  to  by measuring A280*  protein)  1.94.  was  The  calculated as the difference After  washing  off  the  indicated  binding between  with 0.05  0.5 M NaCl, and then with 0.01  when A28O  capacity the  unbound protein  proteins were eluted f i r s t  Saturation  of  the  M acetate-Tris  M imidazole.  o  the  amounts of with  of f  the  column ( i . e . ,  the  eluted  no  fractions  copper-loaded gel  was  OVT applied and unbound.  starting  buffer,  buffer at pH 4.0  the  bound  containing  The recovery of OVT was calculated  as the percentage of eluted protein compared to bound p r o t e i n .  40  3.  Transferrin A s o l u t i o n of 0.2%  buffer,  pH 8.2,  absorbance  at  (w/v)  transferrin  was passed through 280  nm (A280)  o  f  t  n  the  e  in 0.05  M T r i s - a c e t i c a c i d / 0 . 5 M NaCl  column charged with  effluent  from  the  copper i o n .  column was  The binding capacity was calculated from the differences  The  monitored.  of the absorbance of  the eluted protein as compared to the absorbance at the saturation point of the column.  The percentage of protein eluted was calculated by comparing the amount  of protein bound to the column with the protein eluted from the column.  L. PRETREATMENT OF EGG WHITE Eggs were Farm.  obtained  To obtain  a  from  the  University  homogeneous and  less  of  British  viscous  Columbia  sample with  Experimental  suitable  flow  p r o p e r t i e s , the separated egg whites were blended (2000-2500 rpm, 7-10 sec) in a Lourdes MM-1A MultiMixer reported by Li-Chan et a l .  (Lourdes Instrument Corporation, Old Bethpage, NY) as (1986).  M. PREPARATION OF APO, DIFERRIC AND DICUPRIC OVOTRANSFERRIN Iron free 0.1 to  (apo)  OVT was prepared by d i a l y z i n g  standard OVT f i r s t  against  M c i t r i c a c i d , pH 2-3 f o r 36 hr at 4 ° C , then against deionized water p r i o r lyophilization.  Diferric  OVT was prepared by d i a l y z i n g the apo-OVT against  1.7 mM ferrous ammonium s u l f a t e for 36 h r , then excess iron was removed by gel f i l t r a t i o n on a Shephadex G-25 column e q u i l i b r a t e d with 0.05 M Nacl,  pH 8.2  (Cole  et  al.,  1976).  In  a similar  prepared by d i a l y z i n g against 0.01M cupric c h l o r i d e .  manner,  M Tris-acetate/0.5 d i c u p r i c OVT was  41-  N. HISTIDINE MODIFICATION OF PROTEINS Histidine according  residues  to  the  method  Diethyl pyrocarbonate directly  to  containing  a 5-10 8  ethoxyformyl reaction  (DEP)  Ig,  TF,  of  Rogers  while  histidine  OVT and  to make the  mg/mL protein  M urea  solution  of  et  al.  final  solution  stirring.  casein  in  After  =  5.9xl0  3  L  M  - 1  (1977)  concentration 0.05 20  formation was determined  (E240  fractions  were  with  modified  modifications.  of  20 mM was added  M phosphate buffer min  stirring  the  pH  6.6  extent  of  by an increase in A 4 o of 2  cm ) -1  (Roosemont,  1978).  the The  p u r i t y of DEP used was determined according to Holbrook and Ingram, (1973).  0.  ISOELECTRIC FOCUSING Analytical  horizontal  polyacrylamide  was c a r r i e d but in a Bio-Rad Model manufacturer's  instructions.  1415  gel  isoelectric  focusing  electrophoresis c e l l ,  (IEF-PAGE)  according to  Gel slabs were 45 mm x 125 mm and 0.8  Bands were located by means of a Coomassie Blue protein  the  mm t h i c k .  stain.  P. PREPARATION OF ANTISERA Antiserum to egg white and other proteins were produced by immunizing adult female New Zealand white rabbits of antigen  emulsified  given in multiple  (UBC, Animal Care Unit) each time with 1-10 mg  in Freund's complete adjuvant  (FCA).  Immunizations  subcutaneous s i t e s , and repeated intravenously  (I.V.)  in a two  to six week period by replacing FCA with phosphate buffered s a l i n e , pH 7.2, carrier until  a satisfactory  response was obtained.  were  as a  Serum was tested by double  d i f f u s i o n in gel against egg white proteins as reported by Garvey et a l .  (1977).  42  Q. MEASUREMENT OF BACTERIOSTATIC ACTIVITY The method of Dolby and Stephens (1983) was used for the determination of bacteriostatic transferrin  activity  of  the  isolated  proteins.  prepared by the MCIC method were added to  Immunoglobulin  and  5 mL of Trypticase Soy  Broth (TSB) at concentrations of 10 mg/mL each or in a mixture of 5 mg/mL each. A half mL of 0.05 M NaHC0 was added to each broth and s t e r i l i z e d by  filtration  3  (Millex-HA, control the hr.  0.45  um, M i l l i p o r e  (TSB only)  test  culture;  were  Corp.  Bedford, MA).  inoculated with  10  4  The broths  including  colony forming units  samples of the broth cultures were taken  the  (cfu)/mL  after  1,  of  3, and 5  S e r i a l d i l u t i o n s of the bacteria in the broth cultures was accomplished by  plating  on Trypticase Soy Agar (TSA) with the s p i r a l  plater  (Anonymous, 1985).  The inoculated plates were incubated at 37°C for 18 hr.  R. EXTRACTION OF LIPOPOLYSACCHARIDES of JE^. col i ,  Lipopolysaccharides  S. typhi murium and B^ parapertussi s were  extracted by using phenol/water according to the method of Jann (1985). cultivation  in  TSB, the  bacteria  were  killed  by the  addition  of  After  1% phenol,  centrifuged at 5000 x g for 30 min and washed with 0.15 M s a l i n e and centrifuged again.  They were then f r e e z e - d r i e d .  20 mL of water at 68°C. to  the  bacterial  stirring  for  suspension  15  was  formation  of  bacterial  pellet  Twenty mL of 90% phenol, prewarmed to 68°C were added  suspension and the min.  After  centrifuged  two phases. in the  One gram of dry bacteria was suspended in  at  mixture  cooling 5000  to  x g  A precipitate  lower phase.  was  about for  30  kept 10°C min.  at  68°C with vigorous  in This  was formed between  mL water  at  68°C  ice-bath,  resulted the  in  layers  the the  and a  The upper aqueous phase was c o l l e c t e d by  s u c t i o n , then the lower phenol phase together with the p e l l e t 20  an  as described above.  was treated  with  The combined aqueous phases were  43  dialyzed against d i s t i l l e d water for 48 hr in the cold room to remove phenol and low-molecular weight m a t e r i a l .  The solution was then  freeze-dried  to  give a  white powder.  S. LACTOPEROXIDASE ASSAY The  lactoperoxidase  content  of  fractions  obtained  by  MCIC method were  analyzed by using the procedure described by Sigma Chemical Co. ( B u l l . No. for  peroxidase  prod.  phosphate b u f f e r , (w/v) The  pyrogallol initial  as the  (0.32  (0.32  buffer,  pH 6.0)  in  in A420 was  A substrate  mL),  0.147  until  To t h i s mixture,  mixture  constant with a cuvette at  zero time,  M potassium  (0.16  mL), 5%  0.1  containing H£0  mL of a lactoperoxidse M potassium phosphate  The solution was mixed by inversion and the increase  recorded every  Spectrophotometer  0.1  mL) was mixed by i n v e r s i o n .  (10 mg lactoperoxidase per mL of 0.1  was added.  of  M hydrogen peroxide  mL) and d i s t i l l e d water (2.1  A420 was monitored  containing f r a c t i o n  Cary 210  p.8250).  pH 6.0  reference.  the  No.  8-84  10 seconds f o r  (Varian Canada I n c . ) .  increase in absorbance was determined using l i n e a r  about two minutes,  using a  The i n i t i a l  rate of  linear  r e g r e s s i o n , and was used to  determine the units of lactoperoxidase a c t i v i t y per mg s o l i d :  A420/20 second Units/mg s o l i d = (12)* 12* =  Units  Extinction c o e f f i c i e n t as determined by Sigma  obtained  protein  x (mg enzyme as solid/mL reaction mix)  were compared to  using pyrogallol  that  as substrate).  of  bovine  lactoperoxidase  (80  units/mg  Lactoperoxidase was calculated as the  percentage of lactoperoxidase content in the pooled f r a c t i o n s .  44  T . SEPARATION OF HEAVY AND LIGHT CHAINS OF IMMUNOGLOBULINS Reduction  and  alkylation  of  S-S  groups  in  the  performed according to the method of Garvey et a l . 2% Ig mL of  in 0.55 0.15  M Tris-HCl  M dithiothreitol  mixture was allowed  to  react  of 0.25 M 2-iodoacetamide to the  reduced Ig  M T r i s - H C l buffer  fractionated respectively.  in for  (IAA)  pH 8.2 0.55  rich  (1977).  M Tris-HCl  buffer  1 hr under a p o s i t i v e N  fraction  were  A 10 mL solution of  was bubbled with N  2  for  15 min.  were added, 2  Five  and  atmosphere.  the  Ten mL  in 0.55 M T r i s - H C l buffer pH 8.2 were then added  sample and the mixture was kept  The reduced and alkylated 0.1  buffer  Ig  Ig was then e q u i l i b r a t e d  containing 4 M guanidine-HCl  on a Sephadex G-75 and an Ultrogel  in the  cold room for  1 hr.  with 1 M propionic acid or and 1 mM IAA,  ACA 54 column (40  pH 8.2 x 2.6  and cm),  RESULTS AND DISCUSSIONS  REDUCTION OF 0-LACTOGLOBULIN CONTENT OF CHEESE WHEY BY USING SODIUM HEXAMETAPHOSPHATE  46  Since al.  elimination  of  B - l a c t o g l o b u l i n by f e r r i c  1985; Kuwata et a l . 1985)  and may r e s u l t  in  loss of  can saturate  as a d d i t i v e s to  extract  in food processing.  whey proteins  investigated  as  a  et  iron binding proteins present in whey  the antimicrobial  methods were investigated (Appendix 1).  chloride methods (Kaneko  activity  of l a c t o f e r r i n ,  non-ferric  Polyphosphates have been extensively used  Gordon (1945) in his patent used polyphosphate  from cheese whey.  possible  means  In  for  this  the  part,  polyphosphates were  selective  precipitation  of  B - l a c t o g l o b u l i n s from cheese whey leaving immunoglobulins in the supernatant.  A. OPTIMUM CONDITIONS FOR SEPARATION OF IMMUNOGLOBULINS AND B-LACTOGLOBULIN Mapping  simplex  c a l l e d the " i n i t i a l our  case,  the  optimization  simplex".  separation  with  two  factors  generated  After  the  repetitive  performing  nine  experiments  A f t e r obtaining the response values which were,  efficiency  (SE)  of  immunoglobulins,  reported back to the computer to obtain new v e r t i c e s a form of  three  sequences of  vertices  for  centroid,  the  the  (experimental  reflection,  values were  conditions)  and  in  curve-fitting.  MSO, mapping was done by p l o t t i n g  response values against the factor l e v e l s .  in  the  A crude approximation of the response  surface appeared to d i r e c t the search for higher SE towards more a c i d i c conditions (pH 4 . 0 - 4 . 2 ) ; down to  therefore,  4.0-4.2.  polyphosphate  In  the  range for  lower and upper l i m i t of pH was narrowed  a s i m i l a r manner,  concentrations  (1.2-1.4  higher  SE could be expected at  mg/mL);  therefore,  the  higher  range  of  concentration of SHMP was r e s t r i c t e d to 1.3-1.4 mg/mL. With the new lower and upper l i m i t s for the pH and SHMP, the CMO program was applied. was  After  created.  reported.  entering The  Upon  the new ranges, a new i n i t i a l  experiments  improvement  were in  the  carried  out  response  and  simplex the  values,  implemented a f t e r six experiments in the centroid search.  (three  vertices)  response values were  simultaneous  shift  was  Figures 4A and 4B show  gure 4. Approximate response surface patterns for (A) pH and (B) SHMP concentration obtained by mapping accumulated data from simplex optimization (Vertices 1-9) and centroid optimization (Vertices 10-15). T target values of pH and SHMP.  48  the approximate response surface for both factors from which, based on the present best values of 4.07 and 1.33,  the target values (T)  pH and SHMP,  However,  respectively.  because of  of 4.03 the  and 1.36  failure  were set for  to achieve  further  improvement in response values, further experimentation was discontinued. Examining the best response value (Vertex 15 Figures 4A and 4B), i t was found that pH 4.07  and 1.33 mg SHMP/mL y i e l d e d about 80% elimination  from Cheddar cheese whey (into the p r e c i p i t a t e ) , immunoglobulins in the supernatant.  with almost complete recovery of  The majority  of a-lactalbumin  the supernatant as indicated by SDS-PAGE (Figure 5). serum albumin was p r e c i p i t a t e d amount  remaining  evident outer  in  the  along with the  supernatant.  line  Recovery of  corresponding  p r e c i p i t a t e s showed no such p r e c i p i t a t i n g the  sample  application  well)  might  to  precipitating application  lines, well.  a-lactalbumin weight  was  The rather  weak  and consequently lower  (not  DIMENSIONAL  toward  anti-bovine shown),  OF  supernatant  IgG  lactoferrin,  the  whey  probably  antigenicity.  ILLUSTRATION  the  standard  arc;  EFFECTS  inner and  anode,  protein to  Approximately (not  was  showed a long, while  transferrin  due  supernatant were determined to be IgG by means of R.I.D.  B. THREE  in  indicated by the  formed  response of  Igs  The inner p r e c i p i t a t i n g  represent  respectively,  in  However, most of the bovine'  The supernatants  the  line.  Serum albumin and 8 - l a c t o g l o b u l i n arcs are  was found  B-lactoglobul in with only a small  immunochemically as shown in Figure 6. precipitating  of B-lactoglobulin  far  lines  (by IgM.  and the  outer  from  antiserum its  the  the  toward  low molecular  90 % of  Igs  in  the  shown).  OF pH AND HEXAMETAPHOSPHATE ON  SEPARATION EFFICIENCY Contour and 3-dimensional visualization  of the  Cheddar cheese whey.  surface plots were generated by computer to aid in  relationship  between  pH, SHMP and SE of  Figures 7A and 7B showed t h a t ,  immunoglobulins  in  in general, combinations of  49  CCW  p  s = BSA m IgG-HC OVA * IgG-LC  +  m  B-Lg  4ft<*-La  Figure 5. SDS-PAGE of supernatant (S) and p r e c i p i t a t e (P) obtained a f t e r treatment with 1.33 mg/mL SHMP at pH 4.07. CCW, Cheddar cheese whey; a - L a , a-lactalbumin; B-Lg, B - l a c t o g l o b u l i n ; IgG-HC, immunoglobulin G heavy chain; IgG-LC, immunoglobulin G l i g h t chain; BSA, bovine serum albumin; OVA, ovalbumin.  50  s CCW p IgG 8-L9  abwp abwp abwp abwp abwp abwp  Figure 6. Immunoelectrophoretic pattern of Cheddar cheese whey. S, supernatant; CCW, Cheddar cheese whey; P, p r e c i p i t a t e ; IgG, immunoglobulin G; B-Lg! 8 - l a c t o g l o b u l i n ; abwp, antibovine whey p r o t e i n s .  51  (A)  Figure 7. Contour (A) and 3-dimensional (B) surface plots of relationship between pH, SHMP and Separation effeciency (SE) of cheese whey treatment, ("about" angle=60 and "above" angle=35 for 3-dimensional p l o t ) .  52  53  low  pH  and  high  SHMP  concentration  Separation  efficiency  increasing  SHMP concentration  isoelectric interact  point  was  improved  the  polyphosphates  act  negative as  interact d i f f e r e n t l y  in  good  separation  by decreasing pH values  above  of whey p r o t e i n s ,  with  resulted  below  4.25  and by  1.2  mg/mL.  By lowering  the  positive  side chain amino groups could  groups  cross-linking  surrounding agents.  phosphate  However,  the  efficiency.  molecules  different  v i a polyphosphates to form aggregates.  pH below  by  the  which  proteins  may  Surface exposed amino  groups and unfolding or expansion of protein molecules when the polyphosphate is bound may play  an  important  role  in  that  interaction,  leading  to  preferential  p r e c i p i t a t i o n of p - l a c t o g l o b u l i n s (Melachouris, 1972). The contour p l o t  (Figure  7A)  shows three humps with SE of 0.508, 0.577 and  0.302, which can be seen also in Figure 7B.  Multiple peaks might have been caused  by the absence of data points between SE 0.508 and the other two peaks with SE's of 0.302 and 0.577; closer intervals  in other words, more experiments  under  the  conditions with  between pH 4.2 and 4.08 might be required in order to obtained a  smoother s u r f a c e .  C. ELIMINATION OF PHOSPHORUS The phosphorus d i s t r i b u t i o n in  Table  5.  supernatant  Removal and the  of  72.27. and  precipitate,  d i s t i l l e d water for 48 hr. by d i a l y s i s might  in the supernatant and the p r e c i p i t a t e 45.3%  of  the  total  are shown  phosphorus  from  r e s p e c t i v e l y , was achieved by d i a l y s i s  the  against  F a c i l e removal of polyphosphate from whey preparations  indicate weak binding of phosphorus with whey p r o t e i n s .  Since  no p r e c i p i t a t i o n of whey proteins by SHMP was observed at pH values higher than 5, i t was assumed that i o n i c interaction might be involved in the interaction SHMP and p r o t e i n s . charged groups  At  (basic  pH values  amino acid  lower  than  residues)  in  the  isoelectric  protein  point,  between  positively  molecules might  interact  Table 5. Phosphorus d i s t r i b u t i o n precipitate  in  supernatant  obtained by SHMP treatment.  and P,  phosphorus.  Fraction  mg P/100 mL  mg P/100 mL after  % removed by d i a l y s i s  dialysis Supernatant  65.0  Precipitate Cheese whey  16.0 50.0  18.1 8.75 8.6  72.2 45.3 82.8  55  with each other v i a SHMP and cause the aggregation of the p r o t e i n s . the  pH above  interaction dialysis  the  isoelectric  might  thereby  content  (Friend et a l . ,  and  be disrupted and the  process,  phosphorus  point  free  separating  within  the  increasing  a  level  net  By increasing  negative  charges,  this  SHMP could be removed by a simple  whey  protein  recommended  for  fraction infant  containing  formulas  a  (0.033%)  1983).  D. PROPOSAL OF NEW INFANT FORMULA Table 6 compares the composition of the new infant and cow's milk  and  the  commercial SMA formula, has  been  changed to  However,  simple  compositional  current the  commercial  ratio  of  In  to  found  order  adjustment  of  casein/whey  contents of a-lactalbumin,  formula.  of cow's milk  in  between  SMA (whey-based)  casein/whey proteins  40/60  differences  formula to that of human  cow  lactoferrin,  mimic  the  protein  and  human  ratio  in  ratio  does  not  milk  proteins,  the  (79/21)  human  milk.  minimize i.e.  the  higher  immunoglobulins and lysozyme in human milk  as compared to cow's m i l k . By eliminating the whey protein Table closer  6.  In  B-Lg completely with f u l l  composition of  this  the  their  quantities  to that  separated  from  egg white  can  proposed by Friend et infant  formula.  immunoglobulins benefit to infant  al.  Thus, when  new B-Lg-free  proposed formula,  in  be  (1983), the  incorporated  feeding.  retention  of other whey proteins,  formula would be as shown in  immunoglobulins and  found in human m i l k . incorporated in order  to  B-Lg-reduced into  infant  in  this  lactoferrin In  new  are much  addition, infant  lysozyme  formula  as  improve the  therapeutic  value  of  supernatant  which  rich  in  formulae,  may  be  is an  additional  56  Table 6. Protein  composition  of  human  and  proposed p- l a c t o g l o b u l i n (P-Lg)-free  Human  Cow  Total %  Total %  3  Protein Total  100  100  cow's  milks  infant  formula.  Whey-based  P-Lg free  formula Total %  Total %  100 40. OC  Caseins  35.0*  79.0  Total whey  65.0  21.0  3  2.8  b  8.0  11.2  b  32.0  a-lactalbumin p-lactoglobulin  —  immunoglobul ins  11.0  2.3  6.0  3  17.0  3  lysozyme  6.0  3  others  8.0  3  lactoferrins  b  c  d  3  3  serum albumin  3  17.0  3  3  and whey-based  60.0  C  formula  100 40.0 60.0 17.2  d  —  14.l  d  1.8  6.6 5.id  10.9  d  1.7b  4.9  d  10.5  d  3.4  d  d  —  1.2  b  —  —  Gurr, 1981 Calculated from electrophoretic scanning of Cheddar cheese whey Friend et a l . , 1983 Whey protein composition i s calculated based on our data (b)  7.3d  and  57  PART  II  SEPARATION OF BOVINE IMMUNOGLOBULINS AND LACTOFERRIN FROM WHEY PROTEINS BY GEL FILTRATION TECHNIQUES  58  Although  benefits  expected  immunoglobulins compared to 1975; B a l l a b r i g a , 1982; justified  by  feeding  feeding  infants  hyperimmunized  Ebina et a l . ,  1984,  1985)  Ig  with  (Hilpert  non-immunized et  al.,  1974/  are unknown, some uses may be  in c o l l a b o r a t i o n with other antimicrobial  components in cow's milk as  discussed by Packard (1982).  L a c t o f e r r i n together with secretory immunoglobulin  A from  considerable b a c t e r i o s t a t i c  human milk  enteropathogenic 1980).  showed a  s t r a i n s of E^ c o l i  effect  (Dolby and Stephens 1983;  against  human  Stephens et  al.,  In t h i s part of the t h e s i s , gel f i l t r a t i o n techniques were assessed for  immunoglobulins and l a c t o f e r r i n  fractionation.  A. GEL FILTRATION ON SEPHACRYL S-300 Gel proteins range  filtration  is  the method of  (Van Oss, 1982-1983).  of  1X10 ). 6  molecular Gel  filtration  porous and r i g i d therefore,  sizes  gel  Sephacryl  immunoglobulin  choice f o r  preparative  isolation  of  Bovine colostrum contains proteins with a wide  from a-lactalbumin of  the  (MW c a .  high molecular weight  14,500)  proteins  to  IgM  (MW ca.  requires  a  highly times,  to provide a good s t a b i l i t y  and short  separation  S-300  experiment.  The  yield  90%  (Van Oss,  isolated  by  was  chosen  for  gel  filtration  this can  be  as  high  as  of  1982-1983). Figures colostral  8  and  9  whey and the  show the  separation  ammonium s u l f a t e  patterns  of  treated whey,  the  untreated  respectively.  bovine Results  indicated that two major peaks were obtained in the e l u t i o n p r o f i l e of treated whey (Figure 9) which were also in the e l u t i o n p r o f i l e of untreated whey 8).  The f i r s t  fraction  (Fl)  appeared  in the void volume of  indicated that the molecular weight was in the v i c i n i t y second peak indicated a lower molecular weight p r o t e i n . untreated  bovine c o l o s t r a l whey and the ammonium s u l f a t e  (Figure  the column which  of a m i l l i o n ,  and the  Fraction 3 (F3)  of both  treated whey contained  59  ELUTION VOLUME, ml  Figure 8. Gel f i l t r a t i o n of bovine c o l o s t r a l whey on Sephacryl S-300 Superfine column (94 x 2.5 cm) eluted with 0.1 M T r i s - H C l buffer pH 8.0 containing 0.5 M NaCl. Flow r a t e , 12 mL/hr. 1 and 3 are f r a c t i o n s 1 and 3, respectively.  60  Figure 9. Gel f i l t r a t i o n of crude Ig obtained from ammonium sulfate treatment on Sephacryl S-300 Superfine column (94 x 2.5 cm), eluted with 0.1 M T r i s - H C l buffer pH 8.0 containing 0.5 M NaCl, flow rate 12 mL/hr. 1 and 3 are f r a c t i o n s 1 and 3, r e s p e c t i v e l y .  61 99% IgG when analyzed by radial  immunodiffusion  (Table  7)  and double sandwich  ELISA. Figure  10  shows  the  electrophoretic  f r a c t i o n s obtained by gel f i l t r a t i o n observed  that  F3,  containing IgG.  which  patterns  fractionation  represented  IgG,  colostral  (Figure 8:  was  a  However, the less pure F l f r a c t i o n  which moved the same distance as l a c t o f e r r i n .  of  highly  whey  F l , F3).  and  It  purified  was  fraction  contained some contaminants  L a c t o f e r r i n may have bound firmly  to the high molecular weight compound (IgM)  through hydrogen-bonding since both  proteins are g l y c o p r o t e i n s . Immunoelectrophoresis identification because  of  and its  Interpretation  is  of  the  characterization sensitivity  of  one  and  of  most  important  immunoglobulins  specificity  immunoelectrophoretic  (Ohtani  patterns  and the potency of the a n t i - s e r a employed.  techniques and  for  their  and  classes,  Kawai,  depends upon the  the  1981).  specificity  Immunoelecrophoretically,  F l and F3  showed a single p r e c i p i t i n arc against anti-whole bovine antiserum, a t t e s t i n g the p u r i t y of these f r a c t i o n s it  (Figure  11).  represents a high molecular weight  distance may represent  the  Since F l d i f f u s e d a short distance,  (IgM)  while  lower molecular weight  antibody or antigen i s inversely proportional al.,  to  F3 which d i f f u s e d IgG.  a  The d i f f u s i o n  to the molecular weight  longer rate of  (Atassi et  1984).  B. GEL FILTRATION ON TSK HW-55 Figure  12 i s an e l u t i o n  pattern  of  c o l o s t r a l whey on a TSK HW-55 column  which i s b a s i c a l l y s i m i l a r to the e l u t i o n p r o f i l e of c o l o s t r a l whey on Sephacryl S-300.  SDS-PAGE  immunoglobulins. Sephacryl  S-300  indicated  that  the  major  portion  of  Fl  and  F2  were  However, F l and F2 were not as pure as F l and F3 obtained from (Figure  13).  Immunoelectrophoresis  clearly  showed that  the  Table 7.  Immunoglobulin  G  contents*  of  fractions  obtained  from  gel  f i l t r a t i o n on Sephacryl S-300 and crude Ig prepared by ammonium s u l f a t e treatment.  Protein content**  Sample  (mg/mL)  IgG content  Purity  (mg/mL)  %  F3 (Figure 8)  10  9.90  99  F3 (Figure 9)  10  9.90  99  Crude Ig  10  5.10  51  7.2  6.00  83.3  7.6  7.00  92.1  AW-fraction 2 (Figure  15)  CCW-fraction 2 (Figure  *  Determined by radial  16)  immunodiffusion a n a l y s i s .  * * Determined by BioRad reagent.  63  F1  F3  Ig  LF  CW  Figure 10. SDS-PAGE of f r a c t i o n s obtained from gel f i l t r a t i o n on Sephacryl S-300. F l , f r a c t i o n 1; F3, f r a c t i o n 3 (Figure 8 ) ; Ig, crude immunoglobulin; LF, l a c t o f e r r i n ; CW, untreated c o l o s t r a l whey; HC, LC, immunoglobulin heavy and l i g h t chains, r e s p e c t i v e l y .  64  F3 F3  a  F1 F1 BSA TF  Figure 11. Immunoelectrophoretic analysis against anti-whole bovine antiserum of f r a c t i o n s obtained from Figure 8. F3, f r a c t i o n 3; F l , f r a c t i o n 1; ; BSA, bovine serum albumin; TF, t r a n s f e r r i n .  65  0  120 240 ELUTION VOLUME, ml  Figure 12. Gel f i l t r a t i o n pattern of c o l o s t r a l whey on TSK column (40 x 2.6 cm) eluted with 0.07 M imidazole-0.05 M KC1 b u f f e r , pH 6.5; flow rate, 50 mL/hr. 1 and 2 are f r a c t i o n s 1 and 2, r e s p e c t i v e l y .  66  F1  F2  1  2 3  4  M AW  CW  -  n  * m  W  HC LC  Figure 13. SDS-PAGE of f r a c t i o n s (Fl and F2) obtained from Figure 12 as compared to standards. Lane 1, a-lactalbumin; Lane 2, B - l a c t o g l o b u l i n ; Lane 3, bovine serum albumin; Lane 4, t r a n s f e r r i n ; M, standard mixture; AW, a c i d whey, CW, c o l o s t r a l whey; HC and LC, immunoglobulin heavy and l i g h t chain, respectively.  67  f r a c t i o n s obtained by TSK column contained some i m p u r i t i e s , mainly bovine serum albumin, not found in the f r a c t i o n s  obtained by Sephacryl S-300 column  (Figure  14). By comparing f r a c t i o n s obtained from gel f i l t r a t i o n on Sephacryl S-300 ( F l , F3) with those obtained on TSK HW 55 contains  immunoglobulins of  (Figure 14).  higher  (Fl,  F2),  it  i s obvious that the  purity  than  those  former  obtained from TSK HW 55  Therefore, the Sephacryl S-300 type column was chosen to  isolate  Ig from a c i d and cheese whey.  C. ISOLATION OF IMMUNOGLOBULINS FROM WHEY PROTEINS Figure 15 shows the e l u t i o n  profile  of acid whey on Sephacryl S-300 while  Figure 16 i s the e l u t i o n p r o f i l e of Cheddar cheese whey on the same column. first  fraction  (Fl)  which  eluted  containing high molecular weight acid whey  than  lipoprotein fraction  3,  that  fraction  for  the  at  void  components.  cheese whey.  in both wheys.  contained  the  immunoglobulins  whey  contained  albumin (Figure  mainly  turbid  The F l f r a c t i o n The F l f r a c t i o n  from  acid  whey  was sharper  for  may represent  the  (F2), and  solution  a shoulder of cheese  whey  as  The immunoglobulin f r a c t i o n contained  some impurities which were probably l a c t o f e r r i n Immunoelectrophoresis  was a  The second f r a c t i o n  indicated by SDS-PAGE (Figure 17 and 18).  7).  volume,  The  and bovine serum albumin  (Table  showed that f r a c t i o n F2 of both acid whey and cheese  immunoglobulins  with  smaller  amounts  of  bovine  serum  19).  D. ISOLATION OF LACTOFERRIN FROM WHEY PROTEINS Figure 20 shows the e l u t i o n p r o f i l e of Cheddar cheese whey when eluted from a haparin-Sepharose column with a l i n e a r NaCl gradient. tively  adsorbed to  the  L a c t o f e r r i n was s e l e c -  column from cheese whey and was eluted at  about 0.5 M  68  Figure 14. Immunoelectrophoresis of f r a c t i o n s obtained by Sephacryl S-300 and Fractogel TSK column against anti-whole bovine serum antiserum. (Fl-S and F3-S, f r a c t i o n 1 and 3 of Figure 8, respectively) ( F l - T and F2-T f r a c t i o n 1 and 2 of Figure 12, respectively) IgG, immunoglobulin G.  69  Figure 15. Gel filtration of acid whey on Sephacryl S-300 Superfine column (94 x 2.5 cm), eluted with 0.1 M T r i s - H C l b u f f e r , pH 8.0 containing 0.5 M NaCl. Flow rate 18 mL/hr. 1, 2, 3 and 4 are the f r a c t i o n s obtained.  70  ELUTION VOLUME, ml  Figure 16. Gel f i l t r a t i o n of Cheddar cheese whey on Sepharyl S-300 Superfine column (94 x 2.5 cm), eluted with 0.1 M T r i s - H C l b u f f e r , pH 8.0 containing 0.5 M NaCl. Flow rate 18 mL/hr. 1, 2, 3 and 4 are the f r a c t i o n s obtained.  71  IgG AW  4  3  2  1  AW  Figure 17. SDS-PAGE of f r a c t i o n s (1, 2, 3, 4) obtained from Figure 15. AW, acid whey; IgG, immunoglobulin G; HC and LC, heavy and l i g h t chains of immunoglobulins, r e s p e c t i v e l y .  72  Figure  18. SDS-PAGE of f r a c t i o n (1, 2, 3, 4) obtained from Figure 16. IgG, immunoglobulin G; CCW, Cheddar cheese whey; HC and LC, heavy and l i g h t chains of immunoglobulins, r e s p e c t i v e l y .  73.  Figure 19. Immunoelectrophoresis of f r a c t i o n s obtained from gel f i l t r a t i o n of whey protein against anti-whey protein antiserum. AW, acid whey; Fl-H f r a c t i o n 1 from Figure 20; F2-A, f r a c t i o n 2 from Figure 15; F2-C, f r a c t i o n 2 from Figure 16; IgG, immunoglobulin G.  74  ELUTION VOLUME, ml  Figure 20. Heparin-Sepharose chromatography of Cheddar cheese whey. Cheese whey (400 mL) dialyzed against 0.05 M NaCl in 5 mM veronal-HCl, pH 7.4 was applied to the column (10 mL s e t t l e d g e l ) . The column was washed with the same buffer and then eluted with a l i n e a r gradient of NaCl ( ) as indicated. The flow rate was 50 mL/hr. UB, unbound p r o t e i n s ; 1, fraction  75  NaCl  isocratically.  The i s o l a t e d  with Immunoelectrophoresis fraction.  SDS-PAGE  p u r i t y of t h i s  lactoferrin  (Figure 19),  (Figure  21)  gave only one p r e c i p i t a n t  which indicated the high purity of  also y i e l d e d  a  single  band,  line this  confirming  the  fraction.  E. ANTI-LIPOPOLYSACCHARIDES ACTIVITY OF ISOLATED IMMUNOGLOBULINS Figure  22  shows the  anti-1ipopolysaccharide  i s o l a t e d from colostrum using a gel were  extracted  from  the  pathogenic  Bordetella parapertussis and t h e i r an enzyme  linked  filtration  technique.  bacteria,  E^  of  immunoglobulins  Lipopolysaccharides  col i ,  S_;_ typhimurium  and  binding with i s o l a t e d Ig was measured using  immunosorbent assay (ELISA).  have binding a c t i v i t y  activity  The i s o l a t e d  IgG was shown to  against LPS i s o l a t e d from E^ c o l i and to a l e s s e r extent  against S_j. typhimurium.  Higher recognition of c o l o s t r a l  Ig to LPS from E± c o l i  as compared LPS from S_j. typhimurium may indicate that the dairy cow was infected more often activity  by E^ c o l i  and  than by S. typhimurium.  recognition  of  LPS i s o l a t e d  S u r p r i s i n g l y i s o l a t e d Ig showed  from EL parapertussis  whooping cough in i n f a n t s ,  and which may indicate the presence of  in the  of  antigenic  structure  LPS between  this  which causes similarities  bacterium and those  extracted  from E_j_ col i . The  strain  in infants enterotoxin therefore, diarrhea  of  coli  used  in  this  by producing one or both of mainly  affects  fluid  study  is  two classes  transport  known  to  cause  diarrhea  of enterotoxins.  processes of  the  small  E^ col 1 intestine,  any changes in either or both absorption and secretion may result (Holmgren,  1985).  Holmgren  (1985) discussed possible approaches  in for  the prevention of and treatment of E_j. c o l i pathogenic a c t i o n .  One approach was  to use receptor blockade by using a non-toxic binding agent.  This approach was  to prevent binding of toxin to the epithelium.  Therefore, high binding  activity  76  CCW UB  F1 CCW LF  Figure 21. SDS-PAGE of f r a c t i o n s obtained from Figure 20, CCW, Cheddar cheese whey; UB; unbound whey proteins to Heparin-Sepharose column; Fl, lactoferrin rich fraction; LF, lactoferrin.  77  2.0r  Pg  Ig  Figure 22. Anti-1ipopolysaccharide a c t i v i t y of c o l o s t r a l IgG i s o l a t e d by gel f i l t r a t i o n on Sephacryl S-300. m-m , E i c o l i LPS; D-O , S i typhimurium LPS; o - 0 > l i parapertussis LPS.  78  of i s o l a t e d Ig toward LPS extracted of b a c t e r i a l  toxin to the epithelium thus preventing diarrheal  i s used for infant  for  the  lactoferrin  binding fraction  2)  of  immunoglobulins and  isolated  from cheese whey as an  can be incorporated into  activity to  isolation  that Cheddar cheese whey could be an lactoferrin.  important  Importance  as a b a c t e r i o s t a t i c agent has been well established ( R e i t e r ,  Immunoglobulins (Fraction  diseases when Ig  feeding.  These studies demonstrate source  from g± c o l i may i n t e r f e r e with the binding  of  formulae.  immunoglobulins may give  commercial  gastrointestinal  infant  infant  infection.  formulae  in  immunoglobulin  a  clear  order  to  rich  of  1983).  fraction  The 1 ipopolysaccharide evidence protect  to  add  infants  this from  SEPARATION OF IMMUNOGLOBULINS AND LACTOFERRIN FROM CHEESE WHEY BY ADSORPTION AND CHELATING CHROMATOGRAPHY TECHNIQUES  80  When  considering  difficulties  in  the  treatment  mechanization  of  of  gel  a  large  amount  "filtration  renders a large  and more d i f f i c u l t .  adsorption and metal  methods were  investigated  to  extract  these  cheese whey,  techniques  immunoglobulins and l a c t o f e r r i n Therefore,  of  scale operation  proteins  chelating  for  the  extracting  less  feasible  chromatographic  by an easy and  efficient  immunoglobulins and other  bioactive  method.  A. ADSORPTION CHROMATOGRAPHY METHODS In  searching for a method to  isolate  compounds from whey p r o t e i n , the economic d r i v e , ease to mechanize and capacity of the method were used as c r i t e r i a . Figure  23  shows  the  elution  profiles  of  adsorbed  proteins  from  the  chromatographic treatment of 1 l i t r e of Cheddar cheese whey (adjusted to pH 8.2) on s i l i c a ,  c o n t r o l l e d pore glass and alumina, eluted with a c e t i c acid solution  followed by T r i s - H C l  buffer.  The amount of protein small, (Figure  as 24)  lactoferrin too  small  (i.e.,  indicated shows  by  the  low A28O  that  the  acetic  (Lane 2 ) . to  in these f r a c t i o n s values  acid  from the s i l i c a column was very and small  eluted  peak areas.  fraction  contained  primarily  The amount of protein in the T r i s - H C l eluted f r a c t i o n was  be i d e n t i f i e d  unadsorbed protein  (lanes  5 and 12).  fraction,  lane 8)  The s i l i c a - t r e a t e d showed l i t t l e  were obtained when the chromatography was c a r r i e d out at  cheese whey  difference  e l e c t r o p h o r e t i c pattern of untreated cheese whey (lane 1 & 11).  instead of  SDS-PAGE  from  the  S i m i l a r results  the s t a r t i n g  pH of  7.5  8.2.  As shown by SDS-PAGE p r o f i l e s  (Figure  24),  the  large A28O P  a c e t i c acid from c o n t r o l l e d pore glass contained mainly serum albumin  (BSA)  (lane  3),  while  the  peak  eluted  mixture of proteins including l a c t o f e r r i n , BSA and Ig  e a k  lactoferrin  by T r i s - H C l (lane  6).  eluted by and bovine  contained a  81  Figure 23. E l u t i o n p r o f i l e s of adsorbed proteins from s i l i c a (S) (close to the b a s e l i n e ) , c o n t r o l l e d pore glass (C) and alumina (A) chromatographic treatment of Cheddar cheese whey. One l i t r e of Cheddar cheese whey in 0.005 M Na2HP04, pH 8.2 was passed through 1.3 x 7.0 cm column of (S), (C) or (A) e q u i l i b r a t e d with 0.005 M phosphate buffer at pH 8.2. After washing with 30 mL of e q u i l i b r a t i n g b u f f e r , the adsorbed proteins were eluted with E l (50 mL 0.1 M a c e t i c acid pH 2.77 containing 0.5 M NaCl), then E2 (60 mL 0.1 M T r i s - H C l pH 9.0 containing 0.5 M NaCl). The flow rate was 1 mL/min.  82  1 2 3 4  5  6  7 8 9 10 11 12  Figure 24. SDS-PAGE p r o f i l e s of cheese whey and f r a c t i o n s obtained from Figure 23. Lane 1, untreated Cheddar cheese whey; Lane 2, a c e t i c acid f r a c t i o n from s i l i c a sand; Lane 3, a c e t i c acid f r a c t i o n from c o n t r o l l e d pore g l a s s ; Lane 4, a c e t i c acid f r a c t i o n from alumina; Lane 5, T r i s - H C l f r a c t i o n from s i l i c a sand; Lane 6, T r i s - H C l f r a c t i o n from c o n t r o l l e d pore g l a s s ; Lane 7 , T r i s - H C l f r a c t i o n from alumina; Lane 8, unbound f r a c t i o n from s i l i c a ; Lane 9, unbound fraction from controlled pore g l a s s ; Lane 10, unbound fraction from alumina; Lane 11, untreated Cheddar cheese whey; Lane 12, acetic acid f r a c t i o n from alumina, L F , l a c t o f e r r i n ; HC and LC, immunoglobulin heavy and l i g h t chains, r e s p e c t i v e l y .  83  The a c e t i c  acid e l u t i o n  of  alumina  c l o s e l y by a second larger peak.  column y i e l d e d a small  The larger  peak  peak contained mainly  followed  lactoferrin  and BSA according to SDS-PAGE (Figure 24 lane 4 ) .  T r i s - H C l elution yielded only  a very  contained mainly  broad peak with  (Figure 24,  lane 7).  to be t u r b i d ,  low A280;  t  n  i  s  fraction  lactoferrin  The T r i s - H C l eluant emerging from the column was observed  starting  at  the 80 mL e l u t i o n  volume, which may be due to  lipid  f r a c t i o n s eluted from the alumina. Of  the  adsorption  chromatographic  support  materials  c o n t r o l l e d pore glass appeared to be the most promising f o r However,  of  Ig.  reducing the amount of cheese whey sample to 250 mL and analyzing  the  unbound f r a c t i o n to  investigated,  indicated that appreciable quantities  the CPG column.  of  isolation  Ig were not adsorbed  This suggested that the capacity of CPG for  was not very high.  Increasing  the CPG bed volume from  recovery somewhat, but the capacity was s t i l l  10  to  Ig  adsorption  20 mL improved  insufficient for e f f i c i e n t  removal  of Ig from whey.  B. METAL CHELATE INTERACTION CHROMATOGRAPHY 1. Acid whey Acid whey column  (obtained  containing  from a c i d i f i c a t i o n  copper  ions  immobilized  unbound proteins with the s t a r t i n g eluted using a l i n e a r gradient.  alkaline  from MCIC treatment  of  1 litre  detected  by monitoring  of  the  first (Figure  peak 26  fraction;  were  yellowish,  lane  1)  however,  while  indicated it  raw skimmilk)  on  Sepharose  buffer,  was applied to a  6B.  After  washing  the adsorbed proteins were  Figure 25 shows the e l u t i o n p r o f i l e of adsorbed  proteins  A28O  of  that  of  acid whey.  effluent.  the the  The  second peak first  peak  Two major  fractions  peaks were  comprising  was  colorless.  was  the  the  SDS-PAGE  lactoferrin  rich  also contained other proteins which were adsorbed to the  84  Figure 25. Elution p r o f i l e s of adsorbed proteins from MCIC on Sepharose 6B treatment of 1 L Cheddar cheese whey (CCW) and 1 L acid whey (AW) (obtained from raw m i l k ) , using l i n e a r gradient e l u t i o n of 0.05 M T r i s - a c e t a t e containing 0.5 M NaCl, pH 8.0 to 2.8. Flow rate was 0.8 mL/min. CCW, Cheddar cheese whey; AW, acid whey.  85  Figure 26. SDS-PAGE p r o f i l e s of acid whey and f r a c t i o n s obtained by MCIC and gel f i l t r a t i o n . Lanes 1 and 2, are f r a c t i o n s obtained by MCIC; Lane 3, unbound material to MCIC column; Lanes 4 and 5, peak 1 and 2 obtained by gel f i l t r a t i o n on Sephacryl column; Lane 6, mixture of standard proteins ( t r a n s f e r r i n , bovine serum albumin, B-lactoglobulins and a-lactalbumin); Lane 7, acid whey. LF, l a c t o f e r r i n ; HC and LC, heavy and l i g h t chains of immunoglobulins, r e s p e c t i v e l y .  86  column and eluted  in the  same f r a c t i o n ;  these proteins were Ig  second smaller peak was mainly composed of (lane 3)  also  indicated  that the  Ig  (Figure  unbound f r a c t i o n  26  and BSA.  lane 2).  The  Figure 26  contained no Ig  but mainly  a-La and B-Lg. Immunochemical  of active  IgG  ( i . e . , over 77%), was present in the second pooled peak, over 20% of active  IgG  was  present  in  analyses (Table  the  first  pooled  showed that  8)  peak  and  was  the majority  undetectable  in  the  unbound  fractions. Table 9 shows the d i s t r i b u t i o n of whey proteins in comparison to the pooled fractions  obtained  by the  scanning of SDS-PAGE. in  peak  one  (Fl)  the  as  calculated  from  (48%  and  20%,  fraction.  respectively)  while  bovine  proteins  densitometric  in t h i s  serum  albumin  This f r a c t i o n was almost free from a-La and  Immunoglobulins were the major proteins in peak 2,  total  the  L a c t o f e r r i n and immunoglobulins were predominant proteins  represented 27% of t h i s P-Lg.  MCIC process  fraction.  representing 88.4%  Immunoglobulins and l a c t o f e r r i n  of  in both  f r a c t i o n s represented more than 75% of the total p r o t e i n s . Immunoelectrophoresis conducted against anti-bovine whole serum antibodies (Figure  27)  also  immunoglobulins  in  indicated bovine  the milk)  presence as  well  of  IgG  (the  predominant  as  the  presence  of  type  BSA in  of both  fractions.  2. Cheddar cheese whey Figure 25 shows an e l u t i o n p r o f i l e of adsorbed proteins from MCIC treatment of  1 L Cheddar cheese whey at pH 8.2.  from  acid  effluent.  whey.  Two major  The f r a c t i o n s  A s i m i l a r e l u t i o n pattern  peaks were  comprising the  detected first  by monitoring  peak were  while the f r a c t i o n s from the second peak were c l e a r .  was obtained A28O  slightly  o  f  the  yellowish,  Table 8.  IgG a c t i v i t y  of peak and unbound f r a c t i o n s from MCIC treatment  of whey.  Concentration mg/mL IgG  protein  IgG purity  Acid whey unbound  ND  80.3  ND  1st peak  5.0  20.3  24.6  2nd peak  18.0  23.4  77.2  ND  79.3  Cheddar cheese whey unbound 1st peak 2nd peak  1.25  ND  51.4  2.4  40.0  75.4  53.0  ND  65.2  ED whey powder unbound  ND  1st peak  5.00  31.3  16.0  2nd peak  20.00  70.7  28.3  ND not detectable a  IgG a c t i v i t y of dialyzed f r a c t i o n s was determined by RID  D  Protein concentration was determined by Kjeldahl N x 6.38  88  Table 9.  Whey proteins d i s t r i b u t i o n * in acid whey, f r a c t i o n s obtained from MCIC of bovine acid whey, and the unbound materials to MCIC column.  Protein components  %  Acid whey  Fl  F2  Unbound  %  %  %  Materials %  a-lactalbumin  13.9  B-lactoglobulin  56.9  4.8  2.1  Immunoglobulins  11.4  20.1  88.4  —  9.2 8.6  27.1  —  9.5  3.3  Bovine serum albumin Lactoferrin  *  27.9  48.0  Calculated from peak area of the electrophoretic  patterns.  67.8  —  89  Figure 27. Immunoelectrophoretic analysis of f r a c t i o n s obtained by MCIC of acid whey. IgG, immunoglobulin G; P2 and PI are Peak 2 and 1, respectively of Figure 25; T F , t r a n s f e r r i n ; BSA, bovine serum albumin.  90  SDS-PAGE (Figure lactoferrin  28)  indicated that the  first  large  and bovine serum albumin (lanes 5 and 7)  peak was composed of  while  the  peak was mainly composed of immunoglobulins (lanes 6 and 8 ) .  Some B-Lg and a-La  were also adsorbed on C u - i m m o b i l i z e d Sepharose, as indicated 2+  f r a c t i o n at in  the  the early  unbound  However, a f t e r and 4)  in the unbound  stage of whey a p p l i c a t i o n ; no whey proteins were present  fraction  after  120  mL of  whey  had  been  applied  (lane  i n d i c a t i n g that the column had lower a f f i n i t y  for these components than  The absence of p-Lg and a-La in the eluted peaks (lanes 5  can be explained by the fact that they were weakly bound and were  displaced  2).  460 mL of applied whey, p-Lg and a-La were not adsorbed (lane 3  for L F , BSA and IgG. to 8)  second smaller  by L F , BSA and IgG during  later  either  stages of whey a p p l i c a t i o n or were  eluted during the washing stage p r i o r to the e l u t i o n of adsorbed p r o t e i n s . Immunochemical  analysis  (Table  8)  showed that  the majority  was present in the second peak with very minor a c t i v i t y undetectable amount peak (Ig  in the  rich fraction)  unbound f r a c t i o n s .  of active  in the f i r s t  The IgG a c t i v i t y  IgG  peak and  of the second  of Cheddar cheese whey was lower than that of acid whey  obtained from raw skimmilk.  This may be due to pasteurization ( 7 3 ° C , 15 sec) of  milk used f o r cheese manufacturing.  3. E l e c t r o d i a l y z e d and sweet whey powders a . IgG a c t i v i t y and protein content IgG a c t i v i t y and protein content of reconstituted e l e c t r o d i a l y z e d (ED) whey and sweet whey ( a f t e r reconstitution to 6.5% total s o l i d s solution) are compared to those of  l i q u i d cheese whey (Table  10).  S i m i l a r values for % protein were  obtained f o r l i q u i d whey and the reconstituted wheys. was much lower  for  1iquid cheese whey.  the  reconstituted  wheys  However, the IgG a c t i v i t y  (especially  the  sweet whey)  than  91  1  2  3  4  5  6  7  8  w c «i f . , f r a c t i o n s obtained by MCIC on Sepharose 6B treatment. Lane l , control cheese whey; Lanes 2, 3 and 4 are unbound f r a c t i o n ; Lanes 5 and 7, f i r s t eluted peak; Lanes 6 and 8 second P  ?™ , K i immunoglobulin, t  6  d  P  a  ;  f  L F  C  h e d d  a r  C  * respectively. l  a  c  t  o  f  e  r  r  i  h  n  e  ;  e  s  e  w  HC  h  e  y  and  a  n  d  LC  heavy  and  light  chains *.»ama  of ur  Table 10.  IgG and protein contents of reconstituted powders compared to l i q u i d cheese whey.  g IgG/lOOg p r o t e i n  3  ED and  sweet  % protein  Liquid cheese whey  4.0  0.88  Sweet whey  1.2  0.84  ED whey  2.2  0.87  a  IgG content was determined by R.I.D.  b  Protein content was determined as Kjeldahl are g protein/100  N x 6.38;  whey  0  units f o r % protein  mL for l i q u i d cheese whey, and for sweet and ED whey  powders a f t e r reconstitution to 6.5% (total s o l i d s ) s o l u t i o n .  93 b. E f f e c t of pH adjustment Figure  29  shows the  SDS-PAGE p r o f i l e  of  the  supernatant  f r a c t i o n s of fresh l i q u i d cheese whey a f t e r pH adjustment range.  and  precipitate  in the pH 4.5  -  8.5  No p r e c i p i t a t e s were observed and only small amounts of p e l l e t s were  obtained a f t e r centrifugation of the pH adjusted l i q u i d whey.  The p r e c i p i t a t e s  contained trace amounts of B-Lg and an u n i d e n t i f i e d component "C" (presumed to be a casein f r a c t i o n , from i t s p o s i t i o n on the e l e c t r o p h o r e t i c p r o f i l e ) . Figure  30  shows the  SDS-PAGE p r o f i l e  f r a c t i o n s of ED whey at pH 4.5 and 8.2. (lane 3)  of  the  supernatant  of the LF.  At pH 8.2,  immunoglobulins, some BSA,  the p r e c i p i t a t e from ED whey (lane 1)  contained some L F , BSA, B-Lg and only a small amount of It  precipitate  As i n d i c a t e d , the p r e c i p i t a t e at pH 4.5  from ED whey contained a large amount of  B-Lg, a-La and a l l  and  was found from immunochemical analyses (RID,  Igs.  Table 11)  that  the  Ig  in  the p r e c i p i t a t e s from sweet whey at both pH values were i n a c t i v e while those in the  supernatant  difference whey.  In  fractions  showed very  low  immunochemical  activity.  Little  in IgG a c t i v i t y was observed between the two pH treatments of sweet fact,  supernatant and p r e c i p i t a t e  f r a c t i o n s from sweet whey adjusted  to varying pH values from 4.5 to 8.5 showed l i t t l e e f f e c t of pH, i n d i c a t i n g that pH may not  be  the  only  factor  proteins during processing of  causing p r e c i p i t a t i o n .  Denaturation  of  the  the whey powder may be the other f a c t o r causing  precipitation. On the other hand, for ED whey the amount of total p r e c i p i t a t e as well as IgG a c t i v i t y in the p r e c i p i t a t e was dependent on the pH.  SDS-PAGE p r o f i l e s show  much greater tendency for p r e c i p i t a t i o n of Ig from ED whey at pH 4.5 to 5.5 than at higher pH.  94  Figure 29. SDS-PAGE p r o f i l e of l i q u i d cheese whey a f t e r pH adjustment and centrifugation. Lanes 1, 3, 5, 7, 9 and 11 are the p r e c i p i t a t e and Lanes 2, 4, 6, 8, 10 and 12 are the supernatant of samples treated at pH 8.5, 8.0, 7.0, 6.0, 5.0 and 4 . 5 , r e s p e c t i v e l y , L F , l a c t o f e r r i n ; BSA, bovine serum albumin; C, c a s e i n .  95  1  2  3  4  5  Figure 30. SDS-PAGE p r o f i l e s of f r a c t i o n s from ED whey a f t e r pH adjustment and centriguation. Lanes 1 and 3, are p r e c i p i t a t e at pH 8.2 and 4.5 respectively; Lanes 2 and 4, are supernatant at pH 8.2 and 4.5 r e s p e c t i v e l y ; Lane 5, e l e c t r o d i a l y z e d whey; L F , l a c t o f e r r i n , HC and LC, heavy and l i g h t chains of immunoglobulins, r e s p e c t i v e l y .  96  Table 11.  IgG  activity  precipitate  of  pH  4.5  and  pH  8.2  supernatant  (S)  and  (P) f r a c t i o n s from sweet whey and ED whey . a  Concentration, mg/mL IgG  protein  D  0  glg/lOOg protein  Sweet whey 18.3  ND  77.0  1.6  16.9  ND  70.2  1.8  12.4  ND  5.00 1.10  80.6 29.9  6.2 3.7  1.41  57.6  2.5  pH 8.2 P  ND  S  1.25  pH 4.5 P  ND  S  1.25  pH 8.2 P  ND  S pH 4.5 P S  d  d  d  d  ED whey  a  b  No data were obtained  for  d  l i q u i d cheese whey,  which  d  showed almost no  p r e c i p i t a t i o n over the entire pH 4 - 8 range. IgG a c t i v i t y of dialyzed f r a c t i o n s (10X concentrated) was determined by RID.  c  Protein concentration was determined by Kjeldahl N x 6.38.  d  ND = not detectable.  97.  The  supernatant  immunochemical  fraction  activity  detectable  IgG a c t i v i t y  However at  pH 4 . 5 ,  activity suggest  was that  found at  than  from the  due to the  activity  ED  whey  precipitated  at  pH  fraction,  low amount of total  was higher  in  the  supernatant  pH 4.5  IgG  is  in  the  showed  which  higher  showed  no  protein as well as IgG.  precipitated  fraction  preferentially  8.2  (Table  fraction  11).  precipitated  These  from the  and  less  results ED whey.  Unlike the p r e c i p i t a t e d Ig from sweet whey which was immunochemically i n a c t i v e , p r e c i p i t a t e d Ig from ED whey remained a c t i v e .  c . I s o l a t i o n of immunoglobulins by c o n t r o l l e d pore glass Figure 31 shows the e l u t i o n p r o f i l e s of adsorbed proteins from controlled pore glass (CPG) (10 mL bed volume) chromatography of 250 mL of sweet whey (SW) and EDI whey, r e s p e c t i v e l y . (0.1  N acetic  acid pH 2.8  eluted by buffer E2 (0.1 the f i r s t the  In each case, a s i n g l e peak was eluted by buffer El -  2.9  containing 0.5M NaCl)  and a s i n g l e peak was  N T r i s - H C l pH 9.0 containing 0.5 M NaCl).  The size of  peak was decreased f o r EDI and SW compared to ED2, while the size of  second peak  remained f a i r l y  constant,  despite  the  four-fold  reduction  in  volume of applied whey (250 mL in EDI and SW v s . 1 l i t r e in ED2, Figure 31). Electrophoretic a n a l y s i s of the unbound f r a c t i o n of whey from CPG indicated that appreciable quantities in the unbound f r a c t i o n CPG f o r  of Ig were not adsorbed to the CPG but were eluted  (Figure not shown).  Ig adsorption i s not very high.  This suggests that the capacity of  Although the quantity of whey applied  in these studies had already been reduced to 250 mL (EDI), compared to 1 l i t r e (ED2),  Ig  recovery was s t i l l  far  from q u a n t i t a t i v e .  Increasing  the  CPG bed  volume from 10 to 20 mL improved recovery somewhat, but the capacity was i n s u f f i c i e n t f o r quantitative  removal of Ig from whey.  still  98  Figure 31. Elution p r o f i l e of adsorbed proteins from CPG (10 mL) treatment of 250 mL of e l e c t r o d i a l y z e d whey (EDI), 250 mL of sweet whey (SW), and 1 L of ED whey (ED2). Arrows indicate s t a r t of e l u t i o n with El (0.1 N acetic acid pH 2.8 containing 0.5 M NaCl) and E2 (0.1 M T r i s - H C l , pH 9.0 containing 0.5 M NaCl) b u f f e r s .  99  d. Isolation of immunoglobulins by metal c h e l a t e - i n t e r a c t i o n chromatography Figure 32 i s an e l u t i o n p r o f i l e of adsorbed proteins from MCIC treatment 960 mL ED whey reconstituted in water (pH adjusted to 8 . 2 ) .  An elution  s i m i l a r to that from fresh l i q u i d cheese whey was obtained. peaks  were  broader  SDS-PAGE (Figure 33)  and  smaller  than  those  showed that the f i r s t  from  fresh  However,  liquid  of  profile the  cheese  two  whey.  large peak was composed of BSA, some  Ig and P-Lg while the second smaller peak was composed mainly of Igs and a trace amount of BSA (lanes 6 and 7,  respectively).  fractions  2,  adsorbed. BSA,  (Figure  33,  Even at  lanes  the early  L F , P-Lg and trace  The p r o f i l e s of the unbound whey  3 and 4)  indicated  that  some  Igs  were  stage with 120 mL of applied whey (lane 2 ) ,  amounts  of  Igs  were  suggesting that MCIC may have less a f f i n i t y  found in  the  not some  unbound fractions  for these proteins from the ED whey  than from fresh l i q u i d cheese whey. Radial MCIC were  immunodiffusion showed the p u r i t y not  as good as from fresh  of Igs  cheese whey  recovered from ED whey by  (Table  8).  The f i r s t  peak  contained about 16% IgG while the second peak was composed of about 28% IgG on a protein b a s i s .  Unbound f r a c t i o n s showed no detectable IgG a c t i v i t y , despite the  presence of Ig band in the SDS-PAGE p r o f i l e The e l u t i o n  profile  sweet whey reconstituted  of  adsorbed proteins  in s t a r t i n g  buffer  Possibly two peaks were e l u t e d , with the from the f i r s t  larger peak.  (Figure  at  33).  from MCIC treatment pH 8.2  is  720 mL  shown in Figure  Both the large and the shoulder peak were composed  The d i f f e r e n c e  between  lanes 10 and  the two peaks was that the shoulder  peak (lane 11) contained less P-Lg and more a-La than the larger peak (lane The wash f r a c t i o n s  (lanes  32.  second peak appearing as a shoulder  of Igs, BSA, L F , some p-Lg and a - L a , based on SDS-PAGE (Figure 34, 11 r e s p e c t i v e l y ) .  of  6,  7,  8 and 9)  some BSA and trace amounts of LF and Igs.  were  10).  composed mainly of p - L g , a - L a ,  Unbound f r a c t i o n s  (lanes  3,  4,  5)  100  Figure 32. E l u t i o n p r o f i l e of adsorbed proteins from MCIC on Sepharose 6B treatment of 960 mL e l e c t r o d i a l y z e d whey (EDW) and 720 mL sweet whey (SW) powders reconstituted in water, using l i n e a r gradient e l u t i o n of 0.05 M T r i s - a c e t a t e containing 0.5 M NaCl, pH 8.2 to 2.8. Flow rate was 0.8 mL/min. 1 and 2 are f r a c t i o n s obtained.  101  1 2  55 =  3 4  =  -  •  5 6  -  7  «r  BSA HC  •••• •  mm  Figure 33. SDS-PAGE p r o f i l e s of ED whey and f r a c t i o n s obtained by MCIC on Sepharose 6B treatment. Lane 1, control untreated whey; Lanes 2, 3 and 4, are unbound f r a c t i o n s ; Lane 5, wash f r a c t i o n ; Lane 6, f i r s t eluted peak; Lane 7, second eluted peak; BSA, bovine serum albumin; HC, heavy chain of immunoglobulins.  102  1  2 3 4  5 6  7 8  9 10 11 12  Figure 34. SDS-PAGE p r o f i l e s of sweet whey and f r a c t i o n s obtained by MCIC on Sepharose 6B treatment. Lanes 1, 12, control untreated whey; Lane 2 p r e c i p i t a t e ; Lanes 3, 4 and 5; unbound f r a c t i o n s ; Lanes 6, 7, 8 and 9, wash f r a c t i o n s ; Lane 10, f i r s t eluted peak; Lane 11, second (shoulder) eluted peak; BSA, bovine serum albumin.  103  were composed mainly of p-Lg and a-La with some BSA, LF and Ig at a l l whey  application,  relatively  with  the  exception  that  at  the  early  stage  stages of there  less p-Lg and a-La (lane 3).  P r e c i p i t a t i o n occurred in the sweet whey which was made up in the buffer  (pH 8.2  (lane 2)  containing 0.5  M NaCl) p r i o r to a p p l i c a t i o n to MCIC.  and sweet  reconstituted wheys was the b u i l d up of pressure.  cm to 8.0  cm.  The bed height increased  of  applied  with sweet whey i t A28O  affinity  t  0  whey  to  stage.  obtain  9.0  and swelled back only s l i g h t l y during  a p p l i c a t i o n were normally washed from the volume  The Sepharose gel  packed during whey a p p l i c a t i o n , shrinking the bed height from  subsequent washing and e l u t i n g  reduce  Figure 34  Another problem that arose during the chromatography of both ED  became t i g h t l y  the  starting  shows the p r o f i l e of the p r e c i p i t a t e containing a l l major whey proteins  including Ig.  the  was  Unbound proteins  from  column with s t a r t i n g  effluent  A28O  o  °«  f  3  o  r  l i q u i d whey  buffer less;  of  half  whereas  required almost twice the volume of applied whey in order to  °« «  This  9  indicates  that  sweet  whey  proteins  have  less  to bind to the Cu-loaded column than l i q u i d cheese whey and can e a s i l y  be removed during the washing step. IgG  activity  of  Cheddar cheese whey. powders  resulted  conditions. as  other  in  ED and sweet  powders were much  Adjustment of the pH of reconstituted large  amounts  The p r e c i p i t a t e s  proteins.  whey  of  precipitate,  a  higher  pH ( 8 . 2 ) ,  observed unless the whey was c e n t r i f u g e d ,  the  where  than  liquid  ED and sweet whey  particularly  contained large quantities  Even at  lower  under  of LF and Ig,  acidic as well  no p r e c i p i t a t i o n  reconstituted  was  ED and sweet whey  samples had a tendency to clog the MCIC columns. These r e s u l t s suggest that these powders ( i . e . , good s t a r t i n g  materials  for  i s o l a t i o n of active  Ig.  process during the manufacture of these powders ( e . g .  ED and sweet whey) are not It  i s possible that some  heating or drying) caused  104  denaturation and aggregation of the whey p r o t e i n s . that e i t h e r  It  i s therefore recommended  l i q u i d whey or powders produced by a milder process should be used.  C. BINDING CAPACITY AND RECOVERY OF IMMUNOGLOBULINS FROM METAL CHELATE-INTERACTION CHROMATOGRAPHY COLUMN Crude  Ig  (0.3%)  separated  from  colostral  whey  by  ammonium  sulfate  p r e c i p i t a t i o n was passed through a 2.2 mL Cu-loaded Sepharose 6B column as shown in Figure 35. column.  It  was found that  The proportion  the capacity of the column was 101 mg Ig/mL  of immunoglobulin eluted  from the column under a c i d i c  conditions was 94% of the amount bound to the column. the  capacity  lower  than  Figure 35 also shows that  of 62 mg Ig/mL of Cu-Loaded IDA-BGE Sephacryl  that  of the Sepharose 6B counterpart.  However,  S-300  column was  the proportion of  immunoglobulins eluted from t h i s column was 100% of the amount adsorbed to the column.  Thus, the binding capacity f o r bovine Ig appears to f a l l  in the same  range as that reported by Lonnerdal et a l . (1977) f o r human l a c t o f e r r i n ,  which  was 70 mg LF/mL gel containing 50 umole copper i o n s . Using Sepharose  a 2.5 X 9 cm column containing g e l , approximately  near-quantitative application IgG).  liquid  82.2%  mL) of  which  chelating  was copper-loaded g e l ,  whey  (6.5% t o t a l  solids,  containing  from  250 mg  immunodiffusion indicated that peak 1 and peak 2 contained 6 and  200 mg IgG r e s p e c t i v e l y , while detectable  (25  of  recovery of IgG was obtained in the peak containing Ig,  of 750 mL of  Radial  half  a 50 mL bed volume  the IgG content of the unbound f r a c t i o n was not  (below the lower detection l i m i t of R . I . D . ) .  f o r l i q u i d whey.  The purity  The recovery of IgG was  of IgG was also increased from about 4% in  cheese whey to 53% in the IgG rich f r a c t i o n  (F2).  These r e s u l t s  indicate  that  MCIC capacity f o r Ig i s o l a t i o n from cheese whey i s at least 200 mg IgG per 25 mL of copper-loaded gel or about 8 mg IgG/mL copper-loaded g e l ; thus,  approximately  105  0  100  200 300 400 500 ELUTION VOLUME, ml  Figure 35. Saturation point for adsorption of crude Ig (prepared from colostrum by ammonium s u l f a t e method) on Cu-loaded IDA-BGE Sepharose 6B (SROSE) and Sephacryl S-300 (SACRYL). 0.3% crude Ig was passed through a 10 mL column (7.0 x 1.4 cm) e q u i l i b r a t e d with 0.05 M T r i s - a c e t a t e / 0 . 5 M NaCl, pH 8.2. W, washing with the s t a r t i n g b u f f e r s ; E, e l u t i o n with 0.05 M T r i s - a c e t a t e / 0 . 5 M NaCl, pH 4 . 0 . The flow rate was 20 mL/hr.  106  1  litre of whey could be treated with about 25 mL of copper-loaded g e l . The discrepancy between  mg/mL) and for Ig  the  apparent  capacity  MCIC for  crude  Ig  in whey proteins may be due to the presence of other  in cheese whey.  In addition  to  isolation  of  Ig,  whey were also bound to the copper-loaded g e l . may have lowered the apparent and BSA are  of  probably  less  bound to  proteins  both LF and BSA from cheese Binding of these two  binding capacity for  strongly  (101  the  Ig  from whey.  immobilized  since they were eluted before Ig during pH gradient  proteins  However, LF  copper than  Ig,  elution.  D. ANTI-LIPOPOLYSACCHARIDE ACTIVITY OF IMMUNOGLOBULINS RICH FRACTION Figure  36  shows  the  fraction  isolated  isolated  from cheese whey  S^  typhimurium  immunoglobulins  from  and  activity  of  Cheddar cheese whey by MCIC method.  an  parapertussis.  higher  recognition  Among and  the  binding  three ability  i s o l a t e d from E^ c o l i i n d i c a t i n g that t h i s antigen i s f a i r l y cow's from which the wheys were produced. LPS i s o l a t e d from S^. typhimurium.  IgG  rich  Immunoglobulins  recognized and bound to LPS extracted  B^  showed  anti-1ipopolysaccharide  from 0  col i , antigens,  to  the  LPS  common to the dairy  Lower recognition was obtained  The i s o l a t e d Ig showed binding a b i l i t y  for  to LPS  i s o l a t e d from B± parapertussis. LPS, which i s s e r o l o g i c a l l y c a l l e d as 0 antigen and pharmacologically known as  endotoxins,  Gram-negative Potentially  are  bacteria.  all  wall. respect  integral  They contain p r o t e i n ,  to  of  the  outer  membrane  proteins  participate  surface  pathogenicity  of  substances can be exchanged across the in  iron  scavenging or c e l l  Gram-negative  of  l i p i d s and 1 ipopolysaccharides.  with l i v i n g c e l l s or substances in the environment.  form pores through which Other  components  of these components are more or less exposed on the c e l l  and thus can interact proteins  the  bacteria,  adhesion. the  cell  Some cell With wall  107  2.0  Figure 36. Anti-1ipopolysaccharide a c t i v i t y of Ig i s o l a t e d from cheese whey by MCIC method. • - • , g± c o l i LPS; • - • S^. typhimurium LPS; O O , EL parapertussis LPS.  108  1ipopolysaccharides are not only dominant antigens but also mediators of a great many b i o l o g i c a l a c t i v i t i e s though i t  (Jann and Jann, 1985,  Rietschel et a l . ,  1982).  Even  i s hard to speculate on what follows the binding of Ig with LPS, the  binding may i n t e r f e r e with the process by which bacteria adhere to and colonize the i n t e s t i n a l disturb  the  lining  (Packard, 1982).  biological  processes  materials across the b a c t e r i a l  cell  The i n t e r a c t i o n  which  are  of Ig with LPS may also  involved  in  the  transport  of  wall.  E. LACTOPEROXIDASE CONTENT OF LACTOFERRIN RICH FRACTION The l a c t o f e r r i n  rich fraction  obtained by MCIC treatment was yellowish  color and became greenish on f r e e z i n g . presence of  lactoperoxidase which  The color was thought  constitutes  about  1% of  the whey  The color of lactoperoxidase i s due to the iron content ( i t which represents one atom of iron per molecule) lactoperoxidase containing f r a c t i o n citric  acid  presence  for  of  less  lactoperoxidase activity  36  hr.  than in  lost  Moreover,  LF  rich  to the i s o l a t e d f r a c t i o n  the  proteins.  contains 0.071% iron  and Ohlsson, 1985).  The  i t s . color when dialyzed against 0.1 M  the  lactoperoxidase  2% lactoperoxidase  the  (Paul  to be due to  in  in  fraction  assay  LF f r a c t i o n . would  give  indicated  The  extra  presence  the of  antimicrobial  ( P r u i t t and Tenovuo, 1985).  F. IDENTIFICATION OF GLYCOPROTEINS IN LACTOFERRIN-RICH FRACTION  Figure 37 shows the SDS-PAGE of cheese whey, the F l f r a c t i o n obtained from MCIC column, standard l a c t o f e r r i n stained  with  acid-Schiff was  Coomassie  (PAS) stain  shown to  contain  Brilliant  (LF)  Blue  (Figure 37B). Ig,  LP and  and lactoperoxidase (Figure  37A)  as  (LP). well  Using Coomassie s t a i n ,  lactoferrin.  According to  Samples were as  LF rich the  periodic fraction  SDS-PAGE of  109  Figure 37. SDS-PAGE of whey proteins (1), lactoferrin rich fraction (2), lactoferrin (3) and lactoperoxidase (4). (A) stained with Commassie B r i l l a n t Blue and (B) stained with p e r i o d i c acid S c h i f f (PAS). HC and LC heavy and l i g h t chains, r e s p e c t i v e l y .  110  standard LF and LP, l a c t o f e r r i n PAS  technique,  electrophoresis  which  has s l i g h t l y  was  applied  on SDS-PAGE,  indicated  contained covalently  to  detect  glycoproteins  that L F , LP and the  bound carbohydrate.  that the majority of carbohydrate  higher molecular weight than LP. following  heavy chain of  This staining method also  Ig  indicated  in Ig was located in the heavy chains of the  molecule.  G. ISOELECTRIC POINTS OF LACTOFERRIN AND IMMUNOGLOBULINS RICH FRACTIONS The i s o e l e c t r i c whey were found to points of al.,  point be in  of the  standard l a c t o f e r r i n ,  1972).  However,  the  fractions range 6.0,  of  obtained  by MCIC treatment  5.2-6.6 which  covered the  of  cheese  isoelectric  and immunoglobulins 5.5-6.8 (Josephson et  i s o e l e c t r i c point  of  was lower than the reported data of 9.16 - 9.8  lactoperoxidase was 8.63  which  (Righetti and Caravaggio, 1976).  H. HISTIDINE MODIFICATION AND METAL CHELATE-INTERACTION CHROMATOGRAPHY Among a l l  of  the  amino acids comprising p r o t e i n s ,  Rassi & Horvath  (1986)  found that h i s t i d i n e and cysteine gave the highest retention factors when passed through a Cu-IDA column.  However, studying the retention behavior of free amino  acids might not represent the real in p r o t e i n . fraction  The role of h i s t i d i n e  behavior of that amino acid when i t  i s found  in immunoglobulins and the l a c t o f e r r i n  i s o l a t e d by MCIC treatment was investigated  using MCIC.  rich  When control  Ig was applied to MCIC column, almost no protein was eluted in the washing step (Figure 38),  i n d i c a t i n g that a l l  the sample applied was adsorbed to the column.  This f r a c t i o n was subsequently eluted with 0.01  M imidazole s o l u t i o n .  However,  modification of h i s t i d i n e groups of Ig by diethyl  pyrocarbonate (DEP-Ig)  inhibited  Most of h i s t i d i n e - m o d i f i e d  the  was found in  interaction  with the  copper i o n .  the washing s o l u t i o n , while  only a small  amount  of  greatly  protein  Ig was  Ill  Figure 38. E l u t i o n p r o f i l e s of control (Ig) and diethyl pyrocarbonate treated immunoglobulins (DEP Ig). Samples (30 mg/5 mL 0.05M Tris-acetate containing 0.5 M NaCl, pH 8.2) were applied to the column (1.4 x 7.0 cm) and washed (W) with the s t a r t i n g buffer then eluted (E) with 0.01 M imidazole. Flow rate was 30 mL/hr.  112 adsorbed.  These data strongly suggest the involvement of h i s t i d i n e groups of  in the i n t e r a c t i o n with copper ion immobilized on agarose.  Ig  The small amount of  DEP-Ig adsorbed on the column may suggest the  involvement of other amino acids  in the  is  interaction,  e s p e c i a l l y cysteine which  major force contributing to the i n t e r a c t i o n Figure obtained  39  from  shows  the  elution  cheese whey  histidine modification, all column of MCIC, however, applied  were  Appreciable indicate  desorbed  amounts  that  modification  some  I.  of  after  the  lactoferrin  histidine  rich  fraction  modification.  Before  proteins in the sample applied were adsorbed to the  after by  other  blocking h i s t i d i n e  washing  protein  process of  mixture of Ig, per mole  of  and  be the second  (Rassi & Horvath, 1986).  profiles  before  considered to  the  column  were eluted  forces  are  histidine  with  with 0.01  involved  was not  LF and BSA, i t was d i f f i c u l t  groups most of the starting  M imidazole.  in  complete  the  the  proteins  This may  interaction  (since  this  buffer.  or  the  fraction  was  to c a l c u l a t e mole modified h i s t i d i n e  protein).  SEPARATION OF HEAVY AND LIGHT CHAINS OF IMMUNOGLOBULINS A typical  elution  pattern  column i s shown in Figure 40.  of  reduced and alkylated  The f i r s t  Ig  from Sephadex G-75  peak which eluted at  the void volume  represents the heavy chain while the second peak consists of l i g h t chains. third  peak i s eluted  therefore agents.  represents The  demonstrated  at  a volume corresponding to the  small molecules, s p e c i f i c a l l y the  homogeneity  by SDS-PAGE  of  the  (Figure  chains was obtained when Ultrogel indicated percentage  in of  Figure 42. heavy  heavy  41).  (first  Better  light  total  peak)  and  chain  resolution  of  absorbance unit the  light  bed volume, and  reducing and  ACA 54 was used instead of  Based on the  chains  and  total  The  alkylating  preparation  was  heavy and  light  Sephadex G-75 as calculation,  chains  obtained from these figures were 70-75% and 25-30% r e s p e c t i v e l y .  (second  the  peak)  These values  113  Figure 39. E l u t i o n p r o f i l e s of Fl-MCIC f r a c t i o n before (Fl) and a f t e r diethyl pyrocarbonate treatment (DEP F l ) . Samples (30 mg/5 mL 0.05 M T r i s - a c e t a t e containing 0.5 M NaCl, pH 8.2) were applied to the column (1.4 x 7.0 cm) and washed (W) with the s t a r t i n g buffer then eluted (E) with 0.01 M imidazole. Flow rate was 30 mL/h.  114  Figure 40. E l u t i o n p r o f i l e s of reduced and alkylated heavy and l i g h t chains of immunoglobulin on Sephadex G-75 eluted with 1 M propionic a c i d . 1 and 2 are f r a c t i o n s obtained.  115  1 2  3  4  Figure 41. SDS-PAGE p r o f i l e s of heavy and l i g h t chains of Immunoglobulins Isolated by gel f i l t r a t i o n . Lanes 1 and 2, crude Immunoglobulins; Lanes 3 and 4, Immunoglobulin l i g h t and heavy chains, respectively obtained from Figure 40.  116  3.5  100 200 300 ELUTION VOLUME, ml  Figure 42. E l u t i o n p r o f i l e of reduced and alkylated heavy and l i g h t chains of Ig on Ultrogel ACA 54 eluted with 0.1 M T r i s - H C l buffer containing 4 M Guanidine-HCl and 1 mM iodoacetamide, pH 8.2. 1 and 2 are fractions obtained.  117  are within the range of reported data for Ig of d i f f e r e n t al.,  species (Fleischman et  1962; Small and Lamm, 1966).  J . SEPARATION OF LACTOFERRIN AND LACTOPEROXIDASE IN LACTOFERRIN RICH FRACTION In  addition  to  the gel  f i l t r a t i o n process used to  i s o l a t e the LF from LF  r i c h f r a c t i o n , several attempts were made in order to obtain better  resolution  of LF from other protein contaminants.  1. Gel f i l t r a t i o n method Figure 43 shows the e l u t i o n pattern  of l a c t o f e r r i n  rich fraction  from MCIC of bovine acid whey on Sephacryl S-300 column. obtained,  the  first  peak was c o l o r l e s s while  SDS-PAGE a n a l y s i s (Figure 26)  the  (peak 1)  Two major peaks were  second peak was y e l l o w i s h .  indicated that the f i r s t  peak consisted mainly of  immunoglobulins while the second peak contained predominantly l a c t o f e r r i n .  2. Stepwise pH e l u t i o n Figure 44  represents a stepwise e l u t i o n  f r a c t i o n on MCIC column loaded with copper.  process for  separating  LF rich  Elution with the s t a r t i n g  buffer,  0.5 M NaCl in 0.05 M T r i s - a c e t a t e at pH 7 and 6 did not remove any of the bound proteins from the column; however, elution with the same buffer at pH 5 removed some of  the bovine serum albumin.  4.0 gave two peaks, i . e . , as  a cleaning step  gradient  (5.0-2.8)  Figure 45.  for  Further e l u t i o n with the same buffer at pH  LF and Ig, which were not wel1-separated. eluting  slightly  bovine serum albumin,  improved the  then  Using pH 5  elution  separation of LF and Ig  with a pH  as shown in  118  1.4  0  0  ' 60 120 180 240 300 ELUTION VOLUME, ml  Figure 43. Sephacryl S-300 column chromatography of l a c t o f e r r i n rich f r a c t i o n obtained by MCIC of acid whey. 100 mg sample was applied to Sephacryl column (83 x 2.5 cm) and eluted with 0.05 M potassium phosphate b u f f e r , pH 7.4 containing 0.01 M NaCl. 1 and 2, are f r a c t i o n s obtained. The flow rate was 30 mL/hr.  119  280 560 840 1260 ELUTION VOLUME, ml  Figure 44. Stepwise e l u t i o n p r o f i l e of acid whey on MCIC eluted by decreasing pH values. Arrows indicate pHs 7, 6, 5 and 4 of 0.05 M T r i s - a c e t a t e containing 0.5 M NaCl. 1 and 2 are f r a c t i o n s obtained.  120  2.5  0  0  70  140 210 280 350 ELUTION VOLUME, ml  Figure 45. Elution p r o f i l e of bound proteins of acid whey on MCIC column, eluted (E) by using pH gradient (5-2.8) of 0.05 M T r i s - a c e t a t e containing 0.5 M NaCl. 1 and 2 are f r a c t i o n s obtained.  121  3. Imidazole gradient Fraction LF.  elution  1 obtained  by MCIC process contained Ig  This f r a c t i o n was pooled and rechromatographed on MCIC column and  with a 0-0.01 M imidazole gradient.  After washing o f f  wel1-separated peaks were obtained (Figure 46). Ig,  and BSA in addition  while  (Figure  the  47).  However,  gave an extra analysis.  second peak was mainly  is,  subsequent e l u t i o n  therefore,  eluted  the unbound proteins two  The f i r s t  peak contained mainly  lactoperoxidase as indicated by SDS-PAGE with the  peak which was highly p u r i f i e d  It  to  possible  to  starting  lactoferrin separate  buffer  at  pH  2.8  according to SDS-PAGE  these  three  biologically  active p r o t e i n s . The r e s u l t s source  for  of  this  extracting  study demonstrate  immunoglobulins  that cheese whey can be a  and  chromatography techniques i n v e s t i g a t e d , metal is  the  best method as  important  it  is  simple  immunoglobulins, l a c t o f e r r i n  in  lactoferrin.  to  the  immunological  activity  operation  for  Based on a n t i l i p o p o l y s a c c h a r i d e a c t i v i t y  the of  proteins isolated  and easy  in  formulae or infant  feeding.  be  useful  regeneration.  immunoglobulins and  bioactive  therefore,  this  recovery without detectable  (Packard, 1982)  may,  biologically  Furthermore,  well known b a c t e r i o s t a t i c a c t i v i t y of l a c t o f e r r i n , proteins  absorption  chromatography  separating  and lactoperoxidase.  of  the  chelate-interaction  method has advantages of high c a p a c i t y , quantitative damage  Of  reliable  the  the separated  fortification  of  infant  122  Figure 46. Elution p r o f i l e of l a c t o f e r r i n r i c h f r a c t i o n on MCIC column. El, e l u t i o n with l i n e a r gradient of 0-10 mM imidazole solution ( . . . . ) ; E2, e l u t i o n with 0.05 M T r i s - a c e t a t e containing 0.5 M NaCl, pH 2.8. 1, 2 and 3 are f r a c t i o n s obtained.  123  6  5  4  3  2  1  Figure 47. SDS-PAGE p r o f i l e s of f r a c t i o n s obtained from Figure 46. Lane 1, whey p r o t e i n s ; Lane 2, control Fl-MCIC; Lane 3, unbound f r a c t i o n ; Lanes 4, 5 and 6 are peak 1, 2 and 3 of Figure 46; L F , l a c t o f e r r i n , LP, lactoperoxidase.  PART IV  METAL CHELATE INTERACTION CHROMATOGRAPHY OF SKIMMILK  125  In skimmilk there are two major f r a c t i o n s of milk p r o t e i n , caseins and whey or  serum  proteins,  respectively. easily  and  supports;  which  In Part III, efficiently  however,  utilization  are  of  if  the  pH 4.6  insoluble  and  soluble  fractions,  immunoglobulins and l a c t o f e r r i n were i s o l a t e d quite from  whey  skimmilk  proteins  by  Cu-chelate  can be used as a  these a n t i - m i c r o b i a l  starting  chromatographic  material,  compounds may be f e a s i b l e .  broader  Two types  of  buffer were used f o r MCIC column chromatography in t h i s study.  A. MCIC WITH TRIS-ACETATE BUFFER The  possibility  of  utilizing  skimmilk  immunoglobulins and l a c t o f e r r i n was i n v e s t i g a t e d .  for  directly  recovering  Figure 48 shows the  elution  p r o f i l e of skimmilk before and a f t e r 50% d i l u t i o n with 0.05 M T r i s - a c e t a t e / 0 . 5 M NaCl, pH 8.2.  A f t e r washing the unbound proteins with the s t a r t i n g b u f f e r ,  adsorbed  proteins  However,  the  were  amount  s m a l l , and the flow  eluted  of  with  proteins  0.05  eluted  rate became quite  r e s u l t i n g in clogging of the column. whether  skimmilk  was d i l u t e d  or  M acetate-Tris/0.5  M NaCl  under a c i d i c conditions  pH  (peak  slow i n d i c a t i n g p r e c i p i t a t i o n  the 4.0.  1)  was  of casein  This behaviour was observed regardless of  undiluted.  Subsequent e l u t i o n  with  0.01  M  imidazole recovered the bound proteins which appeared as the main f r a c t i o n (peak 2)  in  the  eluted  in  profiles. the  SDS-PAGE analysis  washing  step  were  (Figure  49)  immunoglobulin,  indicated  lactoferrin,  that  proteins  a-lactalbumin,  B - l a c t o g l o b u l i n and c a s e i n , while proteins eluted under a c i d i c conditions were mainly  immunoglobulin  and  lactoferrin.  The  fraction  eluted  with  0.01  M  imidazole, however, was mainly c a s e i n . Table  12  stages of the  shows the  IgG d i s t r i b u t i o n  of  fractions  i s o l a t i o n process of IgG from skimmilk.  showed that the majority  of active  IgG was present  obtained  at  different  Immunochemical analysis  in the  fraction  eluted  at  126  0  70 140 210 280 ELUTION VOLUME, ml  Figure 48. Elution p r o f i l e of skimmilk on MCIC column. 100 mL skimmilk undiluted (SM) or 50% d i l u t e d (DSM) with 0.05 M T r i s - a c e t a t e / 0 . 5 M NaCl) was passed through Cu-loaded Sepharose 6B (1.4 x 7.0 cm), and washed (W) with same b u f f e r . E l , elution with the same buffer at pH 4.0; E2, elution with 0.01 M imidazole s o l u t i o n . 1 and 2 are eluted f r a c t i o n s . The flow rate was 21 mL/hr.  127  Figure 49. SDS-PAGE of f r a c t i o n s obtained in Figure 48. Lanes 1 and 2, skimmilk; Lane 3, unbound skimmilk to MCIC column; Lane 4, washing f r a c t i o n ; Lanes 5 and 6, are peak 1 and 2, r e s p e c t i v e l y ; Lane 7, standard IgG; Lane 8, a - c a s e i n .  128  Table 12.  IgG content of d i f f e r e n t skimmilk on MCIC column.  stages of the i s o l a t i o n of  P r o t e i n Cone, of selected f r a c t i o n mg/mL  IgG from  3  Sample  Skimmilk  (control)  IgG Cone. mg/mL D  IgG Purity %  37.8  0.562  1.49  Unbound skimmilk  18.0  0.246  1.36  Washing f r a c t i o n  4.0  0.828  20.70  Peak 1 (Figure 48)  1.08  0.911  84.35  0.272  1.38  Peak 2 (Figure 48)  a  D  Determined by Bio-•Rad Mississauga, Ont.) Determined by R.I.D.  19.6  Protein  Assay  Kit  (Bio-Rad  Laboratories,  129  a c i d i c pH (pH 4 . 0 ) . of  This f r a c t i o n was more than 84% pure.  IgG bound to the column was quite  unbound  fraction  of  washing  fraction  indicating  column.  By  skimmilk.  eluting  the  electron-donor  solution  almost  but  no  IgG  was  More  that  rich  than  IgG of  strongly (0.01  small  (less  20% pure  skimmilk  casein  the  10%)  as compared to  IgG was  bound  bound material  M imidazole) in  than  However, the amount  a  fraction  fractions  detected  in  the  weakly  to  the  rather  with  (Figure  the  strongly  competing  obtained  contained  49).  These  results  suggest that there was a competition between IgG and caseins to bind copper ions linked  to  agarose,  and  the  casein  fraction  bound  more  strongly  than  immunoglobulins to the column under these c o n d i t i o n s .  B. MCIC WITH PHOSPHATE BUFFER An attempt was made to f i n d conditions under which casein could be eluted while retaining the  elution  loaded  buffer,  profile  column  containing  immunoglobulins and l a c t o f e r r i n  0.5  of  of  a mixture  MCIC  M NaCl,  after  of  skimmilk  equilibration  pH 7.0.  After  M imidazole and 0.05  and F2,  respectively.  unbound  proteins  proteins  eluted  and with  washing  the unbound proteins were removed.  with 0.01  on the column.  with 0.01  eluted with T r i s - a c e t a t e  (lane  M imidazole  0.02  with  M Tris-acetate/0.5  fraction)  immunoglobulins M  the  on copper  phosphate starting  3)  (lane 4)  M NaCl, pH 3.8  (Figure were  51)  casein  eluted  to obtain  indicated that fractions,  were immunoglobulins.  containing 0.5 M NaCl, pH 3.0  buffer  phosphate  The bound proteins were then  Electrophoretic analysis  (turbid  Figure 50 shows  was too small  Fl the  while Peak 2  to  detect  by SDS-PAGE. Figure 50 shows the e l u t i o n  profile  the presence of caseins in skimmilk, A similar  pattern  to  that  of  of a mixture of Ig and l a c t o f e r r i n  in  under the same conditions on MCIC column.  skimmilk-Ig  mixture was obtained;  however,  the  130  Figure 50. E l u t i o n p r o f i l e s of skimmilk (SM), Ig and LF mixture on MCIC column. 60 mg of Ig and LF was mixed with 1 mL skimmilk (SM+Ig+LF) and 30 mg Ig was mixed with 1 mL skimmilk (SM+Ig) and passed through MCIC column (1.4 x 7.0 cm). W, washing with 0.02 M phosphate buffer containing 0.5 M NaCl, pH 7.0; E l , e l u t i o n with 0.01 M imidazole; E2, elution with T r i s - a c e t a t e containing 0.5 M NaCl, pH 3.0.  131  10  9 8 7  6 5 4  3 2 1  Figure 51. SDS-PAGE of f r a c t i o n s obtained in Figure 50. Lane 1, skimmilk; Lane 2, skimmilk and Ig mixture; Lanes 3 and 4 are unbound and peak 1 of SM-Ig mixture a p p l i c a t i o n , r e s p e c t i v e l y ; Lane 5, SM-Ig-LF mixture; Lanes 6 and 7 are unbound and peak 1 of SM-Ig-LF mixture a p p l i c a t i o n , r e s p e c t i v e l y ; Lane 8, immunoglobulins; Lane 9, l a c t o f e r r i n and Lane 10, a - c a s e i n .  132  amount of protein 51)  indicated  bound to the column was increased. the  unbound f r a c t i o n  while  proteins eluted with 0.01 M imidazole were a mixture of Ig and l a c t o f e r r i n  (lane  The bound protein  mixture  (Ig  (lane  and  LF)  6)  may  was b a s i c a l l y  (Figure  casein  7).  that  SDS-PAGE p r o f i l e  be  separated  by  using  gel  f i l t r a t i o n or 0-0.01 M imidazole gradient. Results suggest that the type of ions in the buffer used f o r i n i t i a l e q u i l i b r a t i o n and washing had a great unbound  or  bound  phosphoproteins buffer  in  the  to  the  (Whitney  MCIC  et  equilibration  copper ion (with d i f f e r e n t  influence on whether or not proteins were  column.  al.,  1976),  step  of  column  Since it  the  is  caseins  believed  are  that  classified  as  using phosphate  column can form a complex with  the  color from that formed with T r i s - a c e t i c acid buffer)  which may prevent the phosphoproteins from binding to the column.  These results  also suggest the involvement of phosphoserine groups in the i n t e r a c t i o n with the copper i o n s .  C. MECHANISM OF CASEIN-METAL INTERACTION The p r i n c i p l e of protein affinities this  metal  of proteins to bind to immobilized metal  binding  tryptophan  separation by MCIC process l i e s  is  dependent  on  the  availability  ions. of  It  in the  different  i s suggested that  histidine,  cysteine  and  residues of the proteins to form stable coordination complexes with  ions  (Sulkowski,  performed  at  decrease  non-specific  accomplished metal-chelate  a  by  1985).  slightly  In  alkaline  general, pH with  electrostatic  lowering  and r e s u l t s  the  pH, which  adsorption of high  ionic  strength  interactions. reverses  in protein displacement.  electron donor as a mild chelating agent ( e . g . ,  protein  Elution  protein  agent ( e . g . EDTA) may be used to purge bound p r o t e i n s .  MCIC  is  solutions  to  is  coordination  Alternatively,  imidazole)  to  commonly to  the  a competing  or a strong chelating  Since h i s t i d i n e has been  133  suggested to e x h i b i t at pH 6 . 0 ,  the strongest  (Rassi and Horvath,  retention  factor  on a copper chelate column  1986), the e l u t i o n p r o f i l e  of h i s t i d i n e - m o d i f i e d  casein f r a c t i o n s was i n v e s t i g a t e d .  1. q-Casein a-Casein represents more than 62.5% of t o t a l casein f r a c t i o n s in cow's milk and i s composed of a Figure  52  and K - c a s e i n in r a t i o of 4:1 (Whitney et a l . , 1976).  s  shows  the  histidine-modified a-casein.  elution After  and washing with the s t a r t i n g  profile  loading control  alkaline  pH b u f f e r ,  protein could be eluted under these c o n d i t i o n s . displaced However, total  by using  blocking 3.7  of  amount  four  of  chelating  histidine  histidines  unbound  involvement than  a mild  of h i s t i d i n e  agent  from  10.7% to  control  a-casein  only 10.7% of total  The rest of protein  i.e.  0.01M imidazole  et a l ,  1974)  82.2%  (Table  13)  applied  (89.2%) was (Table  pyrocarbonate  (Webb  groups in the i n t e r a c t i o n  and  a-casein on Cu-chelate gel  residues by diethyl  per mole a-casein  protein  of  13).  out of the  increased the indicating  with the copper i o n .  the Less  18% of the protein was bound and could be eluted with 0.01 M imidazole,  which might indicate the involvement of other amino acids i . e . , T r p , C y s , Tyr in the  interaction.  2. a ^ | - and B-casein a i-Casein s  slightly  is  less than  profiles  of  modification  subfraction  of  50% of the total  a i-casein s  of h i s t i d i n e  of the protein of protein  a  and  the  casein.  histidine  residues  whole  which  represents  Figure 53 indicates blocked  of a i - c a s e i n , s  casein  the elution  a i-casein. s  only a small  a  amount  Without (6.1%)  applied was eluted o f f the column in the washing step and 93.9%  interacted  with the copper under  alkaline  pH and was subsequently  134  Figure 52. Metal chelate i n t e r a c t i o n chromatography of a - c a s e i n . 3 mL of protein (10 mg/mL) before (a-CAS) and after diethylpyrocarborate modification (DEP-a-CAS) e q u i l i b r a t e d with 0.05 M T r i s - a c e t a t e / 0 . 5 M NaCl, pH 8.2 and applied to copper chelate Sepharose 6B (1.4 x 7.0 cm). W, washing with the same e q u i l i b r a t i n g b u f f e r ; E, e l u t i o n with 0.01 M imidazole. Flow rate was 30 mL/hr.  135  Table 13.  Binding  of  modification  Proteins  casein  fractions  3  to  MCIC  column before  Washing step  0  (W)  Eluting step %  10.7  89.2  82.2  17.8  6.1  93.9  DEP-a i-casein  94.3  5.7  Control  13.6  86.4  DEP-8-casein  54.7  45.3  Control polymer K - c a s e i n  87.9  12.1  DEP-Polymer-K-casei n  94.3  5.7  SSS-K-Casein  38.5  61.5  DEP-SSS-K-Casein  98.9  1.1  a-casein  DEP-a-casein Control  after  of h i s t i d i n e groups.  % Control  and  a i-casein s  s  B-casein  a  Calculated based on t o t a l absorbance units  D  See Figures 52, 53, 54 and 55 for  abbreviation i d e n t i t y  (E)  136  Figure 53. Metal chelate i n t e r a c t i o n chromatography of a i - c a s e i n before (a -CAS) and a f t e r diethylpyrocarbonate modification (DEP a - C A S ) . See Figure 52 for conditions of separation. s  s  s  137  eluted with the  eluting  residues  by  DEP out  (Whitney  et  al,  of  1976)  solution  (Table  a  of  total  destroyed  immobilized on agarose.  the  13).  However,  5 histidine protein's  blocking 4.2  histidine  residues per mole ability  to  Based on the c a l c u l a t i o n of t o t a l  bind  a i-casein s  to  copper  ion  UV absorbance, 94.3%  of applied protein was recovered in the washing step and a small amount of applied protein  the  (5.7%) was bound and subsequently eluted with 0.01 M imidazole.  The second major protein of bovine casein i s p-casein which represents 30% of t o t a l  casein f r a c t i o n  (Whitney et a l . ,  1976).  Figure 54  shows the  p r o f i l e s of p-casein before and a f t e r h i s t i d i n e modification with DEP. to  13.6%  of  modification  unbound  control  (Table 13).  p-casein,  al,  1976),  45.3%  was  of  5 histidine  protein  applied  was  ion  bound  binding  modification  (Table  13)  histidine  may be due  histidine  (Whitney  M imidazole s o l u t i o n .  after  after  residues per mole p-casein  subsequently eluted with 0.01 rate  unbound  Compared  This indicates that even though 4.4 h i s t i d i n e residues  were modified out of the total et  54.7%  elution  to  the  to  the  copper  The reason for  compared to  temperature-dependent  other  this  casein  and high  fractions  association-dissociation  properties of P - c a s e i n .  3. K - c a s e i n K - c a s e i n which represents 12.5% of the t o t a l casein in cow's milk occurs in the form of a mixture of aggregates of ic-caseins held together by intermolecular disulfide  bonds  (Whitney  Z i t t l e and Custer  et  al,  1976).  prepared  by  (1963) was considered to assume an aggregated  55A shows the e l u t i o n  the  method  form.  of  Figure  behaviour on Cu-chelate agarose of t h i s preparation with  and without h i s t i d i n e m o d i f i c a t i o n . and h i s t i d i n e  K-Casein  The amount of unbound K - c a s e i n for  modified K - c a s e i n were 87.9%  and 94.3%  respectively  control  (Table  13).  This indicated that the amount of K - c a s e i n adsorbed on Cu-chelate and recovered  138  Figure 54. Metal chelate i n t e r a c t i o n chromatography of p-casein before (p-CAS) and a f t e r diethylpyrocarbonate modification (DEP p-CAS). See Figure 52 for conditions of separation.  139  Figure 5 5 .  Metal chelate i n t e r a c t i o n chromatography of (B) monomer K-caseins (MK-CAS) diethylpyrocarbonate modification (DEP K-CAS). separation c o n d i t i o n s . (K-CAS)  ( A ) aggregated K-casein before and after See Figure 52 for  140  by  0.01  M imidazole  modified K - c a s e i n .  was  quite  low  for  both  control  K-casein  and  histidine  This behavior of aggregated K - c a s e i n on the MCIC column may  be due to i t s aggregated structure which probably r e s t r i c t e d the access to metal ions.  Whether the aggregated structure of K - c a s e i n blocked the  i n t e r a c t i o n was determined.  Figure 55B shows the e l u t i o n  monomers formed by reducing the tetrathionate. more  than  K-casein  the  adsorbed  histidine  onto  residues  (Whitney et a l . , i n d i c a t i n g the  out  1976)  total the of  protein same 3  for  applied  column  histidine  reduced K - c a s e i n  compared to  (Figure  55B).  residues  per  ions  and  aggregated  Modification mole  monomer  of  2.7  K-casein 1.1%  This suggests that d i s u l f i d e bonds of the aggregated K - c a s e i n  conclusion,  buffer  is  feasible.  Using  induced competition between  decreased  immunoglobulins.  the  However,  capacity  of  the  Tris-acetate  phosphoproteins from binding  collected  in  unbound f r a c t i o n  to  while  the  for Ig and  buffer  as  an  Ig and caseins to bind copper MCIC  using phosphate buffer  prevented  the  ions.  use of skimmilk d i r e c t l y as a s t a r t i n g material  separation  equilibrating  of  decreased the amount of bound modified K - c a s e i n to  had l i t t l e or no e f f e c t on the i n t e r a c t i o n with metal  lactoferrin  12.1%  (MK-CAS) was  involvement of h i s t i d i n e residues in the i n t e r a c t i o n of K - c a s e i n  with copper i o n s .  In  p r o f i l e s of K - c a s e i n  d i s u l f i d e bonds and blocking them with sodium  The amount of bound protein  61% of  copper-K-casein  column  in  binding  as an e q u i l i b r a t i n g  column and allowed  immunoglobulins and  of  buffer  them to  lactoferrin  be are  retained on the column. Chemical  modification  studies with T r i s - a c e t a t e  columns, indicated the involvement of h i s t i d y l in the i n t e r a c t i o n with copper ions.  buffer  equilibrated  MCIC  residues of some casein f r a c t i o n s  SEPARATION OF IMMUNOGLOBULINS AND TRANSFERRIN FROM BLOOD SERUM AND PLASMA BY METAL CHELATE INTERACTION CHROMATOGRAPHY  142  It sewage  was  reported  systems  potentially  food  throughout  valuable  (Alexander,  that  1984).  selective  difficult  to  1982,  Europe.  protein  If  that  800,000 tonnes of This  feeding.  precipitation  mechanize.  The  immunoglobulins and t r a n s f e r r i n  is  was  one could extract  processing and animal  involve  in  equivalent  literally  to  140,000  flushed  down  into  tonnes the  of  drain  these proteins they could be used for  However,  methods  blood were dumped  which  possibility  of  most of are  the  batch  using  available  methods  processes and  MCIC  method  to  thus  extract  from blood plasma and serum was assessed in this  part of the t h e s i s .  A. METAL CHELATE INTERACTION CHROMATOGRAPHY 1. Blood serum on Cu-loaded MCIC When blood serum (obtained by incubating blood samples overnight at 5°C) in 0.05  M Tris-acetate/0.5  M NaCl  buffer,  IDA-BGE Sepharose 6B column (Figure 56),  pH 8.2,  was  applied  to  a Cu-loaded  the major portion of the blood proteins  (mainly albumin) did not bind and were recovered when the column was washed with the s t a r t i n g b u f f e r . (Fl)  The bound proteins were eluted with the pH gradient  and i d e n t i f i e d to be mainly immunoglobulins.  imidazole (F2) and 7 ) .  Subsequent e l u t i o n with 10 mM  recovered the major portion of t r a n s f e r r i n  (Figure 57; lanes 5, 6  Fraction 1 gave a long arc by Immunoelectrophoresis which was s i m i l a r  to that of standard IgG, while  fraction  2 y i e l d e d arcs around the well  to that of standard t r a n s f e r r i n and immunoglobulins (Figure  2. Blood serum on MCIC columns loaded with other metal Figure packed  buffer  with  59  represents  Zn-,  Ni-  and  the  elution  Co-loaded  patterns  of  similar  58).  ions blood  IDA-BGE Sepharose  6B.  serum The  from columns capacity  of  Zn-loaded column to adsorb protein was found to be higher than that of N i - and  143  Figure 56. Immobilized copper a f f i n i t y chromatography of blood serum. Blood serum (1 g in 10 mL 0.05 M T r i s - a c e t a t e / 0 . 5 M NaCl, pH 8.2) was applied to the column (1.4 x 7 cm). The column was washed with the s t a r t i n g buffer and then eluted with E l , 0.05 M T r i s - a c e t a t e 0.5 M NaCl, pH 4 . 0 , and with E2, 0.1 M imidazole. The flow rate was 30 mL/hr. Fl and Fz are fractions obtained.  144  11 109 8 7 —  654  32 1  —  TF BSA  Ig-HC Ig-LC  Figure 57. SDS-PAGE p r o f i l e s of blood f r a c t i o n s from MCIC on Sepharose 6B column. Sample i d e n t i f i c a t i o n : Lanes 1, 2 and 3 are F l of Figure 59 from Zn, Ni and Co loaded columns r e s p e c t i v e l y ; Lane 4, plasma protein eluted from Cu-loaded column with pH 4 b u f f e r ; Lanes 5, 6 and 7 are unbound, F l , and F2 in Figure 56, r e s p e c t i v e l y ; Lanes 8, 9 and 10 are standard transferrin(TF), bovine serum albumin (BSA), and immunoglobulins (Ig) r e s p e c t i v e l y ; Lane 11, blood plasma.  145  Figure 58. Immunoelectrophoresis of f r a c t i o n s obtained in Figure 56. P, blood plasma; F l and F2 f r a c t i o n s obtained in Figure 56; T F , t r a n s f e r r i n ; Ig, immunoglobulins, BSA, bovine serum albumin; abws, rabbit antibovine whole serum.  146  ELUTION VOLUME, ml  Figure 59. Immobilized Z n - , N i - and Co- a f f i n i t y chromatography of blood serum. Blood serum (2 g in 20 mL 0.05 M T r i s - H C l / 0 . 1 5 M NaCl, pH 8.0) was applied to the column (2.8 x 8.5 cm). The column was washed with the s t a r t i n g buffer then eluted (E) with 0.1 M Na-acetate/0.8 M NaCl, pH 4.6. The flow rate was 30 mL/h. 1, i s f r a c t i o n obtained.  147  Co-loaded  columns.  Fl  Figure 57  (lanes  2 and 3).  1,  fraction  obtained  from these  They 'are  columns are  composed of  Igs,  compared  TF and other  in  high  molecular weight proteins and appear to contain a small amount of albumin.  3. Blood plasma on MCIC column The plasma supplied had a red hue which was probably due to the hemolysis of sodium  red blood c e l l s during centrifugation  citrate  as  buffer,  pH 8.2,  portion  of  Igs  indicated  as  biological  anticoagulant.  was  applied  the proteins  that 0.1 2.8, from  R.I.D.  M acetate-Tris  To i d e n t i f y  t h i s colored m a t e r i a l ,  pH 4 . 5 ,  IDA-BGE Sepharose 6B; hemoglobin  removal.  other  metal  than  It  0.1  ions, zinc,  was found  M a c e t i c a c i d / . 5 M NaCl, pH  f o r e l u t i n g the adsorbed hemoglobin  however, is  It  elution  interesting  with to  50%  note  ethanol that  source of hemoglobin.  chicken  differences between  Figure 60 also shows the behavior of hemoglobin cobalt  and  nickel  from the columns loaded with metals other than Cu. interactions  was  e a s i l y with a c i d i c  ions.  It  was  found  hemoglobin was r e a d i l y eluted by using 0.1 M a c e t a t e / 0 . 8 M NaCl b u f f e r ,  hemoglobin-metal  957.  standard bovine hemoglobin  (our unpublished data) which may indicate s t r u c t u r a l  chicken and bovine hemoglobins. towards  major  pH 4.0 were  (with more  hemoglobin bound to a copper-loaded column was eluted quite buffers  at  the  Figure 60 shows the e l u t i o n patterns of hemoglobin  M imidazole were not e f f e c t i v e  for  buffer  and ELISA a n a l y s i s  of  M Tris-acetate  Co- and Cu-loaded IDA-BGE Sepharose 6B columns.  Cu-loaded  effective  0.05  a Cu-loaded IDA-BGE Sepharose 6B,  M a c e t a t e / 0 . 8 M NaCl b u f f e r ,  or 0.01  in  addition  The upper part of the Cu-loaded column, however, became  was applied to the column. from Z n - , N i - ,  When blood plasma  eluted with 0.05  by SDS-PAGE,  activity).  strongly reddish.  to  of blood a f t e r  partial  are dependent  on the  that  pH 4.5,  These r e s u l t s indicated that kind of metal  ions and the  148  Figure 60. E l u t i o n p r o f i l e s of adsorbed hemoglobin from MCIC columns (1.4 x 7.0 cm) loaded with Zn, N i , Co and Cu. 2 mL of hemoglobin (3 mg/mL in 0.05 M T r i s - H C l / 0 . 1 5 M NH C1, pH 8.0) was applied to the column and washed (W) with 2-3 times bed volumes of the s t a r t i n g b u f f e r . E l , 0.1 M Na-acetate /0.8 M NaCl, pH 4 . 5 ; E4, 50% ethanol. 4  149  B. IMMUNOCHEMICAL ASSAYS Table  14  compares the  IgG  contents  MCIC columns loaded with d i f f e r e n t the  highest  contained 58).  IgG  purity  in  Fl  different  ions.  (Figure  This indicated that not a l l  Ni-,  metal  some IgG which was evident  subclasses (IgGi,  of  However,  the  F2  (Figure  Fl  IgG was eluted at pH 4.0  obtained  from  56)  immunoelectrophoretogram (Figure 56);  IgG2) may bind more strongly than others.  and Co-loaded columns,  obtained  The copper-loaded column gave  56).  in  fractions  from  the  also  (Figure some IgG  By comparing Z n - ,  Ni-loaded  column gave  highest IgG p u r i t y while the Zn-loaded column gave the lowest IgG content. though  Co-loaded column gave  isolation  of  Igs  since i t s  high  IgG  capacity  purity  it  is  bind  Igs  was  to  not  the Even  recommended for  low  the  and decreased with  repeated use.  C. BACTERIOSTATIC ACTIVITY OF BLOOD IMMUNOGLOBULINS AND TRANSFERRIN Figure  61  shows  that  immunoglobulins  and  transferrin  isolated  by MCIC  method had i n h i b i t o r y e f f e c t s on the growth of E^ c o l i during the f i r s t compared to the c o n t r o l . Stephens  et  al.  (1980)  This r e s u l t s were in agreement with that reported by who  found  that  the  bacteriostatic  against E i c o l i could be enhanced by addition of l a c t o f e r r i n milk.  The i n h i b i t o r y  effect  of t r a n s f e r r i n  coli.  Transferrin,  microbial  growth  by v i r t u e  by making  this  of  its  element  activity  of  IgGi  i s o l a t e d from human  alone was higher than that of  However, mixing Igs with TF may have a s y n e r g i s t i c e f f e c t Ei  3 hr as  high  affinity  relatively  Igs.  on the i n h i b i t i o n for  iron,  unavailable  can  of  retard  (Harrison,  1985).  D. ANTI-LIPOPOLYSACCHARIDE ACTIVITY OF BLOOD IMMUNOGLOBULINS It the  i s well  known that antibodies are considered to be the architecture  immune system (Packard,  1982).  of  Human and animals are defenseless without  150  Table 14.  IgG contents* of blood serum or plasma f r a c t i o n s obtained MCIC column loaded with d i f f e r e n t metal  Fraction***  Protein  (mg/mL)**  from  ions.  IgG (mg/mL)  %IgG  Cu (Figure 56 Fl)  26.5  26.0  98.1  Cu (Figure 56 F2)  23.3  10.2  43.8  Cu (plasma) Zn (Figure 59 Fl)  26.0  25.0  96.2  21.6  5.0  23.2  Ni (Figure 59 Fl) Co (Figure 59 Fl)  24.6  20.0  81.3  25.2  20.0  79.4  immunodiffusion was used for the  determination.  *  Radial  **  Bio-Rad Protein Assay K i t used f o r the  (Bio-Rad Laboratories, Mississauga, Ont) was  determination.  * * * Serum f r a c t i o n unless otherwise noted.  151  Figure 61. B a c t e r i o s t a t i c a c t i v i t y of i s o l a t e d immunoglobulins and t r a n s f e r r i n against E. c o l i . C, c o n t r o l ; T F , t r a n s f e r r i n (10 mg/mL); M, mixture of TF (5 mg/mL) and Ig (5 mg/mL); Ig, immunoglobulins (10 mg/mL); CFU, c e l l forming unit ,  152 them.  The key c h a r a c t e r i s t i c s of the immune system are s p e c i f i c i t y , memory and  ability  to recognize foreign bodies.  isolated  from  blood  by  MCIC method  typhimurium and EL parapertussis. ^  Figure 62 shows the a c t i v i t y toward  infections,  whopping  cough  in  however,  infants  IgG  may  from  coli.  5^  Blood IgG recognition and binding to LPS from  typhimurium and E^. c o l i may indicate  bacterial  LPS extracted  of bovine IgG  that dairy  binding  indicate  to  JL  cows had experienced these parapertussis  similarities  in  the  which  surface  causes exposed  antigens to those of Enterobacteriaceae family.  The binding of blood IgG with  these  physiological  antigens  may  interfere  with  some of  the  activity  change the adhesion properties of these bacteria with the i n t e s t i n a l thus prevent,  to some extent, the  infection  or may  surface and  caused by these bacteria  (Packard,  1982).  E. CAPACITY OF MCIC COLUMN FOR TRANSFERRIN Figure  63  transferrin Saturation  shows the  solution of  the  calculated  binding  to  capacity  Lonnerdal  et  containing  50  recovered  only  copper  15%  loaded  bovine for  copper of  recovered by using 0.01  the  167  TF appears human ions.  eluent.  (acidic  pH)  application  Sepharose volume  At t h i s p o i n t ,  to  be  higher  lactoferrin,  protein;  than  which  was with  however,  M imidazole as an eluent.  of  of  6B  about  0.2%  column. 184 mL  the capacity for TF  mg/mL copper-loaded  Subsequent e l u t i o n  applied  and the  the  elution  column may indicate the biphasic nature of TF. protonation  from  chelating  an  protein was adsorbed.  (1977)  ymole  profile  reached at  be approximately for  al.  a  column was  i n d i c a t i n g no further was  to  elution  A28O  the  gel.  Thus,  the  that  reported  by  70  mg LF/mL  buffer  at  residual  pH TF  gel 4.0 was  This behavior of TF on MCIC  One phase was eluted  second phase was eluted  by using a  simply by stronger  153  1.0r  10  100  1000  ^9 ig  Figure 62. A n t i - l i p o p o l y s a c c h a r i d e a c t i v i t y of blood IgG i s o l a t e d chelate i n t e r a c t i o n chromatography method. E_j. c o l i LPS; typhimurium LPS; O - O , i L parapertussis LPS.  by metal • - • , S.  154  Figure 63. Saturation point of adsorption of standard TF on Cu-loaded IDA-BGE Sepharose 6B. 0.2% TF was passed through 10 mL column (7 x 1.4 cm) e q u i l i b r a t e d with 0.05 M T r i s - a c e t a t e containing 0.5 M NaCl, pH 8.2, W, wash with s t a r t i n g b u f f e r , E l , e l u t i o n with 0.05M T r i s - a c e t a t e containing 0.5M N a c l , pH 4 . 0 ; E2, e l u t i o n with 0.01 M imidazole.  155  F. MECHANISM OF PROTEIN-METAL INTERACTION Porath et a l . ,  in t h e i r pioneering work (1975), postulated that  cysteine and tryptophan  residues of a protein were most l i k e l y  coordination bonds with metal  ions at a neutral  or a l k a l i n e  pH.  have been no d e t a i l e d data published on the role of these in  the  interaction  with metal  ions.  Figure  64  to  histidine, form  stable  However,  there  amino acid residues  shows the  elution  profile  of  standard bovine t r a n s f e r r i n on Cu-loaded column before and a f t e r modification of h i s t i d i n e groups with DEP. starting alkaline buffer, eluted with 0.01  By washing the column, with three bed volumes of the no TF bound to the column was e l u t e d ; the bound TF was  M imidazole.  Modification of  16.6  histidine  the t o t a l  18 residues per mole TF (Sutton and Jamieson, 1972)  inhibited  the  interaction  protein  introduced into  binding  to  the  column.  copper ion would c l e a r l y  of  the  the  protein  column was restored  indicate  the  ions.  serum and column  yielded  mildness  of  regenerate starting  plasma. the  Radial highest  t h i s method for  of  the  without TF with  residues in  the  behavior was observed when  passed through  Cu-chelate  gel.  These  (1975).  that immunoglobulins can be i s o l a t e d from blood  immunodiffusion IgG  activity  i s o l a t i o n of  analysis (higher  Igs.  the Cu-loaded column with 50% ethanol  material.  histidine  Similar  r e s u l t s would support the theory of Porath et a l .  Almost a l l  histidine-modified  importance of  immunoglobulins were  These studies demonstrate  ion.  almost completely  in the washing buffer  The decreased binding of  coordination binding with the metal histidine-modified  with metal  residues out of  indicated  than  However,  95%), it  is  that  Cu-loaded  indicating  the  recommended to  when blood plasma i s used as  156  1.2  T  T  9  E e  O oo  .8 •  CM  6  a.  <  CD G£  O in  CD  <  30  60  ELUTION VOLUME.  Figure 64. Elution p r o f i l e s of control (TF) and diethyl pyrocarbonate treated t r a n s f e r r i n (DEP-TF). Samples (30 mg/5 mL 0.05 M T r i s - a c e t a t e / 0 . 5 M NaCl, pH 8.2) were applied to the column (1.4 x 7.0 cm) and washed (W) with the s t a r t i n g buffer then eluted (E) with 0.01 M imidazole. The flow rate was 30 mL/hr.  SEPARATION OF OVOTRANSFERRIN FROM EGG WHITE BY METAL CHELATE INTERACTION CHROMATOGRAPHY  158  The greater  resistance against  enterobacterial  fed with breast milk than those fed with a r t i f i c i a l to a great milk  extent,  activity  antimicrobial al.,  1983;  (Packard, 1982).  between  in human  in structure  lactoferrin  and  justify  the  e f f e c t of ovotransferrin being added to infant formula (Valenti et  Giacco-Del  thesis  and  infants  attributed,  of l a c t o f e r r i n  The s i m i l a r i t y  ovotransferrin  et  al.,  1985).  In  ovotransferrin does not s e n s i t i z e infants ofthe  formula has been  to the presence of a large quantity  compared to cow's milk  biological  i n f e c t i o n of human  deals with  the  addition,  it  has been  (Giacco-Del et a l . ,  separation  and the  found  1985).  mechanism of  that  This part  the  binding  of  ovotransferrin by using MCIC method.  A. METAL CHELATE-INTERACTION CHROMATOGRAPHY OF EGG WHITE It rich  i s known that the  in electrons  heterocyclic,  (Porath  transition et  al.,  including proteins  metals can form a complex with compounds  1975). due to  These compounds may be aromatic their  However, the binding of these groups to metal  contents  or  of Cys, H i s , and Tyr.  ions depends on the  availability  of these groups or the topography of the protein molecule (Sulkowski, 1985). Figure 65 represents the elution p r o f i l e of undiluted, blended egg white on copper-loaded Sepharose 6B. stage the unbound material  As indicated by SDS-PAGE (Figure 66), was mainly ovalbumin, however,  at  the  at an early later  stage,  most of egg white proteins passed through suggesting the column had reached i t s saturation  point.  During the washing step, another  eluant which, based on electrophoretic pattern, f e r r i n and lysozyme . be pure eluted  The bound material  ovotransferrin, with  0.01M  Immunoelectrophoresis  purer  imidazole  than (peak  conducted  the  commercial was  (FW)  appeared in  could be a mixture  eluted at pH 4.0  2),  against  peak  anti-egg  of ovotrans-  (peak 1) appeared to  ovotransferrin.  likely  to  white  the  be  The  protein  ovotransferrin.  proteins  antiserum  159  Figure 65. Metal c h e l a t e - i n t e r a c t i o n chromatography of egg white. 2 mL of undiluted blended egg white was passed through Cu-loaded Sepharose 6B MCIC column (7 x 1.4 cm). UB, unbound p r o t e i n s ; FW, f r a c t i o n eluted with washing step; E l , e l u t i o n with s t a r t i n g b u f f e r , pH 4 . 0 ; 1, f r a c t i o n eluted with E l ; E2, e l u t i o n with 0.01M imidazole; 2, f r a c t i o n eluted with E2.  160  1 2 3  4 5 6 7 8 9  10  Figure 66. SDS-PAGE of f r a c t i o n s of egg white obtained by MCIC column shown in Figure 65. Lanes 1, 2 and 3, are unbound f r a c t i o n s ; Lane 4, washing f r a c t i o n ; Lane 5, peak 1; Lane 6, peak 2; Lanes 7 and 8, standard ovotransferrin and ovalbumin, r e s p e c t i v e l y ; Lanes 9 and 10, control egg white.  161  indicated that the ovotransferrin f r a c t i o n pure  being  free  ovotransferrin  from  (Figure  contaminants  (peak 1) obtained by MCIC was  which  were  observed  in  the  fairly  commercial  67).  B. CAPACITY OF MCIC COLUMN FOR OVOTRANSFERRIN Figure solution  68  shows  (A28O = 1*94)  The column was eluted  the  calculated  to  at  reached  be  elution  1.94.  appeared  the  bound OVT,  to be lower  lactoferrin  than  volume of  At  approximately  e l u t i o n with buffer at pH 4.0 and 30% of  profile  when  20  (El)  this  commercial  point,  60 mL when A28O  the  capacity  for  mg OVT/mL copper-loaded g e l . and with 0.01  respectively. that  about  reported  M imidazole (E2)  Thus,  the  OVT  o  f  t  n  e  OVT was  Subsequent recovered 65%  binding  capacity  et  (1977) for human  by Lonnerdal  al.  of OVT  ( L F ) , which was 70 mg LF/mL gel containing 50 umol copper ions.  The desorption of OVT in two steps may indicate two forms of OVT. weakens  0.2%  was applied to a copper-loaded chelating Sepharose 6B.  saturated  fraction  elution  A28O  the  One form can be eluted  binding  of  proteins  with  the  the presence of at  by lowering metal  ions  the by  pH.  The  protonation  least  acidity of  the  protein electron donor groups which are responsible f o r binding with the metal ions.  The  second form  imidazole.  Imidazole  can  be  can e f f i c i e n t l y  protein for metal binding.  in multipoint  by  using  a  strong  competitor,  i.e.  compete with exposed His groups on the  As reported by Sulkowski (1985), the presence of one  h i s t i d i n e residue i s s u f f i c i e n t results  eluted  attachment  for retention while to  the presence of 2 or 3 His  IDA-Cu gel and a stronger  retention.  The  presence of two forms of conalbumin reported by Clark et al.(1963) and Feeney et al.  (1963)  reported  was discussed  two  forms of  DEP-non-reactive.  by Powrie  histidine  in  and Nakai  (1986).  OVT, one was  Rogers et  reactive  and  the  al.  (1977)  other  was  162  Figure 67. Immunoelectrophoresis against anti whole egg white antiserum of ovotransferrin f r a c t i o n (F) prepared by the MCIC method as compared to commercial ovotransferrin (OVT), and egg white (EW).  163  Figure 68. Saturation p r o f i l e of commercial ovotransferrin on Cu-loaded Sepharose 6B column. 0.2% ovotransferrin was passed through 3 mL of a Cu-loaded column (7 x 1.4 cm). E l , e l u t i o n with 0.05 M T r i s - a c e t a t e / 0 . 5 M NaCl, pH 4 . 0 ; E2, elution with 0.01 M imidazole. 1 and 2 are eluted ovotransferrin.  .164  C. MECHANISM OF OVOTRANSFERRIN SEPARATION BY MCIC The binding of OVT to metal-chelate gel i s believed to be the result of the a b i l i t y of e l e c t r o n - r i c h groups such as h i s t i d i n e and tryptophan weakly bonded water or buffer The s t a b i l i t y  to  substitute  in the metal complex (Lonnerdal and Keen, 1982).  of the binding even in 1M NaCl would rule out the p o s s i b i l i t y of  ionic interaction  being the p r i n c i p a l  force in the i n t e r a c t i o n .  whether the metal binding a b i l i t y of OVT has any role  To demonstrate  in the mechanism of OVT  separated by MCIC, metal bound OVT was applied to the MCIC column.  Figure 69  represents the e l u t i o n p r o f i l e of metal free OVT (apo-OVT), f e r r i c - and coppersaturated OVT from the MCIC column.  E v i d e n t l y , an i r o n - or copper- containing  OVT was adsorbed on a Cu-chelate gel and subsequently e l u t e d . shows  that  metal-saturated  apo-form of protein  OVT.  Before and a f t e r  contained  two  (Lonnerdal  and  subsequent  dialysis,  1985).  Keen,  However,  tendency  OVT bound with  atoms  of  1982). OVT has  Fe  chromatography 3 +  After two  MCIC  and  Cu  addition  bound metal  on Cu-chelate g e l ,  2 +  of  column of per  as  Figure 69 also strongly  as  the  Fe-OVT and Cu-OVT,  the  molecule,  excess  Fe  3 +  respectively or  atoms per mole protein  and  (Brock,  surface-exposed groups on OVT may have a  if  h i s t i d i n e groups were responsible f o r the copper-chelate  gel and OVT i n t e r a c t i o n , DEP modified OVT was applied to the column. shows the e l u t i o n  profile  of OVT before and a f t e r  Figure 70  h i s t i d i n e group modification  Without m o d i f i c a t i o n , OVT was adsorbed strongly to the Cu-chelate gel  and no OVT was eluted  in  the washing  step at  subsequently be recovered by 0.01M imidazole. did  2 +  to bind copper i o n s .  To investigate  by DEP.  Cu  alkaline  pH.  Bound OVT could  H i s t i d i n e modified OVT (DEP-OVT)  not bind to Cu-chelate gel and was almost completely washed out during the  washing step at a l k a l i n e pH.  This behavior of DEP-OVT on Cu-chelate gel  indicated  of  that  modification  11.7  histidine  residues  out  of  13  clearly  histidine  165  o  1  0  •  30 60 ELUTION VOLUME, ml  Figure 69. Metal chelate i n t e r a c t i o n chromatography of apo-ovotransferrin (APO-OVT), F e - o v o t r a n s f e r r i n (Fe-OVT) and Cu-ovotransferrin (Cu-OVT). 3 mL (8 mg/mL) was applied to Cu-loaded Sepharose 6B (7 x 1.4 cm) a f t e r e q u i l i b r a t i o n with 0.05 M T r i s - a c e t a t e / 0 . 5 M NaCl, pH 8.2. W, Washing with the e q u i l i b r a t i n g b u f f e r ; E, e l u t i o n with 0.01 M imidazole; flow rate was 30 mL/hr.  166  o • • • 0  •  • • , ,  30 60 ELUTION VOLUME, ml  Figure 70. Metal chelate i n t e r a c t i o n chromatography of control ovotransferrin (OVT) and diethyl pyrocarbonate treated ovotransferrin (DEP-OVT). 3 mL (8 mg/mL) was applied to Cu-loaded Sepharose 6B (7 x 1.4 cm) after e q u i l i b r a t i o n with 0.05 M T r i s - a c e t a t e / 0 . 5 M N a c l , pH 8.2. W, washing with the e q u i l i b r a t i n g b u f f e r ; E, e l u t i o n with 0.01 M imidazole; flow rate was 30 mL/hr.  167 residue  per  mole  OVT  Cu-chelate matrix. binding  in  the  drastically  inhibited  the  proteins'  Fe- or Cu- saturated  OVT, there are  h i s t i d i n e groups would destroy the a b i l i t y  separate  conclusion,  this  ovotransferrin  specificity  to  bind  Considering two h i s t i d i n e residues are involved in the metal still  11 h i s t i d i n e  l e f t free to i n t e r a c t with immpbi1ized. metal on Sepharose 6B.  In  ability  part  of  However, blocking  of OVT to bind MCIC column.  demonstrates  for  the  from egg white by a single  and capacity  groups  MCIC column for  first  time  a  chromatographic  ovotransferrin  are  method step.  high,  should be easy to adapt the method f o r i s o l a t i o n of ovotransferrin  to The  and  it  to a larger  scale operation.  The i s o l a t e d OVT from egg white may be incorporated in  infant  formula,  it  babies  since,  has  (Giacco-Del et a l . , 1985). enriched milk determined  was fed  by the  to  not  had  sensitizing  effects  on  OVT-treated  Giacco-Del (1985) reported that when o v o t r a n s f e r r i n 15 babies  for  60 days the  radioimmunoassay method,  remained  values within  of the  total normal  IgE,  as  range.  168  CONCLUSIONS AND RECOMMENDATIONS A hexametaphosphate method was developed for minimizing p-Lg and maximizing Ig  in whey.  The new method avoids saturation  of  iron binding proteins  abolishes t h e i r b a c t e r i o s t a t i c a c t i v i t y and also avoids u l t i l i z a t i o n  which  of non-food  grade chemicals which would require regulatory approval, compared to SHMP which i s already approved as a food grade chemical. For  isolation  skimmilk,  of  bioactive  proteins,  MCIC  blood plasma and serum and egg white  treatment is  of  cheese  whey,  recommended, based on the  following f i n d i n g s :  (1)  Ig  of almost 90% p u r i t y  can be recovered from cheese whey using a simple  process, with p r a c t i c a l l y no pre-treatment of whey. (2)  The MCIC has high capacity for  Ig  i s o l a t i o n from cheese whey, at  least 1  l i t r e of whey per 25 ml copper-loaded g e l . (3)  Lactoferrin  and bovine  serum albumin may also  be  separated  from cheese  whey. (4)  The treated whey or unbound f r a c t i o n  contains mainly p - l a c t o g l o b u l i n  and  a-lactalbumin in concentrations s i m i l a r to the untreated wheys. (5)  This  method  can be  ovotransferrin potential (6)  used for  extraction  from egg white.  of  Ig  The preliminary  TF  from  experiments  blood,  and  indicate  the  of t h i s method to i s o l a t e Ig d i r e c t l y from skimmilk.  The fact that chemical modification of h i s t i d i n e groups in Ig, rich  fraction  and casein  columns supports the interaction  fractions,  idea of  the  destroys coordinate  involvement  of  The  isolated  immunoglobulins  from  colostrum,  T F , OVT, LF  binding  histidine  with copper immobilized on g e l ; however,  conditions may activate the other mechanism of the (7)  and  to MCIC  groups  changing the  in  the  elution  interaction. cheese  whey  and  blood  169  recognize LPS i s o l a t e d from EL col i , These  results  may  encourage  the  S_j_ typhimurium and B_j_ parapertussis.  addition  of  the  bioactive  proteins  to  infant formula to give s i m i l a r performance as human m i l k . (8)  The MCIC column can be e a s i l y regenerated f o r r e - u s e .  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V . , Morr, C.V. and Swaisgood, H.E. 1976. Nomenclature of the proteins of cow's milk: Fourth r e v i s i o n . J . Dairy S c i . 59:795. Wilkinson, York.  A.W.  1981.  The  Immunology  of  Infant  Feeding.  Plenum Press,  W i l l i a m s , J . 1962. A comparison of conalbumin and t r a n s f e r r i n fowl. Biochem. J . 83:355.  New  in the domestic  Williams, C A . and Chase, M.W. 1971. Methods in Immunology and Immunochemistry v o l . 3. Academic Press, New York, NY. W i l l i s , A . T . , B u l l e n , C . L . , Williams, K . , Fagg, C . G . , Bourne, A. and Vignon, M. 1973. Breast milk s u b s t i t u t e . A b a c t e r i o l o g i c a l study. Br. Med. J . 4:64. Workshop P a r t i c i p a n t s . 1976. Human milk in premature a workshop. P e d i a t r i c s 57:741. World Health organization Geneva.  1981.  Contemporary patterns  infant feeding: Summary of  of breast feeding.  Zacharius, R.M. and Zell, T . E . , Morrison, J . H . , Woodlock, J.J. Glycoprotein staining following electrophoresis on acrylamide g e l s . Biochem. 30:148.  WHO,  1969. Anal.  Z i t t l e , C A . and Custer, J . H . 1963. P u r i f i c a t i o n and some of the properties of a i - c a s e i n and K - c a s e i n . J . Dairy S c i . 46:1183. s  184  APPENDIX: NON-FERRIC METHODS FOR B-LACTOGLOBULIN REMOVAL FROM CHEDDAR CHEESE WHEY  185 Different  approaches were t r i e d  to s e l e c t i v e l y eliminate  B-Lg from cheese  whey.  1. Amundson and Watanawamchakorn approach Amundson conductivity adjustment when  and of  of  this  the pH to 4.65 would r e s u l t  acidification)  Inc.,  (1982)  claimed  that  adjustment  80% volume reduced e l e c t r o d i a l y z e d whey to  method  conductivity  Watanawamchakorn  was  tried  on  and cheese whey a f t e r (conductivity  acid  in 53.3% protein whey  (prepared  of  100-200 uMHOS and removal.  from  raw  However, milk  extensive d i a l y s i s and adjustment  Bridge Model  31,  Yellow  the  by  of  Springs Instruments,  the Co.,  Ohio) by addition of 1 M NaCl, i t did not work.  2. S o l u b i l i t y d i f f e r e n c e s of P-Lg and Ig at d i f f e r e n t pHs The pH of range 4-8,  1% p-Lg or Ig was adjusted with 0.1  N NaOH or 0.1  N HC1 to the  and the protein content of the supernatant was determined according  to the method of Nakai and Le (1970) a f t e r  10,000 x g .  The  lowest s o l u b i l i t y of p-Lg was found at pH 4.7 and i t was 46% of the total  p-Lg  in the o r i g i n a l s o l u t i o n .  The lowest s o l u b i l i t y of crude Ig was found around pH  5.8 and i t was about 50% of the total To  check  performed. rate  the  c e n t r i f u g a t i o n at  possibility  F i r s t , gel f i l t r a t i o n  of  Ig in the o r i g i n a l s o l u t i o n . denaturation  of  P-Lg,  two  tests  were  of p-Lg on TSK HW-55 column (15 x 1.5 cm, flow  1 mL/min, eluted with 50 mM i m i d a z o l e - K C l , pH 6.5)  showed no evidence of  aggregation in the e l u t i o n patterns of standard p-Lg used, which suggested that P-Lg was n a t i v e .  The second denaturation  test  depended on the  solubility  globular proteins at t h e i r i s o e l e c t r i c point in the presence of 0.2 M NaCl.  of It  was found that more than 90% of P-Lg was soluble as shown by absorbance at 280nm under the above conditions a f t e r  centrifugation at 10,000 X g .  186  To study the the e f f e c t  of pH on the s o l u b i l i t y , another attempt was made  to d i s s o l v e 1% B^Lg and Ig at 5.2,  5.6,  However,  and the  6.0,  results  with  citrate  buffer  conductivity  indicated  in  in d i f f e r e n t  the  range  that no p r e c i p i t a t i o n  pH values 4.2,  of of  4000  -  4.6,  8000 uMHOS.  p-Lg occurred whereas  minor t u r b i d i t y was noticed in the Ig s o l u t i o n s .  3. Combination of Amundson and Watanawamchakorn and Pearce methods According  to Amundson and Watanawamchakorn  (1982) pH, i o n i c strength and  the concentration of whey are the major factors influencing the p r e c i p i t a t i o n of B-Lg, while according to Pearce (1983) the pH, temperature and the concentration of  whey  are  fraction. to  the  major  factors  influencing  the  preparation  of  enriched  p-Lg  In our study an attempt was made to combine these factors in addition  other  factors  i.e.,  c y s , KI  and CaCl£.  The ranges of  the  values of  these  factors were as f o l l o w s :  Ionic total  strength  (I)  = 0.0-0.2,  pH = 4 . 5 - 5 . 0 ,  s o l i d (TS) = 6.5-20%, cysteine  temperature  (T)  = 25-50°C,  (Cys) = 0.0-0.2%, potassium iodide  (KI)  =  0.0-0.2%, calcium chloride (CaCl2) = 0.0-0.2%.  According to Taguchi's scheme (1957) for f r a c t i o n a l possible  interactions  were chosen to  be IXpH,  TXpH,  factorial  IXT,  KIXI,  a n a l y s i s , the IXCa,  CaXCys,  IXCys. Calcium free  acid whey was prepared by the  addition of potassium oxalate  followed by d i a l y s i s for 48hr against d i s t i l l e d water. performed  (Taguchi,  1957,  L15  for  two  levels).  A set of experiments was  Experiments were carried  out  in random order and SDS-PAGE was used to evaluate the separation e f f i c i e n c y of P-Lg and a-La in each experiment.  187 ANOVA was interactions,  performed  and i t  (using  a  Monroe  was concluded that I,  1880  Calculator)  for  factors  TS, pH, Cys, and Ca were  factors while KI and temperature were n o n s i g n i f i c a n t .  and  important  Simplex optimization was  then applied for the f i v e factors with lower l i m i t s and upper l i m i t s as follows: 1(0.0-1.00),  TS (6.5-20%),  v e r t i c e s were experimented  pH (4.5-5.0)  and (0.0-0.2 M) f o r  and SDS-PAGE showed that the  Cys and Ca.  optimization  of  Six these  factors f a i l e d , therefore we could not s e l e c t i v e l y p r e c i p i t a t e p - L g . In an attempt to an Amber!ite  remove Ca-phosphate from whey under a l k a l i n e  anion exchanger was used to adjust  condition,  the pH of cheese whey to  8.0.  However,the amount of Ca-phosphate removed by t h i s process was much lower  than  that obtained by adjusting the pH using sodium hydroxide.  4. P r e c i p i t a t i o n of B-Lg by polyethylene glycol The behaviour glycol(PEG) 1984).  has  of  been  solutions containing globular investigated  by many  PEG provides an a t t r a c t i v e  precipitation.  It  allows  alternative  regulation  of the pKa of the  (Atha and Inghams,  1981).  In  chemists  polyethylene  (Haire  to other agents used f o r  of protein  e f f e c t s on protein structure and f u n c t i o n . PEG i s the s h i f t  protein  proteins and  et  al.,  protein  s o l u b i l i t y without observable  However the only noticable e f f e c t of  ionizable groups at  some s i t u a t i o n s ,  the  high PEG concentrations  use of  PEG has a  definite  advantage over the use of high s a l t concentrations, where s a l t i n g in and s a l t i n g out phenomena and s p e c i f i c interactions with both cation and anion both the relevant  interpretation state  An a l i q u o t  and the extrapolation  to a well  defined,  complicate  biologically  (Roth et a l . , 1979). of  forty  percent  (W/V)  PEG was added to  with good mixing to give f i n a l  PEG concentrations of 6.7,  20%.  at  Samples were  then  kept  room temperature  for  20 mL of cheese whey 9.2,  11.4,  13.3  an hour and were  and then  

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