Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Characterization of the very low density lipoproteins and apoproteins of egg yolk granules Kocal, James Thomas 1977

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata


831-UBC_1978_A1 K62.pdf [ 6.64MB ]
JSON: 831-1.0094640.json
JSON-LD: 831-1.0094640-ld.json
RDF/XML (Pretty): 831-1.0094640-rdf.xml
RDF/JSON: 831-1.0094640-rdf.json
Turtle: 831-1.0094640-turtle.txt
N-Triples: 831-1.0094640-rdf-ntriples.txt
Original Record: 831-1.0094640-source.json
Full Text

Full Text

CHARACTERIZATION OF THE VERY LOW DENSITY L I P O P R O T E I N S AND APOPROTEINS OF EGG YOLK GRANULES  by  JAMES THOMAS KOCAL  B.Sc, M.Sc,  University  i  of I l l i n o i s ,  1972  U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1974  A T H E S I S SUBMITTED I N P A R T I A L F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF i  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF FOOD SCIENCE U N I V E R S I T Y OF B R I T I S H COLUMBIA  We a c c e p t t h i s to  t h e s i s as conforming  the required  standard  THE U N I V E R S I T Y OF B R I T I S H COLUMBIA O c t o b e r , 1977 (P)  J a m e s Thomas K o c a l ,  1977  In  presenting  an  advanced  the I  Library  further  for  this  thesis  degree shall  agree  scholarly  at the University make  that  p u r p o s e s may  h i s representatives.  of  this  thesis  University  2075  Wesbrook  Vancouver, V6T  Date  of  1W5  gain  Rn'ftnpp  Place  Columbia  Columbia,  f o r reference copying  by t h e Head  I t i s understood  of British  Canada  of British  f o rextensive  be g r a n t e d  f o r financial  Fooci  f u l f i l m e n t o f the requirements f o r  available  permission.  Department The  i t freely  permission  by  written  in partial  shall  that  n o t be a l l o w e d  and  thesis  Department or  that  study.  of this  o f my  copying  I agree  or  publication  without  my  -  i  -  ABSTRACT  The of  very  hen's  egg  low d e n s i t y yolk  were  lipoproteins  isolated  by  ultracentrifugation  and  electrophoretic  chromatographic  characterize The  and  and  assess  Beckman in  Prep  was  used  the  determination  software system An in  that  is  gel  purity  Scanner with  an  (MF))  combination  of  the  VLDL  accessory on-line  to  to  permit  rapid  granules  preparative  performed  (MF) the  data  the  Ultracentrifugal,  were  and  to  apoVLDL.  ultracentrifuge  acquisition  flotation/sedimentation  developed  of  filtration. analyses  from  system  coefficients. data  analysis  for  The using  this  described.  agarose  combination  allowed  was  the  UV  conjunction of  agarose  a  (VLDL  for  disc  gel  with  rapid  the  electrophoretic use  of  s y s t e m was  sudan b l a c k  electrophoretic  developed  B prestained  analysis  of  large  which,  lipoproteins,  VLDL  (MF)  particles. Flotation preparation Alkylation due of  to  velocity  with of  (MF)  of  egg  (MF)  particles  with  to  exist  of  floating  S^ in  VLDL  (MF)  41  groups and  indicated  boundaries  iodoacetamide  sulfhydryl  the macromolecules  VLDL  to  (S^  prevent  decreased  108S.  a partially  It  75  is  the  a  and  207S).  aggregation  flotation  possible  aggregated  heterogeneous  state  rates  that  the  in  fresh  yolk. Concanavalin  a  two m a j o r  VLDL  oxidation  analysis  retained  and  an  A affinity  chromatography  of  unretained  fraction.  retained  The  VLDL  (MF)  produced  fraction  -  contained VLDL  twice  (MF),  as  while  much  the  A l l  hexosamine  sialic  VLDL  (MF)  was  preferentially solubility were  of  fractions  extracted  by  and  (MF)  gel  was  ethanol-ether  produced  could  That  be  presence  aggregate  of  extracted  from  heterogeneous,  9,600-136,000 ethanol  (BME).  staining only  18  weight  from  in  the  TMU  gels  in  positively  with  but  used  of  of  by  by  was  to  analytical ratios. (NaDOC)  both  methods remained  precipitated, (SDS)  or  d e l i p i d a t e VLDL analysis one  that  the  of  but  urea. (MF)  and  indicated  w h i c h was of  each  the band  an  proteins was  polypeptides. 22  polypeptides  presence  these  Schiff  present  hexose,  fractions  deoxycholate  electrophoresis  into  the  much  which  t h e NaDOC a p o p r o t e i n  demonstrated  many  Two  lipids  subunits,  SDS  resolved, not  sodium  Electrophoretic  two.  Fourteen  aggregates  as  preserved  characterized  dodecylsulfate  was  dissociated  daltons)  and  phospholipid/protein  apoprotein  containing  bands were  half  containing  residues.  ethanol-ether  sodium  (TMU)  other  ApoVLDL was  contained  lipids  Removal  apoprotein,  three  the  and  their  apoprotein.  of  unfractionated  residues.  neutral  procedures.  Tetramethylurea  the  the  by  obtained  the  the  glycoproteins  delipidated using  solubilized  dissociate  sugar  filtration and  aggregated  soluble.  were  (MF)  phospholipid-protein  ultracentrifugation VLDL  VLDL  as  p a r t i a l l y d e l i p i d a t e d w i t h n-heptane  the  isolated  acid  -  carbohydrate  unretained  carbohydrate. and  total  i i  of  bands  were  reagent.  most  of  in  the  urea,  which  In  (MW  SDS  and  glycoprotein the  absence  contained  reduced  ranging  samples.  from  2-mercaptoin of  higher  nature, BME, molecular  -  The 8M u r e a SDS  apoVLDL was or  void  and  volume  proteins. from  A  the  SDS  The  molecular  were  from  urea  column  the  weights  analyzed  by  6%  was  column. an  found  the  columns  were The  aggregate  between  proteins of  agarose  fractions  the-urea  column  and  on  apoprotein  c o r r e l a t i o n was  the  column  Two  three  of  -  fractionated  2mM S D S .  column,  i i i  the  extracted  two  eluted  protein of  the  from  the  from  equilibrium  from from  other  fractions  apoproteins  sedimentation  containing the the two  eluted TMU  the  gels. SDS  techniques.  2 As  plots  weights  of were  meniscus  to  ln  c vs.  r  were  calculated the  bottom  for of  ranged  from  molecular  weight  component  corresponded  polypeptides  33,000  well  weight  each measured  the  component  values  nonlinear,  cell. to  166,000  ranged  with  c h a r a c t e r i z e d by  The  from  radial  daltons 6,000  gel  to  molecular  distance  high molecular  the molecular SDS  average  and  the  low  ranges  electrophoresis.  the  weight  28,000.  weight  from  These of  the  -  iv  TABLE  OF  -  CONTENTS  Page  ABSTRACT TABLE  i  OF CONTENTS  LIST  OF T A B L E S  LIST  OF F I G U R E S  .  .  „  v i i i  SYMBOLS .  .  . x i i  ACKNOWLEDGEMENTS  II. III.  iv  ix  A B B R E V I A T I O N S AND  I.  .  xiv  INTRODUCTION  1  LITERATURE REVIEW  3  EXPERIMENTAL PROCEDURES A.l.  A . 2.  Isolation  of  VLDL  (MF):  Preparative ultracentrifugation.  14  c.  Agarose  14  d.  Preparation in  e.  A l k y l a t i o n of of  VLDL  granules  .12  b.  gel  yolk  lipoproteins.  Isolation  Isolation  of  P r e p a r a t i o n of  a.  the presence VLDL  (MF):  filtration  A f f i n i t y chromatography  n-heptane  reducing  agents.  fractionations.  c o n t r o l l e d pore on  glass  Concanavalin  . . .  beads  .  17 .17  A-  4B VLDL(MF):  16 .17  Further  b.  using  on  of  (MF)  Gel  D e l i p i d a t i o n of  12  chromatography  a.  Sepharose B. l .  10  18 Partial  delipidation 19  - v -  B.2.  C l .  D.l.  Delipidation  VLDL  (MF) : T o t a l  a.  Ethanol-ether  b.  Sodium deoxycholate  Agarose  gel  delipidation  21  delipidation  filtration  21  detergent  of  delipidation . . . . .  apoVLDL  25  Gel  filtration  on  Sepharose  6B  containing  8M u r e s . .  .25  b.  Gel  filtration  on  Sepharose  6B  containing  SDS  .25  A n a l y t i c a l procedures: prestained  Analytical of  VLDL  Electrophoretic  .  .  .  separation  lipoproteins  procedures:  26  Analytical  ultracentrifugation  (MF)  a.  Data  acquisition  and  handling  b.  Flotation/sedimentation  of  data  velocity  „  .28  .  .29  ultra-  centrifugation D. 3 .  23  a.  of D.2.  of  Analytical  33  procedures:  Physical  studies  of  VLDL  (MF)  apoproteins a.  35  Calibration weight  of  Sedimentation  c.  Polyacrylamide  d.  for  molecular  disc  Extraction  Staining  35  equilibrium  Electrophoresis i.  columns  determinations  b.  i.  Sepharose  of  gel  studies  electrophoresis  of  protein  in  the  glycoproteins  Chemical  analyses:  Amino  E.2.  Chemical  analyses:  Carbohydrate  Hexose  and  b.  Sialic  acid  from urea  presence  E. l .  a.  acid  hexosamine  37  of on  in  TMU . . .  acrylamide  gels.42  SDS acrylamide  analysis  38  42 gels .  .45 45  analysis  46  determination  46  determination  47  - v i -  IV.  E. 3.  Chemical analyses:  SH a n d SS d e t e r m i n a t i o n  F. 1.  UV a b s o r p t i o n s t u d i e s  .48 49  RESULTS AND D I S C U S S I O N  50  A.  D a t a a c q u i s i t i o n and h a n d l i n g o f d a t a  50  B.  I s o l a t i o n o f VLDL  54  C.  (MF)  1.  Isolation of F I I fraction  54  2.  Agarose g e l chromatography  55  3.  Alkylation  59  4.  Gel filtration  on porous g l a s s beads  Flotation velocity ultracentrifugation 1.  UV a b s o r p t i o n s p e c t r u m  2.  Flotation velocity analysis o f VLDL  o f VLDL  (MF) . . .  66  Electrophoretic behavior  E.  Affinity  (MF)  F.  Delipidation  o f VLDL  1.  Partial  d e l i p i d a t i o n u s i n g n-heptane  2.  Total delipidation with ethanol-ether  3.  Sodium d e o x y c h o l a t e  chromatography on Concanavalin  74 A - S e p h a r o s e 4B . .78  (MF)  detergent  64  . . 64  D.  85 85 . .89  delipidation  91  G.  Amino a c i d  o f apoVIDL  94  H.  E l e c t r o p h o r e s i s o f V L D L (MF) i n t h e p r e s e n c e o f TMU . . .  96  I.  Molecular by  J.  composition  63  w e i g h t e s t i m a t i o n o f apoVLDL p o l y p e p t i d e s  SDS-polyacrylamide  Agarose g e l f i l t r a t i o n  gel electrophoresis o f apoVLDL  100 105  - vii -  V.  SUMMARY A N D  LITERATURE APPENDIX  CITED  CONCLUSIONS  113 116 125  -  LIST  v i i i  OF  -  TABLES Page  Classification Commercial -  UV  used  Weight of  SH  Ellman's  Comparative  Markers  Ultracentrifuge  System  for  and  30  C a l i b r a t i o n of  Buffers  Markers and  SS  for  for SDS  Gel  TMU E l e c t r o p h o r e s i s . Electrophoresis.  Contents  of  VLDL  .40 43  (MF) 62  (S^)  VLDL  Composition  of  of  Native,  Reduced  (MF)  70  Apoproteins  from  Fractionated Lipoproteins  Sedimentation  . . .  Reagent  F l o t a t i o n Rates  Carbohydrate and  the  4  36  Chemically Modified  Whole  in  Lipoproteins  Columns  Estimation  and  Serum  Mini-Computer  Solut ions  Molecular  using  -  Weight  Filtration Reagent  Human  Equipment  Scanner  Molecular  of  Coefficients  (S  ,  )  of  .80 VLDL  (MF)  obs and  VLDL  Affinity Amino  Fractions  from Concanavalin  A  Chromatography  Acid  Granule, Human  (MF)  Composition  Yolk  Serum  Plasma,  VLDL  82 of  Apoproteins  Whole  Yolk,  Hen  from  Yolk  Serum, 95  - ix-  LIST OF FIGURES Figure  Page  1  P r e p a r a t i o n o f VLDL (MF)  2  U l t r a c e n t r i f u g a l r e d i s t r i b u t i o n o f egg y o l k  13  granule l i p o p r o t e i n s  15  3  Ethanol-ether  22  4  Data f l o w from t h e Beckman Prep UV Scanner and  d e l i p i d a t i o n o f VLDL (MF)  Ultracentrifuge 5  UV Scanner t r a c e s and d e r i v a t i v e c u r v e s f o r (A) one and (B) two component systems  6  53  E l u t i o n p r o f i l e s o f y o l k g r a n u l e s , F I and F I I f i l t e r e d on B i o g e l A-50M columns  8  52  L i n e a r r e g r e s s i o n p l o t f o r two component system i n f l o t a t i o n v e l o c i t y run  7  31  55  E l u t i o n p r o f i l e s o f (A) a l k y l a t e d VLDL (MF) and (B) 2 - m e r c a p t o e t h a n o l reduced VLDL (MF) from porous g l a s s bead columns  9  UV a b s o r p t i o n spectrum o f VLDL (MF) (A) as a f u n c t i o n o f w a v e l e n g t h and (B) as l o g - l o g f u n c t i o n . . .  10  68  H y d r a t e d d e n s i t y o f VLDL (MF) e s t i m a t e d from ^ s and s o l v e n t d e n s i t y a t 20°C  12  67  F l o t a t i o n v e l o c i t y a n a l y s i s o f VLDL (MF) (A) f l o t a t i o n p a t t e r n (B) d e r i v a t i v e c u r v e  11  55  71  Agarose g e l e l e c t r o p h o r e s i s p a t t e r n o f (A) sudan b l a c k B p r e s t a i n e d and (B) Coomassie B l u e s t a i n e d VLDL (MF)  76  13  Elution Con A  14  SDS in  profile  of  alkylated  Sepharose  4B  column  polyacrylamide the  presence  retained stained 15  16  18  fractions with  Sepharose  4B  VLDL  „'  (MF)  of  Coomassie  Sepharose  Blue of  and  filtration  in  the  of  tetramethylurea  (TMU)  (B)  TMU  SDS  polyacrylamide  insoluble  of  BME  and  (B)  (R)  column  PAS  84  delipidated  VLDL  (MF)  of  NaDOC.  apoVLDL by  VLDL  (MF)  (A)  VLDL, gel  in  whole  and  (C)  the  filtration  VLDL  .92  .  .  VLDL . . . .  97  of  (MF), soluble  Fraction  TMU  soluble  Band  3/Sepharose  (C)  TMU  soluble  Band  2  SDS  polyacrylamide  Band  6B,  6B,  the  Fraction  gel  electrophoresis  of  BME  and  stained PAS  Blue  and  (B)  SDS  polyacrylamide  gel  electrophoresis  absence and  B  of  (B)  BME PAS  of  apoVLDL  with  Coomassie  Blue  A  99  (A)  Coomassie  .  1/Sepharose  (B)  the  ,  of  insoluble  in  (B)  presence  TMU  by  in  TMU  presence  gel  lipids  electrophoresis  (A)  (A)  and 4B  heptane  presence  NaDOC f r o m  Electrophoresis  conducted  apoVLDL  86  gel  the  of  unretained  A  chromatography  apoVLDL  of  (U)  Con  of  in  20  BME from  from 79  electrophoresis  Separation  presence  19  (A)  (MF)  gel  (A)  Removal 17  of  VLDL  102  and  of  apoVLDL  stained  with 103  -  21  Gel  filtration  containing 22  Gel from  23  Low  8M u r e a  gel  apoVLDL  on  Sepharose  6B  of  106 apoVLDL  column  on  sedimentation  centrifugation SDS  -  8M u r e a  filtration  speed  of  x i  of  SDS  or f r a c t i o n Sepharose  equilibrium  apoproteins  chromatography  V 6B  107  ultra-  isolated  by 110  - xii-  ABBREVIATIONS AND SYMBOLS  apoB  the protein component of human serum low density l i p o p r o t e i n ; also present i n the very low density lipoproteins.  apoC  the major protein component of human serum very low density l i p o p r o t e i n  apoprotein  delipidated protein moiety of a l i p o p r o t e i n  apoVLDL  delipidated protein moiety of VLDL  BIS  methylenebisacrylamide  BME  2-mercaptoethanol  BSA  bovine serum albumin  Con A  Concanavalin A  CPG  controlled pore glass  DTE  dithioerythritol  DTU  data transfer u n i t  DVM  digital  EDTA  disodium ethylenediamine tetraacetate  F  uncorrected  FI  f l o a t i n g f r a c t i o n from yolk granules  FII  s u b p e l l i c l e , f l o a t i n g f r a c t i o n from yolk granules  HDL  high density l i p o p r o t e i n  LDL  low density l i p o p r o t e i n  LDFG  granule, low density f r a c t i o n , composed of LDFG-1 and LDFG-2  voltmeter  f l o t a t i o n rate at any given density  - xiii  LDF  plasma  low d e n s i t y  (LPL )  and  1  LDI^  f r a c t i o n , composed  of  LDL^  (LPL ) 2  LDL  granule  low density  lp  lipoprotein  MF  myelin  figure  NaDOC  sodium  deoxycholate  NaN^  sodium  azide  Pv  phosvitin acid  -  l i p o p r o t e i n f r a c t i o n of  f r a c t i o n of  PAS  periodic  Schiff  PTS  p-toluenesulfonic  SDS  sodium  Sj. r  uncorrected  FII  FII  stain  procedure  for. g l y c o p r o t e i n  acid  dodecylsulfate flotation rate,  expressed  in  svedbergs °  —13 cm/sec/dyne/g)  (10  in  an N a C l  medium  of  1.063  density S°  flotation rate concentration  fully  corrected  dependence  SH  sulfhydryl  SS  disulfide  TBA  thiobarbituric  TCA  trichloroacetic  to  for  effects  standard  acid  N,N,N',N'-tetramethylethylenediamine  TMU  1,1,3,3-tetramethylurea  TTY  teletype  VLDL  very  WSF  water  low density soluble  conditions  acid  TEMED  lipoprotein  f r a c t i o n from y o l k  of  plasma  g/ml  -  xiv  -  ACKNOWLEDGEMENTS  I  wish  excellent  I  thank  Dr.  W.D.  and  sincere  adviser,  for  gratitude  his  Dr.  M.A.  the  members  Powrie,  Tung,  and Dr.  my g r a d u a t e J.  throughout  t o whom  p r e p a r a t i o n of of  to Dr.  S.  encouragement,  constructive criticism  during  those  my  I  course  for  committee, Dr. for  their  of  this  advice,  the manuscript.  Vanderstoep  an  guidance,  the  owe m u c h  Nakai,  I  also  R.J.  valuable  wish  Bose, suggestions  assistance.  Esta years of  and thank  especially to  express  research  assistance study.  to  And  finally,  for  her  of  this  I  would  like  encouragement,  graduate  study,  manuscript.  and  to  dedicate this  cheerfulness,  and  especially for  thesis  t o my  patience during  her  work  in  wife my  preparation  - 1 -  I.  INTRODUCTION  Egg yolk i s composed of approximately 16% protein, 33% and  50% water.  Less than 2% of t h i s material  lipid  i s d i a l y s a b l e ; most  of the yolk s o l i d s are macromolecular lipoproteins  (Cook, 1968).  Yolk i s considered a mixture of 77% soluble plasma and  23%  p a r t i c u l a t e granules. The  yolk plasma can be fractionated by  into a low density (10.6%). WSF  The LDF  ultracentrifugation  f r a c t i o n (66%), and a water soluble f r a c t i o n consists of two  contains <*-,/ff-, and y-  low density  livetins.  lipoproteins;  Ultracentrifugation  the of  the  granules dispersed i n solutions of high s a l t concentration, separates 3% f l o a t i n g low density material f r a c t i o n which contains 3.7% lipoprotein.  These HDL  from the sedimenting  phosvitin, and 16.3%  are termed <*- and  high  density  /3-lipovitellin  (Saito  et a l . , 1965). The LDFG has recently been further separated i n t o two termed the myelin f i g u r e and (Garland, 1973). and  the granule low density  Electron microscopy was  fractions  lipoprotein  used to assess the  purity  the s t r u c t u r a l i n t e g r i t y of these l i p o p r o t e i n p a r t i c l e s p r i o r  to t h e i r chemical analysis. The  l i p o p r o t e i n i s o l a t e d i n t h i s study has been designated  as a very low density  l i p o p r o t e i n (VLDL), rather than the "myelin  f i g u r e " of Garland (1973), because physicochemical analysis that i t s properties low density  indicates  c l o s e l y resemble those of the blood serum very  lipoproteins.  However, i t i s acknowledged that  the  - 2 -  structural In  features  the  present  chromatographic experimental of  the  protein  native  of  these  work,  analyses  conditions, VLDL  moieties.  two  types  of  lipoprotein  ultracentrifugal, were to  fraction  performed,  characterize (MF),  and  the  not  electrophoretic  under and  are  a variety  assess  the  delipidated  similar.  and  of purity  (apoVLDL)  -  II.  LITERATURE  lipids  are  and  salt  be  complex  Lipoproteins  solutions.  different can  are  noncovalently  interactions.  lipoproteins  separated  of  into  density  low density, et  a l .  serum  while  the  to t*-lp,  classes  serum  was  same d e n s i t y amount  of the  The  I).  These  and  a  classes  level  of  of in  plasma  high  energy  form  energy  into  the  density  gel to  show  t0/5-lp, the  Chicken  was and  S^  triglycerides  four  the  the  whose  which  reserve.  can  fatty be  lipoproteins  classes  on  HDL  VLDL  agarose  was  as  on  the  the  very  Immarino these  human  fraction  to  pre-^-lp  found  gel  electrophoresis  of  the  to  contain  human s e r u m ;  a  class  was  (VLDL)  lipoprotein the  substantial found,  low.  the in  thus  chylomicrons,  that  20-400S  are  of  origin.  relatively VLDL  aqueous  lipoproteins.  and  serum  lipoproteins the  hydrophobic  in  and  electrophoresis  performed  in  moieties  ultracentrifugation.  quantitative distribution  LDL  or  are  (1973)  triglycerides  for  divided  at  of  lipoprotein  by  remain  determined.  tissues as  Day  chylomicrons  transport rich  and  dispersible  densities  classes  agarose  protein  e l e c t r o s t a t i c and  their  been  and  which  protein-to-lipid ratios  have  t h e LDL  chylomicrons  chicken  are  (Table  by  in  generally  different  performed  Alexander  while  are  ultracentrifugal products  corresponds  on  bound  determine  low density  (1969)  compounds  The v a r i o u s  Serum l i p o p r o t e i n s basis  -  REVIEW  Lipoproteins and  3  principal vehicles  man  (Nichols,  acids  stored  in  1969).  can be the  for  oxidized  the  They by  triglyceride  Table  I.  C l a s s i f i c a t i o n o f Human Serum  Lipoproteins  , Class  Chylo VLDL  Density  l e s s than  (g/ml)  S  0.95  greater  0.95  -  1.006  LDL  1.006  -  1.063  0-20  HDL  1.063  -  1.21  0 -  20  -  o (A)  Diameter  f  than  400  greater  than  300  -  900  170  -  260  £-lp  40  -  100  oC-lp  400  9  Electrophoretic^ mobility  e  1000  origin pre-^-lp  I  I  a)  from Scanu et a l . ,  b)  chylomicrons,  very  c)  S^  the n e g a t i v e  designates  of d e n s i t y  1.063  1974 low  density,  low  density,  sedimentation  and  high  density  c o e f f i c i e n t i n Svedberg u n i t s i n an N a C l s o l u t i o n  g/ml  d)  agarose  gel  e)  in salt  s o l u t i o n of d e n s i t y  1.21  g/ml  lipoproteins  rather  t h a n 1.063  g/ml  Human s e r u m hydrated for  VLDL  densities  the weight  phospholipid, Negatively spherical  span a wide  and  flotation rates  composition 15%  of  VLDL  cholesterol  stained  spectrum  VLDL  are  macromolecular  and  seen  particle  (Table  are 10%  of  55%  I).  sizes,  Average  triglyceride,  protein  values 20%  (Skipski,  1972).  electron microscopically  complexes  with  diameters  as  ranging  from  o 300-900 A.  The  transparent  centers  halo due  was to  Garland  10%  dense The  et. a l . ,  myelin (1973)  myelin  figures and  of  structures  yolk  myelin  Hillyard lipoproteins  near  the  membrane-like an  easily  isolated  86%  lipid  indicated  46%  these lead  the  and  have  electron  periphery.  layer  but  The  is  likely  deformed  particle  egg  granules  and  14%  surrounding  the  of  a weakly  560-1300  "myelin  o A in  about  one  analysis  30%  or  phospholipids  more  of  figure". were  the  electron  osmiophilic  phospholipid  length  by  Lipid  Electron microscopy  with brain  name  yolk  protein.  fraction revealed  figures to  from  presence  triglyceride.  lamellae  o 480-1100 A i n w i d t h egg  (MF)  t e t r o x i d e - s t a i n e d MF  similarity  figure  outer  halos"  but  1975) .  contained  concentric  substructure  "grayish an  figures  cholesterol  osmium  as  show no  a r t i f a c t u a l f l a t t e n i n g of  The  the  with  interpreted  (Morrisett  of  particles  core.  myelin  Dimensions  reported  for  of the  figures.  _et a l . isolated  (1972) by  examined  chicken  ultracentrifugal  methods,  analytical ultracentrifugation,  acid  end  group  yolk  VLDL.  analysis  showed  serum  and  techniques.  for  yolk  Immunochemical  electrophoresis  identical results  egg  and  serum  amino  and  egg  - 6 -  The physicochemical evidence presented i n t h i s thesis, the r e s u l t s of H i l l y a r d e_t a l . (1972)  and Alexander and Day  (1973)  mentioned above, and the physical properties of blood serum VLDL, suggest that the term VLDL (MF) i s the most suitable operational d e f i n i t i o n of t h i s l i p o p r o t e i n . Burley and Cook (1961) developed a procedure for the i s o l a t i o n of yolk granules by d i l u t i n g yolk with an i s o t o n i c s a l t solution. The granules were found to disrupt i n NaCl solutions of 0.3M  or higher.  U l t r a c e n t r i f u g a l patterns of such a dispersion i n sodium b a r b i t a l buffer (pH 9.0)  indicated the presence of four boundaries, three  sedimenting and one f l o a t i n g .  The f l o a t i n g boundary was polydisperse  and represented about 12% of the yolk granules; f l o t a t i o n c o e f f i c i e n t s were not calculated f o r t h i s f r a c t i o n . Martin et_ a l .  (1963) prepared the LDFG from washed yolk granules  by d i s s o l v i n g them i n 10% NaCl p r i o r to u l t r a c e n t r i f u g a t i o n . LDFG showed two f l o a t i n g boundaries with S^ 20,w rt  Svedbergs,  = -16.8  and  Their  -9.4  and the t o t a l l i p i d and cholesterol contents of LDFG and  LDF of yolk plasma were s i m i l a r .  The nitrogen and phosphorus  content of the protein portion of LDFG was v a r i a b l e and i t was thought t h i s f r a c t i o n was contaminated with phosvitin. In another study, Saito e_t a l . (1965)  studied the changes i n the  LDF and LDFG l i p o p r o t e i n f r a c t i o n s of yolk during  embryogenesis.  U l t r a c e n t r i f u g a l boundary patterns showed the presence of two components i n each f r a c t i o n although poor r e s o l u t i o n and broad peaks prevented the detection of trends i n f l o t a t i o n rate with  -  incubation  time.  components  in  buffer  (30%  infertile to  those  of  in  but  present  in  too  small  of  and  to  occurred. two  removed  resolved  the  in  IM  two  remained  at  the  origin  layer  plate.  triglyceride 30%  15%  and The less  respectively).  The  of 88%  (73%)  LDFG i n t o  and  proportion  The  to  veronal  be  for  similar  faster  various  amount  maturation,  boundaries  chromatography  resolve  and  in  to values  the  at  LDF  phases  on  with  During were  of  but  any  LDFG was  of  was  present  changes  indicated  of  that the  flotation coefficients  maturation, found  the  to decrease  to  respectively.  to  thin  yolks  comment  NaCl.  and  found with  of  lipoprotein composition  yolk  components  1971b)  the  the  permit v a l i d  -13.9S of  -18S)  Ultracentrifugal patterns  poorly  and  LDFG c o n t a i n e d and  (1967)  slightly  Hydroxylapatite  the  amount.  (F)  comparable  variable,  greater  during  of  LDFG w e r e  increase  -11.7S  (57%  of  maturation.  -7.3S  up  were  of  -5.6S  more  the  70%  stages  rates  way  -11S)  w e r e much more  and M a r t i n  flotation  (23,%)  of  and  successive  presence  (1971a,  70%  -33S  study  have  of  of  flotation rates  to  amounts  (F)  and  and  ovaries  to  might  (30%  -  Flotation rates  from hen's  at  found  -24S  LDF  MacKenzie  yolk  NaCl  yolk.  component  growth  Proportions  IM  of  7  cholesterol lipid  protein, while  and  LDFG-2  had  two  Gornall  faster  (77%)  LDFG-1,  LDFG-2.  protein; 92%  than  and  LDFG-1  about  half  contained LDFG-1  contained  twice  Unfractionated  LDFG-1 had  lipid  The  LDFG-2  (21%)  however,  Kuksis  moved  migrating  phospholipid  in  and  subfractions.  the LDFG-2  found 12%  enabled  8%  85%  lipid  protein.  LDFG  - 8 -  and  LDF  from  the  results  when  separated  concentrations  plasma  of  were on  salt  said  to  give  hydroxylapatite  were  required  to  essentially  the  except  higher  that  solubilize  the  same  LDFG  fraction. Evans egg in  yolk the  et_ a l .  (1973)  lipoprotein  were  isolated  ultracentrifuge  should  f i r s t  be  designated  than  composition  of  l i p o p r o t e i n more  blood  VLDL  Although column were  the  chromatography  a l l  with  Bacon VLDL and  fraction resolved  A-50M. and void  et  The  In  from  cold  for  more  molecular  (1973)  to  and  second  was  peak  weight not  months.  of  a  density  The  chemical that  lipoproteins.  separated  A-15M),  five  fractions  with  lipid  triglyceride  and  of  larger  lipoproteins.  by  fractions  molecular than  The  the  proteins  by  and  Musser  (1974)  preparative peaks  the  a  ultracentrifugation  on  a  column  flotation coefficient  3 million;  isolated  f i r s t  peak,  (F)  of  Biogel  of  -21S  eluted  in  studied.  study, eggs  had  low  similar.  Bacon  plasma  VLDL were  the  solutions  approximates  Biogel  weight  be  very  low density  the  or  two w e l l - d e f i n e d p r o t e i n  fresh six  4B  that  salt  lipoprotein.  serum  when  contained  from yolk  another  VLDL  poor  The  appeared  a l .  a molecular volume,  was  high  as  closely  blood  peaks.  smaller  fractions  of  density  (Sepharose  two  lipoproteins  fractions of  from  low  that  resolution  isolated  weight  than  recognize  flotation in  rather  serum  to  by  lipoprotein  the  as  the  Evans  and No  from  et  al.  (1974,  eggs which  changes  in  1975a)  had  been  structure,  isolated stored  lipid  in  the the  content,  the  - 9 -  molecular  weight  detected  after  was  to  used  separated  ranges  cold  the  VLDL  size  by  gel  chromatography  10  and  17  overlap  Chang floating yolk MF  molecular (1969)  ^DL^  or  components  therefore, of  egg  yolk  techniques  the was  of  4B.  six  three  Molecular  (1973)  the  Chang's  their  lipid  were  estimated  weights  of  5,  considerable  three  fractions.  studies  fractions  separated  studied  was  although  among  sedimenting  were  fractions  molecules  determined, observed  VLDL  ultracentrifugation  plasma; these  total  electron microscopic  the  t h e VLDL  l i t e r a t u r e to to  assess  granule of  was  concerning  protein moiety in  were  and  the  on  isolated  FII  two from  fraction  composition  into  and  capacities.  reports  appeared  and  of  Sepharose  size  Garland  emulsification No  on  three  performed  fractions  granules.  and  of  of  Preparative  from yolk  m i l l i o n daltons  of  proportions  storage.  isolate  and  or  VLDL  physical  aspects  f r a c t i o n of date.  The and  egg  of  yolk  o b j e c t of  the  purity  and  apoVLDL u s i n g  the  granules this  physicochemical the various  ultracentrifugation, electrophoresis  lipoprotein  and  have  study, properties experimental gel  filtration.  -  III.  .EXPERIMENTAL  In unless water  a l l a  was  used  chemicals,  purity  in  a n a l y t i c a l grade was  Biorad Burdick  required.  a l l buffers  and  Glass  company,  PM 1 0 , Science  Laboratories  Laboratories and  Jackson  Carbowax  were  distilled, The were  PM 30  Biogel Lab.  chemicals  solutions.  l i s t e d by m a n u f a c t u r i n g  Amicon Applied  -  PROCEDURES  experiments,  special  10  used deionized  following used:  u l t r a f i l t r a t i o n membranes  20M  A-50M,  Dowex  50W-X2  tetramethylurea  Calbiochem  m e t h y l e<-D  Eastman  N,N -methylenebisacrylamide,  mannopyranoside p-dimethyl-  1  aminobenzaldehyde, thiobarbituric o Electro-nucleonics,  riboflavin,  TEMED,  acid  Inc. CPG-10  (3000  A)  Fisher 2-mercaptoethanol, insoluble glycol, black ICN  Pharmaceuticals  Matheson  Coleman  Bell  propylene  deoxycholate,  B r i l l i a n t Blue persulfate,  acrylamide,  R,  sudan  acid,  ovalbumin  2,4-pentanedione  3-(2-aminoethyl)  mercaptoacetic acid.  starch,  blue,  B  ammonium and  sodium  Coomassie  Mallinckrodt  potato  bromphenol  indole  HCl,  p-toluenesulfonic  -  Nutritional  Biochem.  Corp.  Pierce  11  -  agarose thiodiglycol Concanavalin Dextran  Pharmacia  Fine  2000,  A-Sepharose Sepharose  4B, 2B,  Blue 4B  and  Chemicals Sephadex bovine  G-75  serum  and  G-200  albumin,  (superfine)  conalbumin,  dithioerythritol,  glucosamine  HC1,  human y - g l o b u l i n ,  iodoacetamide,  Sigma  sialic Worthington  Biochem.  Corp.  acid,  lysozyme  chymotrypsinogen  6B,  -  A.1.  Isolation  of  Infertile  eggs  ration  were  Department than  24  on  of  absorbent  prior was  paper  yolk  membrane  from  the v i t e l l i n e  pooled  into  a.  was  a  The at  20,000  x  g  centrifuge.  The  the  sedimented  (pH  7),  remove  was  was  to  punctured  membrane.  of  was  The  EDTA  (pH  standard  1)  laying  Columbia  eggs were  were  at  (Evans  a  1:1  spatula yolk  the 4°C  (v/v) and yolk  in  (plasma)  a was  resuspended  30 m i n u t e s 2-3  the  stored  albumen  adhering  liquid  7.0)  from  supernatant  removed  a  British  from  any  with  diluted  minutes  repeated  fed  (Figure  for  less  egg to  of  and  rolled  white.  The  separate  the  several  eggs  yolk  was  granules  separated  for  lipoproteins  beaker.  yolk  granules  stirred  procedure plasma  towels  30  of  The  separated  0.02%  for  hens  University Farm.  of  use.  then  were  Leghorn  carefully  yolk  containing  -  Preparation  Science  to  Isolation  granules  the  collecting  Liquid water  by  Poultry  yolk  (MF):  from White  provided  hours  Each  VLDL  12  more  and  cold  stirred  for  plasma  by  Sorvall removed in  until  more  1968) .  a l l  (4°C) 30  distilled  minutes.  centrifugation  RC2-B by  refrigerated  decanting,  EDTA  recentrifuged.  times  et^ a l . ,  with  This  and  solution washing  the water  soluble  -  DILUTED  13  -  YOLK  c e n t r i f u g e 20,000 30 m i n  SEDIMENT  -  GRANULES  SUPERNATANT  resuspend stir  PLASMA  (discard)  30 m i n  3X  recentrifuge ~1  WASHED  GRANULES resuspend stir  1  FII  i n NaCl  £o=1.063  g/ml)  78,000  x g16-18  hr  I  FRACTION  Biogel  (discard)  hr  centrifuge FLOATING  SUPERNATANT  A-50M  FI, column  F i l l ,  FIV,  (discard)  1 VLDL  (MF)  LDLg (discard)  Figure  1.  Preparation  of  VLDL  (MF).  FV  - 14 -  b.  Preparative ultracentrifugation  Washed g r a n u l e s w e r e s u s p e n d e d i n 0.025M s o d i u m buffer containing was a d j u s t e d  0 . 0 1 % EDTA a n d 0 . 0 2 % NaN^ (pH 7 . 0 ) .  to a density  were r o u t i n e l y b u f f e r e d peroxidation  o f 1.063 g / m l w i t h N a C l .  t o pH 7 a n d c o n t a i n e d  or aggregation  phosphate  The s o l u t i o n  A l l solutions  EDTA t o i n h i b i t  of l i p o p r o t e i n s (Mauldin  and F i s h e r ,  1970). The d i s p e r s e d for a period  a t 78,000 x g  o f 1 6 - 1 8 h o u r s a t 4°C i n a B e c k m a n L 2 - 6 5 B o r L 3 - 5 0  ultracentrifuge. of  g r a n u l e s were then c e n t r i f u g e d  This  c e n t r i f u g a t i o n ensured q u a n t i t a t i v e  flotation  l i p o p r o t e i n s h a v i n g h y d r a t e d d e n s i t i e s o f 1.04 g / m l o r l e s s i n t o  the upper p a r t  of the preparative  tube contents.  Those macro-  m o l e c u l a r components o f y o l k h a v i n g h y d r a t e d d e n s i t i e s g r e a t e r 1.063 g / m l w e r e s e d i m e n t e d  ( F i g u r e 2 ) . The c o n t e n t s o f t h e t u b e s  were r e d i s t r i b u t e d i n t o f i v e d i s t i n c t sedimenting f r a c t i o n s . was p e e l e d  zones:  s p a t u l a , and t h e opaque s o l u t i o n b e n e a t h  c.  two f l o a t i n g a n d  three  The s e m i s o l i d l a y e r o n t h e s u r f a c e ( F I )  from the side of t h e c e n t r i f u g e  pasteur pipet  than  tube w i t h  the t i p of a  ( F I I ) was r e m o v e d w i t h  a  f o r further fractionation.  Agarose g e l chromatography  A 1 0 0 x 50 cm c o l u m n o f B i o g e l A-50M 5 0 - 1 0 0 mesh) was f i t t e d  with  ( 2 % w/v  coarse sintered glass  agarose,  filters  a t each  -  F i g u r e 2.  15  -  U l t r a c e n t r i f u g a l r e d i s t r i b u t i o n of egg granule  lipoproteins.  yolk  - 16 -  end.  Glass  filters run  at  beads  ( 2 mm d i a m e t e r )  prevented room  pressure  clogging  temperature  head  of  100  Lipoprotein directly 113  from  ml/hr  collector  1.0M  0.01% was  carried  eluated was  out  in  NaCl,  to  with  the void  concentrated  4°C.  Samples  at  4°C,  as  described  of  in  the  an  Isco Model  VLDL  boiled  2.5  3  g NaHC0  d.  per  in  Preparation  The  chromatography, prepared  containing  with  0.1%  UA-2  contained  (MF)  as  a  was  hydrostatic  LKB  UV  in  60 m l  was  eluted  buffer  UltroRac of  99  (MF)  (pH  drops  analyser.  at The  g  were  dialysed  against  and  analysed  for  7.0)  (6.6 280  for  nm peak,  and  8 hours  distilled  amino  ml).  first  fraction  78,000 x  at  fraction  eluates  t h e VLDL  aliquots  acid  at  water  content  E.l.  distilled  in  the  with  water  (Mauldin  VLDL were  and  protein-containing  performed  liter  applied  fractions  u l t r a c e n t r i f u g a t i o n at  was  the  column  under  phosphate An  eluate  of  section  under  The  fashion  sodium NaN^.  lyophilized, delipidated  which were  were  0.02%  collect  this  use.  preparation  0.025M  volume,  by  Dialysis  by  ascending  c o n c e n t r a t e was  EDTA a n d  used  spaces  water.  FII  Monitoring was  an  the void  prolonged  the u l t r a c e n t r i f u g e  with  containing  during  in  cm  in  and  commercially containing  Fisher,  presence  isolated  described  oxygen-free  of  water  2-mercaptoethanol.  mg  EDTA  membranes and  1970).  reducing  from y o l k above,  100  available  granules  except  saturated  that  agents.  and a l l  separated buffers  with nitrogen,  and  - 17 -  e.  Alkylation  VLDL the  procedure  in  1.0M  (MF) of  NaCl,  of  VLDL  was  alkylated  Battel et  0.025M  a  with  mM i o d o a c e t a m i d e  or  reducing  added was of  in  agents  excess  separated Sephadex  chemically Ellman's  A.2.  from  G-75.  Gel  A  added  stop  the  lipoprotein  as  on  1%  purity  of Gel  used  in  an  agarose the  18  7.0) and  (10  was  was  passed  then  hours.  No  by  reacted  denaturants was  a l k y l a t e d VLDL  (MF)  desalting  column  on  content  controls  were  1970)  described  as  to  mg/ml)  2-mercaptoethanol  the  a  of  the  determined in  using  section  E.3.  fractionations  pore  by  glass  beads  analytical  peak  preparation.  column was  extremely  from Gel  the  ultracentrifugation, 2%  agarose  filtration  slow  and  did  of  column,  VLDL  not  (MF)  improve  lipoprotein.  filtration  attempt  volume  BME  disulfide  controlled  examination,  through  and  and  and  (pH  time.  Further  the void  for  reagents  et: a l . ,  (MF):  an heterogeneous  the  reaction  sulfhydryl  preliminary  remove  according  lipoprotein  buffer  this  The  indicated a  at  unreacted  VLDL  to  The  nitrogen  the  filtration  isolated  G-75  were  iodoacetamide  (1968).  under  (Beveridge  of  with  phosphate  Sephadex  modified  Isolation  VLDL  to  reagent  a.  of  of  al.  sodium  through 10  column  (MF)  to  through  further  controlled pore  purify  the  glass  lipoprotein.  beads  was  Treatment  of  - 18 -  the glass beads with a 1% (w/v) s o l u t i o n of Carbowax 20M i n d i s t i l l e d water, followed by a d i s t i l l e d water wash (4X) eliminated a l l adsorption problems. Two ml aliquots of VLDL (MF) or alkylated VLDL (MF) were applied to a 81 x 1.5 cm c o n t r o l l e d pore glass bead column o (3000 A, 20-80 mesh), and eluted at 60 ml/hr with 1.0M NaCl, 0.025M sodium phosphate and 0.01% EDTA buffer (pH 7.0).  The eluted  samples were concentrated by u l t r a c e n t r i f u g a t i o n when necessary, then analysed by agarose d i s c g e l electrophoresis (section D.l.) and a n a l y t i c a l u l t r a c e n t r i f u g a t i o n (section D.2b). b.  A f f i n i t y chromatography on Concanavalin A-Sepharose 4B  A f f i n i t y chromatography was carried out according to the procedure of Shore and Shore (1973).  A 9 x 1 cm column of Concanavalin  A-Sepharose 4B was e q u i l i b r a t e d at room temperature with a 0.2M NaCl, 0.1M Na-acetate buffer containing 1 mM CaC^, ImM MgCI^j and 1 mM MnCl . 2  The pH was adjusted to 6.8 with a c e t i c a c i d and 0.02%  was added as a preservative.  NaN^  The alkylated VLDL (MF) (approximately  4-6 mg protein) was dialysed against the same buffer f o r 36 hours at 4°C, then layered onto the column bed.  The column was thoroughly  washed with the e q u i l i b r a t i n g b u f f e r , and the adsorbed substances were eluted at a flow rate of 8 ml/hr with the e q u i l i b r a t i n g buffer made to 0.2M with respect to methyl ©t-D mannopyranoside.  The  e l u t i o n pattern was monitored by absorbance at 280 nm and 2 ml  -In-  fractions  were  collected.  The  VLDL  (MF)  were  extensively  dialysed  EDTA  (pH  7.0)  at  methanol  (2:1  v/v)  section by  B.2a.  4°C,  samples  (section The  (section  (section  D.3d.).  B.l.  order  homogeneous to  to  of  Alkylated changes  of  lyophilized  in  an  nitrogen freeze  to  Insoluble small  molecular  water  and  v/v)  as  by  0.01%  chloroform-  described  d e l i p i d a t e d but  analyzed  unretained  containing  delipidated with  (3:1  not  the  VLDL  Gustafson  VLDL  (MF)  the  in  concentrated  sedimentation  velocity  potato weight  and  The  carbohydrate  electrophoresis  separated for  48  VLDL  by  hours  0.01%  were  VLDL  times,  starch  gel  for  isolated  insoluble  samples  lipid  used  n-heptane  (MF)  into  partially delipidated  containing  atmosphere.  prevent  was  dialysed  of  dried  SDS  previously  (1965),  was  a l l  and  was  P a r t i a l delipidation using  (MF)  presence  at  above  E.2b.)  (MF):  separate  atmosphere  dryer  and  VLDL  Freeze  inert  and  obtained  d i s t i l l e d water  lipoprotein). in  of  fractions,  the method  4°C  E.2a.  Delipidation  In  later  retained  D.2b).  apoprotein  analysis  were  of  against  ethanol-ether  u l t r a c e n t r i f u g a t i o n and  analysis  4  lyophilized  or  Some  fractions  EDTA  gel at (pH  potato  starch  stored  in  (MF)  was  especially  filtration.  4°C  against  7.0), (100  then mg/ml  a desiccator  handled  when  according  under  opening  at  a  the  oxidation. was  purified  ultraviolet  prior  absorbing  to  use  material,  to' remove which  the  -  interferes samples.  water  Partial  were  of  4°C  starch  was  then  by  five  at  resuspended  or  NaCl  protein  residue  g  as  were  for  8 hours  at  ml/hr  monitored  at  0.2M 280  for  4B  was by  analysis  determined the method  by of  pH  7.0  cold  dried  2 hours from  potato  After a  0.2M  The  50  ml samples  decanted final nitrogen  and  insoluble  buffer  soluble  insoluble  was  the  of  (MF)  Tris-HCl  desiccator.  in  the  stream  (VLDL  4°C.  a  starch  supernatant  under  the  in  extraction,  material  at  (3X) ,  stored  heptane.  with  water  extractions  the  dried  the  n-heptane.  and  each  -10°C,  was  and  minute  After  either  and  (4%  nm,  and  at  then  (pH  8.2)  phospholipid-  potato  starch  pooled  required, rpm  the method  heptane-delipidated  of  and (pH  to  a  77  x  2.5  eluted  at  a  flow  rate  eluates  were  8.2).  fractions then  (section Lowry  (1959).  VLDL were  u l t r a c e n t r i f u g a t i o n at  applied  buffer  the  45,000  Bartlett  by  agarose)  Tris-HCl  u l t r a c e n t r i f u g a t i o n when velocity  30  concentrated  4°C,  Sepharose with  The  distilled  NaCl  (MF)  of  by  above.  samples  of  in  separated  The  column 68  7.0) was  VLDL  at  sediment  extracted  (pH  centrifugation  x  the  then  the  g  1.0M  lyophilized  -10°C.  3000 x  60 m i n u t e s .  was  1.0M  at  analysis  extracted with  consecutive  n-heptane  starch)  (3X),  finally  samples  for  8.2  velocity  washed w i t h  and  centrifugation, at  pH  was  (3X)  centrifuged the  starch  d e l i p i d a t i o n of  accomplished  -  sedimentation  Tris-HCl  insoluble  aliquots  UV  potato  0.2M  distilled  and  the  The  (3-5X),  The  in  20  The  were  analyzed D.2b.).  et_ al.  Samples  of  extracted with  calibrated of  concentrated  by  by  sedimentation  Protein  (1951) the  cm  78,000  and  content phospholipids  unfractionated  chloroform-methanol  -  (2:1  v/v).  neutral using  B.2.  The  lipids  by  Delipidation  extract  thin  a petroleum  a.  layer  against  0.15M  conditions  of  VLDL  was  to  0.001M  EDTA  by  Scanu  diagram  of  the  in  (3:1  for  50 m l  of  solvent  2 hr  at  -10°C  separated  2000 x was  g  for  15 m i n  from -10  o  (MF)  Edelstein is  v/v)  Gel  solvent  G system.  mixture  with  slow  was  dialysed  for  The  standard  delipidation  (1971)  shown i n  containing  2  to  containing  were  supernatant  C.  The  f i r s t  by  48  3.  5 mg p r o t e i n 95%  hr  followed;  Figure  stirring.  the  p r e c i p i t a t e d p r o t e i n was  ethanol-ether  supernatant for  Silica  of  were  ethanol-ether  The  precipitated  centrifugation  at  supernatant-solvent  mixture  saved. The  the  at  presence  delipidation  7.0).  procedure  extracted  1  (pH  and  solutions  p r o t e i n was  Total  the  on  (95:5  d e l i p i d a t i o n , VLDL  NaCl,  for  delipidation  Lipoprotein  v/v)  examined  chromatography  (MF):  outlined  schematic  -  ether-diethyl ether  Ethanol-ether  Prior  a  lipid  21  16  hr  was  at  solvent  discarded  -10°C,  then  then  re-extracted  mixture  and  and  precipitate extracted with  the  recentrifuged  recentrifuged  to  separate  as  for  the  2 hr  above.  The ether  precipitated  protein. The adjusted  with  supernatant ether  to  a  from ratio  the of  first  solvent  ethanol-ether  extraction of  3:5  with  (v/v)  was and  -  VLDL  (MF)  22  -  (2-5  rag  protein/ml)  Ethanol-ether 1 v/v)  2 hr  a  SUPERNATANT  PROTEIN  Ethanol-ether  Ethanol-ether  (3:5  50 m l ( 3 : 1  16 h r  v/v) at  -10°C  2 hr  SUPERNATANT-  at  PROTEIN  PRECIPITATE  v/v)  -10°C  PRECIPITATE-  50 m l  ether  16 h r  at  SUPERNATANT (discard)  -10°C  -PRECIPITATE  PROTEIN-  SUPERNATANT (discard)  PRECIPITATE  Wash 3X w i t h  Dry  under  TOTAL apoVLDL (store under N at 2  Figure  3.  Ethanol-ether (from  Scanu  Ether  N„  -10°C)  d e l i p i d a t i o n of  & Edelstein,  1971)  VLDL  (MF)  -  allowed  to  extract  centrifuged 3X w i t h in  as  above  and  ether,  dried  under  a desiccator. The  (v/v).  thin  b.  was The  layer  This  amount  delipidation 2:1  (pH  3  10)  of  sodium  (11.5  mg N a D O C / m g V L D L  2 hours  determined SDS  (0.1%  deoxycholate  prior by  w/v)  to  gel  the method was  added  which  approximate  VLDL  the  concentration  (protein  +  column hr  with  of  VLDL  Sephadex  (MF) G-200  0.05M NaHCO^,  were  for  detergent  stored  at  4°C  apoVLDL  the in  the  -10°C  after  chloroform-methanol  presence  against  to  the  of  and  at  Protein  ejt a l .  lipids  by  1971). solution  temperature  using to  a  was  BSA  clear  standard. lipid  determination.  c a l c u l a t e d by  factor  0.05M  concentration  (1951)  protein was  NaCl,  Simons,  room  reaction mixture the  0.15M  lipoprotein  incubated  to  at  apoVLDL.  the  (Helenius  added and  washed  delipidation  dialysed  Lowry  by  and  the  then  combined,  total in  was  1966).  concentration  lipid)/protein The  analyzed  of  interfered (MF)  mixture  re-extraction with  filtration.  opalescence  protein  by  was  (MF))  the  remaining  (Scanu,  hours  Solid  for  lipid  VLDL were  48  The  precipitates  deoxych'olate  for  -10°C.  f r a c t i o n was  chromatography  of  -  purified nitrogen  e x t r a c t was  Sodium  at  the  determined  Samples NaHC0  overnight  23  7.14,  The  multiplying  obtained  from  the  ratio.  plus  NaDOC  superfine  0.15M  NaCl  sample and  (pH  was  eluted  10)  applied at  a  containing  to  flow  a  40  rate  x  2.5  cm  of  4.4  ml/  1 0 mM N a D O C .  -  The  gel  filtration  ascending  was  fashion.  bed volume.  Blue  to  the  determine  and  2000  V  o  to  (1959)  Bartlett  multiplying from et  the  a l .  and  bile  salt  in  detergent  to  a 38  x  2  ml/hr  with  eluted  using  a PM  0.1M  in  D.3d.).  the  gel  NaDOC i n  procedure The the method presence  for  than  of  the  in  2%  of  an the  were  total  used  column.  absorbance  from of  the  at  280  eluted  assayed  by  by  nm;  fractions  phospholipid  phosphorus  of  by  30 membrane) The  of  void  according  calculated  25.  Total  the method  of  Sephadex  volume,  column  Mosbach  et  and  G-75  buffer  This  of  Folch lipids  was  by  cholesterol  Pearson  a l .  lipid  The  layer  as  free and  peak  gel  filtration  complex  was  applied  at  The  a  flow rate  protein  of  peak,  by u l t r a - f i l t r a t i o n  then  subjected  presence  of  SDS  to (section  determined  according  apoprotein  was  was  the  extract  chromatography  9 0 : 1 0 : 1 and  apoprotein  classes  was  protein  to  (1954).  (1957)  thin  was  the  i t  eluted  9.0).  the to  concentrated  in  eluates  acid,  and  apoprotein  e_t a l . by  subjecting  (pH  delipidated-detergent of  concentrating  protein-detergent  electrophoresis the  75:25:4).  specific  removed  Tris-HCl  (hexane:ether:acetic  NH^OH,  was  10 membrane.  polyacrylamide  G  was  f r e e medium. cm c o l u m n  which  the  by  amount lipid  fractions  u l t r a f i l t r a t i o n (PM  by  less  mercaptoethanol  directly  of  by  the  temperature  (1953). The  180  and  positions  the  amount  eluant  were  determined  measured  the  room  t  P r o t e i n was was  at  volumes  Dextran  phosphorus  -  performed  Sample  V  24  also  mentioned  extracted  examined  on  Silica  for Gel  chloroform:methanol:conc. chemically above.  examined  -  C l .  Agarose  a.  gel  Gel  filtration  filtration  Following NaDOC,  the  0.003M  EDTA b u f f e r  1972). 80 and  x  VLDL  The  Protein  in  the  the  (Herbert  et  to  formation  through  a mixed  carried  out  was  rapidly  b.  in  Gel  EDTA b u f f e r and  this  prior  to  (pH  mg  at  a  was  to  urea  cyanate were  ion-exchange buffers urea  at  on  The  and  1972;  6B  solution  gel  (Brown  et  was  the  made  280  a  urea  at  10°C.  nm. of  cyanates 1976).  and  In of  order  urea  filtered  Fractionation  0.1M  0.1M  1969).  to  protein  was  fraction  water.  incubated  a l . ,  a l . ,  8M  solutions  eluted  in  et  carbamylation  containing  solubilized was  at  Fidge,  use.  the  Tris-HCl,  80 m l / h r  made  against  Sepharose  sample  on  freshly  dialysis  was  of  or  applied  containing  before  10°C  0.1M  v/v)  containing  concentrated  always  apoprotein-SDS filtration  a l . ,  the  resin  by  apoprotein 8.0).  in  et  (3:1  was  absorbance  solutions  Edelstein  6B  reported  urea  (Edelstein  flow rate  d e t e c t e d by have  in  protein),  same b u f f e r  filtration  VLDL  8M u r e a  Sepharose  of  of  solubilized  (25  8M  ethanol-ether  of  solutions  freed  containing  column  1973;  Tris  was  solution  exposure  these  6B  containing  laboratories  after  a l . ,  Sepharose  8.6)  eluates  peptides  employed,  (pH  jacketed  Several  avoid  on  apoVLDL  apoprotein  apoprotein  eluted with  of  -  delipidation with  (MF)  2 cm w a t e r  25  SDS  Tris-HCl,  with for The  respect  6 hr  at  0.01% to  SDS,  37°C  apoprotein-SDS  - 26 -  mixture  ( 2 5 mg p r o t e i n ) , w a s a p p l i e d t o a 1 0 0 x 2 cm c a l i b r a t e d  c o l u m n o f S e p h a r o s e 6B c o n t a i n i n g 2 mM  SDS.  The s a m p l e was  e l u t e d a t a f l o w r a t e o f 3 0 m l / h r w i t h 0.002M SDS i n 0.1M H C l b u f f e r a n d a b s o r b a n c e w a s m o n i t o r e d a t 2 8 0 nm. were concentrated and  Tris-  The f r a c t i o n s  b y u l t r a c e n t r i f u g a t i o n a t 78,000 x g f o r 8 h r  d i a l y z e d a g a i n s t T r i s - H C l b u f f e r i n t h e a b s e n c e o f SDS o r  against d i s t i l l e d  water.  produced were s u b j e c t e d  The T r i s s o l u b l e , d e t e r g e n t - f r e e t o molecular  weight determination  apoproteins i nthe  a n a l y t i c a l u l t r a c e n t r i f u g e u s i n g UV a b s o r p t i o n o p t i c s a s d e s c r i b e d in and  s e c t i o n D.3b. subjected  F r a c t i o n s d i a l y z e d a g a i n s t water were  to polyacrylamide  lyophilized  g e l e l e c t r o p h o r e s i s i n the presence  o f SDS ( s e c t i o n D . 3 d ) .  D.l.  A n a l y t i c a l procedures:  Electrophoretic separation of prestained  lipoproteins  A GE-4 g e l e l e c t r o p h o r e s i s a p p a r a t u s was u s e d f o r d i s c g e l e l e c t r o p h o r e s i s .  R i b i e r o and McDonald (1963).  Ethylene  Chemicals)  A l l experiments were conducted  u s i n g a n a g a r o s e g e l c o n c e n t r a t i o n o f 0.5% Sudan b l a c k B s t a i n was p r e p a r e d  (Pharmacia Fine  (w/v).  according  t o t h e method o f  o r propylene  g l y c o l was  h e a t e d t o 100-110°C a n d t h e n 0.1 g o f t h e d y e w a s a d d e d p e r 10 m l of t h e s o l v e n t .  (The s o l v e n t t e m p e r a t u r e must n o t e x c e e d  or a gelatinous mixture stirred cooled  will  result.)  f o r 5 min and f i l t e r e d ,  The m i x t u r e  was  110°C  thoroughly  f i r s t w h i l e h o t , t h e n a g a i n when  t o r o o m t e m p e r a t u r e , t h r o u g h Whatman No. 2 f i l t e r  paper.  -  The  dye  concentration  and  was  stable  A  0.5%  for  (w/v)  electrophoretic refluxing  for  agarose  solution  Tris,  solution glass  ml)  to  allow  glycine  (pH  0.1  ml  sealed prior  for to  (Fisher of  the  slowly  Ornstein were ml,  room  Davis  omitted.  at  hr  a  sampled  (1964)  was  was  B  (48  55°C  then 18.1  The warm  very  to  into  ensure  slowly  g  agarose  delivered  with water  ml  was then  g  in  a  a  0.0016M T r i s ,  dye for  sample  flat  water  flushed stored was  with for  each  conducted  at  pH  ml  IN  diluted with  a  sample  HCl,  5.98  20%  24  (w/v)  to  hr  by  at  4°C  centrifugation of  10-30  gel.  9.5  the  (MF)  nitrogen,  Aliquots  to  that  VLDL  removed  5 min.  0.0079M  Prestaining  of  applied  except  and  The  IN H C l ,  pipet,  against  0.5  excess  were  of  to  1968).  ml  added.  g  water,  to prestaining.  vial  6000 x  (24  0.5%  gel.  temperature, The  A  0.116  vigorously  Noble,  40-45°C  prior  The  Solution  6.6-6.8)  48  adding  approximately  layered  adding  electrophoresis  and  pH  4°C  solution.  prestained gel  for  dark.  stirring  a warm  agarose  the  distilled  1968;  were  to  approximately  by  of  buffer  into  and  the  at  mini-centrifuge)  this  100  at  up  cooled  electrophoresis.  Disc  to  1 hr  ml  to  8.8-9.0)  dialyzed  by  dye  3.2  ml  while  Dingman,  tubes,  8.6)  22.6  was  in  prepared  slowly  drawn  of  accomplished  gels  pH  setting  was  by  ml,  were  buffer  was to  solution stored  100°C  and  tubes  was  of  at  and  was  (MF)  gel  cooled  water  100  The  VLDL  pi  was  when  agarose  electrophoresis  meniscus. bath  to  (1.5  agarose  -  final  month  (Peacock  distilled water  the  15 m i n u t e s  burning  ml  one  grade  prevent  6.2  of  27  as  and  described stacking  g Tris, sucrose  water solution  -  and  water  upper  to  gel The  obtain  solution VLDL  pi  of  overlayered ml  20%  with  sucrose  electrode from  +  was  performed  was  negligible.  achieved, (v/v)  acetic  study  mobility  of  VLDL  in  a Pharmacia  (v/v)  D.2.  on  solution  was  tracking  were  used  dye  place  of  the  equal  60 m l top  from  20%  of  sucrose).  the  gel  and  solution  (Narayan  et  a l . ,  the  lower  and  blue)  30-45 min; of  the  the  tubes  B +  omitted  Electrophoresis heat  generated  (MF)  stored  was in  7.5%  run  in  solution. stained  the  effect  (MF).  The  and of  non-stained the  latter  dye gels  samples  solution were  then  Coomassie  Blue,  and  GD-4  destainer  using  7.5%  (v/v)  gels  were  then  The  destained  on  were the  electrophoretic  stained  for  electrophoretically acetic stored  acid in  A n a l y t i c a l procedures:  and  7.5%  (MF)  Analytical ultracentrifugation  2-3  destained 5% (v/v)  acid.  VLDL  20  1966).  was  VLDL  and  carefully  upper  lipoproteins.  the  of  ml  (Bromphenol  for  volume  (10  resolution  removed  an  B-1X  in  prestained tube  in  (w/v)  methanol.  acetic  8.6)  mA p e r  both  to  B +  applied  When s u i t a b l e  parallel  0.25%  (pH  2.5  acid  with  solution  d i s t i l l e d water)  containing  Initially,  with  50 m l  gels  hr  were mixed ml  used  layer.  sucrose  The  at  the  sample  light  buffer  gels  (20  -  which were  mixture were  baths.  those  the  samples  B-2X  this  Tris-glycine  for  (MF)  sucrose-solution Thirty  solutions  28  of  -  a.  Data  An developed system,  on-line  for  use  which  centrifuge. analyzing  is A  the  analyses  of  II  the  lists  acquisition  an  acquisition  accessory of  obtained  equipment  from  used  and  of  source  a  was  scanner UV  the  light  a narrow  across  the  the  cell  scanner  into  The  output  via  from  digitized  at  in  absorption  4  was  optical  preparative  programs were  the  ultra-  written  shows  a block  of  data  for  velocity  ultracentrifuge.  analysis  a  the  signal  to  measured  protein lamp  Table  diagram  from  constant  signal  radial to  the  of  the  the  each  to  the  within  electronics teletype.  and  separate  The which  unit  tube of  output  and the  centrifuge  the  which  cell.  from  cell.  data  unit The  carriage  absorbance the  light  f i l t e r  electronics  u l t r a c e n t r i f u g e was  d i g i t a l voltmeter scanner  as  The  1976).  photomultiplier  recorder  and  scanner The  the  solution.  (Chervenka,  the  the  in  as  interference  speed.  distance  from  an nm  on  from  identify  components from  280  mounted  concentration  sample  with  around  p l o t t e d by  used  output the  the  was  electrical  with respect  teletype  system  flotation/sedimentation  and  mercury  assembly  A m u l t i p l e x e r was the  by  wavelength  i n f o r m a t i o n was  sample  UV  Beckman  Figure  accessory  short-arc  photomultiplier  processed  data  ultracentrifuge.  absorbance  moved  the  mini-computer  lipoproteins/proteins  The  isolated  the  of  mini-computer  with  to  the output, c o l l e c t i o n , storage analytical  -  handling  conjunction  series data  and  data  in  29  was  relayed  transfer  unit;  transferred  t e l e t y p e was  to  the  the  as  equipped  mv  a with  -  Table  II.  Commercial UV  30  -  Equipment  used  in  Scanner-Mini-Computer  1)  Beckman  L2-65B  2)  Beckman  Prep  preparative  UV  Scanner,  a)  Optical  unit  inside  b)  Door mounted  c)  Electronics  d)  Potentiometric  scanner  the  Ultracentrifuge-  System.  ultracentrifuge  equipped the  with:-  ultracentrifuge  chamber  unit  unit recorder  3)  Schlumberger-Solartron  A220  digital  4)  Schlumberger-Solartron  data  transfer  5)  Texas  6)  Monroe  (model  1880)  s c i e n t i f i c programmable  7)  Monroe  (model  PL-2)  d i g i t a l X-Y  Instruments  (model  733  ASR)  voltmeter  unit  Silent  plotter  700  teletype  printing  terminal  calculator  ULTRACENTRIFUGE PREP.  U.V.  SCANNER  MULTIPLEXER  POTENTIOMETRIC  SCANNER  RECORDER  ELECTRONICS  UNIT  DIGITAL  VOLTMETER  (DVM)  DATA TRANSFER  UNIT  (DTTJ)  TELETYPE (TTY)  HIGH  SPEED  PRINTER CASSETTE  TAPE  STORAGE  MONROE C A L C U L A T O R  X-Y  gure  4.  Data and  flow  from  PLOTTER  t h e Beckman Prep  Ultracentrifuge.  U.V.  Scanner  -  a  high  (hard of  speed copy)  data  silent printer, and  for  into  to  which  cassette  subsequent  The process  on  32  tape  -  recorded  (soft  manipulation  incoming  information  flotation/sedimentation  copy)  with  following mini-computer from  the  data  permitting  the Monroe  programs the  of  paper retrieval  calculator.  were w r i t t e n  analytical  coefficients  on  the  to  ultracentrifuge  lipoprotein/protein  samples:  (1)  TTY-subroutine: tape  and  the  registers (2)  transfer  of  Derivative  program  with  respect  time  in  location  of  apparent  from  Linear  The  the  using  the  the  ln  derivative  the data  observed 1.  the  of  The  in  from  point  short  from  derivative for  not  be  the  concentration  from  (a)  the  center curve  of  for  easy readily  from  U t i l i z e d the plotting  which  regression  flotation/sedimentation A  storage  the  UV  Appendix).  derivative  plotted,  of  (cm)  obtained  previous  was  cassette  data  determined  w h i c h may  routine:  the  and  distance  curve  data  derivative  plotted, allowing  position  plot  on  the main  Recalled  (dc/dx)  point.  concentration  with  to  stored  (Monroe program) .  routine:  distance  (b)  data  information  (program l i s t e d  position  equation  plot  boundary  from  calculated  the  this  simultaneously  regression  boundary and  and  output  calculated  r e t r i e v a l of  calculated  radial  each  s c a n was  scanner (3)  to  for  with  memory,  seconds  rotation  of  for  the mini-computer  mini-computer  each  Allowed  subroutine  at  was  the  data  program. center  analysis  was  coefficient the  end  of  of  the  performed  calculated the  program  - 33  converted to  the  standard  with  where  , obs  performed  time  x  =  radial  in  a  or  subroutine  din x — dt  from  velocity  added).  (Equation  AN-F  three  the  center  of  rotation  quartz  windows  V L D L (MF) and prior  diluted to  7.0).  ultracentrifugation  were  made  rotor.  The  with  to  be  epon  used  were  the  analytical  flotation analysis (pH  were  samples with  runs  samples  cells  for  dialyzed  dialysate  to  0.025M  Centrifugation  was  equipped using use  run  were with  either  of  the  a l l  UV an  AN-F  simultaneously.  aluminium-filled  center-  runs.  against  the  0.5-0.9  ultracentrifugation.  contained  (cm)  (radians/sec)  (4-hole)  separate  sector  1)  M  L2-65B u l t r a c e n t r i f u g e  Analytical  an  program,  seconds distance  Beckman  optics.  allowed  EDTA  1 — — ^2  S„_  v e l o c i t y measurements  and  0.01%  f  regression  Flotation/sedimentation  pieces  for  in  and  S  linear  velocity  mm d o u b l e  units  and  , .= obs  angular  Twelve  buffer  F  =  (2-hole)  rotor  or  (Monroe  coefficient  Flotation/sedimentation  absorption AN-D  routine  t  (*> =  flotation/sedimentation  conditions.  plotting  S  b.  observed  -  sodium  appropriate  absorbance A l l  buffers  phosphate  performed  at  and  18,000  used  - 34 -  rpm, i n NaCl s o l u t i o n s Sedimentation (a)  0.2M  (pH  analysis  Tris-HCl  density  was  (pH  1.0630  performed  8.2)  and  (b)  g/ml  in  the  0.01M  or  1.0385  following  NaCl,  g/ml  at  20°C.  buffers:  0.025M  Tris-HCl  7.2). UV  on  of  paper  scans  and  cassette  coefficients by  were  plotting  calculated  time. S^  The  units  coefficients  of  the  equations the data v,  to  was  /0 =  7=  sodium the  2 and  zero  1.07  x x  3  _ 2  10~^c?  was  the  -  3.227  +  7.90  x  derived  from  the  by  M  2  -  10~^/> +  plus  center  plot)  by  solutions The  Tables,  the  1.585 1.2735  10~ M 4  by  and  Values s?f  specific  3  +  0.01%  polynomial  the  (Lindgren  x  flotation  obtained  1927).  partial  l/^o  program.  containing  from  a  using  the  densities  derived  as  (1954)  measuring  of  converted  plotting  extrapolating  for =  Gofman  densities,  7.0). were  relation v  _ 4  NaCl  Critical  value  10  line  (derivative  and  solvent  (pH  obtained  x  distance  determined  solutions  The  the  regression  various  (International  from  M  in  were  of  DeLalla  linear  phosphate  flotation.  10  by  4 different  buffer  density  obtained  4.067  the  lipoprotein  of  hydrated  at  the  recorded  flotation/sedimentation  slope  of  and  f l o t a t i o n c o e f f i c i e n t s were  outlined  in  the  intervals,  l i p o p r o t e i n peak  density,^,  VLDL  0.025M  viscosities  from  observed  as  subroutines Hydrated  EDTA and  of  minute  observed  the  of  dialyzing  The  apex  function  short  2-4  logarithm  the  the  tape.  at  natural  to  standard  taken  the  rotation  to  were  et_ al.,  1.000  for  v s /a volume, 1969).  (Equation  2)  (Equation  3)  - 35  where  ^  = solvent density  -  (g/ml)  = solvent viscosity (centipoises) M = m o l a r i t y of NaCl  D.3.  A n a l y t i c a l procedures:  a.  solution  P h y s i c a l s t u d i e s o f VLDL (MF)  C a l i b r a t i o n of Sepharose columns f o r m o l e c u l a r  apoproteins  weight  determinations  The  approximate  l i p o p r o t e i n and  molecular weights  of the p a r t i a l l y  a p o p r o t e i n f r a c t i o n s were determined  delipidated  w i t h agarose  gel  f i l t r a t i o n c o l u m n s c a l i b r a t e d w i t h n o n - e n z y m a t i c p r o t e i n s o f known molecular weights. molecular weights  The  s t a n d a r d p r o t e i n s and  are l i s t e d  bed volume were d e t e r m i n e d respectively. and  The  i n T a b l e I I I . The  using Blue Dextran  V  K  K V V V  av g  o  v o i d v o l u m e and  2000 and  total  tryptophan,  d i s t r i b u t i o n c o e f f i c i e n t s f o r the standard proteins  samples were c a l c u l a t e d u s i n g e q u a t i o n 4  where  their respective  e  -  av  = distribution  V  (Ackers,  o  (Equation  o  coefficient  = e l u t i o n volume f o r the p r o t e i n = v o i d volume of the column = t h e t o t a l bed volume  (ml)  1967).  (ml)  (ml)  4)  -  Table  III.  36  Molecular Weight of  Gel Filtration  D , „ „ . Protein f  P  -  Markers  for Calibration  Columns.  Molecular Weight ,, , f (daltons)  Source  Ovalbumin  egg  white  45,000  Conalbumin  egg w h i t e ,  ^-globulin  human c o h n F I I  Type  I  85,000  310,000  -  A of  each  the  as  standard  gels  from K  plot  least  versus  b.  Beckman  versus  p r o t e i n was  log  f i t  12  Sedimentation  equilibrium  place  AN-F  rotor.  absorbance  units  after  in  0.1M  successive  equilibrium  pattern.  The  Mw  obtained  from  , were  M  w  R = Universal  studies  studied  scans  280  at  values  for  equation  the  of  calculated  data  and  plotted  windows  at  out  absorption  15,000  in  a  optics.  rpm a t  20°C  fitted  in  a  concentrations  of  0.2-0.3  (pH  8.0).  nm s h o w e d the  carried  no  apparent  2RT  din c o — r — . 2 2 ( 1 - v / j ) a) d r  constant  temperature  v  =  partial  specific  volume  /O =  solvent  density  (g/ml)  concentration  of  were  Equilibrium change  4-  was  in  molecular  weight,  5.  T =  c =  samples  were  quartz  buffer  gas  weight  f r a c t i o n a t i o n range  w i t h UV  10,000  Tris-HCl  v  where  the  the molecular  studies  with  were  =  app *  at  cells  Proteins  the  equation  equipped  performed  sector  of  of  weight,  equilibrium  mm d o u b l e  within  regression  molecular  runs were  logarithm  weights  L2-65B u l t r a c e n t r i f u g e  using  app  the  linear  Molecular  squares  -  Sedimentation  Equilibrium  assumed  K  employed.  the av  of  37  , (Equation ^ f r  (8.313  x  lO^ergs/degree/mole)  (°K) of  (absorbance  the  at  5)  protein  280  nm)  (ml/g)  -  r  =  radial  o5 =  38  distance  angular  -  from  velocity  the  center  of  rotation  (cm)  (radians/sec)  2 The  term d i n  c/dr  was  obtained  the  data  from  the  slope  of  the  least  squares  2 regression  line  of  plotted  as  ln  c vs.  r  .  A l l  data  were  <,  recorded D.2a.  using  A l l  the  TTY-data  computations  calculator  using  acquisition  were  programs  system  described  carried  out  in  the Monroe  w r i t t e n by  van  de  Voort  in  section  1880  (1976).  2 Data curvature second  were  order  obtained  from p l o t s  by  computer  (ln  measured  weight,  M  ,  =  taking  squared  w  c  at  the  In  fitted bx  +  f i r s t  cx  c vs. to 2  a  ),  each  point  Polyacrylamide  exhibiting  least-squares and  was  The  disc  gel  }  2  substantial polynomial  values  of  of  polynomial  the  din  c/dr  weight-average  calculated  = { 2RT/(l-v>)«j »  w  r  derivative  radial distance.  M  c.  a +  of  from  {din  2  electrophoresis  at  were each  molecular  equation  c/dr  2  of  6.  }  (Equation  6)  in  tetramethylurea  (TMU)  Urea  gels  Kane  et  (1973)  or  (1964)  method. Gels  tubes  with  were  a l .  were  prepared  (1975),  prepared  6 5 - 7 5 mm r u n n i n g  according  which  in gels  120  is  x  (5%,  to  the  method  a m o d i f i c a t i o n of  the  6 mm o r  105  x  7.5%  10%  acrylamide)  or  8 mm  of  Kane Davis  glass and  - 39  10-15 and  mm s t a c k i n g  buffers  used  The of  monomer and  volumes  of  also  avoid gel 45  Table  gels  through  0.64 prior  were  were and  layer  level  surface.  The  upper  stacking  reactant  by  adding No.  mixture  to mixing A  formed  Whatman  g urea/ml,  The  solutions  IV.  solutions,  a  0.64  5  and  Urea of  was  gels  distilled occurred  solution,  in  in  Equal  added  to  order  water  volumes  of  paper.  persulfate  pouring  Gelation  equal  g urea/ml  filter  mixed.  of  mixing  on  which  the  to  top  of  the  approximately  minutes.  t h e monomer  of  mixture.  (1:1  v/v)  mixture. with  and  An  This  allow  8.6)  either  on  equal  0.64  water  for  VLDL 72  top to  hr  of  and  the  gels. volume  (MF) at  the  Dialyzed  one-tenth  were  of  formed  solutions  was  and  mixing adding  poured  exposed  to  a  added over  to  the  equal 0.64  riboflavin-persulfate  g u r e a / m l was  gel  by  g  urea/ml  mixture  t h e monomer lower  fluorescent  volumes  gel,  buffer overlayered  light  for  60  NaCl,  0.001M  minutes  photopolymerization.  application  stacking  volume  stacking  distilled  gels  Tris-phosphate  containing  The (pH  running  formation.  of  to  in  monomer-buffer  just  cyanate  acrylamide).  listed  filtering this  assured  (3.3%  Tris-TEMED  contained  solutions  are  lower  and  mixture  gels  -  An of  was  4°C.  dialyzed  against  Delipidation  stacking  gel  or  in  of a  0.4M VLDL  test  (MF)  tube  was  prior  carried to  gel. samples  (50-200  equal volume freshly  of  ul) pure  prepared  were  pipetted  glass  standard  onto  distilled reducing  EDTA  the  TMU  and  solution  out  -  Table  IV.  Reagent  Running (a)  Solutions  (separating)  Monomer  40  -  and  Buffers  for  TMU  Electrophoresis.  gels  solution  acrylamide  0.4 0.3 0.2  g/ml g/ml g/ml  (10%) (7.5%) (5%)  methylenebisacrylamide 0.011 (b)  Tris-TEMED  solution  2M T r i s - H C l  buffer  containing (c)  Persulfate  Stacking (a)  (upper)  Monomer  g/ml  pH  9.1  0.003 ml  TEMED/ml  -2 6.25 x 10 M ammonium ( 0 . 1 4 2 6 g/100 ml)  solution  (0.28%)  persulfate  gels  solution  acrylamide  0.133  g/ml  (3.3%)  g/ml  (0.28%)  methylenebisacrylamide 0.011 (b)  (c) (d)  Tris-TEMED  0 . 2 4 M T r i s w i t h 0 . 0 0 1 3 m l TEMED m l a d j u s t e d t o pH 6.7 w i t h phosphoric acid  Photocatalyst Persulfate  Electrode (a)  solution  Upper  tank  0.027  mg/ml -3  solution  4.5  x 10 (0.1027  Lower  Reducing (a)  M ammonium g/100 ml)  persulfate  buffers  tank  buffer  Tris  0.0425  Glycine (b)  riboflavin  tank  buffer  Tris-HCl  M  0.0465 M, 0.12  M,  pH pH  8.91 8.07  solutions  Standard  reducing  agents  (dissolved i n upper 8 . 5 w i t h 4N NaOH)  0.010  ml/ml  thiodiglycol  0.025 ml/ml m e r c a p t o a c e t i c 0.02 g/ml EDTA e l e c t r o d e b u f f e r , pH a d j u s t e d t o  (b)  0.01  M dithioerythritol  (c)  0.1%  2-mercaptoethanol  acid  per  -  (or  10  mM D T E  or  for  60  min  ambient  at  one-tenth  volume  volume  80%  of  density  of  layered  over  0.1%  BME)  tracking  dye  (w/v)  TMU-water  the  reducing and  the  by  sample  in  insoluble  following applied  10  the  x  reduced  agents  as  prepared was  until to  to w i t h i n  stopped  (w/v)  10  then  destained  at  of  to  and  one-tenth  increase  buffer  was  reduction  solution  centrifuge  rapidly  5000 x  g  mA p e r  gels  in  0.1%  the  with  tubes.  separated  and  7.5%  in  the  then  could the  and  supernatant  each  supernatant  be  TMU  The  from  (v/v)  denatured with  the  other  was  then  dye  When t h e  Following (w/v)  acetic  gel  entered  bottom  of for 16  the  8M o r  10M  standard  was  the  the  gels,  immediate hr  of  and  5%  reducing  applied  dye  urea  agent.  stacking  tracking  Coomassie acid  with  dissociating  mA p e r  removed  0.25%  or  as  1.25  gel. the  were  BME, TMU  of  were  acid.  4 hr  (MF)  tracking  f e w mm o f  the  for  VLDL  to using  the  sulfosalicylic stained  (MF)  1 cm g l a s s  current  2.5 a  and  were  tank  incubated  delipidation,  blue)  and m i x e d  upper  t h e VLDL  mM D T E ,  constant  increased  moved was  with  gels  added The  this  was  gel.  alternatives A  mixture  Bromphenol  d e l i p i d a t i o n and  p r o t e i n were  Some s a m p l e s and  this  Following  (0.001%  were  the  contrifugation  to  and  mixture.  incubating  agents  added  phase.  Alternatively, accomplished  were  -  temperatures.  sucrose  the  41  to  the  gel,  band  then  had  electrophoresis fixation in  fixation Brilliant (v/v)  the  gels  Blue  R,  methanol.  20%  -  c.  i .  Extraction  A stained  but  were  Gel  slices  the  p r o t e i n was  et  a l . ,  set  prepared  and  the  acrylamide  for  10 m i n .  gel  electrophoresis  The  removed  (1969) were  Samples  Weber in  (pH, 7 . 2 ) of  gels,  boiled of  and  the  prepared  buffer  TMU  and  in  disc  8M u r e a ,  0.01  ml  gel  cut  out  and  3  speed  and  0.05%  incubated  not  at  subjected  to  of  protein.  crushed, SDS  37°C  centrifugation  (Weiner for  at  SDS  and  12  hr  3000 x  g  polyacrylamide  known  BME,  and  that  tubes  SDS,  (10  tubes  SDS  in  (model  Weber  gels  0.1M  of  and  sodium  or  samples  molecular 0.1  weights  ml  0.015-0.03  g  sample SDS  for  or  10%  phosphate from  extracted (Table  ethanol. from  V),  buffer, 5-10  GE-4)  Osborne  7.5%  r e c r y s t a l l i z e d 3X  mg)  with  system  of  Acrylamide  0.1%  test  of  electrophoresis  f o l l o w e d was  protein of  were  presence  6 mm g l a s s  proteins  stoppered  the  x  containing  were  D.3d).  (1972).  120  were  then  a l .  lipid-free and  in  procedure et  was  gels  electrophoretic analysis  0 . 0 5 M NaHCC>  low  (section  electrophoresis  band  gels  by  supernatant  Electrophoresis  used  each  crushed  A Pharmacia was  SDS  extracted with The  from urea-acrylamide  f r o m TMU  for to  -  protein  gels  corresponding  1972).  d.  of  of  42  min  the  were  0.9  ml  (Deutsch,  1976). The were  applied  Bromphenol  samples  or  the molecular  individually  blue  and  one  to  drop  the of  gels;  weight 8-10  glycerol  pl  were  marker of  proteins  0.001%  added  and  (w/v) mixed.  (5-10  pl)  - 43 -  Table  V.  Molecular  Weight  Protein  Markers  Source  for  SDS  Electrophoresis  Molecular  Weight  BSA  dimer  bovine  134,000  BSA  monomer  bovine  67,000  Ovalbumin  egg  45,000  et-chymotrypsinogen  bovine  lysozyme  egg  white  white  25,000  14,000  (daltons)  -  The  gels  were  2 mA p e r  gel  subjected for  30 m i n  phosphate  buffer,  phoresis,  the  tip  dipped  India  sulfosalicylic  acid  ink. for  Coomassie  B r i l l i a n t Blue  diffusion  in  scanned  in  5%  (v/v)  a Transidyne  mobilities  from  densitometric  logarithm A  linear  through from  the  of  the  data  equation  was  fixed  16  hr,  then  stained  R  for  4 hr.  The  and  2980) to  7.5%  analysis  of  with  (v/v)  Apparent  of  0.25%  molecular  a  needle  (w/v)  destained  acetic  acid,  dye were  yielded  sodium  (w/v)  by  then  at  550  nm.  calculated  plotted  relative mobility data  of  electro-  with  20%  were  the data were  the  0.1M  densitometer  tracking  versus  in  current  in  marked  gels  scanning  the  constant  thereafter  dye  were  weight  as  the  (R ). m m  the  best  f i t  line  weights  were  estimated  7.  molecular  a  intercept  b  slope m  gels  and  MW  R  at  Upon c o m p l e t i o n  The  traces  Log  where  7.2). tracking  (TG  points.  gel  the  compared  molecular  regression  (pH  methanol  Relative the  5 mA p e r  SDS of  -  electrophoresis  and  0.1%  position  in  to  44  of  relative  MW =  a +  bR m  (Equation  weight  the  regression  mobility  of  the  line protein  7)  - 45 -  d.  i .  Staining  of glycoproteins  Unstained SDS  prior  to s t a i n i n g .  temperature (v/v) 10%  acid.  The  was  alcohol,  incubation  0.5%  arsenite,  the  acid  containing  Each  acid  5%  of  8 g basic  hr incubation acid  then  and a  oxidized  incubated  f o r 60 m i n . acid  The g e l s left HC1  S c h i f f r e a g e n t was  in 2 liters  fuchsin,  to stand  D a r c o G60  E.l.  alcohol,  10% in  further  and s t a i n e d  i n at least  f o r 2 hr, followed  acid  0.01N  a t ambient  Two  80 m l  b y 0.5%  changes  f o r 20 m i n e a c h  i n the  (w/v)  o f 1%  completed  were then  transferred  to tubes  overnight,  then r i n s e d  i n 0.1%  t o remove e x c e s s  s t a i n and  fading.  Na-metabisulfite  allowed  c  20 m l S c h i f f r e a g e n t ,  and bound  acid.  (H I0^) -> o  treatment.  The  and  (v/v) a c e t i c  (v/v) a c e t i c  (w/v) s o d i u m m e t a b i s u l f i t e , retard  b y a 6-9  g e l was  (v/v) a c e t i c  (w/v) s o d i u m periodic  10%  overnight  (v/v) i s o p r o p y l  followed  gels  t o remove f r e e  incubated  c a r b o h y d r a t e s were  (w/v) p e r i o d i c  s o d i u m a r s e n i t e , 5%  g e l was  i n 10% a c e t i c  d a r k a t room t e m p e r a t u r e . of  were t r e a t e d  80 m l o f 2 5 %  This  /(v/v) i s o p r o p y l  overnight  Each  i n at least  acetic  gels  on a c r y l a m i d e  then  water, stirring  f o r 2 hr prior  charcoal  filter  Chemical Analyses:  Freeze dried,  prepared  adding  Amino  delipidated  21 m l c o n c e n t r a t e d  for 2 hr.  to decolorizing  (Fairbanks  acid  by d i s s o l v i n g  16 g HC1  The s o l u t i o n  was  with  amount  a small  e t a l , , 1971).  analysis  samples were h y d r o l y z e d  with  -  p-toluenesulfonic mixture  was  acid  prepared  aminoethyl)  indole  to  2.5  5 ml.  this in  A  20-30  hydrolyzed  for  amino  utilizing  Chemical  a.  exchange of  each  plug  of  column  25  distilled  hydrolysis  tube  110°C,  then  (Phoenix  Carbohydrate  and  3.3  at  1.7 ml  100°C.  Each  g Dowex  50W-X2  of  transferred to  and  prevent  hexoses the  (hydrogen  to  glass  column  resin eluted  hexosamines  on  a  Phoenix  Instruments  hydrolysis  After  were  to  were  hydrolyzed  HCl.  the  frozen  evacuated  Samples  0.1N a  and  analysis  p r o t e i n were  hr  of  technique.  lipid-free 24  diluting  1 ml  analyzed  of for  in  was  Precision  elution  3-(2-  then  tubing,  with nitrogen.  column  1 0 mg  dissolved  hydrolysis  at  hydrolysis  water,  determination  The  water,  hr  and  hexosamine  wool  outlet.  boiling  in  The  g PTS  apoVLDL was  times  single  2.85  of  Each  36  1971).  and  were  glass  ml  analyzer  mg p r o t e i n ,  resin, tube  of  3  Analyses:  tubes  Chang,  sealed  or  acid  Samples  contained  2.5  bath.  24  a  Hexose  hydrolysis  in  flushed 22,  and  -  dissolving  mixture,  and  m o d e l M6800  E.2.  HCl  ice-acetone  um H g  Corp.)  by  mg s a m p l e  hydrolysis  a dry  (Liu  46  with  form)  equipped  the  2N H C l  sealed  mixture  hydrolysis  from b l o c k i n g from  in  the with  the  resin  cation contents a  lower  with  (Abraham  et  a l . ,  1960). The  water  eluate  was  lyophilized  and  analyzed  for  hexose  -  content  using  1960).  A  of  100  a modified  galactose  pg/ml  was  The HC1  Morgan  (1933).  and  1 ml  tubes. was at  in  One  90°C.  One  1:1  b.  of  Twenty for of The of  mg  1 hr a  as  2%  acid  (v/v)  room  reagent  addition was  the  acid  of  concentration  the  in  lined, IN  of  in  incubated of  remove  Elson  2 ml  screwcap  sodium  4 ml  to  and  water test  carbonate for  45  min  95%  ethanol  p-dimethylaminobenzaldehyde  HC1)  Ehrlich's at  procedure  dissolved  teflon  was  evaporator  540  were  added  reagent nm.  the  and  mixed  thoroughly.  absorbance  Glucosamine  HC1  of (2  the  ug/ul)  determination  was  (1959).  80°C.  were  by  temperature,  determined  lipid-free  of  to  ( 6 7 7 . 5 mg  the  a  at  Burnett,  standard.  Sialic Warren  of  w/w)  f r a c t i o n was  the mixture  to  and  a rotary  acetylacetone  and  (Shields  (1:1  in  hexosamine  ethanol-concentrated  solution  sodium  dried  transferred  tube  acid  samples  reaction  standard.  was  dried  Sialic  at  the  for  cooling  solution  used  assay  of  each  after  colored was  to  as  were  Ehrlich's  ml  hr  ml  Anthrone  eluate  The  -  mannose m i x t u r e  analyzed  After  1 ml 25  and  aliquots  added  and  used  acid  excess  and  47  A 0.2M  0.2  determined Sialic  aliquot  sodium  incubated  arsenite  acid  p r o t e i n was ml  solution  using (0-5  pg)  hydrolyzed of  sample  metaperiodate  for  20 m i n  (10%  the  (w/v)  at  in  room  sodium  thiobarbituric was  used  as  the  in  10 m l  of  was  mixed  with  temperature, in  standard.  0.1N  9M p h o s p h o r i c  arsenite  acid  0.1  ml  acid. then  a  H^SO^  1  ml  solution  -  of  0.5M  tubes  sodium  and  sulfate until  the  (0.6%  (w/v)  TBA  solution  and  the  tubes  The  tubes  2 min  was  read  E.3.  cooled  at at  1000 549  Chemical  x  sulfhydryl  absorbance  the  VLDL and  SH  (MF)  Tris-glycine  by  0.02  absorbance  M  of  SH/g  ml 412  added of  SS  for  the  15  were  of  were  added  min.  added  was  top  ml  to  centrifuged  (solvent)  layer  were  buffer,  pH  per  0.1  ml  x  A '412  8.0, of  were  analysed  Ellman's  diluted and  to 2.9  for  reagent  10  ml with  1%  ml  of  (w/v)  0.5%  the  diluted  sample.  SH  content  was  reagent,  x  (MF)  using  Samples  (equation  73.53  VLDL  content  Ellman's  nm  determination  alkylated  disulfide  1974).  was  and  or  in  of  bath  the  Three  sulfate),  reaction mixture of  to  nm.  (w/v)  addition  added  cyclohexanone  g  and  sodium  water  The  a l . ,  Tris-glycine  0.5M  of  was  disappeared.  complex.  et  in  in  ml  acid)  color  a boiling  4.3  (Beveridge NaCl  sulfuric  TBA  in  and  analyses:  of  -  yellow  placed  colored  Samples their  were  were  the  for  0.1N  shaken  the  extract  in  48  Following  determined  8).  D  (Equation  8)  C where  A  '412  absorbance  at  412  C  protein  D  dilution  factor:  73.53=  10  x  /1.36  10  absorptivity molar  nm  concentration  basis  30.2  ; where and  to  10  the  in  mg/ml  for  SH  or  150  for  total  x  10  is  the  molar  1.36 is  pM/ml  the  conversion  basis  and  from  f r o m mg  SDS  SH  the to  g.  - 49 -  Diluted 1 ml  of  samples  10M u r e a  temperature.  and  Ten m l  protein,  w h i c h was  10 m i n .  This  The to  0.05 SS  ml  of  samples  the  UV  was  a  Beckman  a hydrogen  of  12%  by  =  in  and  by  and  TCA w e r e  added  repeated  3 ml Color  of  to  was  the method  uM  SH 2  9.  (w/v)  added  for  1 hr  to  at  precipitate 5000 x  twice to  0.5%  absorbance  equation  total  incubated  centrifugation at  solvent.  reagent  d e t e r m i n a t i o n were  BME,  (w/v)  dissolved  g  and  BME. diluted  developed by  adding  was  at  measured  412  Protein concentration  of  Lowry  uM *  SH  et  a l .  the  for  remove  SDS,  room  nm. in  (1951).  _. (Equation  9)  studies  spectra DB  SS  p r o c e d u r e was  measured  SS/g  for of  determined by  absorption  Absorbance with  ml  same  Ellman's  uM  F.l.  0.02  washing  c o n t e n t was  the  ml)  sedimented  p r e c i p i t a t e was 10 m l w i t h  (0.2  of  u n m o d i f i e d VLDL  spectrophotometer  lamp w i t h a  in  (MF)  standard  2 0 0 - 4 0 0 nm w a v e l e n g t h  samples quartz  range.  were  measured  cuvettes  using  - 50  IV.  R E S U L T S AND  A.  Data  one  of  DISCUSSION  acquisition  Flotation  and  the most  common  properties  The  and  subsequent on-line was UV  labor  data  Scanner  The  use  f i r s t  from  centre  of  from  a plot  rotation  curve that  which are  (x)  the  value is  of  in  especially to  and  be  greatly  reduced  processing  system.  research.  described  of  this  the  ln  (c)  seconds The  of  X-Y  useful  manually  of  in  was scan  the  resolve  Beckman  Prep  UV  of  versus  dc/dx  from  of  mv  the  rotation.  the  point,  the  was  scanner from  two  two  the  distance  center  drew  data.  A program  the  calculated  the  permitted  calculate  distance  into  and  system  the  data.  the  resolving  data  automatic  D.2a.  to  curve  plotter  of  conjunction with  versus  from  the  in  of  converted  derivative  the  an  Such a  use  is  solution.  experimental  system  distance  by  section  the  in  study  c o l l e c t i o n of  The  in  of  the  proteins  ultracentrifuge  the  for  the massive  concentration to  ultracentrifugation  used  calculator which  (dc/dx).'  difficult  methods  ultracentrifuge  included time  velocity  processing  the  of  logarithm  rotation,  derivative  and  the Monroe  natural  the  data  in  this  equipment  The program o u t p u t of  in  application  plot  for  can  to  recording  signal  the  for  of  lipoproteins  involved  accessory  derivative written  of  c o l l e c t i n g and  acquisition  automatic  handling  physical  calculations  developed  data  and  sedimentation  physical time  -  center and  the  derivative  component boundaries  systems  -  (Figure  5).  A minor  velocity  analysis  The  on  axis  desired  permitted  the X-Y  plot  to  and  one  c a l c u l a t i o n of  These  data  position  (In  of  x  the  f i t  the vs.  -  of  the  program  sedimentation  plotter  dimensions,  derivative the  revision  51  could the  set  of  be  time)  scaled Each  points  which  were  lipoprotein  velocity  data.  f l o t a t i o n or  written  and scan  floating  analysis.  yielded  were  points  later  or  to  one  any  such  used  for  coefficient.  associated  boundary  flotation  calibrated  sedimentation  the  for  with  protein  the  apex  sedimenting  boundary. The  data  collected  from  and  observed  several  entered  into  flotation rate  lipoprotein regression previous  or  protein  plus  a  or  the  The  of  (Figure  6).  average  X  and  Y value,  equation,  and  the  series  regression  of  plotted  the  program  line rates.  and  to  short  best  f i t  slope  data  (In  squares  intercept  to  correct entry  calculate  the of  the  from  was  the  and  data  points  pairs,  the  standard  added  at  observed slope  coefficients the  data  from  the  regression deviation  points.  the the  of  the  of  linear  time)  regression  through  to  the  termed  x vs.  the X-Y  were  calculated  program,  line  and  which  intervals  This  included  subroutines  coefficients  Manual  least  output  the  the  time  c o e f f i c i e n t of  s t a t i s t i c a l parameters  program  sedimentation  program  sedimentation  c o r r e l a t i o n c o e f f i c i e n t of A  different  respectively.  plotting,  slope  at  second  c a l c u l a t i o n , performed  calculated  and  scans  to  revolutions  of the  per  end  of  flotation  or  the  the  derived  standard minute  S  c  r (rpm),  the  regression or  S„_. 20,w  the  ln  Figure  5.  UV  Scanner  x  (cm)  traces  and d e r i v a t i v e c u r v e s  In  for  (A)  one  and  (B)  x  (cm)  two  component  systems.  - 53 -  Figure 6.  Linear regression plot for two component system in f l o t a t i o n v e l o c i t y run.  - 54  -  density and v i s c o s i t y of the buffer solution, and the p a r t i a l s p e c i f i c volume C v ) of the l i p o p r o t e i n or protein was  required  for these c a l c u l a t i o n s . This l i n e a r regression plus p l o t t i n g program i s very v e r s a t i l e . It could be used for manual p l o t t i n g , or p l o t t i n g plus regression of the data i n the preparation of c a l i b r a t i o n curves from gel filtration,  SDS gel electrophoresis experiments or Lowry protein  determinations.  B.  I s o l a t i o n of VLDL  1.  (MF)  I s o l a t i o n of FII f r a c t i o n  This i n v e s t i g a t i o n was physicochemical  concerned with the i s o l a t i o n and  c h a r a c t e r i z a t i o n of the very low density l i p o p r o t e i n  from the granule f r a c t i o n of egg yolk. Yolk granules were separated  from the plasma by low speed  centrifugation of whole yolk d i l u t e d with pH 7 water containing EDTA.  0.01%  The granules were found to sediment r a p i d l y and t h i s proved  to be a more e f f i c i e n t means of their i s o l a t i o n than u l t r a c e n t r i f u g a t i o n of whole undiluted yolk as performed by Garland  (1973).  Washed granules were dispersed i n 0.025M sodium phosphate buffer containing NaCl (density = 1.063  g/ml), EDTA to i n h i b i t  peroxidation of the l i p o p r o t e i n s and sodium azide to i n h i b i t b a c t e r i a l growth.  A l l buffer solutions were r o u t i n e l y adjusted  to  - 55 -  pH  7.  The  fraction  which  The to  of  Biogel was  neutrality. to  (1970)  the  isolation  isolation  1.71M  was  granules  starting  large  A-50M.  1.0M  salt  NaCl  by  no  effect  effect  coefficients  when  evaluating  human p l a s m a  VLDL  (from  differing  2.  of  of  protein  with  VLDL but  Biogel  (MF) also  FI  is of  a  dispersions.  Absorbance  represent  absolute  the Blue the  the  d e t e c t i o n of Dextran column  polysaccharide  FII  gave  used  light 280  two  to  peaks  particles.  eluant  scattering nm,  was  determine indicating  of  (100  5  cm)  from  et  al.  distribution  eluants  280  nm  of of  to  absorbance  concentration  does  from  x  at  isolation  effects  but  (MF).  during  subjected  the  the void the  a  Sata  in  The  concentration,  VLDL  reduced  or  were  therefore,  isolated  on  for  FII  modified  pattern.  7).  only  of  maintained  fractions  not  the  lipoprotein  pH was  (Figure  l i p o p r o t e i n peaks  2000 was  and  and  protein  of  (MF)  chromatography  function  at  VLDL  the  of  was  lipoprotein classes)  A-50M  the  (1973)  elution patterns  chromatography  granules,  of  source  isolation  elution  Agarose  filtration  pattern  other  on  on  gel  of  concentrations.  Yolk gel  the  electrolyte  gel  for  decomposition  EDTA and  no  the  Garland  concentration  with  observed  of  quantities  The  retarded The  also  of  were  material  p u r i f i c a t i o n procedure  allow  column  ultracentrifuged  of  these  not  is this  turbid  truly  suitable  for  column.  volume  position  inhomogeneity  of  of  these  - 56 -  0  1  60 F i g u r e 7.  '  90  1  120  >  150  I  I  180 210 Tube Number  I  1—  I  240  270  300  E l u t i o n p r o f i l e s o f y o l k g r a n u l e s , F I and F I I f i l t e r e d on B i o g e l A-50M columns. ( B u f f e r = 1.0M N a C l , 0.025M sodium phosphate,0.01% EDTA, pH 7 . 0 ) .  -  The preparations the  first  in  volume.  The of  size.  the  use  LDL  (1969)  larger  the  and  VLDL  the  limited  the volume  FII  allowing be  more in  Gel  peak  eluted  present VLDL  the  sample  in  gel  an  experiments  FI  was  and  FII  and  FII  contained  be  from  that  performed  at  3X  was  also  of  contained  earlier  greater  fractions  contained  in  than  molecular  by  globules  which  only  the  the  whole  and  FII  starting  of  VLDL  FI  fraction,  be  (MF).  separated  to  the  of  column  i t  was  decided  material for  lipoproteins.  (not of  The  gel  early  however, without  The  LDL^  the VLDL  peak  (MF)  than would  ultracentrifuged) isolation.  f o l l o w e d by  granules.  the  study.  FII  to  The  t h e LDL  Since  close  from  this  FI  peak,  normally  experiment.  volume, yolk  both  the volume  applied  of  for  i t  from both  could  other  e l u t i o n volumes  isolated  that  FI  a l t e r n a t e method  of  fraction eluted  isolation  filtration  the void  small was  to  FI  present  t h e VLDL w i t h  fractions  in  (MF)  for  filtration  a t t e m p t e d as  phosvitin  of  sample a  FI  l i p o p r o t e i n peak  LDL  that  l i p o p r o t e i n peak  of  both  (MF),  f r a c t i o n as  fraction, eluted  possible  was  is  floating  of  contamination  the  micelles.  (MF)  elution  the  the  found  t h a n LDL  entire  of  With  broad  indicating  observed  experiments  the  each  fraction.  filtration  of  second  fraction,  Since to  The  microscopy  were much  from  t h e VLDL  l i p o p r o t e i n from  FII  Chang  electron  produced  lipoprotein,  polydisperse  that  peaks  -  indicated heterogeneity.  eluted  the v o i d  the  two  57  the  the  yolk VLDL  , HDL  (MF)  fraction in  a l l  (MF)  and  lipoproteins  VLDL  granules  peak, the  were  - 58 -  Evans plasma Three 280  by  The  highest  were  large  percentage  lipid  million  (81%).  agarose  column  crude  VLDL)  which  was  reported  VLDL  of  They  isolated  for  The range of  and  VLDL  of  gel  the  galactose  in  or  first  those was  in  4B,  to  from  Biogel  yolk  A-15M  estimated  the  afterward to  columns.  at  contained  eluting  range  by  and Musser  contained  from  separate  low  a  fraction  (presumably  of  the  floated  plasma  5-17  agarose  two m a j o r  to  by  using  components  (Hjerten,  the  Evans as  of  1964).  When  or  The et  ultratube  LDF^.  No  data  fraction.  upon  gel  for  VLDL  is  the  column. filtration  in  the  fraction  void  probably  (1973) . expanded  macromolecules.  agar,  plasma  the  eluting  al_.  2%  the  a procedure  first  a  of  bed m a t e r i a l has  large  yolk  a DEAE-cellulose  peak.  gels  very  this  f r a c t i o n and  broad  isolated  LDF^  used  lipoproteins  gradient  developed  two p e a k s ,  second  filtration  either  (1974)  the unadsorbed  of  column.  the meniscus  c h a r a c t e r i z a t i o n of  Mahadevan  to  to  the  a  density  analytical density i t  (1974)  to  correspond  that  polymer  Bacon  not  yielded  use  and  the v o i d volume  from yolk  f o l l o w e d by  corresponded  one  A-50M)  studied  eluted  Sepharose  volume  size  (Biogel  therefore did  isolation  on  eluted  (89%);  (1973)  techniques,  Raju  The  lipid  AB  VLDL  u l t r a v i o l e t absorbance  f r a c t i o n which of  isolated  Sepharose  having  Molecular  eluted  centrifugation  were  eluted  1975b)  on  et_ a l .  LDF.  lipoprotein  and  (1973,  daltons. Bacon  from  al.  chromatography  fractions  nm.  less  gel  et  an  Nonspecific  uncharged  Agarose, linear  adsorption  of  the  -  protein with A  the  high  the  and  ion-exchange  relationship  ionic  VLDL  agarose  so  gels. The  of  drastic  and  constantly widely  serum VLDL Several  procedure Sata  was  Raju  in was  structure  VLDL  in  is  these  In  a buffered  subfractions utilize  the  or  as  an  1966;  Margolis,  1967;  1972;  Quarfordt  et  1974;  and  to  the  Rudel  et  in  of  of  classes  and  1972;  the  the  use  VLDL of  gel  by diffusion  filtration  native of  human  lipoproteins.  conjunction  Kalab  a l . ,  to  forces  alternative  al. ,  elute  unnecessary.  Agarose  other  technique  to  isolation  only  size.  precludes  characterization  from  this  molecular  probably  the  interfere  lipoproteins  solution.  and  to  studies  of  was  subjected  isolation  and  lipoproteins  procedures.  sample  the  of  likely  with  isolation  Martin,  Fidge,  1968b;  1973;  1974).  Alkylation  preliminary  observed  and  plasma  the  Kirsch,  In it  in  however,  1970,  and  used  precaution,  (Werner,  3.  was  ultracentrifugation  et_ a l . ,  Glickman  e l u t i o n volume  This  laboratories  preparative  not  adsorption  in  and  thus  to minimize  remains  used  are  as  fractionation  filtration,  is  between  delicate  gel  -  effects  strength'buffer  (MF)  59  that  Mahadevan  VLDL were  a buffer  VLDL  (1976)  insoluble  solution  suggested  as  ultracentrifugal  that  (MF)  discovered in  that  aqueous  containing  the  samples  0.01M  insolubility  of  flotation velocity aged, the  buffer  aggregates  apoproteins systems,  but  2-mercaptoethanol the  proteins  experiments,  were from  yolk  were  soluble  (BME).  might  formed.  be  It  partly  -  due  to  which been  the  hinder due  bridges that  to  oxidation  to  It  not  the  with  BME  a l l  by  be  the  lipoprotein.  diluted and  took  in  place  gel  formation gel  of  a  isolation  of  VLDL  The  volume,  followed  material.  caused  of  turbid  filtration,  lipid  fresh  a  exceeds  that  an  It  was  (Means wide  and  range  Feeney, of  could  samples  have  disulfide  subsequently  of  observed  under  nitrogen  VLDL  protein  lipid  was  1971).  conditions  and  egg  yolk  or  granules  was  and  0.1%  VLDL  as  yolk  containing  0.1%  After  the  iodoacetamide. in  in  the  contained  acrylonitrile  the  sulfhydryl-blocking  under  obtain  with  moiety.  iodoacetamide  almost to  to  void  protein  groups  the  suspension  probably  the  also  isolation  the  possible  s t i l l  that  oxidation  resulted  eluted  the  from  BME.  (MF)  was  of  lipoproteins  subjecting  thought from  cause  water  exist  any  prepared  f r a c t i o n which  groups is  could  i f  alkylated with  solution  sulfhydryl  It  particles  (MF)  Upon  chosen  protein  (MF)  saturated  (MF)  therefore the  VLDL  containing  opalescent  of  other  of  suspension.  by  (MF)  forming  therefore  a c r y l o n i t r i l e to  r e a c t i v i t y of of  was  nitrogen  the  Iodoacetamide the  (MF)  buffers  clear  dissociation  because  VLDL  handling  chromatography, Addition  i f  in  degassed  was  storage  state  subsequent  groups,  It  determined  deoxygenated,  in  and  VLDL  bridges,  effect.  a p a r t i a l l y aggregated steps  of  sulfhydryl  particles.  oxygen  this  aggregation  free  (MF)  diminish could  The  of  of  -  inter/intra-molecular disulfide  solubility.  exclusion  numerous of  of  between VLDL  the  helped  in  existence  60  a l l  employ  reaction  agent far  conditions  an with  extremely sulfhydryl  -  groups, There  although  have  been  pH no  6.0-8.5  such  a  the  (MF)  (MF)  and  0.01%  sulfhydryls  guanidine have  and  only  Cook,  the  conditions native  7.0).  addition  might  buried  1960).  accessible  not  be  in  native  groups. content Even  As of  after  the  SH  of  on  It  not  the  the  buffer  during of  the  for  modifying  of  1.0M  VLDL  (MF)  as  this  can  only  s u c h as  SDS,  urea  or  agents  have  been  shown  causing  (Nichols  purpose  of  loss  et  this  rather  of  a l . ,  197 6;  be  than  to  involved  the  lipoprotein molecule  be  lipid  experiment  which might  oxidation,  the  containing  attempted  groups  normally  that  used  reaction  lipoproteins,  the  such  buffer  dissociating  was  concerning  delipidated  chosen  denaturants  sulfhydryl  interior  and  control  determinations the  were  reaction.  iodoacetamide;  on  Quantitative was  the  sulfhydryl  which  reactive.  The unmodified  with  state;  for  literature  the protein molecules  lipoprotein aggregation  groups  optimal  the  phosphate  These  effects  of  in  be  performed  sodium  hydrochloride.  and  is  iodoacetamide the  dissociation  block  (pH  deleterious  Bernardi  in  by  its  0.025M  EDTA  with  accomplished  to  was  in  to  lipoproteins  Experimental  remained  VLDL  NaCl  reports  chemical modification  lipoprotein. VLDL  native  -  reported  previous  the m o d i f i c a t i o n of  61  content  samples  were  protein  SS  are  carried  out  of  listed  disulfide  the  (MF)  (MF)  to had  VLDL  VI.  addition  exposing  compared  the VLDL  Table  the  c o n t r o l VLDL  groups  alkylation,  alkylated in  after  configuration,  expected,  the  the  SH of  previously sample  (MF)  content SDS  which  buried  had  alkylated  a much  and  greater  a  alters  SH  high  sample. SH  content  -  Table  VI.  Estimation Using  Alkylated  A  of  VLDL  VLDL  data  Ellman's  •,  s  Unmodified  of  (MF)  (MF)  reported  the  mean  as  a mean  62  -  S H a n d SS  Contents  of  VLDL  Reagent.  SH c o n t e n t * (uM/g p r o t e i n )  SS c o n t e n t * (pM/g p r o t e i n )  71.05+  2.50  308.85+  434.53+20.25  83.47+  of  6 determinations  +  standard  15.62  4.40  error  - 63 -  than  that  of  the  VLDL  (MF)  would  inaccessible out  in  the  control. indicate  to  the  the  of  groups,  resulting  molecules.  The  should  used VLDL  with  expected  lipoproteins. which  were  alkylated to  those  w i l l  be  4.  of  Gel  this  to  samples  and  of  had  larger  filtration  on  were carried  groups  in  oxidized  VLDL,  on  and  that  present  and  The  of  BME  reduced  alkylated  the  the  porous  glass  in  VLDL  of  the  (MF)  flotation (MF).  rates,  The corresponding  ultracentrifugal  section  results  C.2.  beads  o Glass to  separate  preparation  beads  the VLDL than  (3000 A (MF)  could  be  pore  particles obtained  size) into from  were  used  a more the  in  an  attempt  homogeneous  Biogel  (MF)  congruent  VLDL  flotation coefficients  discussed  hand,  Ultracentrifugal  alkylated  VLDL  These  lipoprotein  modified  aggregates  of  SS  other  the  flotation coefficients combinations  the  to  the  state.  unmodified  lipoproteins.  further  alkylated  aggregates  unaggregated  the  had  SH  were  alkylated  0.1%  of  groups  the  hypothesis.  of  those  of  the  distinct  reduced  presented  of  the  r e a c t i o n was  sample  were  the  usually for  (MF)  the  in  addition  similar VLDL  groups  test  (MF)  The  SS  of  SH  the  most  (MF)  content  the  of  agents.  formation  analysis  to  of  content  when  that  VLDL  remain  unmodified those  SS  few  velocity  was  the  low  lipoprotein  samples  suggest  in  that  most  SH  dissociating  unmodified  demonstrates  flotation  of  data  protein  that  high  iodoacetamide  absence These  The  columns  (1%  -  or  2%  was  agarose).  VLDL The  (Figure  (MF)  8A).  prior  first  to  peak  (V  contain  =  only  centrifugation,  be  further  using size  the had  it  may b e  large  C.  the  eluted at  been the that  of  chosen.  have  of  the use  porous  as  BME  peaks  nm  (Figure  9A)  shows  a  (Figure  containing (V  =  BME w a s  80 m l ) ;  position  8B).  the  found  second  and was  found  ultra-  and  beads  glass  These  section  results  p a r t i c l e s was  even  though  reports on  beads  the in  glass for  always  w i l l  D.  (MF)  been no  proper  the  bead  gel  pore  literature columns;  filtration  proved  VLDL  achieved  of  VLDL  (MF)  (MF)  f r o m 220  exponential curve  of  unsuccessful.  spectrum  spectrum smooth  unmodified  flotation velocity  l i p o p r o t e i n s had  absorption  the  (MF)  phosphate  ultra-  lipoproteins  Ultraviolet absorption  UV  sodium  to  F l o t a t i o n v e l o c i t y u l t r a c e n t r i f u g a t i o n of  The  a l k y l a t e d VLDL  concentrated by  t h e VLDL  separation of  C.2.  glass  There  the V  both  NaCl  two  column  when  electrophoresis.  section  porous  0.1%  peak was  by  gel  f r a c t i o n a t i o n of  1.0M  fraction  The m a j o r  in  in  CPG  (MF)  agarose  p a r t i c l e s such  1.  360  and  observed  produced  then analyzed  column  concerning  ml)  discussed No  from  BME.  was  beads  filtration  134  -  a d d i t i o n of  e n t i r e VLDL  e  centrifugation  peak  glass  The  t u r b i d peak  contain the  clear to  one  eluted from porous  buffer  to  Only  64  without  nm  to  any  - 65  -  Tube  Figure  8.  E l u t i o n p r o f i l e s of (B)  2-mercaptoethanol  g l a s s bead  columns  (A)  Number  a l k y l a t e d VLDL  r e d u c e d VLDL  (MF)  (MF)  and  from  porous  -  obvious vs  absorption  wavelength  Extrapolation contribution large peak to  VLDL at  only  9B)  of  line  of  this  nm i s  of  2.  280  nm.  shows  a  absorbance  the  to  is  (Figure  The  (MF)  of  VLDL  buffered NaCl  carried g/ml  out or  coefficients  f  =  S  (MF)  (Figure seen 10B),  in  1.0630  were  18,000  g/ml  these  to  UV  of  The  small  280  nm  due  represents  flotation A l l  7.0)  observed  standard  residual  particles.  by  (pH  nm.  the  contribution  optics.  solutions  20°C.  scattering  large  examined  360  the  A  at  to  represents  this  peak  absorption  f r o m 300  1975).  when  rpm u s i n g  at  corrected  was  light  of  of  analyses densities  flotation  conditions  using  10.  1.0385  The VLDL  of  analysis  were  S  Ma,  Flotation velocity  purity  segment  from  The  absorption  plot  wavelengths  9A)  subtracted.  total  log-log  linear  1967;  at  equation  The  resulting  ultracentrifugation  1.0385  -  lower  (Tanford,  detected  scattering  8-11%  at  (Figure  particles  280  light  peak  66  or  10A)  more  ( 1  ~ /° 1.063 V  ) / ( 1  - / ° 1.0385 V  typical flotation patterns VLDL  (MF)  indicated  readily  in  c a l c u l a t e d from  recovered that  the  a l l  from  computer  t h e UV  of  the  samples  (Equation  )  porous  were  generated  optics  native  VLDL  glass  (MF), bead  polydisperse. derivative  concentration  scan.  10)  alkylated column  This  plot  is  (Figure  Native  -  220  240  67  260  -  280  7\.  0.36  0.38  0.40  0.42 log  Figure  9.  300  320  340  (nm)  0.44  0.46  ( ^  /100)nm  0.48  0.50  UV a b s o r p t i o n s p e c t r u m o f V L D L (MF) ( A ) a s w a v e l e n g t h a n d (B) as l o g - l o g f u n c t i o n .  a  0.52  0.54  function  of  - 68 -  70  60  (A)  504  c  AO 4  o  u 4J fi  <1J O  C  o u  30 7 20 4 10 4  6.9  7.0  7.1 x  7.2  (cm)  500 1 A00  x  300  1  4  T3  U  2001  100 4 1—  -+-  1.931  Figure  10.  F l o t a t i o n v e l o c i t y a n a l y s i s of p a t t e r n (B) D e r i v a t i v e curve.  1.97A  VLDL  (MF)  (A)  Flotation  - 69 -  VLDL  (MF)  being were  floated  apparent  40-120S.  decreased added rate S  f  the  S  75  f  alkylated with  heterogeneity S^  at  in  to of  the  of  41  and  slow  VII).  (section  density  was  obtained  by  of  as  component  specific  comparative  different plot  of  of  (1.014 v  g/ml)  =  was  .  results.  (MF) using  peaks  of  were BME  decrease  was  in  flotation  the  alkylation  the  disulfide  these VLDL  (MF)  porous that  the  the  to  with  BME.  confirm  this  beads  showed occurred.  component  flotation  coefficients  densities  (Figure  of  solvent  partial  due  exposed  alkylated  floating  and  Accurate  the  fractionation  of  for  to  native  bonds  or  glass  no  slow  of  fractions  density  and  range  that  product  impossible  VLDL  volume  of  the  indicate  solvent  the  marked  When 0 . 1 % a  which  component  of  measuring  a function  where  insolubility  partial  the  density  (0.986 ml/g), floating  of  of  (MF)  fast  of  indicating  hydrated  (^s)  for  column  in  peaks  the  of  Samples a  and  by  of  analysis  through  lipoprotein at  flotation  The  reduction  a  2 major  t h e r e was  disrupted  B.3).  floated  45S  VLDL  showed  the  results  The  Extrapolation  values  by  flotation rates,  (MF)  the  were  S^  with major  of  alkylation.  (MF),  to  These  disulfide  passed  by  VLDL  component  indication  of  108S  unmodified  and  VLDL  also  flotation rates  Sulfhydryl  similar  Samples  The  f  70-250S,  f  and  aggregates  (MF)  207S.  iodoacetamide  s u l f h y d r y l groups or  VLDL  S  flotation rates  (Table  (MF)  and  of  in  S  the  116S  VLDL  to  range  the  its  inaccuracy  a pycnometer  high of  of  volume  the  faster  flotation  measuring  prevented  times  produced  specific  measurement  11).  viscosity  solvent  of  rate.  the.  determination  -  Table  VII.  70  -  Comparative F l o t a t i o n Rates Reduced  and  (S^)  of  C h e m i c a l l y M o d i f i e d VLDL  Flotation Sample  Native,  Slow  (MF).  Rate  Component  (S ) f  Fast  Component  native  VLDL  (MF)'  74.84  +  4.5  207.30  native  VLDL  (MF)  45.39 +  2.5  116.43 +  40.51  2.1  107.69  +  0.1%  BME  alkylated  +14.3  6.9  b  VLDL  (MF)  a  mean o f  20 d e t e r m i n a t i o n s +  b  mean o f  6 determinations +  +  standard  standard  error  error  of  of  the  the  +6.2  mean  mean  -  l  1.01  I  1.02  l  11.  1.04 of  Hydrated-density and  solvent  -  i  i  i  1.05  1.06  1.07  l  1.03  Density  Figure  71  NaCl  of  density  solution  VLDL at  (MF)  20°C.  ,  1.08  1.09  (g/ml)  estimated  from  r^s  -  Turner on  the  partial  increase in  the  and a  in  partial  lipid  an  a hydrated  theoretical the  to  values  the  partial  values the  may  be  of  300-900  VLDL  to  arisen  of  the  VLDL  larger  A  (Scanu  plasma yolk  VLDL  plasma  (Hillyard  distribution analyzed  in  The  flotation  et  a l . ,  (Koga VLDL  et_ a l . ,  schlieren  has  been  rates  of  use  of  protein.  a decrease 1966);  would of  factor,  of  have  the  the  average  the  calculated  is  considered  to  rabbit  VLDL  (Shore  optical  optical  lipoproteins.  from  or  1973), et_ al.,  chicken  on  use  the  in of  analytical  the  peaks  a  the  Beckman  Prep  broad  when  ultra-  determination  1974),  serum  showing  three major  from  system.  used  The  and  heterogeneous,  system  conventionally  1976),  two  partial  possess  varying  1969),  with  bound  apoprotein.  (Fidge,  also  the  theoretical  VLDL  were  as  carbohydrate  with diameters  a l . ,  values;  0.724 ml/g  plasma  et  ml/g,  These  derived  thus  been  0.724  corresponding  The in  1%  this  Pig  (Yamauchi  schlieren  the  molecules  flotation rates the  the  a  increase  calculated.  human s e r u m  1974).  e_t a l . , 1 9 7 2 )  using  centrifuge  f r a c t i o n of  d i s t r i b u t i o n of  be  content  an  volume  0.982 ml/g  (Gibbons, than  lipid  that  Using  experimentally  causes  protein  of  from  of  of  found  specific  may  the  a c t u a l p a r t i a l s p e c i f i c volume  continuous o  egg  g/ml  somewhat  The VLDL  rat  of  close  and  0.003 ml/g.  volume  1.018  effect  l i p o p r o t e i n causes  partial  s p e c i f i c volume  volume  a  by  protein  have  protein moiety  specific  of  the  proteins  specific  are  could  studied  of  volume  average  density  discrepancy  average  volume  specific  -  (1958)  portion  theoretical partial  to  a  Cook  specific  the  assuming  and  72  of UV  - 73 -  optical  system  absorption VLDL  (MF)  (1977)  this  optical  described with  coefficient  use  calling  VLDL  schlieren  particles.  from  the  The for  the  it  was  in  this  in  effect  are  likely  coefficients  the  of  et  a l .  ultracentrifuge the  flotation particles.  "turbidimetric solutions  results  agreement  be  in  or  of  the  high  of  the  conventional  with  the  results  study  were  of  any  (less  than  optics.  is  even  though not  corrected  several  advantages  of  negligible  lipoprotein)  observed  approximation  since  component  effect  2 mg/ml  The  corrected  effects,  single  concentration  good  not  concentration  amount  The  a  this  for  the  w i t h UV to  analysis  turbidity  concentration  combination  E  human s e r u m VLDL  calculated  system.  used  Model  Ma  of  system.  heterogeneous low  progress,  of  good  quantify  since  in  samples  found  in  was  determine  of  an  flotation rates  to  optical  rates  of  for  flotation the  true  these  effects  1975). There  determine  such  be  of  scanner  the  to  flotation  to  UV  impossible  coefficients  (Ma,  to  application  the  Beckman  method  to  first  work  the  They  Johnston-Ogston  however, were  due  technique  obtained  this  for  this  the  determine  ultraviolet  ultracentrifugation" lipid  to  of  distribution  proposed  was  While  the  an  study  system  particles.  equipped  They  in  as  VLDL  absorption (Figure directly  of  9A),  are  flotation (MF) UV the  related  in  coefficients  the  light  large,  ultracentrifuge.  due  reflection to  of  using  to of  the UV  concentration  low  and  was  UV  there  content  measured.  particle  optical  heterogeneous  Since  protein  light  the  size,  was of  system  particles l i t t l e  VLDL  Turbidity which  is  greatly  -  magnifies optical 2%  is  apparent  system  usually  the  diluted also  UV  Lower  that  and  minimize  diffusive  of  recording used  in  labor  D.  from 1% One  UV  scans and  boundary  system to  be  conjunction in  scans  with  taking  of the  agarose broad  origin  film band,  strips which  corresponds  lipoprotein multiple  bead  which  do  components  of  of  (section  electrophoretic  system.  short by  be  (MF)  used had  to  be  concentrations and  (30-45  the  large  minutes)  of  at the  VLDL  C.2)  (MF)  could  allowing  time.  tedious  the  a  lar  Automatic  acquisition the  performing  system manual  calculations.  (MF)  (MF),  or  i n i t i a l l y  origin,  chylomicrons in  of  TTY-data  cassette  the  seconds,  eliminates  VLDL  were  75  period  the  and  VLDL  t h e ACI  migrate  the  ultracentrifuge  a  alkylated  using  not  low  of  VLDL  usually  negligible,  every  scanner,  column,  that  are  of  the  could  samples  use  cell  data  UV  remained  to  (MF)  measurements  (MF),  glass  of  centrifugation  the  the  Samples  VLDL  of  concentration  lipoprotein  The  collected in  behavior  a porous  VLDL  schlieren  spreading.  Electrophoretic  of  of  effects  period  processing  involved  the  a  concentration  concentrated.  short  When t h e  lipoproteins,  requiring  concentration  size  The  study  system;  than  particle  number  to  -  concentration.  concentrations  optical  rather  means  used  necessary,  preparation. with  is  protein  74  agarose  VLDL  be  isolated  electrophoresed  electrophoresis was of  gel.  observed. human (Table  fraction detected not  (MF)  resolved  kit. This  serum I). in  using  on  The the this  -  Migration by  two  types  of  of  material.  to  of  passage  interactions affect were  the  large  affected  the A  secondly,  lipoproteins Since  agarose,  series  of  gels  gels  to  strength  did  not  the  pore  add  resolve size  was of the  the  was  particles. eliminated  (Figure  s t i l l  t h e way  and  Two to  lipoproteins  with  than  size  that of  the  offer  resistance  physicochemical  medium  the  the  small  of  could  lipoproteins  gel  must  have  were The  3%)  polymerized  the w/v)  of  the  performed using B  was  to  VLDL  (MF)  samples;  the  acrylamide  only  0.5%  trace  samples  agarose.  in  VLDL  The  the m i g r a t i o n  run  parallel  of  the  with  the  for  protein with  Coomassie  Blue  d e t e c t e d , no  matter which  VLDL  sample  first  band  migrated  the  agarose.  same  Electrophoresis  in which  enter  the  into  large  second  showed  acrylamide  the  the  not  low  of  while  did  At  incorporated  entry  gel,  and  size.  handling.  permit  stained  composite  pore  any  Unstained  using  optimum  easy  to  formed  later  gels. the  were  performed  (0.5%  sudan b l a c k  12).  bands  the  was  permit  were  were  through gel  and  too  gels  samples  running  pore  agarose  Experiments  (Figure  12).  (less  lipoproteins  prestained  applied  supporting  unlikely  co d e t e r m i n e  the  prestained  the  experiments  concentrations  lipoproteins  is  may  modified  resolution.  therefore  VLDL was  i t  and  is  and  and  fluid,  was  lipoproteins  lipoprotein molecules  remained  (MF)  size  field  gel  the  acrylamide-agcrose acrylamide  the  electric  the  the  pore  an  of  resolution. to  in  between  First,  between  adsorbed  -  lipoproteins  interactions  supporting  75  results,  band  indicating  about  three-quarters  remained  Coomassie that  (MF)  on Blue  top  of  stained  prestaining  of  - 76 -  (A)  Figure  12.  (B)  Agarose B  gel  prestained  electrophoresis and  (B)  pattern  Coomassie  Blue  of  (A)  stained  sudan  black  VLDL  (MF).  - l i -  the. V L D L  (MF)  with  sudan b l a c k  B had  no  effect  on  electrophoretic  an  electrophoretic  mobility. The  use  facilitated Molecular  of the  was  apparently  Agarose the a  had  gels.  gels  agarose  Immarino was  et  usually  less  has  was  been  a l . ,  the  in  combination  and  application with  requiring  black the  requires  B prestaining bands  staining  and  are  a  of  many  with or  seen  years  and  and  the  the the  gel  agarose  gel.  to  handle;  this  method  the  slides  has  of  Noble,  to  B  been  gel  followed used by The  1%  through  normally  1968; the  film by  electro-  Narayan  study  provides  an  the  thus  carried  performed The  film  concentration.  gel,  a l .  disc  electrophoretic Agarose  sample  et  present  lipoproteins.  agarose  strip.  electrophoresis  lipoprotein. a  electrophoretic  however  a plastic  sensitive  lipoproteins  steps  1968a;  (1963).  disc  the most  use  for  or  B prestained  of  a medium  1976),  sudan b l a c k  agarose  is  as  a l . ,  McDonald  migrating  destaining  adapt  Martin, et  paper  few pg  the  the  impossible to  on  enter  of  of  successful.  sudan b l a c k  only  charge  not  pores  were  Yamauchi  of  the  medium  components.  obtained  did  Attempts  (Kalab  gels  two  electric  which  on microscope  Ribeiro  the  (w/v)  for  lipoproteins  electrophoresis  as  and  into  resolution  low.  not  e l e c t r o p h o r e t i c method  system,  the  0.5%  used  1969;  supported  is  as  penetrate  too  were  acrylamide  gel  to  than  phoresis  first  in  lipoproteins  1966b)  well  (MF)  large  of  (1966a,  VLDL  too  Prestaining on  as  as  l i p o p r o t e i n band  scale  of  of  some r o l e  content  Agarose separation  effects  gels  The  of  preparatory  disc  resolution  sieving  lipoproteins agarose  agarose  out  immediate  strip Sudan result  eliminating  after  the  electrophoretic  -  separation. to  the  E.  The  entire  completion of  and  the  VLDL  fractions,  Alaupovic, samples  which  only  would  have  1974)  of  apoVLDL  contained  three  to  almost  and from may  s i a l i c the be  of  Raju  the  acid  three  a  size,  and Mahadevan  composition  of  yolk  but  sugars,  of  have  VLDL.  obtained.  A-Sepharose  separated  The  the  affinity  sugar  c o l u m n may  residues  be  unavailable  lesser of  revealed  (2.51%);  VLDL-U had  ratios  only  detected  quantities these  indicating  the  found  of  sugars these  different a f f i n i t i e s for  They  (McConathy  lipoprotein.  and  determined  o(-D  1976). to  carbohydrate  similar,  hr.  4B  VLDL  applied  A  gels  (retained).  al.,  carbohydrate  with  The  very  (1976),  plasma  were  et  2  methyl  delipidated fractions  major  residues.  than  was  serum  (Azuma  the  (4.83%),  The  are  of  amount  much  (MF)  w i t h human  (MF)  0.2M  VLDL-R  Concanavalin  the  small  hexose  fractions  similar  of  VIII).  sugar  the  aggregation  t w i c e as  was  VLDL  and  VLDL  less  agarose  Concanavalin  eluted with  f r a c t i o n was  to  analysis  (Table  fractions  serum  the  A-Sepharose  A l k y l a t e d VLDL  observed  pig  bind  contained  carbohydrate  been and  the  (unretained)  the unadsorbed  Carbohydrate the  13).  non-alkylated  due  to  of  requires  Concanavalin  m a t e r i a l was  VLDL-U  normally  binding  was  (Figure  patterns  column,  (MF)  adsorbed  formation  electrophoresis  applied  Similar  for  the  from  Alkylated  two  When  experiment  on  mannopyranoside  and  -  A f f i n i t y chromatography  column  into  78  that  VLDL-R 1.03%  in  a l l  hexosamine obtained particles  Concanavalin  carbohydrate  the  sugars  present  in  a  A.  0  5  10  15 Tube  Figure  13.  Elution (  i  profile  indicates  of  alkylated  application  of  20  25  Number  VLDL  (MF)  from  0.2M  methyl  Con-A  c*. - D  Sepharose  4B  mannopyranoside)  column  Table  VIII.  Carbohydrate  Composition  of Apoproteins  from Whole  and F r a c t i o n a t e d  Lipoproteins.  Hexo s e/Hexo s amine/ Sample  Hexose* (%  VLDL  (MF)'  Sialic  Acid*  Total (%)  Sialic  Acid  Ratio  +  0.08  0.58 +  0.03  0.25 +  0.03  2.51  1.0/0.35/0.15  VLDL-U  0.67 +  0.03  0.22 +  0.02  0.14 +  0.01  1.03  1.0/0.33/0.21  VLDL-R  3.16  0.15  1.08  0.06  0.59 +  0.03  4.83  1.0/0.34/0.19  VLDL (yolk  1.68  hexosamine* of protein)  +  +  1.91  0.52  0.39  2.82  1.0/0.27/0.20  1.00  0.53  0.33  1.86  1.0/0.50/0.30  1.30  0.67  0.38  2.35  1.0/0.50/0.30  2.23  1.67  0.83  4.73  1.0/0.75/0.37  VLDL-U° (pig serum)  0.93  0.47  0.14  1.54  1.0/0.50/0.15  VLDL-R° (pig serum)  4.20  3.21  2.05  9.46  1.0/0.76/0.49  3  plasma)  VLDL (chicken b  VLDL (whole  serum)  b  VLDL (pig  egg  yolk)  C  serum)  *  mean  of  3 determinations  a  from Raju  b  from Abraham  c  f r o m Azuma  +  and Mahadevan, e t a l . , 1960  e_t a l . , 1 9 7 6  standard  1976  error  of  the  mean  -  hexose/hexosamine/sialic to  the  results  Abraham class to  of  the  The  ratios  a  was  study  of  egg  studied  and  source  of  egg  composition  A  column  of  (Azuma  and  retained  f r a c t i o n , and  when The  compared  ratios  fraction  particle sizes  Concanavalin velocity  tabulated  the  another,  in  the  pig  (MF),  yolk  indicating serum  VLDL-R  with  IX).  whole  61.45S  lipoproteins  data  for  the  VLDL-R  are  of  system were  and  fraction.  similar  serum  fractions  indicated  the  the  unfractionated  amount  in  fractions  the of  were  unretained the  not  t h e r e may  by  be  present  constant a  variety  optics and  the  the  53.16S  for  used  results in  (S  a l l  , ) obs  Different  are  13.87  VLDL-U,  and  in  particles amounts  in  three  of  similarity  a t t r i b u t e d to  size.  were  detected  The  from  sedimentation  coefficients  13.29  c o e f f i c i e n t s c o u l d be which  the  lipoproteins  absorption  components  (MF),  in  that  examined  sedimentation  VLDL  bound in  lipoprotein  however,  VLDL-U  acquisition  Two  observed  for  sedimentation  the  lipoprotein  VLDL.  and  UV  (MF).  present  that  1976)  (MF)  residues,  higher  were  serum  similar  VLDL  VLDL  lipoproteins.  e_t a l . ,  a f f i n i t y column were  with  yolk  similar  carbohydrates  concluded  pig  a  and  sources  very  granule  The  carbohydrate  sugar  to  VLDL  (Table  56.48S and  egg  ultracentrifugation.  conjunction  samples, '  A  of  total  to  serum.  They  the  yolk 20S  from both  (1.0/0.5/0.3). the  the  chicken  much  one  15.45  present  t w i c e as  The w h o l e  and  1.0/0.27/0.20,  of  fraction study.  of  obtained  Concanavalin  apoVLDL  ratio  yolk  carbohydrate  presence  of  egg  -  acid  (1960)  lipoproteins  lipoprotein  from  the  e_t a _ l .  whole  identical  from  of  81  of of  -  Table  IX.  Sedimentation VLDL  (MF)  82  -  Coefficients  Fractions  from  (S  , ) obs  of  VLDL  Concanavalin  A  (MF)  and  Affinity  Chromatography.  Sedimentation Sample  VLDL  Slow  (MF)  Coefficients  Component*  Fast  (S  , ) obs Component*  13.87  +  0.68  56.48  +  2.52  VLDL-U  13.29  +  0.65  53.16  +  2.34  VLDL-R  15.45  +  0.76  61.45  +  3.15  *  mean  of  3 determinations  +  standard  error  of  the  mean  -  carbohydrates their  bound  behavior The  on  by  SDS  gel  mercaptoethanol. PAS  stained  two  fractions  It  each  can  be  60, 300  and  ;  while  bands  respectively) in  the  SDS  (136,000) Bands  1,  5,  were  bands  were  observed  PAS  8  in  while  containing  proteins.  content  these  groups  sterically  the  related having  VLDL  was  (MF)  only  71,500, but  VLDL-U This  was  in  a  of  in  the  and  evident lesser with  stain  78,700, protein,  other  band for  1 glycoprotein.  PAS  bands  VLDL-R was  amount  bands  respectively)  positive  the  fractions.  35,800  59,500  that  the  VLDL-U  The  Only  PAS  both  and  on  differed  130,000,  71,500  weakly  agreement  obtained  to  fraction.  65,200  contained  is  common  (136,000,  the  other  It  those  protein.  with  2—  compositions  were 8  of  were  chromatography  VLDL-R for  strongly  bands,  with  131,100,  the  VLDL-R  presence  predominant  lightly  fractions.  A has  of  molecules  7 and  were  in  for  of  total  rich  carbohydratecarbohydrate  fractions.  Concanavalin hydroxy  the  compared  (136,100,  (136,000,  both  in  affinity  4,  account  VLDL-U and  polypeptide  by  2,  predominant  positive  glycoprotein  of  1,  10  stained  the  few p o l y p e p t i d e s  5 and  7 and  strong  The  separated  VLDL-U r e a c t e d  were  in  14).  would  column.  of  are  respectively)  2,  gels in  results  although that  1,  A affinity  electrophoresis  VLDL  35,800  -  protein moieties,  compositions  (Figure  of  seen  Con  These  gels  other  the  the  polypeptide  analyzed  from  to  83  a  <*-D  sugar  these  resolved  specificity for  the  C-3,  glucopyranosyl,  <x-D  mannopyranosyl  residues  structures into  (Goldstein are  unadsorbed  bound and  C-4  et_ a l _ . , to  the  adsorbed  and  1965)  C-6 or and  affinity fractions  columns. and  - 84 -  (A)  1  2 3 4 7 8  (B) 1 3  5 6 7  (U)  Band 1 2 3 4 5 6 7 8 9  Band 3 4 6 8  ( R )  Figure 14.  9  9  10  10  11  11  1 2 3 4 5 6 7 8 9 10 11  MW 136,000 130,000 110,700 78,700 74,200 67,600 60,300 54,100 9,200  MW 136,100 131,100 83,200 77,200 71,500 67,000 65,200 59,500 46,300 35,800 23,800  SDS polyacrylamide gel electrophoresis of apoVLDL i n the presence of BME of (U) Unretained f r a c t i o n and (R) Retained f r a c t i o n from Con A Sepharose 4B column stained with (A) Coomassie Blue and (B) PAS.  I  - 85  the  differences  results  of  patterns  F.  the  of  in  of  VLDL  been  several  by  gel  Martin,  reports  1968b  lipoprotein Partial  in  and  usually  results  solubility  of  for  the  detergents  of  the  Alkylated the procedure  of  chloroform-methanol  one  the  a  calibrated  in  particles  a l . ,  the  of  (Brown  other  (MF)  was  extracts of  was  Sepharose  could  1964).  ether  or  neutral  a l l  the  the  Thin  however,  class  residues,  separated  into  2  4B  (Figure  and  of  results  preserves  without  need  n-heptane  according  chromatography  presence  The  partially by  Peak  gel I  of  lipoproteins  the  fractions 15).  and  agents.  layer  and  Kalab  apoproteins.  partially delipidated lipids  have  delipidation  insoluble  lipid  separated  there  1969;  n-heptane,  phospholipids.  column  be  fractionation  a l . ,  delipidated with  neutral to  the  Total of  not  However,  dissociating  (1965). of  et  formation  the  or  Gustafson  (MF)  et  diethyl  corresponding  VLDL  (MF)  phospholipid-protein  VLDL  absence  spot,  delipidated  by  n-heptane  lipoproteins  p r e f e r e n t i a l removal  indicated  supported  electrophoretic  l i t e r a t u r e concerning  Augustyniak  the  on  VLDL  the  delipidation with  use  gel  f i l t r a t i o n techniques.  partially delipidated  only  and  A are  (MF)  heterogenous  subfractions  to  analysis  P a r t i a l delipidation using  into  the  Concanavalin  their.apoproteins.  The  in  for  carbohydrate  Delipidation  1.  of  affinity  -  of  f i l t r a t i o n  eluted  at  1.4  ce o 00  1.2  1  1.0  1  0.8  1  0.6  t  0.4  t  0.2  t  u  c oo  O w  0.0  — i — — i —  40  160  200  Elution Figure  15.  Sepharose  4B  — i —  — i —  240  280  Volume  chromatography  of  300  (ml) heptane  320  360  f  400  V„ d e l i p i d a t e d VLDL  (MF)  -  a  volume  equivalent  to  a volume  at  Partial  d e l i p i d a t i o n produced  corresponding  ratios  Neither  fractions  in  the  HC1  II  (pH  third  of  contained  prior  45,000  i t  did  not  peak by  both  I  and  gel  0.83  two  , ) obs  volume  was  by peak  was  II.  homogeneous  sedimenting  components  (S  II  34,000.  for  filtration  10.45S  components  of  characterized  contained  and  the  weight  peak  in  and  0.2M  Tris-  20°C. void  eluted  from  samples.  This  l i p o p r o t e i n was  appear  VLDL  (MF)  in  samples  non-  likely  that  were  delipidation.  Scanu  (1966)  with diethyl  were  for  rpm a t at  285,700 w h i l e  residues  obtained  5.03S  delipidated  to  and  at  and  of  a molecular  0.59  15.55S  contained  since  delipidation  the  8.2)  heptane  Granda  products  7.22S  peak,  aggregate  alkylated  a l l  I  contained  alkylated an  Peak  buffer A  to  analytical ultracentrifuge;  fractions. peak  above  weight  soluble  phospholipid/protein the  -  a molecular  emerged  of  87  soluble  cholesterol  in  and  subjected  ether  in  alkaline  the  human s e r u m  VLDL  to  presence  0.2M  SDS.  buffers,  triglycerides.  of  with  the  partial  removal  Sedimentation  The of  velocity  almost analysis  a of  the mixture  sedimentation (1966)  isolated  and  4S, 0.7  7S  gel  and  Three  and-their  respectively.  4.5S,  of  10.9S  VLDL w i t h  soluble  filtration.  14S  presence  of  human serum  starch. by  the  coefficients  extracted  insoluble  of  indicated  3 lipoprotein residues and  15.5S.  a l .  presence  of  phospholipid-protein  residues  were  fractions  in  et  the  These  heptane  Gustafson  with  had  phospholipid/protein  sedimentation ratios  were  rates  0.9,  2.4  - 88  Aqueous extracted  with  containing rates the  of  low  30S  were  LDL  and  from  5S  the  the  11S.  The  of  of  extracted  the  without  denaturation  aggregation the  of  removal  act  a protective  molecules. stability and  In of  sucrose  have  protect  serum  1965).  The  Three  4S,  and  and  of  the  water  an  VLDL  (1973)  also  extracted  with of  was  diethyl 10S,  70%  on  ether.  13S,  of  20S  the  and  extracted  components.  used the  fractions  from  agent, case  membrane  water-insoluble  of  by  low  were  Neutral  Martin  et_ a l .  density  lipoprotein  isolated  lipids  having  were  also  the  r e l a t i v e l y nonpolar  phospholipid-protein  lipoproteins  has  been  found  suggesting  part  lipoprotein  of  the  collisions  contributes Compounds  be  adequate  lipoproteins potato  such  the as  in  to  coincide  water  structure  may or  lipoprotein  glucose, for  denaturation  used  (MF)  structural  substitutes  from  starch  to  that  between  solvent  complex.  found  the medium,  water  to  by  2 p a r t i a l l y d e l i p i d a t e d VLDL  preventing  lipoproteins. found  (MF)  residual  essential  been  fractions, sedimentation  heavier  13S.  1969)  with  rates  (1964)  Takats,  lipids,  serum  form  and  three  represented  yolk. 7S  of  Martin  pig  to  et_ a l _ .  (Martin  lipoproteins.  of  either  the  and  preparation  of  constitute  neutral  from  low density  either as  and  component  extraction  allowed  with  13S  egg  heptane  The  composed  extraction procedure  rates  The  VLDL  sedimentation  Augustyniak  plasma  ether  were  reassociated  similar by  porcine  Janado  observed  apparently  and  ether  lipoproteins  found.  sedimentation in  and  with  A  of  phospholipids  density  Components  (.1963)  diethyl  both  3S,  solutions  -  this  glycerol water  to  (Gustafson,  study  has  many  -  hydroxyl  groups  water-soluble  which  offer  starch Partial  with  diethyl  lipid  from VLDL VLDL  (MF)  dried  of  the  increased  of  and  54-100%  a l . ,  can  easily  be  1959).  is  to  VLDL  (MF)  advantage  as  that  separated  the  from  in  Zilversmit from  heptane. on  suspensions  incomplete  Complete  by  a more  surface  depending  the  aqueous  results  achieved  state  cholesterol  buffer  has  The  (1964)  and  the  this  l i p o p r o t e i n monolayers of  the  completeness  neutral lipids  the  starch-  lipoproteins  extraction between  because  the  heptane  by  heptane  extraction  spread  between  phosphate  cholesterol of  of  freeze-dried  contact  demonstrated  amount  of  lipoproteins  neutral  e x t r a c t i o n of  closer  The  of  the  e f f i c i e n t means  area  of  removal  removal  e x t r a c t i o n of  mixture w i t h n-heptane.  the  VLDL.  usually  was  in  and  Starch  d e l i p i d a t i o n of  et  (MF)  same p r o t e c t i o n  centrifugation.  ether  (Hayashi  residues  by  -  the  carbohydrates.  phospholipid-protein insoluble  89  extracted varied  the monolayer  formed  by  from the  lipoprotein.  2.  Total  The in  of  and the  in  the  of  the  protein moieties  of  VLDL.  (1971)  using  standard  methods  for  Small  ethanol-ether  recovery.  the  one  Edelstein  lipoproteins.  ethanol-ether  d e l i p i d a t i o n procedure  characterization of  Scanu one  delipidation with  These  molecular mixture,  proteins  may  is  3:1  (v/v)  however, be  peptides leading  recovered  critical  The  ethanol-ether  d e l i p i d a t i o n of weight  most  by  very  low  from VLDL to  method has  factors of  become  density are  soluble  incomplete  protein  changing  the  solvent  - 90  ratio  to  3:5  extraction  (v/v)  times,  utilization  of  -  ethanol-ether the need  large  (Scanu  to work  volumes  of  at  and  low  solvents  Edelstein,  temperatures are  several  of  this  delipidation procedure.  The  by  this  method,  solubilized with  The (1957)  but  may  renders  apoVLDL  such d i s s o c i a t i n g  as  a matter  of  chemical  carbohydrate  the  by  of  presumably  hydrophobic  lipid  is  ethanol-ether  urea  or  disadvantages  SDS  or  of  even  This  urea.  Folch  et_ a l •  the  presence  in  method  was  d e l i p i d a t i o n of  hydrolysis  the  precipitated  method  SDS.  for  acid  treatment  lipid.  interactions is  v/v)  is  and  Long  used  samples  procedures,  e.g.  analysis.  ethanol-ether removal  (2:1  however,  acid  Solubilization  when  as  or  amino  apoprotein  partially soluble,  involving  protein-protein  the more  agents  convenience,  upon  with  only  analysis  The insoluble  later  chloroform-methanol  of  for  be  1971).  rendered  This  which  are  of  e f f e c t e d by of  the  (Kane  et  al_. ,  d e l i p i d a t i o n was  alkylated  insolubility  areas  removed  even  a  is  effected  reversible  1970).  completedly  The  urea  become apoVLDL  soluble  by  nature.  i n t e r a c t i o n of  protein which  VLDL  in  or  SDS  exposed produced  8M u r e a  or  -3 10  M SDS  were  Tris-HCl  d e t e c t e d by  extracts of  in  the  of  the  over serum  thin  the  years  w i l l  other for  apoproteins.  lipoprotein  layer  apoprotein.  apoprotein Many  buffers.  be  Only  trace  amounts  chromatography The  disc  presented  gel  and  in  chloroform-methanol  discussed have  in  been  the  et  al-  (1968)  the  serum  and  obtained  a  soluble  I.  developed  c h a r a c t e r i z a t i o n of succinylated  patterns  section  and  Gotto  f r o m human  phospholipids  electrophoretic  d e l i p i d a t i o n procedures  isolation  of  various low  density  apoprotein  -  after  delipidation with  added  to  removed  the by  protein  to  Shore  of  the  protein  lipoprotein  by  ethanol-ether  alkylation with  presence the  of  SDS.  presence  0-976) serum  the  a  of  place  in  procedure  containing  the  delipidation  without and  aqueous  phase  only  i f  does  not  egg  systems organic  affect  yolk  the  ionic  (MF),  (0.01M)  lipids  have  and  low  could  the  density  by  be  reduction in  the  solubilized  added.  removal  Cham  of  and  lipids  and  (2:3 the  phase  v/v).  contained or  however,  the  a  remained  the  pH o f  procedure  Knowles  Delipidation  apoproteins  strength  in  from  precipitation, utilizing ether  was  later  described  iodoacetamide  u r e a was  When t h i s  VLDL  and  complete  protein  lipoproteins.  of  6-8M  for  buffer the  SDS  human s e r u m  apoproteins  di-isopropyl  while  of  (1967)  borohydride  technique  butanol  of  v/v).  delipidation followed  modified  SDS  lipoproteins,  aqueous  This  3%  developed  mixture took  of  The  Shore  moiety  sodium  (3:1  extraction  and  preparation  and  -  ethanol-ether  prior  dialysis.  91  was  in  lipids. the  buffer  applied  apoprotein  system  for  was  precipitated.  3.  Sodium  deoxycholate  ApoVLDL was the  prepared  in  NaDOC d e l i p i d a t i o n p r o c e d u r e  produced in  detergent  the  was  applied  to  void  volume  as  cholesterol  eluted  later  contained  no  a  a  column  clear in  a  a and of  delipidation  soluble  aggregated  the viscous, Sephadex  solution  while  separate  peak  detectable neutral  but  lipids  or  clear  G-200. the  form  amber  The  solution  protein  phospholipids  (Figure  using  16A).  c h o l e s t e r o l when  The  eluted  and protein  examined  - 92 -  o Figure  16.  Tube  Number  T  (A) S e p a r a t i o n o f a p o V L D L and VLDL (MF) l i p i d s b y f i l t r a t i o n i n t h e p r e s e n c e o f NaDOC. (B) R e m o v a l o f NaDOC f r o m a p o V L D L b y g e l filtration.  gel  -  by  chemical  assay  phospholipid  eluted total the  of  apoprotein  detergent  bound  Sephadex  the void  volume  thin  detergent  on  at  by  remained  The filtration  or  G-75  volume  the was  free  was  assayed  apoprotein  showed that may or  the  the have  by  by  presence  to  in  for  presence  was  the  highly  by  the  and  of  soluble  0.5%  pH  the  as  gel  The  protein  close  9.0  salt.  boundary,  from  the  sample  The buffer.  however,  which  This  the when  Tris-HCl  apoprotein,  lipid  to  detected  bile  state.  the  stirring  the  in  of  aggregated of  medium.  NaDOC w a s  sedimenting  removal  constant  than  by  eluted very  No  analysis  rapidly  been  pressure  apoprotein  detergent 16B).  a highly  the  the  (Figure  in  caused  from  a detergent-free the  Less  apoprotein.  removed  velocity a  chromatography.  the  p r o t e i n was  indicated  aggregation  the  lipoprotein  was  concentrated  ultrafiltration. The  acrylamide (MF) for  detergent  gel  comparison.  The  identical Chen  deoxycholate obtain  Lipid  a  lipid  an  exchange  in  the  by  free  protein  was  presence  3:1  (v/v)  of  subjected SDS;  was of  a  to  sample  polyof  VLDL  co-electrophoresed the  two  samples  I).  Aladjem  (1974)  and  Helenius  and  delipidation  in  protein  human s e r u m  detergent  in which  the  electrophoretic patterns  (section and  in  apoprotein  ethanol-ether  detergent  removal  delipidated  electrophoresis  delipidated with  were  to  of  -  layer  and  column  Sedimentation  93  from  treatment  deoxycholate  previously  occupied  is by  Simons  conjunction with  was  low  postulated  bound  to  lipid.  gel  density to  be  hydrophobic The  (1972)  presence  used  filtration lipoprotein.  caused regions of  by  - 94 -  micellar  concentrations  of  detergent  apoprotein  and  detergent  micelles.  The  protein-detergent  complex  by  in  mixed m i c e l l e s the  as  because  a  separate  peak.  deoxycholate  therefore  Helenius LDL  filtration  deoxycholate-lipid micelles  elute  and  gel  micelles  caused  and  is  Simons  of  to  be  of  LDL  can be  than  separated  undergo  however  any  caused  lipid from  the  of  the  detergent;  size  and  therefore  organic  that  of  and  was  a non-denaturing  treatment  not  detergent  d e l i p i d a t i o n procedure  thought  did  as  smaller  (1971) d e m o n s t r a t e d  f r o m human s e r u m  SDS-delipidation  well  formation  the presence  are  This  a much m i l d e r  as  the  examined  detergent  solvent  deoxycholate  conformational  denaturation  of  dilipidation. delipidated  changes; the  protein  moiety.  G.  Amino  acid  composition  The r e s u l t s granule of  are The  of  The  lack  for  its  of  isolated included  yolk  glutamic  apoVLDL  acid  analyses  presented  in  from y o l k  plasma,  for  granule  t h e VLDL w i t h  serine,  amino  apoVLDL are  apoVLDL  serum  of  of  apoVLDL  respect  to  has  high  acid , alanine  tryptophan  residues  low  absorbance  at  due  to  X. whole  24 h r The  hydrolysates  amino  yolk,  acid  hen  of  yolk  composition  serum  and  human  comparison.  of  absorbance  Table  of  light  an  amino  levels  and in  of  lysine, yolk  2 8 0 nm a f t e r scattering  acid  composition  aspartic and  granule  l i t t l e  or  threonine, no  apoVLDL would  subtracting (Figure  acid  9).  the  typical  tryptophan. account  contribution  - 95 -  Table  X.  Amino Yolk  Acid  Composition  Plasma,  Whole  of Apoproteins  Yolk,  Hen Serum  Weight  Amino Acid  Yolk granule apoVLDL  Asp  Yolk plasma apoVLDL  from Yolk  a n d Human  Granule,  Serum  VLDL.  % (w/w)  Whole y o l k apoVLDL  Hen serum apoVLDL  Human s e r u m ' apoVLDL  10.65  11.16  11.40  10.90  10.94  Thr*  8.72  5.69  6.48  6.26  6.55  Ser*  8.29  6.57  7.50  7.09  8.35  Glu  12.15  10.53  11.30  11.30  17.27  Pro  3.00  3.36  3.02  3.62  4.50  Gly  4.65  4.24  4.61  4.44  2.79  Ala  7.15  7.35  7.79  7.65  6.10  Val  5.08  6.67  6.73  6.60  5.06  Met  1.79  2.11  1.48  2.02  N.D.**  H e  5.79  6.47  6.85  6.54  3.19  Leu  9.29  11.30  10.30  10.40  11.10  Tyr  4.00  4.60  2.12  3.41  2.81  Phe  4.57  4.09  4.19  4.25  5.50  Lys  8.86  9.93  7.70  7.02  9.20  His  1.43  1.36  1.40  1.12  1.56  Trp  0.00  N.D.**  0.61  1.77  N.D.**  Arg  4.57  5.76  5.35  4.58  1.56  *  values  **  not  a  from Raju  b  from H i l l y a r d  c  from Kane  f o r threonine  and  serine extrapolated  determined and Mahadevan,  1976  e t a l . , 1972  fit a l .,  1975  to  zero hydrolysis  .  1  H.  Electrophoresis  Kane  (1973)  lipoproteins The as  whole well  as  reagent.  that its  (MF)  in  serum  (apoB)  fraction LDL  content  remaining  that  TMU  into  their  of  of  the  presence  delipidates  low  apoprotein  were  found  apoproteins  to  TMU  and  dissociates  characteristic  human s e r u m  the  of  of  be  (apoC)  density  subunits. lipoprotein  human s e r u m  VLDL  p r e c i p i t a t e d by of  human VLDL  which  this  are  soluble  TMU.  gel  and  granule  gels  were  top  Band gel  of  solvent  and  TMU.  10%  d e l i p i d a t e d on acrylamide  Three  gel  stacking  located  at  the  3 appeared running  Bands  2 and  and was  be  gel with  confirmed  from  blue the  a  small  the  3 represented  the  of  the  stacking  presence  were  of  the  d e t e c t e d when  17A).  Band  1  the  remained  f r a c t i o n p r e c i p i t a t e d by  TMU.  the  running  stacking  gel  molecular weight  tracking  experiments  top  the  (Figure  i n t e r f a c e of  to  in  p r o t e i n bands  the  the  3 was  on  was  Coomassie  band  through  used.  (MF)  stained with  2 was  band  VLDL  electrophoresed  denaturing  in  f r o m human  The  Yolk  on  VLDL  -  demonstrated  apoprotein  represents  in  of  -96  dye.  i n which  The no  apoprotein  and  the  protein  presence  tracking  of  dye  fractions  travelling  was  soluble  TMU. Alternatively,  and  the  then  produced  (MF)  was  p r e c i p i t a t e d p r o t e i n was  centrifugation. were  VLDL  Both  subjected only  band  t h e TMU  to 1  delipidated with separated  soluble  and  electrophoresis.  (Figure  17B),  which  from  the  t h e TMU The  TMU  TMU  remained  in  a  soluble  insoluble insoluble on  top  of  test  tube  protein  by  fractions fraction the  (A)  Figure  (B)  17.  Electrophoresis of  (C)  of  VLDL  tetramethylurea VLDL  (MF)  (TMU).  (A)  whole  (MF)  (B)  TMU  insoluble  (C)  TMU  soluble  VLDL  VLDL  in  the  presence  - 98 -  stacking TMU  gel;  soluble  mobilities  and  not  by  2,  the  which gel,  or  protein  7.5%  or A  5%  used  to  eluted  p r o t e i n bands  on  (band  (Figure  was  the  (Figure  aggregate to  of  both  the  The  had  VLDL  (MF)  affect  small  two  fractions  proteins  which  TMU  gel  gels.  and  was  with  6B  dissociate  gels  this  to  contain  protein. from  column  the  were  (Figure sliced  electrophoresed  The  protein  eluted  band  an  to  TMU  by  SDS  fraction  gel  was  found  B from  the  SDS  to  from in  The the  the by  same  filtration  polypeptide B  (band  and  the  of  18).  precipitated  have  identical  interfacial protein  proteins,  to  and  found  weight  of  2-mercaptoethanol  failed  of  and  electrophoretic patterns  fraction A 3 had  dissociation  acrylamide  electrophoresis SDS  (thiodiglycol  p r o t e i n bands  SDS-Sepharose  A and  of  agents  the  the  molecular  ' The  of  these  SDS  acrylamide  as  gel  the m o b i l i t y  between  SDS  stacking  combination  the  in  the  electrophoresis,  extracted with  10%  1 8 B ).  gels.  which  Further  and  the  soluble  the  system. in  of  aggregate  composition  not  TMU-soluble  to  an  reducing  urea,  found  composition  18A).  composition  common  SDS  1)  polypeptide  column  did  from  presence  of  the  from  gels,  17C)  3 from whole  interface  be  this  10M  procedure  urea-acrylamide  an  the  or  in  8M o r  c o r r e l a t i o n was  fractions  three  TMU  at  TMU  Changing  electrophoresis  these  method.  hy  acrylamide  in  (Figure  2 and  d i t h i o e r y t h r i t o l , f o l l o w e d by protein.  evident  2 bands  believed  acid)  were  bands  remained  was  using  this  the  TMU  dissociated  mercaptoacetic this  proteins  identical with  running  were  other  f r a c t i o n produced  delipidated Band  no  from  the  2 appeared contain  column  (Figure  SDS to  be  polypeptides 18C).  - 99 -  (A)  1  (C)  (B)  2  Band 1 2 3 4 5 6 7  2  4 5  5  10 12 13 14  10 13  15 16 17  16  18  1  R  lo 19 -20  20  8 9 10 11 12 13 14 15 16 17 18 19 20 21 22  MW 136,800 130,600 111,200 86,300 82,600 79,200 77,800 76,500 75,000 72,300 68,100 64,900 60,400 54,900 49,800 45,700 41,000 35,300 29,800 24-800 21,600 9,700  -21  22  Figure  18.  22  SDS p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s i n t h e presence o f BME o f (A) TMU i n s o l u b l e Band 1/Sepharose 6B Fraction A (B) TMU s o l u b l e Band 3/Sepharose 6B Fraction B (C) TMU s o l u b l e Band 2  -  The  molecular  section  weights  of  these  studies  of  (Kane  et  1977)  using  of  egg  (band  The  2)  and  with  the  apoB  egg  human  to  be  (Gotto  clarify  et  on  gel  of  with  discussed  the  TMU w a s of  1976;  however  VLDL w h i c h  in  achieved  human  Mahley  that  serum  and  Holcombe,  dissociation  interfacial  protein  was  serum  a l .  is in  a l . ,  VLDL  from  found  VLDL  isolated  chicken  the  This  apoB  apoB  from egg  the  (Scanu  f r o m human,  et_ a l . , rat,  f r a c t i o n from  may as  be  was  Positive  hen  VLDL  similar  Hillyard  yolk  LDL-  studies.  f r o m human  study,  serum.  protein using  a  represent above  as  isolated  this  to  isolated  apoprotein  1972). in  in  classified  (1976)  TMU  appears  to  et_ a l .  identical  identification  immunological  techniques  point.  weight  or  as  the major  estimation  gel  10%  of  apoVLDL  polypeptides  by  the  SDS  electrophoresis  electrophoresis  7.5%  presence  that  apoB-like  this  Molecular  BME  be  t h e VLDL  (Marsh,  appears  present  et  isolated  yolk  of  complete;  of  in  Chan  demonstrated  SDS  the  It  not  resembling  polyacryamide  of  w i l l  apoproteins  plasma  precipitated,  serum.  t h e VLDL  would  I.  rat  component  appears  apoVLDL  was  the  composition  f r a c t i o n p r e c i p i t a t e d by  (1972)  polypeptides  aggregate.  apoprotein  designated the  an  protein  chicken  plasma  of  apoVLDL  was  and  of  technique.  p r o t e i n was  1974),  subunit  1975)  this  yolk  LDL  and  the  a l . ,  The like  -  I.  Complete d i s s o c i a t i o n in  100  of  acrylamide  glycoprotein  apoVLDL  was  conducted  in  gels.  The  gels  stained  for  procedure  and  using  the  PAS  were  staining  presence  -  for  protein with  with  Coomassie  Coomassie  Blue  The most  intensely  131,000;  82,900;  The  remaining  of  most  intensely  136,300;  always  stained  In were  the  absence  130,600;  75,000  of  The  73,500;  BME,  36,500  PAS  bands  had  A l l  other  bands were  18  and  molecular  but  protein  proteins  had  12,500  weights  consistently  (Figure of  136,300,  and  present for  19A).  9,600.  in  a l l  glycoprotein.  to molecular  19B).  The  less  bands  9 glycoprotein  and  molecular  136,800;  The  73,500  detected but  of  bands  136,800;  intensely  and  were  bands  intense.  weights  20A).  weights  other  were much  (Figure  of  were  (Figure  detected  35,200  corresponded  60,200 PAS  45,600; but  BME  weights  positively  bands  and  with  major  60,200;  stained  PAS-reactive  with  molecular  only l i g h t l y  bands  bands were  reduced  had  65,200;  positively  resolved.  bands  stained  111,700;  Twenty-two  apoVLDL was  72,100;  Fourteen  -  Blue.  stained  bands  preparations. The  when  101  36,500  stained  stained  (Figure  20B).  less  intensely. The of  BME  most  from  noticeable  the  sample  protein  (9,600)and  proteins  (12,500;  larger which of  proteins disappear  smaller The  that  range  were  detected,  or  minor  in  bands  was  the  may  be  upon  the  of  indicate  molecular  weight which  by  apoVLDL of  the  several  91,500; joined BME,  pattern  due  to  low molecular  higher  95,200 together  resulting  in  by  omission  weight  molecular  and  the  weight  99,100).  These  disulfide  bonds  the  formation  proteins.  results  caused  of  aggregates  weight  the  absence  62,700;  addition  some o f  in  formation  48,400;  molecular  above  change  that from  could  slight  apoVLDL 9,600  be  to  contains  9 major  136,000.  Other  polymorphic  variations  in  the  forms  of  content  polypeptides components  the of  major  sugar  - 102 -  (B)  (A)  1 3 5 6 9 10 12 13 15 16  18  20  Band 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22  MW  136,300 131,000 111,700 86,200 82,800 79,600 78,000 76,500 75,000 72,000 67,900 65,200 60,200 54,500 50,400 45,600 41,300 35,200 30,000 24,600 21,800 9,600  21  22  Figure 19.  SDS polyacrylamide gel electrophoresis of apoVLDL i n the present of BME and stained with ( A ) Coomassie Blue and (B) P A S .  -  103  -  (B)  (A)  MW  Band  136,800  1 2  130,600 112,000  3 4  4 5  7 8  9 10 11 12 13  95,200  8 9  6 7 8  91,500 87,800 81,300  10  9 10  73,500  11 12  62,700  13 14  54,000 48,400  15 16  36,500  12  14  15  15  16  99,100  5  66,700 59,100  24,100  17  21,000  18  12,500  16  17  18  18  Figure  20.  SDS  polyacrylamide  conducted (A)  in  gel electrophoresis  the absence  Coomassie Blue  and  of  (B)  BME PAS.  and  of  apoVLDL  stained  with  -  residues. that  14  This  upon  havereported otherwise  -  microheterogeneity  bands were  apoprotein  104  PAS-reactive  carbohydrate  that  and  demonstrated  sialic  analysis.  differences  identical  was  in  the  acid  Brown  sialic  polypeptidescould  was  et  the  found  al.  acid  account  by  in  the  (1969)  content  for  fact  this  of  type  of  polymorphism. Hillyard  et _al.  electrophoresis chicken  serum  21,800 which components The  major  9,600)  VLDL.  of  protein be  to  major 9,000  molecular found  in  to  the  plasma  hen  in  4 protein  the  51,200;  Hillyard.  apoVLDL  and  i f  i t  13,000.  presence  of  dithiothreitol,  was  reduced  from  Yamauchi and  detected  which  j^t a l . 18  The  glycoproteins, Raju egg  to  approximately  polypeptides  (ranging  from  components and  similar  to  plasma  was  a l .  that  43%  (135,000;  (1976)  found found  subjected  to  in 13  from  10,000  82,000;  71,000 molecular those  (1976) of in  of  the an  12,000weight  6,000.  apoVLDL  135,000  et  weight  protein  weight  the molecular  isolated  and Mahadevan yolk  a molecular  (1976)  5 were major  16,000).  from  12,000  had  of  70,700.  a protein which migrated  as  other  (molecular  found  field  the  and  in  weight  The  60,800  Chan  electrophoretic In  form.  study  acid  components  a molecular  reduced  present of  of  phenol-urea-acetic  only  weight  that  consisted  a  f r a c t i o n had  the  similar  apoprotein  using  detected  The  dropped  charact erized total  system,  were  may  (1972),  gel  plasma  135,000)  of  62,000  components  and  were  study.  protein  SDS  to  yolk  71,000;  weight  this  egg  bands  when  apoVLDL  electrophoresis  in  -  the  presence  study with in  when  of  BME  J.  The ether  or  as  did  (pH  8.6)  (V^.),  separated to  the  Two urea  peak  calibrated eluted  at  110,400. i t and  were had  2 peaks  or  a  obtained  of  not  stain  weight  in  as  this  intensely  protein  were  observed  equivalent fraction  protein  at  weight  two p r o t e i n s  results which  in  found  volume,  peak  ethanol-  0.1M  Tris-  6B-8M u r e a  3 distinct  to (B)  of  peaks  (V^^-j.)  eluted  gel  (Figure  f o l l o w e d by  fraction V was  The  was  first  eluted  equal The  volume  peak  of  passed  a molecular  26,800.  at  whole  to  21).  a  second  a  volume  through  from  of  the  column  total  from  bed  the  8M  fraction V  was  an  completely dissociated  by  completely dissociated  by  SDS.  a (A)  was  as  i f  volume  e l u t i o n volumes V  8M  approximately  applied  that  the  fraction  weight  the  from  apoVLDL  indicated not  22)  6B.  a volume  the void  was  when  (Figure  i d e n t i c a l to whose  These  into  third  soluble  Sepharose  the void  The  delipidation with  completely  SDS-Sepharose  from  by  8M u r e a .  in  apoVLDL  second  obtained  was  did  found  volume.  peaks  a molecular  which  i t  was  apoVLDL  apoVLDL  (V-j-j.) •  whole  small  the  eluted  bed  a volume The  column.  but  total  column  column were  of  peak  protein  column  of  containing  The  equal  (12,500)  9,600 molecular  d e o x y c h o l a t e was  resolved  well  the  apoprotein  chromatography f i r s t  protein  However,  filtration  (MF)  sodium  buffer  similar  -  apoprotein.  gel  VLDL  A  omitted.  Blue  reduced  Agarose  HCl  was  Coomassie  the  BME.  105  of  the  urea  to  the  SDS  aggregate 8M  urea;  1.5'  Figure  21.  Gel filtration  of  apoVLDL  on  Sepharose  6B  c o n t a i n i n g 8M  urea.  -  107  -  -  These  results  electrophoresis. of  the  two  stacking  proteins  ranging common samples the  band  gel A  extracted  from  using  differences Fraction  A  molecular  running  the  known in  (band  and  3  fractionated of  apoVLDL  in  chromatography Molecular not but  weight  of  and the in  and  29,800.  3  presence  of  Blue  of  the  mainly  proteins  significant fractions. of  the  proteins  were  from  well  Fraction  an  molecular  compared  of  130,600;  protein with of  high  six  stained faintly.  bands  of  calibration  detected,  (136,800;  obtained  apparent  weights with  Sepharose  those 6B  SDS.  determinations  detect  such  using  heterogeneity  molecular  to  their  BME,  the  The  remaining  figures  weight  give  due  and  of  to  indicated  1 major  faint  These  18B).  bands were  The  c a l i b r a t i o n curve the  and  therefore  contained  9,700  A l l  column  presence  weights  Coomassie  and  SDS  were  B).  solutions  consisted  35,500).  quantities gels)  gel)  Eighteen  and  the  the  markers  TMU  with  these  corresponded  18A  from  proteins.  f r o m TMU  from  (Figure  the  weights  (fraction  urea  in  column  molecular  3  weight  SDS  gels  intensely  small  24,800  the  on  other  carbamylation.  apparent  B  fractions  band  only  the  molecular  ( f r a c t i o n A)  molecular  1  of  TMU  interface  of  1  TMU  the  aggregate  Polypeptides  the  the  some o f  in  do  an  at  evident;  present  columns  was  found  from  130,600 were  60,400 ; 45,700  obtained  was  those  to  64,900;  21,600;  gels  18C).  B from  weight  molecular  with  protein which  dissociation  and  stained  (band  agreement  electrophoretic patterns,  fractions  which  "  chemical m o d i f i c a t i o n by  SDS  curve  in  apoVLDL were  incomplete  possible  and  9,700  both  of  The  (Figure  from to  are  108  weights  calibrated  in molecular  corresponding  to  gel  filtration  weights the  of  elution  the  -  volumes power the  of  of  the  the  fractions.  gel  in  accurate  isolated  the  also  amount or  of  analysis  detergent  data,  the  of  of  the  greater  resolving  and  the  dissociation  8M u r e a ,  SDS  and  the molecular  at  (v)  BME  of  provide  weights  may Plots  curvature  the  and  apoprotein  a l l  SDS  was  reflect  ln  the  the  the  the r  2  therefore  molecular  of  of  the  (Figure fitted  23) to  a  was  to  is  bound  not  known  of  to  specific  assumed  be  from  the  to  approximate  exhibited least  The  calculated  partial  an  column  ultracentrifugal  unknown and  this  carbohydrate  prior  The  SDS  techniques.  aggregation  also  that  the  weights  experiment. were  from  fractions  removed  state  realization  c versus  were  The  apoproteins  a f f e c t e d by  of  the  time of  the  with  be  so  B  equilibrium  to  apoproteins.  of  0.724 ml/g,  o f f r a c t i o n s A and  sedimentation  bound  therefore,  apoproteins  which  by  determined, of  volumes  weights  determined  easily  the  method  of  polypeptides.  The m o l e c u l a r were  presence  determination  -  combination  electrophoresis  apoproteins  a more  The  109  be  value  apoproteins.  substantial  squares  quadratic  2 function.  Values  of  derivative  of  polynomial  allowed  the  din  c a l c u l a t i o n of  c/dr  were at  then  obtained  each measured  the weight  average  by  radial  molecular  taking  the  distance. weight  (M  first This  )  at  w each  point. Weight  estimated for  average  to  fractions  the molecular  range A and  molecular from  weights  33,300  to  165,800  B respectively.  weights  of  the  of  protein  the  apoproteins  and  from  These values from near  5,800 were  were to  taken  the meniscus  28,100 from (M)  Figure  23.  Low by  speed SDS  gel  sedimentation  e q u i l i b r i u m u l t r a c e n t r i f u g a t i o n of  chromatography.  apoproteins  isolated  -  to  the  (Mw  bottom  app  )of  one-half  these those  inaccurately since The  (B)  of  determined  represent  c a l c u l a t i o n of  correctly the  sample  weights  of  Raju  at  each  represent and  are  fractions  peak was  apoA,  apoB  weights  of  180,000  agreement B as  ln  average  ApoA  the  molecular  weight  12,500  molecular  weight  component  0.5% of  that  SDS  the  SDS  each  consisted and  2 major  78,000 and  and  was  of a  It  gel  molecular  electrophoresis. by  3 protein third  peak  bands  was the  contained  by  molecular ApoB  proteins  indicated  In  peaks.  designated  of  bands.  contained  formed  of  gel  heterogeneous.  arbitrarily  ApoC  the  of  apoVLDL  the  was  2 major  weight.  gel  to  more  state of  2  weights  electrophoresis  were  was  r  due  cell  obtained but  observed.  molecular  plasma  few minor  bands  a dimer  9,400 m o l e c u l a r  and  peak  64,000.  9,400.  was  c versus  range SDS  yolk  first,  fractions  100,000,  the  determined by  two..  3  with  separated  in  first  weights  of  apoproteins  weights of  about  values  the molecular weights  The w e i g h t  (1976)  molecular  two monomers  plots  the  aggregation  of  of  of  and  consisted  of  weights  or  These  heterogeneity  G-200  and  4 bands;  filtration.  weights  (B)  centrifuge  these  apoC.  13,200  in molecular  into  shoulder  in  and  the  and  the  molecular  in  demonstrated  proteins and  a  from  proteins  The m a j o r  A  Sephadex  separated  error  close  and Mahadevan  The  eluted  the  (A)  from nonlinear  sample.  in  on  well  SDS-gel  radial distance  filtration second  app  the  apparent  55,800  heterogeneity Mw  of  The  were by  -  the molecular  considerable  polydispersity calculated  cell.  fractions  considerable  introduced  the  I l l  of  2 bands  that  the  disulfide  this  study  of  12,500  linkage a  similar  -  range  of  molecular  although This  only  could  compared weights  the  could  apoproteins used.  in It  delipidation of  the  in  weights  Sepharose  gel.  The  higher  molecular  and Mahadevan  (1976)  for  the  the was  Raju  result  of  irreversible  chloroform-methanol found  VLDL  in  (MF)  this  the  study  results  in  B,  column.  G-200  by  of  6B  Sephadex  weights  egg  yolk  present  e_t a l .  of  agarose  column  the  apoVLDL  aggregation  of  the  d e l i p i d a t i o n procedure that  which  chloroform-methanol  irreversible  (1976)  135,000;  et  a l .  Sephadex  G-150  from  column,  the  molecular is  present  of  12,000-13,000, fractions this  in  of  which  one  18  denaturation  in  from  components  fractionated  The  SDS.  eluted  which to  second upon  in  the  by  with  SDS  those  gel  with  molecular  16,000.  from hen peaks  the void  The  135,000  plasma were  dropped  to  apoprotein  This  protein  a molecular 6,000.  on  a  eluted  volume.  LDL-like  f r a c t i o n had  to  (1976)  glycoproteins.  Two  the  reduction  correspond  and  were  a l .  granules.  proteins  apoVLDL  2mM  et  agreement  yolk  62,000  fraction represented  VLDL.  close  polypeptides  71,000;  which  Yamauchi  were major  containing of  are  by  apoVLDL  found  weight  (1976)  appear  study.  plasma  82,000;  column  weight  determined  study  71,000 molecular Chan  in  the  size  e l e c t r o p h o r e s i s , f i v e of  2  on  pore  from  the  Yamauchi  and  resolved  f o r f r a c t i o n s A and  large  be  of  were  observed  the  molecular  apoVLDL  found  to  was  -  apoproteins.  The for  due  determined  fractions  was  2 peaks  be  to  weights  112  large  which  weight These  fractions  reported  -  V.  SUMMARY AND  egg  low density  lipoproteins  yolk  were  isolated  a  characterize  and  preparation were  was  rates  having  storage,  the  this  state  Passing  in  VLDL  a retained  material  was  retained the  contain  therefore  as  hexose, could  be  of  the  VLDL  upon  alkylation  decreased  (MF)  samples  sulfhydryl  to  (MF)  p a r t i c l e may  fresh  an  the  much  VLDL  an  of  of  VLDL  (MF)  41S  and  75S  that  and  the  the  207S.  flotation  108S. aggregates  Alkylation however  exist  apoVLDL.  boundaries  (S^)  in  a f f i n i t y column  bound  a  fraction.  of  of  VLDL  i t  was  (MF)  partially  than  in  twice  much  total  (MF),  while  and  as  the The  sialic  Concanavalin  More  fraction  carbohydrate.  termed  indicated  aggregation,  unretained  hexosamine  and  to  yolk.  through  and in  egg  (MF)  floating  groups.  VLDL  hen's  ultra-  performed  form d i s u l f i d e  this  (MF)  were  VLDL  Two m a j o r  fraction contained  half  of  of  Ultracentrifugal,  analyses  prevented  unfractionated  contained to  found  of  (MF)  produced  as  purity  granules  preparative  filtration.  analysis  that  oxidation  that  of  the  flotation coefficients  VLDL  iodoacetamide  aggregated  The  the  the macromolecules  Upon  believed  gel  heterogeneous.  demonstrated of  to  assess  from  combination  chromatographic  velocity  was  resolved  by  agarose  and  and  Flotation  with  CONCLUSIONS  very  electrophoretic  due  -  The  centrifugation  It  113  the  PAS-reactive unbound  lipoproteins  glycolipoproteins.  fraction.  carbohydrate  unretained  acid  A  residues  VLDL were and  (MF) found  -  VLDL  (MF)  was  preferentially solubility were  the  isolated  by  (MF)  NaDOC w a s  gel  to  the  were  was  found  organic  to  remained  proteins  were  was  and  be  indicated of  the  the  TMU  many  the  other  SDS  of  residues.  Two  NaDOC a n d  of  by  while  analytical  the  the  however  in  comparison  from both the  ethanol-ether  organic  ratios.  ethanol-ether.  apoproteins  lipid,  the  fractions  phospholipid/protein  NaDOC  procedures delipidated  delipidated  solvents.  The  apoprotein  urea. was  used  into  to  d e l i p i d a t e VLDL  subunits.  3 apoproteins,  electrophoresis  demonstrated  preserved  characterized  The  which  that  each  of  band  (MF)  and  Electrophoretic one the was  of  which  analysis  was  proteins  an  aggregate  extracted  heterogeneous,  from  containing  polypeptides.  ranging  in  (TMU)  presence  ApoVLDL was  and  or  and  d e l i p i d a t i o n procedure  system.  apoproteins  two.  gels  a mild  SDS  Tetramethylurea the  lipids  delipidated using  soluble  in  and  their  p r e c i p i t a t e d by  solubilized  dissociate  by  upon removal  proteins  neutral  filtration  solvent  aggregated  the  phospholipid-protein  ultracentrifugation VLDL  -  p a r t i a l l y d e l i p i d a t e d with n-heptane,  extracted  of  114  from  dissociated  9,600  to  2-mercaptoethanol. nature  and  stained  136,000  into  Fourteen  of  positively  differences  carbohydrate  absence  of  2-mercaptoethanol,  the  the  18  to  (molecular  presence  bands were Schiff  composition  bound  only  in  these  with  in•polypeptide  the  polypeptides  daltons)  microheterogeneity in  22  the  is  of  urea,  to  This slight  apoprotein.  bands were  SDS  glycoprotein  reagent. due  weights  resolved,  In  the  most  of  -  which the  contained  reduced The  8M u r e a SDS  apoVLDL  column,  urea was  was  proteins by  SDS  and  the not  found  was  3  urea as  Two  from  the  good the  extracted  by  column.  an  fractions  from  the  electrophoresis  analyzed  urea  agent  gels;  had  The  aggregate  eluted  TMU  of  agarose  fractions  a dissociating  The m o l e c u l a r w e i g h t s were  aggregates  6%  apoprotein  c o l u m n was  between  gel  weight  f r a c t i o n a t e d on  2mM S D S .  of  molecular  not  present  in  samples.  or  volume  higher  115-  identical the  sedimentation  the  A  SDS  the  two  the void  proteins;  correlation column  polypeptides  and  the  identified  electrophoretic  two a p o p r o t e i n s  from  from  other  SDS. the  containing  eluted  protein  as  the  were  of  from  columns  patterns.  from  the  equilibrium techniques.  As  SDS  column  plots  of  ln  2 c versus  r  were  calculated the  bottom of  molecular the with SDS  for  weight  cell.  average  radial  weight  was  from  weight  ranges  These  of  and  values  were  the meniscus  range  33,300-165,800  component.  the molecular weight  molecular weights  distance  The m o l e c u l a r  component  low molecular  for  the  high  5,800-28,100 correspond  the polypeptides  to  for  well  observed  in  electrophoresis.  serum VLDL (1976)  and  et  plasma  study,  e_t a l . egg  (1972)  yolk  i t  yolk  al.  (1976)  VLDL.  appears plasma  From that  and  egg  established  VLDL by  c h a r a c t e r i z e d hen  Yamauchi  egg  weight  each measured the  Hillyard  yolk  nonlinear,  plasma studied these  the  identity  between  immunochemical methods. VLDL, the  and  Raju  polypeptide  results  apoproteins  yolk  the  granules  and  those  isolated have  Chan  and Mahadevan composition obtained  in  from h e n ' s  similar  hen's  protein  et^ a l . (1976)  of  egg  this plasma, moieties.  and  -  116  -  LITERATURE  Alexander, of  C.  and  C.E.  Day.  CITED  1973.  selected vertebrates.  Distribution  Comp.  Biochem.  Abraham, S., L.A. H i l l y a r d and L.I. serum and egg y o l k l i p o p r o t e i n s : and s i a l i c a c i d . Arch. Biochem. A c k e r s , G.K. columns. Augustyniak, of  J . , W.G.  lipoprotein Azuma,  Martin  and  J . , N.  structure.  Kashimura  and  W.H.  Cook.  and  Biochim. T.  W.L.,  of Bacon,  I.  Brown  chicken, W.L.  and  egg  and  and  turkey M.A.  yolk  M.A.  and  1976.  Musser.  egg  Acta  1974.  lipoproteins  B a r t l e t t , G.R. 1959. Phosphorous J . B i o l . Chem. 2 3 4 : 4 6 6 .  agarose  assay  in  gel. column  density  on  pig  serum  native lipoproteins Acta 439:380.  Low d e n s i t y Poultry  Partitioning  using  low  84:721.  Studies  1973. yolk.  filtration  Characterization  r e l a t i o n to  Biophys.  Musser.  quail  lipoproteins  46B:295.  gel  1964.  their  Komano.  for  lipoproteins. III. A f f i n i t y chromatography of on C o n c a n a v a l i n A - S e p h a r o s e . Biochim. Biophys. Bacon,  serum  C h a i k o f f . 1960. Components of g a l a c t o s e , mannose, glucosamine Biophys. 89:74.  1967. A new c a l i b r a t i o n p r o c e d u r e J . B i o l . Chem. 2 4 2 : 3 2 3 7 .  l i p o v i t e l l e n i n components,  of  Physiol.  Sci.  turkey  lipoproteins 52:1741.  blood  Poultry  Sci.  plasma 53:1167.  chromatography.  B a t t e l , M . L . , C.G. Z a r k a d a s , L.B. S m i l l i e and N.B. Madsen. 1968. The s u l f h y d r y l groups of muscle phosphorylase. I d e n t i f i c a t i o n of c y s t e i n y l peptides r e l a t e d to f u n c t i o n . J . B i o l . Chem. 2 4 3 : 6 2 0 2 . B e r n a r d i , G. and W.H. Cook. 1960. M o l e c u l a r w e i g h t and b e h a v i o r o f l i p o v i t e l l i n i n urea solutions. Biochim. Biophys. Acta 44:105. B e v e r i d g e , T . , S . J . Toma a n d S. a n d S S - g r o u p s i n some f o o d J . Food S c i . 39:49. Brown, W.V., R.I. Levy p r o t e i n s i n human Chem. 2 4 4 : 5 6 8 7 . •  Nakai. 1974. proteins using  D e t e r m i n a t i o n of SHEllman's reagent.  and D.S. Fredrickson. 1969. Studies plasma very low density l i p o p r o t e i n s .  of the J . Biol.  B u r l e y , R.W. a n d W . H . C o o k . 1961. I s o l a t i o n and c o m p o s i t i o n of a v i a n e g g y o l k g r a n u l e s a n d t h e i r c o n s t i t u e n t dand / S - l i p o v i t e l l i n s . Can. J . Biochem. P h y s i o l . 39:1295.  r  -  117  -  Cham,  B.E. and of plasma 17:176.  B.R. Knowles. 1976. A solvent system f o r d e l i p i d a t i o n o r serum w i t h o u t p r o t e i n p r e c i p i t a t i o n . J . L i p i d Res.  Chan,  L . , R . L . J a c k s o n , B.W. O ' M a l l e y a n d A . R . M e a n s . of very low d e n s i t y l i p o p r o t e i n s i n the c o c k e r e l . estrogen. J . Clin. Invest. 58:368.  Chang, C M . 1969. Madison, Wis. Chen,  on  egg  C. and F. A l a d j e m . 1974. o f human s e r u m l o w d e n s i t y Comm. 6 0 : 5 4 9 .  Chervenka,  C H .  1976.  preparative DS-452. Cook,  Studies  yolk.  Ph.D.  Thesis.  Univ.  of  Wis.,  Subunit structure of the apoprotein lipoproteins. Biochem. Biophys. Res.  Evaluation  ultracentrifuges.  of  a UV  Beckman  scanning  accessory  Disk electrophoresis proteins. Ann. N.Y.  for  application data,  W.H. 1968. M a c r o m o l e c u l a r components of egg C a r t e r ( E d i t o r ) , Egg Y o l k Q u a l i t y . A Study of and B o y d , E d i n b u r g , p. 109.  Davis, B.J. 1964. t o human s e r u m  1976. Synthesis E f f e c t s of  yolk. Hen's  Spinco  In Egg,  T.C Oliver  II. Methods and a p p l i c a t i o n Acad. Sci. 121:404.  D e L a l l a , O.F. and J.W. Gofman. 1954. Ultracentrifugal analysis of serum l i p o p r o t e i n s . I n D. G l i c k ( E d i t o r ) , M e t h o d s o f B i o c h e m i c a l Analysis, I n t e r s c i e n c e , N.Y. 1:459. Deutsch,  D.  upon  1976.  Effect  p e p t i d e bond  of  prolonged  cleavage.  Anal.  100°C  heat  Biochem.  treatment  in  SDS  71:300.  Edelstein, C , C T . L i m and A.M. Scanu. 1972. On t h e s u b u n i t structure o f t h e p r o t e i n o f human s e r u m h i g h d e n s i t y l i p o p r o t e i n . I.A. s t u d y of i t s major p o l y p e p t i d e component (Sephadex f r a c t i o n III). J . B i o l . Chem. 2 4 7 : 5 8 4 2 . E l s o n , L.A. and W . T . J . Morgan. 1933. C i t e d In E.F. N e u f e l d and V . G i n s b u r g ( E d i t o r s ) , 1966, Methods i n Enzymology, V o l . V I I I , A c a d e m i c P r e s s , New Y o r k , p.56. Evans, R . J . , S.L. B a n d e m e r , J . A . D a v i d s o n , K. H e i n l e i n a n d 1968. B i n d i n g of l i p i d to p r o t e i n i n the low d e n s i t y from the h e n ' s egg. Biochim. Biophys. Acta 164:566. Evans,  R.J.,  1973.  D.H.  Bauer,  Structure  dispersity  of  lipovitellenin  the in  of  S.L. egg  very the  Bandemer, yolk  very  low d e n s i t y structure.  S.B.  Vaghefi  low d e n s i t y  and  C J .  Flegal.  lipoprotein.  l i p o p r o t e i n and  Arch.  S.S. Vaghefi. lipoprotein  Biochem.  the  Poly-  role  Biophys.  of  154:493.  -  Evans,  R.J.,  low  D.H.  density  Poultry  Bauer  and  C.J.  lipoproteins  Sci.  118  -  Flegal.  of  fresh  1974.  and  The  stored  egg  shell  yolk  very  eggs.  53:645.  E v a n s , R . J . , D.H. B a u e r and C . J . F l e g a l . 1975a. The i n f l u e n c e of storage of s h e l l eggs produced by h e n ' s fed crude c o t t o n - s e e d o i l on t h e v e r y l o w d e n s i t y l i p o p r o t e i n s f r o m t h e s e eggs. P o u l t r y S c i . 54:280. E v a n s , R . J . , C . J . F l e g a l and D.H. B a u e r . 1975b. Molecular of egg y o l k v e r y low d e n s i t y l i p o p r o t e i n s f r a c t i o n a t e d ultracentrifugation. P o u l t r y S c i . 54:889. Fairbanks,  G.,  analysis  T.L. of  membrane. Fidge,  N.  Biochem.  1973.  lipoproteins Biochim.  Stack  and  the major  D.R.  Wallach.  polypeptides  of  1971. the  Electrophoretic  human  erythrocyte  10:2606.  The  isolation  and  p a r t i a l c h a r a c t e r i z a t i o n of  Biophys.  sizes by  Acta  and  properties  of  pig  plasma  their  apoproteins.  295:258.  F i d g e , N.H. 1976. C h a r a c t e r i z a t i o n of the small molecular weight a p o l i p o p r o t e i n s from p i g plasma very low density l i p o p r o t e i n . Biochim. Biophys. Acta 424:253. F o l c h , J . , M. L e e s and G . H . S l o a n e - S t a n l e y . 1957. A simple f o r t h e i s o l a t i o n and p u r i f i c a t i o n of t o t a l l i p i d s f r o m tissue. J . B i o l . Chem. 2 2 6 : 4 9 7 . G a r l a n d , T.D. 1973. S t u d i e s on egg low d e n s i t y l i p o p r o t e i n s . Ph.D.  method animal  y o l k m y e l i n f i g u r e s and g r a n u l e Thesis, Univ. of B.C., Vancouver,  B.C. G i b b o n s , R.A. 1966. _In A. G o t t s c h a l k ( E d i t o r ) G l y c o p r o t e i n s C o m p o s i t i o n , S t r u c t u r e and F u n c t i o n , 2nd E d i t i o n , E l s e v i e r , New Y o r k .  Their  Glickman,  size  R.M.  classes Acta  and  of  K.  Kirsch.  human  chylous  1974. fluid  The  apoproteins  lipoproteins.  of  various  Biochim.  Biophys.  371:255.  Goldstein, I.J., C.E. H o l l e r m a n and E.E. Smith. 1965. Proteincarbohydrate i n t e r a c t i o n . II. I n h i b i t i o n studies of Concanavalin A with polysaccharides. Biochem. 4:876. Gornall, by  D.A.  and  A.  Kuksis.  chromatography  Biochem.  49:44.  on  thin  1971a. layers  Resolution of  of  egg  yolk  hydroxylapatite.  lipoproteins  Can.  J .  - 119  Gornall,  D.A.  and  A.  Kuksis.  -  1971b.  g l y c e r o p h o s p h a t i d e s and Can. J . Biochem. 49:51.  Molecular  triglycerides  of  species  egg  yolk  of lipoproteins.  G o t t o , A . M . , W.V. B r o w n , R.I. L e v y , M.E. B i r n b a u n e r and D.S. Fredrickson. 1972. Evidence f o r the i d e n t i t y of the major apoprotein i n low d e n s i t y and v e r y l o w d e n s i t y l i p o p r o t e i n s i n n o r m a l s u b j e c t s and patients with familial hyperlipoproteinemia. J . Clin. Invest. 51:1486. G o t t o , A . M . , R.I. lipoprotein; derivative.  L e v y and D.S. Fredrickson. 1968. Human s e r u m B e t a P r e p a r a t i o n and p r o p e r t i e s o f a d e l i p i d a t e d , s o l u b l e B i o c h e m . B i o p h y s . R e s . Comm. 31:151.  G r a n d a , J . L . and A. Scanu. 1966. apoproteins of the very lowserum. Biochem. 5:3301.  S o l u b i l i z a t i o n and p r o p e r t i e s of t h e and l o w - d e n s i t y l i p o p r o t e i n s o f human  G u s t a f s o n , A. 1965. New m e t h o d f o r p a r t i a l d e l i p i d a t i o n o f lipoproteins. J . L i p i d Res. 6:512.  serum  G u s t a f s o n , A . , P. A l a u p o v i c and R.H. F u r m a n . 1966. Studies of the c o m p o s i t i o n and s t r u c t u r e o f serum l i p o p r o t e i n s . S e p a r a t i o n and c h a r a c t e r i z a t i o n of p h o s p h o l i p i d - p r o t e i n r e s i d u e s obtained by p a r t i a l d e l i p i d a t i o n o f v e r y l o w d e n s i t y l i p o p r o t e i n s o f human serum. Biochem. 5:632. H a y a s h i , S., 200-400 ether.  F. L i n d g r e n a n d A . N i c h o l s . 1959. Degradation of S and h i g h d e n s i t y l i p o p r o t e i n s o f human s e r a b y e t h y l J . Am. Chem. S o c . 81:3793.  H e l e n i u s , A. and low density Helenius,  A.  K. S i m o n s . 1971. R e m o v a l o f l i p i d s f r o m human l i p o p r o t e i n s by d e t e r g e n t s . Biochem. 10:2542.  and  lipophilic  K. and  Simons.  197 2 .  hydrophilic  The  binding  proteins.  J .  of  detergents  Biol.  Chem.  of  L.A.,  H.M.  White  apolipoproteins  H j e r t e n , S. 1964. chromatography Acta  in  and  S.A.  chicken  Pangburn. serum  and  1972. egg  yolk.  to  247:3656.  H e r b e r t , P . N . , R.S. Shulman, R.I. L e v y and D.S. Fredrickson. F r a c t i o n a t i o n o f t h e C - a p o l i p o p r o t e i n s f r o m human p l a s m a low density l i p o p r o t e i n s . J . B i o l . Chem. 248:4941. Hillyard,  plasma  1973. very  Characterization Biochem.  11:511.  The p r e p a r a t i o n of a g a r o s e s p h e r e s for o f m o l e c u l e s and p a r t i c l e s . Biochim. Biophys.  79:393.  I m m a r i n o , R.M., M. Humphrey and P. A n t o l i k . 1969. Agar g e l l i p o p r o t e i n electrophoresis: A correlated study w i t h u l t r a c e n t r i f u g a t i o n . C l i n . Chem. 1 5 : 1 2 1 8 .  -  International and  Critical  Technology.  McGraw-Hill Janado,  M.  in  and  low  Book  W.G.  Tables 1927.  of  Company,  -  Numerical Data.  E.W.  Martin.  density  120  Washburn  New Y o r k .  1973.  Physics  (Editor)  , Chemistry  Volume  II,  p.328.  Evidence  l i p o p r o t e i n from pig  for  protein  serum.  Agr.  subunits  Biol.  Chem.  37:2835. Kalab,  M.  and  W.G.  lipoproteins Kalab,  M.  and  W.G.  modified Kane,  J.P. of  pig  subunit  Kane,  Martin.  Electrophoresis  gel.  Anal.  Biochem.  1968b.  Gel  filtration  lipoproteins.  A rapid  species  of  of  J.Chromatog.  in  serum  serum  native  and  35:230.  electrophoretic technique apoproteins  pig  for  identification  lipoproteins.  Anal.  J . P . , E.G. R i c h a r d s and R . J . H a v e l . 1970. Subunit heterogeneity i n human s e r u m B e t a - l i p o p r o t e i n . Proc. N a t l . Acad. S c i . 66:1075. J.P., J.  T.  Sata,  Clin.  of  R.L. very  Invest.  Hamilton  and  low density  R.J.  density  1975. of  Apoprotein  human  serum.  56:1622.  S., D.L. H o r o w i t z and A.M. of l i p o p r o t e i n s from normal  low  Havel.  lipoproteins  Scanu. 1969. r a t serum. J .  L i n d g r e n , F . T . , L . C . J e n s e n , R.D. W i l l s Flotation rates, molecular weights  Liu,  of  24:218.  53:350.  composition  Koga,  1968a.  agarose  serum  1973.  Biochem. Kane,  Martin. in  lipoproteins.  Lipids  T.Y. and Y . H . Chang. 1971. p-toluenesulfonic acid. J .  I s o l a t i o n and p r o p e r t i e s L i p i d Res. 10:577.  and N.K. F r e e m a n . 1969. and h y d r a t e d d e n s i t i e s of  Hydrolysis B i o l . Chem.  of p r o t e i n s 246:2842.  with  L o w r y , O.H., N . J . R o s e n b r o u g h , A . L . F a r r and R . J . R a n d a l l . P r o t e i n measurement w i t h the F o l i n phenol r e a g e n t . J . Chem. 1 9 3 : 2 6 5 .  1951. Biol.  Ma,  serum  S.K. low  1975. density  Angeles, Ma,  Measurement  of  lipoproteins.  size Ph.D.  the  4:337.  distributions Thesis,  of  Univ.  human  of  Calif.,  very  Los  Calif.  S.K. , V . N . Schumaker and C M . K n o b l e r . 1977. Turbidimetric ultracentrifugation. A p p l i c a t i o n t o t h e s t u d y o f human s e r u m very  low d e n s i t y  lipoprotein distributions.  J.  Biol.  Chem.  252:1728. MacKenzie,  S.L.  composition Can.  J.  and of  W.G. hen's  Biochem.  Martin. egg  45:591.  yolk  1967. at  The  macromolecular  successive  stages  of  maturation.  -  121  -  M a h l e y , R.W. a n d K . S . Holcombe. 1977. A l t e r a t i o n s of the plasma l i p o p r o t e i n s and a p o p r o t e i n s f o l l o w i n g c h o l e s t e r o l f e e d i n g i n the r a t . J . L i p i d Res. 18:314. Margolis, S. 1967. S e p a r a t i o n and s i z e d e t e r m i n a t i o n o f human^ serum l i p o p r o t e i n s by agarose g e l f i l t r a t i o n . J . L i p i d Res. 8:501. Marsh,  J.B.  1976.  recirculating  Apoproteins perfusate  of  of rat  the  lipoproteins  liver.  J.  Lipid  in  a  non-  Res.  17:85.  M a r t i n , W . G . a n d I. Takats. 1969. Physical-chemical properties of the very low d e n s i t y l i p o p r o t e i n of p o r c i n e serum. Biochim. Biophys. Acta 187:328. M a r t i n , W.G., N.H. T a t t r i e and W.H. Cook. and d i s t r i b u t i o n s t u d i e s of egg y o l k Biochem. P h y s i o l . 41:657. Mauldin,  J .  and  aggregation  W.R. of  Fisher. serum  1970.  low  1963. Lipid lipoproteins.  pH and  density  ionic  strength  lipoproteins.  M c C o n a t h y , W . J . and P. A l a u p o v i c . 1974. of Concanavalin A w i t h major density lipoproteins. Evidence f o r the s p e c B i n i t s a s s o c i a t e d and f r e e forms.  extraction Can. J .  dependent  Biochem.  9:2015.  S t u d i e s on the i n t e r a c t i o n c l a s s e s o f human p l a s m a i f i c b i n d i n g of l i p o p r o t e i n FEBS L e t t e r s 41:174.  Means, G.E. and R.E. Feeney. 1971. A l k y l a t i n g and s i m i l a r reagents. In Chemical M o d i f i c a t i o n of P r o t e i n s . C h a p t e r 6. Holden-Day Inc., San F r a n c i s c o , p.105. M o r r i s e t t , J . D . , R.L. J a c k s o n and A.M. G o t t o . 1975. Lipoproteins: S t r u c t u r e and F u n c t i o n . I n E.S. S n e l l ( E d i t o r ) , V.ol. 44, Ann. Rev. Biochem., Annual Reviews, Inc., Palo A l t o , p.183. M o s b a c h , E . H . , H . J . K a l i n s k y , E. H a l p e r n a n d F . E . K e n d a l l . 1954. D e t e r m i n a t i o n of d e o x y c h o l a t i c and c h o l i c a c i d s i n b i l e . Arch. Biochem. Biophys. 51:402. Narayan, K.A., H.L. C r e i n i n e l e c t r o p h o r e s i s of r a t 7:150.  a n d F . A . Kummerow. 1966a. Disk plasma l i p o p r o t e i n s . J . L i p i d Res.  Narayan, K.A., W.E. D u d a c e k and F . A . Kummerow. 1966b. Disk e l e c t r o p h o r e s i s of i s o l a t e d r a t - s e r u m l i p o p r o t e i n s . Biochim. Biophys. Acta 125:581. Nichols,  A.V. • 1969.  classes  of  plasma  Functions  and  lipoproteins.  interrelationships Proc.  Natl.  Acad.  of  different  Sci.  64:1128.  -  122  -  N i c h o l s , A . V . , E . L . G o n g , P . J . B l a n c h e , T . M . F o r t e a n d D.W. Anderson. 1976. E f f e c t s o f g u a n i d i n e h y d r o c h l o r i d e on human p l a s m a high density lipoproteins. Biochim. Biophys. Acta 446:226. N o b l e , R.P. 1968. Electrophoretic in agarose gel. J . L i p i d Res.  separation 9:693.  of  plasma  lipoproteins  O r n s t e i n , L. and B . J . D a v i s . 1964. In D i s c Electrophoresis. D i s t i l l a t i o n Products Industries, Rochester, N.Y. P e a c o c k , A . C . a n d S.W. D i n g m a n . 1968. Molecular weight estimation and s e p a r a t i o n o f r i b o n u c l e i c a c i d by e l e c t r o p h o r e s i s in agarose-acrylamide composite gels. Biochem. 7:668. Pearson,  S.,  method Anal.  S.  Stern  for  the  Chem.  Quarfordt,  T.H.  McGavack. of  1953.  total  A  rapid,  cholesterol  accurate  in  serum.  25:813.  S.H.,  A.  Heterogeneity filtration  and  determination  Nathans, of  M.  Dowdee  human v e r y  chromatography.  and  H.L.  low density J .  Lipid  Hilderman.  lipoproteins  Res.  1972. by  gel  13:435.  Raju,  K.S. a n d S. M a h a d e v a n . low density l i p o p r o t e i n s A n a l . Biochem. 61:538.  1974. I s o l a t i o n of h e n ' s egg y o l k by D E A E - c e l l u l o s e chromatography.  Raju,  K.S. a n d S. M a h a d e v a n . 1976. P r o t e i n components i n the v e r y l o w d e n s i t y l i p o p r o t e i n s of h e n ' s egg y o l k s . I d e n t i f i c a t i o n of highly aggregating ( g e l l i n g ) and l e s s a g g r e g a t i n g (non-gelling) proteins. Biochim. Biophys. A c t a 446:387.  R i b e i r o , L . P . and H . J . McDonald. u s i n g p r e s t a i n e d serum. J . Rudel,  L.L.,  J.A.  Lee,  M.D.  1963. Electrophoresis Chromatog. 10:443.  Morris  and  J.M.  Felts.  of  1974.  very  lipoproteins  *  C h a r a c t e r i z a t i o n of plasma l i p o p r o t e i n s s e p a r a t e d and p u r i f i e d by agarose column chromatography. Biochem. J . 139:89. S a i t o , Z . , W.G. M a r t i n and W.H. Cook. 1965. Changes i n the major m a c r o m o l e c u l a r f r a c t i o n s of egg y o l k d u r i n g embryogenesis. Can. J . Biochem. P h y s i o l . 43:1755. Sata,  Sata,  T.,  D.L.  of  gel  J.  Lipid  T.,  Estrich,  R.J.  Res.  of  and  plasma  L.W.  Kinsell.  lipoprotein  1970.  Evaluation  fractionation.  A.L.  Jones.  1972.  Characterization  triglyceride-rich lipoproteins  chromatography  hyperlipemic  Wood a n d  for  11:331.  Havel  subfractions gel  P.D.  chromatography  from blood  humans.  J .  Lipid  plasma Res.  of  separated  normolipemic  13:757.  and  of by  -  Scanu, A. 1966. protein. J .  Forms Lipid  123  -  o f human s e r u m Res. 7:295.  high  density  lipoprotein  S c a n u , A . M . a n d C. E d e l s t e i n . 1971. S o l u b i l i t y of aqueous s o l u t i o n s of the s m a l l m o l e c u l a r weight p e p t i d e s of the serum v e r y l o w d e n s i t y and h i g h d e n s i t y l i p o p r o t e i n s : Relevance to the recovery problem during d e l i p i d a t i o n of serum lipoproteins. Anal. Biochem. 44:576. Scanu,  A.M.,  L.  Vitello  and  S.  p h y s i c a l methods to the Rev. Biochem. 2:175.  Deganello.  study  of  1974.  serum  S h i e l d s , R. a n d W. B u r n e t t . 1960. Determination c a r b o h y d r a t e i n serum by a m o d i f i e d a n t h r o n e 32:885. S h o r e , B. a n d V . G . S h o r e . 1967. beta-lipoproteins. Biochem. S h o r e , V . G . a n d B. S h o r e . density lipoproteins. components. Biochem. Shore,  V.G.,  B.  Shore  and  and p r o p e r t i e s of of c h o l e s t e r e m i a .  Application  lipoproteins.  of  Critical  of protein-bound method. A n a l . Chem.  The p r o t e i n m o i e t y o f human serum B i o p h y s . R e s . Comm. 2 8 : 1 0 0 3 .  1973. H e t e r o g e n e i t y o f human p l a s m a l o w Separation of species d i f f e r i n g i n p r o t e i n 12:502. R.G.  Hart.  1974.  Changes  rabbit very low density Biochem. 13:1579.  in  apolipoproteins  lipoproteins  on  induction'  Shipski, V.P. 1972. L i p i d c o m p o s i t i o n of l i p o p r o t e i n s i n n o r m a l and diseased states. In G . J . Nelson ( E d i t o r ) , Blood L i p i d s and L i p o p r o t e i n s : Q u a n t i t a t i o n , C o m p o s i t i o n , and M e t a b o l i s m . WileyI n t e r s c i e n c e , New Y o r k . p..471. Tanford,  C.  1967.  Light  Macromolecules.  Scattering.  John  Wiley  and  In  Physical  Sons,  Chemistry  New Y o r k , C h a p t e r  of 5,  p.275.  T u r n e r , K . J . and W.H. Cook. 1958. M o l e c u l a r w e i g h t and p h y s i c a l properties of a l i p o p r o t e i n from the f l o a t i n g f r a c t i o n of egg yolk. Can. J . Biochem. P h y s i o l . 36:937. van  de  Warren, J. Weber,  Voort,  F.  1976.  Personal  communication.  L. 1959. The t h i o b a r b i t u r i c B i o l . Chem. 2 3 4 : 1 9 7 1 . K.  and M.  Osborne.  determinations J . B i o l . Chem.  1969.  by d o d e c y l 244:4406.  The  acid  assay  reliability  of  sialic  of  sulfate-polyacrylamide  acids.  molecular gel  weight  electrophoresis.  -  124  -  W e b e r , K., J . R . P r i n g l e and M. O s b o r n e . 1972. Measurement of m o l e c u l a r w e i g h t s by e l e c t r o p h o r e s i s on S D S - a c r y l a m i d e gels. I n C.H.W. H i r s and J . N . T i m a s h e f f ( E d i t o r s ) , M e t h o d s in E n z y m o l o g y , P a r t C , V o l . X X V I , A c a d e m i c P r e s s , New Y o r k , p.3. W e i n e r , A . M . , T . P i a t t a n d K. W e b e r . a n a l y s i s of p r o t e i n s p u r i f i e d on electrophoresis. J . B i o l . Chem.  1972. Amino-terminal sequence a nanomole s c a l e by g e l 247:3242.  W e r n e r , M. 1966. filtration.  F r a c t i o n a t i o n of l i p o p r o t e i n s J . Chromatog. 25:63.  Yamauchi,  Kurisaki  K.,  J .  a n d <K.  c o m p o s i t i o n of h e n ' s egg y o l k A g r . B i o l . Chem. 40:1581. Z i l v e r s m i t , D.B. lipoprotein  1964. films.  Sasago. very  E x t r a c t i o n of J . L i p i d Res.  1976.  from blood  by  gel  Polypeptide  low density  lipoprotein.  c h o l e s t e r o l f r o m human 5:300.  serum  -  125  -  APPENDIX U.V.  Derivative  with  plotting  program subroutine.  266  - 126 315.  120  160  057  015  222  066'  000  146  115  001  266  022  377  161  121  377  222  013  222  146  001  130  266  001  066  162  023  026  222  024  146  001 000  266  024  013  163  121  000  160  013  010  314  001  352  260  000  021  155  121  121  314  022  013  222  013  000  164  001  on  302  001  020  247  020  120  155  120  015  161  015  000  314  000  001  241  001  121  201  121  015  360  015  000  326  000  001  315  001  127  273  127  022  277  022  067  322  067  053  315  045  222  336  222  134  322  130  066  315  066  053  336  045  000  322  000  120  121  - 127 -  015  001  013  000  004  001 202  127  001 004  067  127  140  054  067  222  127  054  134  067  127  066  077  023  on  127  067  056  023  075  060  067  126  120  075  067  013  066  105  001  073  066  001  121  076  056  013  127  060  001  067  120  005  054  013 001  113 013  127 023  000  001  066  004  067 075  105 .  121  126  166  013  067  202  001 000  073  121  054  120 013  022 013  121 013  001 005  001 004  001 004  060  126  127  066  021  016  101  067  067  121 013  076 121  074 127  001 001  013  067  001  115  120  000  121  013  120  013  140 056  066  001 004 127 067 020 127 016 067 074 127 067 026 057 066 074 060 057 066 075 202 144 202 140 057 000 066 020 065 001 000 120 013 on on 056 110 012 120 013 005  - 128 005 022 001 020 120 013 003 005 021 003 020 120 013 on 010 066 047 121 013 003 005 151 120 013 003 005 121 067 022 007 on 000 020 127 022 067 025 001 121 021  013 005 005 110 012 120 013 005 005 126 067 047 066 025 154 056 no 000 007 012 002 000 000 000 000 000 021 056 020 no 001 012 000 001 001 003 004 007 010 on  - 129 -  no  121  121  002  013.  013  012  on  003  000  010  005  001  020  127  010  120  020  003  013  067  006  003  070  005  005  12i  003  121  013  001  013  003  no  003  005  003  005  127  012  127  022  000  022  067  003  067  030  001  040  121  003  023  013  007  111  003  002  002  005  005  020  127  004  120  021  no  013  067  007  on  052  007  007  066  012  126  070  006  067  005  on  014  no  001  066  004  005  040  126  002  023  067  on  111  072  004  003  066  no  020  030  010  120  121  121  013  013  013  on  003  003  007  005  005  066  013  022  014  no  - 130 -  004  020  007  126  120  020  067  013  no  071  003  on  066  007  155  052  121  m  005  013  on  022  003  154  121  007  121  013  021  000  003  111  000  005  001  127  020  024  067  no  002  115  004  020  121  066  110  013  072  on  003  066  111  007  071  on  no  000  050  001  no  120  121  005  000  067  001  000  021  003  155  111  005  121  000  120  000  020  013  000  120  004  154  013  005  111  002  126  010  on  067  022  001  .  006  121  121  066  013  021  001  on  013  154  007  005  111  022  005  001  m  no  022  on  012  111  024  120  002  111  013  - 131 005  013  006  005  004..  111  121  005  004  067  022  152  021  111  127  111  005  020  000  020  067  022  120  104  121  013  118  013  003  004  002  001  126  011  121  067  024  013  001  111  003  066  002  001  104  020  127  111  120  020  001  000  000  022  000  000  111  155  111  003  121  005  020  000  022  120  000  121  013  154  013  003  121  Oil  007  000  on  005  000  020  no  127  120  004  067  013  121  026  002  013  202  Oil  003  144  121  007  066  013  021  005  002  111  111  on  001  005  127  024  151  020  002  110  067  020  005  103  no  121  066  on  - 132 m  007  000  on  no  050  001  120  021  121  000  067  000  000  021  000  155  111  154  121  000  121  000  020  000  000  120.  000  154  013  127  111  002  067  010  on  026  022  001  202  121  121  144  013  021  126  on  013  067  007  005  005  022  005  066  111  no  103  on  012  111  024  120  005  111  013  176  007  005  060  020  005  176  no  121  121  on  067  013  155  021  on  111  111  on  on  000  021  154  022  001  m  000 155  121  121  000  000  013  020  000  002  120  127  on  013  067  024  on  115  111  on  121  003  026  013  020  067  003  120  006  066 055 117 010 062 202 130 202 134 056 001 000 202 130 202 134 066 102 056 120 013 000 007 202 130 056 120 013 000 on 202 134 066 013 056 120 013 001 000 060  - 133 056 120 013 001 001 060 056 060 120 013 001 004 056 060 120 013 001 005 066 034 056 060 120 013 000 006 056 060 120 013 000 010 121 013 001 000 121 022 013 001  001 353 121 024 013 000 006 060 121 013 001 004 121 022 013 001 005 353 121 024 013 000 010 060 056 060 120 013 001 003 056 060 120 013 001 006 066 100 062 121  013 001 001 041 127 067 115 000 127 067 026 127 067 077 066 050 043 127 067 115 000 127 067 026 043 127 067 115 012 001 121 021 013 000 on 202 134 043 127 067  - 134 115 012 001 121 022 013 000 on 013 202 134 043 127 067 115 000 127 067 026 121 013 001 003 041 121 013 001 000 022 043 020 126 021 067 050 126 020 067 050 202  140 127 067 115 062 121 013 001 005 041 127 067 026 127 067 077 066 051 000 127 067 115 043 127 067 026 012 001 121 021 013 000 007 202 130 043 127 067 026 012  -  135  -  001  051  100  Oil  121  062  101  020  022  202  102  120  013  140  103  015  000  126  104  000  007  067  105  001  013  Oil  106  377  202  066  107  377  130  077  108  377  043  000  109  377  127  051  110  377  067  377  111  377  026  050  112  377  000  051  113  377  127  050  114  222  067  202  115  134  115  114  043  057  127  377  610  002  067  377  611  Oil  026  377  612  024  121  613  111  013  614  002  001  615  020  006  616  013  041  617  120  121  618  000  013  619  000  001  620  155  022  798  003  043  799  020  020  800  013  801  120  802  000  803  000  804  155  805  121  004  126 021 067 051 126 020 067  


Citation Scheme:


Citations by CSL (citeproc-js)

Usage Statistics



Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            async >
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:


Related Items