Coupled Magneto-Electrophoresis For Improved Hemoglobinopathy Resolution by David Chukwudi Jirinzu Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science in the Chemistry Program / Gr-l #$Z2Lf- 8 4,/f/ -. Adviser / Date Dean, ~rabuate School a II, 1484 -Date - YOUNGSTOWN STATE UNIVERSITY June, 1984 Youngstown State University Graduate School Thesis Submitted in Partial Fulfillment of the Requirements For the Degree of Master of Science TITLE: COUPLED MAGNETO-ELECTROPHORESIS FOR IMPROVED HEMOGLOBINOPATHY RESOLUTION ACCEPTED BY THE DEPARTMENT OF CHEMISTRY Major Pofessor ate Q 19 8q Dean, Graduate School v Date ABSTRACT Coupled Magneto-Electrophoresis For Improved Hemoglobinopathy Resolution David Chukwudi Jirinzu Master of Science Youngstown State University, 1984 Misinterpretation of different hemoglobin variants has been the cardinal source of errors in hemoglobinopathies screening prior to genetic counseling, or medical treatment designed to ameliorate a pathological condition. In this study, a magnetic field is coupled toanelectro- phoretic power supply in order to enhance the resolution of separated globin chains over conventional electrophoresis. The diamagnetic susceptibility of hemolysates is utilized to- oppose the mobilities due to the charged globin chains, when an applied voltage is on. Normal hemolysate has been inves--- tigated, and distinct band separation of HbA and HbAZ was ob- served. The resolution was definitely superior to conventional electrophoretic method. The technique is easy to perform and provides time optimization. It is envisaged that coupled magneto-electrophoresis, though a novel idea, may be useful in the establishment of loci for the various hemoglobinopathies. Because the variants have differing net charges, with the iron in a divalent state, a - repulsion or enhancement will result when placed in a magneto- electrophoretic field. The direction may be valuable to the diagnostic clinician. iii ACKNOWLEDGEMENTS I wish to take this opportunity to thank those who have made this study worthwhile. Special thank you to my adviser, Dr. Daryl Mincey, for his gracious efforts and advice towards this study. I wish to thank Dr. Francis Smith for his valuable time used in reading through the manuscript and making the necessary recommendations. I wish to thank Dr. Howard Mettee for his effort and time used in reading through this thesis. His reasonable sugges- tions were valuable in putting this thesis in its final form. Finally, my thanks to all those who have assisted, directly or indirectly, to make this study successful. TABLE OF CONTENTS PAGE ABSTRACT............................................. ii ACKNOWLEDGEMENTS ..................................... iii TABLE OF CONTENTS.................................... iv LIST OF ABBREVIATIONS............................. ... vi LIST OF FIGURES...................................... vii LIST OF TABLES....................................... viii CHAPTER I. INTRODUCTION................................. 1 Basic Theory of Electrophoresis .............. 1 Tiselius Moving Boundary ..................... 2 Electrophoresis .............................. 2 Agar Gel Electrophoresis ..................... 4 Structure of Hemoglobin ...................... 6 Clinical Significance of Hemoglobin .......... 16 Interpretation of Hb Electrophoretic Patterns 19 - Background for the Coupled Magneto-Electro- phoretic Technique ......................... 20 Statement of Problem ......................... 2 8 11. LITERATURE REVIEW .............................. 36 Methods ...................................... 36 Ion-Exchange Chromatography .................. 37 Isoelectrofocusing ........................... 38 Electrophoretic Technique .................... 41 111. MATERIALS AND APPARATUS...................... 43 - Materials .................................... 43 Apparatus .................................... 44 TABLE OF CONTENTS (Continued) PAGE CHAPTER IV . EXPERIMENTAL ................................. 45 Cell Design and Construction ................. 45 Preparation of Solution ...................... 45 ....................................... Method 48 ......... . V RESULTS. DISCUSSION. AND CONCLUSIONS 52 Results and Discussion ....................... 52 .................................. Conclusions 53 Future Work .................................. 56 . APPENDIX A The Guoy Technique ...................... 57 . ... APPENDIX B The Modified Guoy Technique by Quinke 59 REFERENCES ........................................... 62 LIST OF ABBREVIATIONS ABBREVIATIONS Oc 2,3 - DPG DEAE - cellulose ~e~+ HbCO HPLC DEFINITIONS Degree Centigrade Deciliter 2,3 - Diphosphoglycerate Diethylaminoethyl Cellulose Iron (11) Gram Hemoglobin Carboxyhemoglobin High Performance Liquid Chromatography Milliliter of Mercury Milliliter Oxygen Percent Hydrogen Ion Concentration Index - Point of Electrical Neutrality Oxygen Tension Carbon Dioxide Tension - - Micro Volume Volt vii FIGURE LIST OF FIGURES PAGE 1. Tiselius' Electrophoresis Arrangement ............. 3 2. Cellulose Acetate Electrophoresis ................. 5 3. Schematic Tetrameric Composision of Hemoglobin .... 7 4. Three Dimensional Structure of Hb Molecule Based on X-ray Crystallography ........................ 8 5. The Basic Unit of Hemoglobin ...................... 9 6. Idealized Representation of Heme Molecule......... 12 7. Normal Oxyhemoglobin Dissociation Curve and Curve for the Case of a 50% Anemia .................... 15 8. The Normal Oxygen Dissociation Curve.............. 17 9. Hemoglobin Electrophoresis on Cellulose Acetate at pH 8.6 ...................................... 30 10a. Effect of Applied Magnetic Field From a 14.7 Kilogauss NMR Magnet for a Two Hour Duration .... 32 lob. The Effect of the Same Magnetic Field on a Sample of Ferrohemoglobin in an Electrophoretic Cell. .. 32 11. Diamagnetic Shielding of a Nucleus................ 34 - 12. Mini Electrophoretic Cell for Coupled Magneto- Electrophoresis (Vertical Span) ................. 46 13. Mini Electrophoretic Cell for Coupled Magneto- Electrophoresis (Horizontal Span) ............... 47 14. The Set-Up-for Coupled Magneto-Electrophoresis ... ;- 51 15. Effect of Magneto-Electrophoresis on a Normal Hb Hemolysate ...................................... 54 16. Gouy Balance Arrangement for Measuring Magnetic Susceptibility .................................. 58 17. Principle of the Quinke Balance Based on Gouy Technique ....................................... 61 - LIST OF TABLES TABLE PAGE 1. Some Clinically Important Hemoglobinopathies ...... 26 CHAPTER I INTRODUCTION Electrophoresis is a useful tool that a clinician, or investigator could employ to diagnose pathological states or in vitro life processes in the laboratory. Electrophoresis is basically the separation of macromolecules under the in- fluence of an electric field. It is the classical method in use today in the laboratory for the analyses and separations of proteins, lipoprotein, hemoglobin, and other biologic poly- mers since most biologic polymers are electrically charged. Moreover, it has been accepted as the reference method in all diagnostic procedures of charged species furnishing medical investigators with knowledge which would have otherwise been -. difficult to achieve. The most widely used method is zone electrophoresis. A spot or thin layer of the sample is appli- - ed on an electrically conducting medium; an electric field is applied and molecules migrate on or through the supporting medium. The rate of migration of a molecule in an electric field depends on the support, the magnitude of the ne-k charge on the molecule, and the size and shape of the molecules. Other factors that affect electrophoresis include the follow- ing: degree of dissociation, extent of ionization, pH, volt- age applied, distance between electrodes, viscosity of support- ing medium, concentration and ionic strength of electrolyte, - temperature, solubility of sample, and affinity between sepa- rating material and the supporting medium. The work of Tiselius (1920) in his famous U-tube experi- ment, revolutionized the technique conceived by the Russian scientist Reuss (1809), who performed a study of the kinetic behavior of colloids. Tiselius used sliding baffles to esta- blish sharp boundaries between sample and buffers, since Sam- ple - buffer mixing was an inherent defect. Due to the absence of a solid matrix, his technique was termed free - solution 1 I electrophoresis. Here the rate of mobility was found to be dependent on the charge to mass ratio. For this endeavor, he was awarded a Nobel prize in 1948. An illustration of Tiselius' arrangement is shown in Figure 1. The numerous difficulties associated with the free-solution electrophoresis are alleviated or eliminated when separations are carried out in a stabilizing medium such as paper, a layer of finely granulated solid, or a column packed with suitable -. solid. Hence electrophoresis in a stabilizing medium has been tagged electrochromatography and this has been employed in vari- - ous applications in the diagnostic laboratory and biochemistry. Electrochromatography facilitates the separation of proteins and other macromolecules contained in serum, urine, spinal fluid, - - gastric juices, and other body fluids. Electrochromatography has been employed by biochemists to fractionate alkaloids, antibiotics, nucleic acids, vitamins, natural pigments, ster- oids, amino acids, carbohydrates, and other organic acids. Since the property of the medium is pertinent to the sepa- - ration process, results have been primarily due to a combination of the electrophoretic effect and absorption, ion exchange, or other distribution equilibria. Numerous solid media have been 1 Figure 1. Tiselius electrophoresis arrangement. : (11, slid- ing baffle to facilitate unique loading of sample and buffer; (2), sample and buffer; (3), buffer only. employed and these include: paper, cellulose acetate mem- branes, cellulose powders, starch gels, ion exchange resins, glass powders, agar gels, and polyacrylamide gels. The most widely used support medium is cellulose acetate; this is a plastic material which can be made into thin strips. The strips contain pores and tracks through which ions can mi- grate by capillary action. A typical cellulose acetate elec- trophoresis set up is illustrated in Figure 2. Kohn introduced cellulose acetate in 1958. The acetate support medium has the advantage of rapid analysis using small sample volumes, great tensile strength when wet, and a pure and relatively uniform structure. Further, sample absorption is almost absent, and it possesses low affinity for dyes, A 2,3 crystal clear acetate strip facilitates quantitation. Agar Gel Electrophoresis Agarose gel electrophoresis has proven successful in sepa- -- rations involving aqeous buffers supported within a polymerized gel matrix. Agar gel electrophoresis has the distinct advan- tage in that it can contain larger samples than cellulose acetate or celiulose nitrate systems; hence, this prok-ess can be employed in preparative scale electrophoresis of macromole- cules. The property of the gel matrix can be freely manipula- ted since gels enhance the friction as well as molecular siev- ing action. Viscosity and pore size have a strong influence CI L - on electrophoretic mobility. Agarose itself is a polygalactose polymer which has proven successful in its applicability to separating large macromole- Power Supply - Figure 2. Cellulose acetate electrophoresis. Buffer is in - - the anode and cathode compartments. The anode (1) and cathode (2) are platinum wires positioned in the proper compartments (6) and (7) ; (3). strip support; (4). plastic tank; (5). wick; (8). cellulose acetate strip; (9). ice/buffer solution. and 2 (10). is a plastic covering which minimizes evaporation. - cules such as nucleic acids, lipoproteins, lactate dehydro- genases, and bacteriophages. Agar gel electrophoresis has gained prominence in most clinical laboratories in routine diagnosis of macromolecules. Its ease of management coupled with versatility has increased its acceptability as the gel medium of choice and could possi- bly displace cellulose acetate. Structure of Hemoalobin Hemoglobin is a globular protein mass with a central cavity and having a molecular weight of 67,000 daltons. The Hb molecule is radially segmented into four tetrahedrals, each segment consisting of loops of globin chains and each projects to the exterior surface of the sphere an ion-porphrin complex (heme) which is essentially the site of oxygen binding. -. The spheroidal Hb tetramer consists of two groups of iden- tical globin chains and each chain is associated with a heme - molecule. In adult hemoglobin (HbA), these chains are common- ly designated as alpha and beta. The two alpha and two beta chains are arranged as in Figure 3 and Figure 4. Each adja- cent alpha and-beta chain interact via narrow strips between them (alpha l beta contact points and two broad areas of con- 2 tact, alpha beta l ). During periods of oxidative interconver- 1 sion from oxygenated Hb to the deoxygenated state, there is a rotation of the alpha beta contact point such that the 1 1 central cavity enlarges enough to accomodate a molecule of 2,-3 -diphosphoglycerate (DPG), a product of glucose metabolism. Conversely, oxygenation causes a rotation of the two segments Figure 3. Schematic tetrameric composition of hemoglobin 5 showing two pairs of identical globin chains. For HbA, - there are two alpha and two beta chains. Each globin chain has an attached heme group (light gray oval). 2,3-DPG, a product of glucose metabolism, occupies the central cavity - - in the deoxygenated state. Figure 4. Three dimensional structure of Hb molecule based 5 on x-ray crystallography. The alpha chains (light gray) overlie the-beta chains (dark gray). The central cavity (site of DPG binding) is visible. The disc-like objects are heme groups, one in each globin chain. N and C represent the amino and carboxyl terminals of the alpha chains. Figure 5. The basic unit of hemoglobin is a long chain of amino acids so looped to provide a cleft that is occupied by 5 - the heme group. The globin chain consists of a helical arrangemept (A to H) linked by short non-helical chains of - amino ac2ds: For the beta chain,A2 is the site of substitu- tion for Hbs S and C; F8 is the proximal histidine position, which interacts with the iron in the heme group. of Hb molecule resulting in a diminution of the cavity and consequent loss of the DPG. In addition to alpa2 beta2, normal adult Hb also contains HbA2, designated as alpha2 delta 2 , composed of two alpha and two delta chains. This is a minor component of normal red 5 blood cells. There is a fraction in normal red blood cells known as H~A~ whose proportion is accentuated with aging of the Hb. This component is thought to be linked to glutahione molecule at the beta chain. It has the formula alpha2 beta 2 ' In addition to Hbs-A A and A adult Hb contains other 1, 2' 3 minor components which include one having the formula alpha2 X betabeta in which beta X chain has an NH2 terminal group at- tached to a hexose moiety. This type of Hb, the glycosylated hemoglobin, is of significance in diabetic patients and the monitoring of the HbA fraction has proven to be a valuable Ic diagnostic tool. A major hemoglobin component of intrauterine life is fetal hemoglobin, HbF, and trace amounts persist in adult life. HbF consists of two alpha and two gamma chains (alpha gamma 2 ) 2 and is made up of F and F2. While F is associated with two 1 1 gamma chains, F- apparently has no gamma chains since +-he N- 2 terminal end is blocked. Hemoglobin Gower I1 is an embryonic type and has no counter- part in adult life. Each globin chain is a long strand of amino acids, 141 in each alpha chain and 146 in each beta, - delta, or gamma chain. Each chain contains helical segments, seven in an alpha chain and eight in a beta chain, delineated by short non-coiled segments. The homology of the beta, gamma, and delta is unique,with the beta and delta chains differing by only 10 amino acids. Each globin chain is looped about itself so as to form a pocket which embodies the heme group, and this pocket is surrounded by essentially hydrophobic amino acids. The heme moiety is suspended within this cleft by a non-covalent link- age of the iron atom to the imidazole group of the proximal histidine (position 92 of the beta chain or position 87 of the alpha chain). The distal histidine has its imidazole group at position 63 of the beta chain or position 58 of the alpha chain which is continuous with the heme iron, but it is able to swing in and out of this position to permit the bind- ing and release of 02. Whether Hb is oxygenated or deoxygena- ted, the four iron atoms of the tetrameric hemoglobin molecule exist in a divalent state. The structure of a single heme group may be represented as a square planar complex with the four nitrogens of the polyphyrin rings at the angles as illus- - trated in Figure 4. The central iron atom is in a hexavalant coordination state analogous to the inorganic iron complex, ferrocyanide. Of the two remaining coordination bonds, one is closel-y asso- ciated with an imidazole residue from the specific globin chain to which the heme group is attached. The remaining bond is available for the reversible uptake of oxygen. No ligand is 6 known to occupy this latter site in deoxyhemoglobin. - Function of Hemoglobin Hemoglobin is uniquely adapted for the reversible uptake of oxygen within a critical 0 tension. The functional feature 2 imidazole 6 Figure 6. Idealized representation of heme molecule. is a chain-to-chain interaction whereby an alteration in spa- tial arrangement of one molecule to another facilitates the uptake or release of 0 2 ' The dissociation curve for normal blood hemoglobin is sigmoidal. This is a result of interac- tions between the four subunits comprising a hemoglobin mole- cule. These interactions are termed cooperativity. Dissociation of O2 from hemoglobin (deoxygenation) occurs in four distinct steps, each having a dissociation constant because of cooperativity changes that accompany each succes- sice release of 02. Values for each K are not established yet. K values are de- fined as equilibrium constants by - K = (Hb406) (02) units in 1 moles (Hb40,) - - The association constant is the reciprocal of K. The smaller the dissociation constant, the more tightly bound is the oxy- 6 gen and the more stable is the hemoglobin-oxygen complex. The converse is true for the association constant. In the saturated hemoglobin molecule all of the oxygen - are essentially equivalent; a decrease in the ambient oxygen tension may cause any of them to be released first. The release of the fourth O2 doesnot occur at physiologic conditions. The above sequence of events accounts for the sigmoidal shape of the normal oxygen dissociation curve as illustrated in Figure 7. The properties of the Hb molecule facilitate the loading and unloading of large quantities of oxygen over a physiologically critical range of p02. The total oxygen content of normal blood is about 20 mL/100 mL (v/v): the re- lease of 5 mL/100 mL is equivalent to releasing one oxygen molecule from a hemoglobin tetramer which culminates from a p02 of about 60 mmHg (point a to V). The release of a second O2 requires a further decrease of 15 mmHg in the p02 from 40 down to 25 mmHg. The release of the third oxygen molecule can then be effected by a decrease in the p02 of only 10 mmHg. Once the hemoglobin molecule has taken up a molecule of 02 at the heme site, and given up 2,3-diphosphoglycerate, the remaining three sites are oxygenated with a slight increment in p02. Conversely, a molecule with all four heme sites oxy- genated produces oxygen availability at three other sites. - This coordination of oxygen "tension" changes is dependent upon (1) the reversible binding of certain amino acids across the alpha beta contact point, allowing the alpha beta dimers 1 2 to flex back and forth as the Hb molecule is oxygenated and deoxygenated, and (2) the movement of DPG in and out of the central cavity-- DPG stabilizes the deoxygenated configura- tion. In vivo DPG concentration markedly affects the 02 disso- ciation curve of Hb as illustrated in Figure 7. As the con- - centration of 2,3-DPG increases, the dissociation curve shifts to the right manifesting diminished oxygen affinity for hemo- globin and elevated O2 delivery to the tissues. On the contrary, Figure 7. Normal oxyhemoglobin dissociation curve and curve 6 for the case of a 50% anemia. The delivery of 25% of the total oxygen content of fully oxygenated arterial blood (5 mL/100 mL blood requires a drop in pO of about 60 mmHg, from a to v). - 2 a low DPG concentration results in shifting the curve to the left. This indicates an elevated O2 affinity for hemoglobin, with a corresponding decrease in delivery to the tissues. This latter situation is problematic in blood banking, wherein blood preserved in acid-citrate dextrose solution shows pro- gressive diminution in DPG concentration. Changes in blood pH and p02 influence oxygen affinity for hemoglobin. For a decrease in blood pH or an increase in pC02, as in acidosis, there is a corresponding shift of the disso- ciation curve to the right. This phenomenon is a case of the classical acid Bohr effect. Conversely, a decrease in pC02, or an increase in blood pH, may be due to alkalosis. The re- sultant shifting of the curve to the left is termed the alka- line Bohr effect. Clinical Significance of Hemoglobinopathies Synthesis of hemoglobin is under genetic control and numer- -- ous hemoglobin variants, many capable of producing severe he- molytic anemias and even fatality, have been detected. Early detection of certain hemoglobinopathies have proven useful in the avoiding of serious medical problems. Counselinq-of indi- viduals or marital partners to determine the probability of having offspring with abnormal hemoglobins has resulted in broad-based screening programs. All inherited abnormal hemoglobinopathies fall into one of three categories: (a) inherited abnormalities of the struc- ture of one or more of the globin chains (b) inherited abnor- malities relating to the frequency of synthesis of one or more pO2 (mmHg) 5 Figure 8. The normal oxygen dissociation curve is sigmoidal. - - Changes in 2,3-DPG concentration markedly influence the posi- tion of the curve to the right, and the converse is true. The action of 2,3-DPG is a pertinent mechanism for controlling the rate of oxygen delivery to the tissues. of the globin chains, or (c) absence of the usual switch from (F) to adult (A) hemoglobin synthesis. The first type is a result of the substitution of one or more amino acids in a globin chain. A good example is the difference between hemoglobin S and hemoglobin A, where valine is substituted for glutamic acid in the sixth position. Num- erous other hemoglobinopathies belong to this class. The thalassemias fall into the second group and are the result of a genetic defect in the production rate of adult 3 hemoglobin. This reduction in the amount of hemoglobin A1 is due to a variety of alternations in the hemoglobin patterns which have shown the genetic significance of the thalassemias. Some thalassemias interact with beta-chain hemoglobin variants such as HbS, and are also associated with increased amounts of hemoglobin F and A2 due to the effective beta-chain production; these are designated as beta-thalassemia major. -- The heterozygous carrier states are termed beta-thalassemia minor. Beta-thalassemia major is usually severe. The Hb pattern for this homozygous thalassemia is indicative of a variable elevation in HbF with low, normal, or elevated level of hemoglobin A 2 ' Hemoglobin F level may vary from 40 to 90 percent. Clinical distinction of the heterozygous beta-thalassemia minor varies from moderately severe anemia to normal findings. The severe forms are rare and individuals having them are of - mediterranean nations. The sub-clinical forms of beta-thalas- semia minor are known to exist in many individuals. Hemoglobin F is slightly elevated from 2 to 6 percent in 50% of the cases, whereas HbA2 is elevated 3.5 to 7 percent. The balance is due to HbA1. The beta-thalassemias are charac- terized by persistent fetal hemoglobin production, elevation of hemoglobin A2 levels, and interaction with beta-chain structural hemoglobin variants. In some patients with a thalassemia blood semblance there is no elevation of hemoglobins A and F levels; however, there 2 are small amounts of hemoglobins H and Bart's in the cells. Hemoglobin H is a tetramer of normal beta chains (B4), while hemoglobin Bart's is a tetramer of gamma chains ( y4) . Evi- dence suggests that hemoglobins H and Bart's are probably caused by defective alpha-chain synthesis, and this class of thalassemia does not interact as in B-thalassemia. These in- teract with alpha-chain variants and are referred to as alpha- thalassemias. Delta thalassemias and beta-delta thalassemias 5 have also been documented. Interpretation of Hb Electrophoresis Hemoglobin electrophoresis is indispensible for the detec- of hemoglobinopathies. Hemoglobins are genetically controlled and the presence of abnormal hemoglobins is often associated with functional, physical and morphologic abnormalities in the erythrocyte as well as pathological manifestation such as hemolytic anemia. The normal pattern is characterized by an intense band of HbAl and if the concentration of hemolysate is high enough, a weak HbA2 band is demonstrated. HbAl may have a blurred faint band of HbA3 moving in front of it. Normal values are: Sickle Cell Trait Sickle cell trait is a heterozygous state with a distinct electrophoretic pattern of HbA SA 1 2' Hemoglobin values are HbA, 55-65%, HbS 35-45%, Hb F is less than 2%, Hb A2 is normal. This trait confers protection from falciparum malaria and is present in about 10 percent of all blacks in the United States. Blood smears are usually normal, but supravital pre- parations of fresh blood reveal sickling. Sickle Cell Anemia -. Sickle cell anemia is a homozygous state. The hemolysate consists of mostly HbS, about 95 percent, with variable amounts of HbF--usually less than 15 percent in the adult. No HbA is observed. On electrophoresis at alkaline pH, HbS migrates to the same position--HbS can be distinguished from other variants by citrate agar electrophoresis at pH 6.0-6.5. It can- as well be distinguished from HbD and HbG by a positive sickle cell test (Sickledex) . The major clinical manifestations include chronic hemo- lytic anemia, impairment of growth and development with in- creased susceptibility to infection, vasoocclusive "crisis" - and organ damage due to vasoocclusive events, leg ulcers, and renal dysfunction. This is the most common abnormal hemoglobin. Testina for Sickle Cell Disease Sickle cell is usually tested by two methods in the lab- oratory. The first method, the oxygen deprivation method, demonstrates the presence of HbS in erythrocytes. In this technique, a cover slip is used to cover a drop of blood, and the frequency with which the cells sickle is observed. The other test is the solubility test for sickle cell screening. Here whole blood is placed in a hypertonic phos- phate buffer solution to release the sickle Hb from the red cells when saponin is added. The solution turns cloudy due to the formation of insoluble crystals which are unique to HbS as well as HbC ,C , and S . Hemoglobins A, Harlem Zingurchor Travis C, D, F, G, I, J, and 0 Arab yield negative results. Conse- 15 quently, electrophoresis is a better technique for screening. Sickle Cell Disease Sickle cell disease is a heterozygous state with approg- mately equal amounts of HbS and HbC. This form of sickle cell anemia is the most common genetic variant though clinically milder than sickle cell anemia. It has fewer complications - - when it occurs in childhood. Sometimes adults are asympto- matic only to be diagnosed when a routine smear exposes target cells, rare irreversibly sickled cells, and a relative measure of reticulo cytosis (3 to 10 percent). HbC Disease HbC disease is a homozygous state; the hemolysate contains more HbC and variable amounts of HbF. HbAl is lacking. On electrophoresis at alkaline pH, HbC migrates slowly to the same position as HbA2, HbE and HbO. It can be sepa- rated from HbA by column chromatography, and from other Hb 2 variants by acid pH electrophoresis on citrate agar. The homozygous state produces hemolytic anemia that is mild to moderate, with hemoglobin levels from 8 to 12 gram/ 100 mL. The majority of red cells on peripheral blood smear are target cells. HbC cells form intracellular crystals when suspended in a hypertonic solution. Sickle Cell HbDpunjab Disease HbDpunjab hemoglobinopathy causes a severe hemolytic ane- mia, though it is milder than HbSS disease. It is inherited as a double abnormal homozygous trait, one for the sickle hemo- globin trait and the other, a gene for HbDpunjab. It can be distinguished from HbS by electrophoresis on citrate agar at -- acid pH. HbDpUnjab is suspected in heterozygotes when a nega- tive sickle test occurs, yet an Hb component is evident at the S position. HbE Disease HbE disease is due to the homozygous inheritance of the gene for HbE. HbE is the third most prevalent abnormal hemo- globin in the world and occurs primarily amongst orientals. - - HbEE is associated with microcytosis, target cell formation, and a mild hemolytic anemia. At acid pH in agar electrophoresis, HbE migrates with HbA and not with HbO or HbC. Whole blood O2 dissociation curves demonstrate a slight shift to the right. The heterozygous state, HbAE or HbE trait, is commonly encountered and has not shown any hematologic abnormality. Thalassemia Minor The hemolysate consists of HbAl with HbA2 elevated above 4 percent, often with a slight increase in HbF. The erythro- cytes exhibit hypochromia and microcytosis in confirmatory hematologic diagnosis. HbS-Beta Thalassemia The typical pattern is that of HbA1. Clinical and hema- tologic abnormalities are mild. HbC-Beta Thalassemia The hemolysate contains more HbC than HbAl with variable -- amounts of HbF. Typical values are: HbC 65-95 percent, HbA about 20 percent. HbA2 is completely lacking. This pattern is identical to HbC diseases. - - HbC-beta thalassemia is a hematologic disorder with little clinical evidence of disease. The mean corpuscular hemoglobin is reduced, with hypochromic target cells, fragmented red cells, and microspherocytes seen on peripheral blood smears. Thalassemia Major - The clinical diagnosis is uniquely that of HbF electro- phoretic pattern as high as 98 percent with variable amounts of HbA1, or HbAl may be completely absent. The H~A~ value is usually normal. Alpha Thalassemia The clinical condition is characterized by marked micro- cytosis and hypochromia of the red cell associated with mild anemia and erythrocytosis; HbA2 andHbF levels are normal or low. Iron deficiency must be ruled out in alpha thalassemia. A variant of homozygous beta thalassemia is thalassemia intermedia, a less severe impairment in beta-globin synthesis, with fewer alpha-chains inclusion. Individuals with this dis- order maintain a hemoglobin level of 6-10 g/dL. This condi- tion ranges from a very mild disease to a more severe disease state. The electrophoresis pattern manifests hemoglobin values- of 20 to 100 percent HbF, up to 7 percent HbA2 and 0 to 80 percent HbA, depending on the genotype of the patient. Iron - overload is a complication because of the highly accelerated but ineffective erythropoiesis. There is an increase in plasma iron turnover and increased gastrointestinal iron absorption. - - Unstable Hemoglobin Disease Unstable hemoglobin disease is characterized by the pres- ence of unstable hemoglobins. The latter are structural var- iants of HbA; these undergo denaturation within the red cell - which results in irreversible precipitation and the formation of insoluble inclusion (Heinz bodies). Anemias due to unstable hemoglobins are designated as unstablehemoglobin hemolytic anemias. Unstable hemoglobins are inherited as an autosomal domi- nant trait. Of the 70 known Hb mutants that are associated with unstable hemoglobin hemolytic anemia, most are of beta- chain origin; only 8 alpha-chain mutants are known. Many of the substitutions associated with this condition are neutral, and their electrophoretic migration are indicative of HbA. The Heinz bodies are selectively removed in the spleen. Because of the low gene frequency, a homozygous state is in- frequently encountered in life. However, Heinz body anemia is variable, it may be quite mild or moderately severe depending on the variant. Most variants exhibit some degree of varia- bility in oxygen binding affinity; however, inclinations to normal binding exists. Variants exhibiting decreased oxygen affinity tend to manifest lower hemoglobin levels. Hemoglobinopathies with Abnormal Oxygen Hemoglobinopathies with abnormaloxygen prevail as an ery- -- throcytosis in patients with elevated O2 affinity and a reduc- tion in O2 unloading, and as an anemia in patients with de- creased oxygen affinity (Hh Kansas). However, clinical mani- festations are-minor; cyanosis might occur in variantswith decreased oxygen af finity . r15 Some clinically important hemoglobinopathies are illus- trated in Table 1. r: 0 -rl +' C4 k 0 [I) a rd rl rd E 0 a c r6 rn c, d H X E 2 0 Z H a 0 4 w 0 3 X E 3 P: 0 2 H * 4 4 4 U H Z H 4 U 2 0 m rl W 4 a 3 0 U c 0 -4 c, 3 -lJ -rl z+' rn a 7 m r: d 0 -rl .rl Qc, Orb rl r: Do- 0 -4 5: ma C 0 -rl c, rd rlc, rd rn U a, -rl 4-1 r: -4 -rl r: rl rd U z Clinical Hemoglobin Manifestation - Designation Substitution Comments Erythrocytosis Chesapeake alpha arg+leu This group of abnormal 92 J Capetown Malmo Ypsilanti 92 alpha armlu 97 beta his-+gin 99 beta as~tyr globins is characterized by marked leftward dis- placement of the 0 dis- 2 Kempsey 99 beta aswasn sociation curve. Yakima betag9asFhis Rainier Bethesda 145 beta tyrdhis Statement of Problem The study of hemoglobin variants and hemoglobinopathies have been employed in clinical diagnostic settings mainly by electrophoretic techniques. Electrophoresis as defined, is the separation of charged macromolecules by means of an applied potential. The technique of electrophoresis has been success- fully utilized to fragment biologic macromolecules such as serum proteins, enzymes, and hemoglobin. The prime goals in any electrophoresis procedure is the detection and quantitation of the migrated fractions. In tbe normal protein or hemoglobin electrophoresis, a stain, usually Ponceau S is used to enhance the visualization of the resolved fragments. The intense red bands produced correspond to the separated components, and these can be quantitated by means of a densitometer. However, the detection is resolution limited. A distinct resolution of the -. separated bands will obviously yield a more accurate detection 5 result . - A macromolecule such as hemoglobin is associated with numerous variant forms. Each is genetically controlled as well as connected with functional, physical, and morphologic - - abnormalities in the erythrocyte, resulting in pathological manifestations. Each variant form has a distinct elect;ophore- tic mobility, but numerous diagnostically significant variants have similar electrophoretic mobilities. The various mobilities are mainly due to the quantity of the charge/mass of each at a- - particular pH. In the identification of hemoglobinopathies, two basic electrophoretic techniques are usually employed. Electrophoretic separation of hemoglobins on cellulose acetate at pH 8.6 iden- tifies all pathological variants including hemoglobin S. There is increased mobility towards the anode as illustrated in Figure 9 below. Poor resolution is a problem in separating for HbF, HbA, HbD, HbG, HbL, and HbS which all have about the same mo- bility. When agar gel is used to separate hemoglobins under typical electrophoreticconditions HbA and HbF are better resolved, but the cluster of variants A, A2, E, G and D have the same mobil- ity. However, the concentration of Hb applied to the gel is directly proportional to the mobility. Large concentrations of HbA will result in increased electrophoretic mobility. Care is therefore cardinal in adjusting the Hb concentration of samples. Equal concentration is needed for better interpreta- tion. The quest for a better resolution technique has been diffi- cult. It is proposed that a simpler, more precise procedure -- that will combine modern electrophoresis with the magnetic property of hemoglobin should be investigated. The advantage of this technique is believed to be the use of an applied mag- netic field to- fix the hemoglobin molecule while the -applied electric field moves the charged particles in an anodic direc- tion. Unlike a conventional electrophoresis, a better resolu- tion could be achieved in a relatively shorter time. The apparent advantage in this technique is that the need for buffer change is eliminated. Only one buffer type is required, and time optimization will be achieved if the technique proves successful. A*' C, 0, D G S F A I i I/ 8 .igin .ic anhydrase Figure 9. Hemoglobin electrophoresis on cellulose acetate at pH 8.6, showing the relative positions of various h-emoglo- 5 bins. Hemoglobin F is poorly resolved. Background for the Magneto-electrophoresis System A magnetic effect study based on the Gouy technique was carried out using a micro-electrophoretic cell. The micro- cell consists of rectangular plates of plexiglass which have been glued together after wells have been milled into the upper half. A study of the effect of an applied magnetic field alone on a sample of hemolyzed red blood cell containing hemoglobin showed that the attraction of the magnetic field was ineffec- tive in causing the sample to migrate. The illustration of the effect is shown in Figure 10a. Supre Heme buffer was used in this study. When the same study was performed on a 2 mL blood sample which had been treated with 5 grams of sodium thiosulfate, there was significant movement from the point of application - as illustrated in Figure lob. Capillary action of the buffer solution had no significant effect on the mobility of the bl-d sample. Theoretical Basis for a Coupled Magneto-electrophoresis Enhancement Technique - - In this study the induced magnetization and the suscepti- bility of the substance to magnetization are utilized to attempt to enhance the resolution of separated macromolecules. Studies have shown that a hemoglobin molecule solution - that has been reduced to ferrohemoglobin by means of sodium thiosulfate is paramagnetic, whereas oxyhemoglobin is diamag- 20 netic. These findings are all based on the Gouy technique Cellulose acetate plate Origin Blood sample Figure 10a. Effect of applied magnetic field from a 14.7 kilogauss NMR magnet for a two hour duration. Origin Band of ferrohgmoglobin Figure lob. The effect of the same magnetic field on a sample of ferrohemoglobin in an electrophoretic cell. which is relied on in this study. Under the influence of a magnetic field, a diamagnetic molecule will have its bonding electrons in a plane perpendicular to the magnetic field. The result of this motion is the development of a secondary field which opposes the primary field. The resultant is smaller since the nucleus has been shielded from the applied field. Illustra- tion of this is in Figure 11. The intensity of magnetization induced thus is less than that produced in vacuum. A paramag- netic substance such as ferrohemoglobin produces the opposite effect . The result is a repulsion between the substance and the applied field. The susceptibility is therefore negative accord- ing to the equation M =-X H m where M is the magnetization, H is the magnetic field strength^, and Xm is the magnetic susceptibility. Paramagnetic intensity due to the induced magnetization- is greater in the substance than that of the applied field in vacuum. Attraction of the material towards the applied field is characteristic of paramagnetics, and the magnetic sqscepti- bility Xm is always positive. Though all paramagnetic com- pounds have a built-in diamagnetism as a universal property, the magnitude of paramagnetism usually overwhelms the feeble, opposing diamagnetism. Paramagnetic susceptibility is independent of the applied field but is usually inversely related to temperature according to the equation r Circulating electrons field 11 Applied THO field 2 Figure 11. Diamagnetic shielding of a nucleus. where um is the magnetic dipole moment, is the molecular magnetizability. From (7) above magnetism has a contribution 2 proportional to the term um/3KT. Magnetic susceptibility is thus related to the magnetizability, the dipole moment, and 21 temperature. To reiterate, this is usually determined by the Gouy balance. It is apparent that a permanent magnetic moment of a macro- molecule that is paramagnetic can be employed to position the induced moments at a more or less stationary locus. An applied potential from an electrophoretic power source can be used to separate different hemoglobin types in an anodic direction. The differing intensities of magnetic susceptibilities would gene- rate well defined rates of mobilities unique to the variants. In essence, the induced magnetization will retard the migration of mutant Hb, which could result in a better resolution of these variants. CHAPTER I1 LITERATURE REVIEW Methods Hemoglobin variants have been detected, studied and eval- uated by several techniques in the literature, but the tech- nique of diagnostic important in the laboratory is electro- phoresis. It is the technique of choice in most hemoglobino- pathy screening programs. Currently the novel technique of isoelectrofocusing has been employed. These valuable techniques are discussed below. Electrophoretic Techniques The identification of various hemoglobin variants is carried out by electrophoresis at various pH levels using a variety of support media. -. The most commonly used diagnotic procedures for hemoglo- binopathy evaluation are electrophoresis on cellulose acetate at pH 8.6 and electrophoresis in agar gel at pH 6.2. Other support media occasionally used to identify abnormal hemoglo- bin variants include potato starch gel, paper, agarose, and polyacrylamide gel. Because cellulose acetate provides sharp resolution of hemoglobin bands in a relatively short time, it is readily available, it permits staining and clearing, and it allows densitometric quantitation; it is the support medium of choice. Cellulose acetate was first discovered and employed in protein fractionation by Kohn in 1957. He established that cellulose acetate, as a support medium, was amongst all three media in use then, paper (Durum, 19501, agar gel (Gordon, 1949), most similar to Tiselius' moving boundary method. Since then cellulose acetate has proven to be a valuable diagnostic tool for routine investigation superseding paper medium. Commer- cial kits of cellulose acetate have further enhanced its avail- ability, and kits commonly used include Microzone (Beckman In- struments, Inc., Fullerton, California 92634), Sepratek (Gel- man Instrument Co., Ann Arbor, Michigan 48106), and Zip Zone (Helena Laboratories Corp., Beaumont, Texas 77704). These have been evaluated and proven satisfactory with the Zip Zone kit providing the best overall performance in the separation of 7,8 hemoglobin variants. Routine protein fractionation by agar gel was first re- 9 ported by Gordon. It has gained use, parallel to cellulose acetate, in the separation of some hemoglobin variants. Agar gel has superseded paper as a support medium for zone electro- -- phoresis. It has a more homogenous structure and provides faster and sharper resolution, and may be dried to a transparent thin film. This technique is however, concentration limited 9 -- as far as the mobility of hemoglobin is concerned. Ion-Exchanae Chromatoura~hv Since the inception of ion exchange chromatography, num- erous protein oligomers have been fractionated using various - ion-exchange materials. Of importance in hemoglobin screening programs is diethylaminoethyl cellulose (DEAE cellulose), whose backbone is a polysaccharide; DEAE-cellulose is a weak anion- exchanger which may be bound to protein. Eventual elution of the protein is accomplished by varying the pH or salt concen- tration. This technique has been employed in evaluating individuals with Beta-Thalassemia. Efremov et a1 had reported a simplified protocol utilizing ultragranular DEAE-cellulose and Pasteur. 10,ll pipets as columns. Several alternative methods have been discussed in the literature, but these suffer from considerable imprecision. Because ion-exchange column chromatography is primarily employed for the diagnosis of beta-thalassemia, it is restrictive in scope and is less valuable a tool for routine investigation of hemoglobinopathies. A recent advance in hemoglobin analysis has been put for- ward by Hannash et al. This technique incorporates anionic exchange material in the use of DEAE-cellulose with high per: formance liquid chromatography. The procedure has been used to separate Hbs A2, S, F. Details of this technique, anion-- 12 exchange HPLC, are discussed in the literature. Isoelectrofocusing Technique - - Iso-electrofocusing in polyacrylamide gel is gradually gaining acceptance in clinical investigations especially in the fractionation and characterization of proteins including hemoglobin variants. Iso-electrofocusing was first employed 13 for hemoglobin variants by Righetti, P. et a1 (1971). Since then several reports have been documented involving iso-elec- trofocusing in protein separation. It is a simple technique which basically employs the same apparatus as used for electro- phoresis but posesses a greater resolving ability than elec- trophoresis. Other advantages include optimal resolution, and it does not require application of the sample in a narrow zone as in conventional electrophoretic procedure. The concentra- ting property of isolectrofocusing provides a much lower de- tection limit than conventional electrophoresis, and proteins differing slightly in pI values can be resolved. Also the presence of polyacrylamide gel as a support me- dium reduces the effect of electroendosmosis to a minimum for the gel itself is devoid of bound charged groups. Moreover, polyacrylamide gel is a high capacity stabilizer, which is able 14 to withstand the high voltage applied in iso-electrofocusing. Basis of Electrofocusina Every electrophoretic separation of proteins is due to the fact that proteins have a net charge that is dependent on the pH of its surroundings, and this net charge equals the sum of -- the positive and the negative charges on the surface of the pro- tein. A state of continuous change in the net charge exists as the protein traverses a gradient. At a certain well defined pH, the net charge on the protein becomes zero. This-point-is termed the iso-electric point (PI). Electrofocusing employs a stationary and stable pH gra- dient that is incremental in a cathodic direction. If a pro- tein is placed at a pH higher than its PI, the net charge will - be negative and such a protein will be anodic in its migration in an applied electric field. Gradual increases in pH result along the gradient until it reaches its pI where the net charge will be zero and movement will cease. On the other hand, plat- ing such a protein at a pH lower than its pI results in its acquisition of a net positive charge and migration will be cathodic. Likewise, the protein will be static at its pI po- sition. Thus, regardless of where a protein sample is intro- duced into the pH gradient, each one will end up as a narrow zone at a specific pI position unique to the protein. Diffu- sion effects are eliminated by the applied electric field. Separation and Determination As mentioned above, isoelectrofocusing is a special type of electrophoresis. A pH gradient is established along the support medium (polyacrylamide gel), and each protein in the sample migrates to the point where the pH equals the PI. The pH gradient is caused by ampholytes added to the medium, which migrate in the electrophoretic current. Arnpholytes are mix-- tures of molecules carrying positive and negative charges. Commercial ampholytes consist of a mixture of arginine, as-- partic acid, and glumatic acid with synthetic polyamino-poly- carboxylic acids. One end of the gel is immersed in acid and the other in base. Hemoglobin moves within the gel until it - - becomes concentrated at its PI. The relative order of hemo- globins seen after isoelectrofocusing, beginning at the acidic 5,15 anodal end is HbA, HbS, and HbC. Isoelectrofocusing can be done in columns or thin-layer polyacrylamide gels. Thin-layer plates are available comer-- cially as ampholine PAG plate kit, pH 3.5 to 9.5. The LKB 2117 Multiphor concept, a recent advance, has proven successful in hemoglobinopathy screening. Hemoglobin variants with small charge difference, such as HbMalmo (beta 97 his--+ gln) , not detectable by conventional electrophoresis, have been sepa- rated from HbA 1' The more recent application, high voltage electrofocusing, which optimizes time schedule and provides sharper zones, and visual examination of the concentrate, could 16 be used. Electrophoretic Technique Separation and Determination In cellulose acetate electrophoresis, separation and iden- tification of hemoglobinopathies are carried out under alkaline condition. Usually Tris-EDTA-boric acid buffer, pH 8.6 at 25 degrees Centigrade is used and electrophoresis is performed at 450V for 30 minutes. Under these conditions, hemoglobins mi- grate according to their net charge in an anodic direction. The relative orders are HbC , (A2 , E and OA) ; HbN, HbS (D and. G); HbF, HbA and HbK; and HbH, HbI and HbJ. The problem of misinterpretation exists for corresponding mobilities of dif-£- erent variants. Dr. Rose Schneider has introduced a simplified technique for separating globin chains and this can be used to confirm - - other hemoglobins. In this procedure, erythrocyte hemolysate is electrophoresed on cellulose acetate in urea-2-mercaptoe- than01 buffers in the presence of additional 2-mercaptoethanol. The latter severs heme from globin, whereas the urea cleaves the alpha and beta chains; these migrate on cellulose acetate - according to their net charges. The mobilities are dependent upon the pH and buffer composition. This technique is very simple and provides excellent resolution of the globin chains in alkaline as well as acidic buffers, and it requires a small 17-19 amount of hemolysate. The Helena Laboratories electrophoresis procedure has now become the world's model for screening programs. It utilizes Supre Heme alkaline buffer and is capable of detecting hemo- globins S and C and estimating elevated amounts of hemoglo- bins A2 and F. Other hemoglobins such as D move in the same area as S and hemoglobins such as 0 and E have the same mobil- ity as C under alkaline conditions. Nevertheless, the Helena protocol has proven to be the definitive determination for all hemoglobinopathies. It is fast, easy and has the added advan- tage of cost effectiveness. Helena Laboratories has also developed an acidic buffer procedure which distinguishes hemoglobins S and C using citrate agar as the support medium. In the Helena electrophoresis protocol, a drop of packed red blood cells is hemolyzed with hemolyzing reagent, applied -- to a Titan I11 cellulose acetate plate, and electrophoresed in Supre Heme buffer for 25 minutes at 350V. The Supre Heme buffer is reconstituted by dilution to 980 mL using deionized water. This method alleviates the problem of artifacts production on cellulose acetate. The patterns may be interpreted immediately after electrophoresis, or carried through an intensifying stain, usually Ponceau Sf for permanent storage or enhanced reada- bility or quantitation. Quantitation may be carried out by - means of a densitometer and is necessary for the identifica- tion of thalassemia minor. Controls are usually run on the same plate as each group of samples for screening. CHAPTER 111 Materials and ADDaratus Materials needed for the construction of the mini elec- trophoresis cell include a 1/4 plexiglass sheet, manufactured by Baker Plastics, Inc., Duro Epoxie Resin and Hardware, pla- tinum wire strips of about 55 micrometer gauge size and Ersin Multicore Econo real solders (0.040" diameter, 60/40V and ER 220). Other materials are 8/32 inch tap for milling threads for screws, a Drill Press Motor tool model 210, Panavise sol- dering pen #338G, and alpha colored wires from Alpha Wire Corporation #22-7/30 PVC wire of type B Mil-w-16878D. All materials were obtained from the Chemistry Department of Youngs- town State University. Other materials needed for the experiment are a Fisher - Scientific Kodak Timer by Eastman Kodak Company, disposable pipets, Whatman Filter papers (~shless type #42), permanent-_ broad tip marker, Sargent magnetic stirrer for proper stirr- ing of the buffer solution during preparation, the Helena Lab- oratories micro-dispenser, Zip Zone applicatorrand a pair of tongs - - for plate handling. In carrying out this study, Titan IIIH 1" by 3" cellulose acetate plates from Helena Laboratories were used. The blood specimens used were obtained from Saint Elizabeth Hospital Medical Center. The blood was hemolyzed by adding hemolysate - - reagent from Helena ~aboratories and preserved in the refriger- ator until needed for use. The buffer used in this study (Tris-EDTA-Boric Acid, pH 8.2 to 8.6) was obtained from Helena Laboratories of Beau- mont, Texas. The buffer was reconstituted to specification using the required amount of deionized water as outlined in Chapter IV. The staining dye, Ponceau S, used for the affin- ity coloration of the hemoglobin was prepared from capsules. Ponceau S has a very long shelf life. It was properly capped when not in use. All water used in this study, either for buffer recon- struction or solution preparation, was deionized water. Apparatus A Heath Schlumberger Regulated Power Supply model #SP- 17A capable of producing 400V of Direct Current electricity was available for this study. A magnet from the Chemistry Department's DA60 Nuclear Magnetic Resonance Spectrometer, capable of producing 14.7 kilogauss field strength, was used. The radio frequency components of the NMR were not involved in -- this study. The micro-electrophoresis cells were constructed in the machine shop at Youngstown State University. The metal sander, drill press, band saw and vertical mill located in B-27 were useful in putting the cells together. CHAPTER IV Experimental Cell Design and Construction The electrophoresis cell was drawn to scale and cut into dimension by means of a band saw. The cut specimen of plexi- glass was properly smoothed out by use of a sander. A vertical mill was used to engrave the desired wells according to the design in Figure 12 and 13. Once the main body of the cell was completed, the components including the lid were soaked overnight in tap water to remove the glued paper label, which indicates the plexiglass manufactur- er. Then the cells were dried by means of a lintless cloth. A drill press was used to make holes for the insertion of the platinum electrodes. The platinum wires were cut and put in place by means of Epoxy glue and allowed to dry. Threads for screws were made by means of a 8/32 inch Tap. Thereafter, the wires were soldered to the colored alpha wires for positive (red) and negative (black) leads; a soldering iron was used for this purpose. The cell was tested for proper functioning. A vertical span electrophoretic cell as well as a hori- zontal one was constructed for the study. Illustrations are in Figure 12 and 13. Preparation of Solutions The Supre Heme buffer (~ris-EDTA-Boric Acid, pH 8.2 - 8.6) - from Helena Laboratories, Beaumont, Texas was reconstituted , (G- - Screw hole - Platinum wire - Plexiglass - Well - - Figure 12. Mini electrophoretic cell for coupled magneto- electrophoresis (vertical span). screw Figure 13. Mini electrophoretic cell for coupled magreto- electrophoresis (horizontal span) . according to manufacturers instructions. Each packet was re- constituted in 980mL of deionized water. The buffer solution is stable for a month at temperature of 2 to 80'~. Conse- quently, when not in use the buffer solution was stored in the refrigerator. The 5 percent acetic acid solution for destaining was freshly prepared by mixing 5mL of glacial acetic acid with 95 mL of deionized water. Ponceau S stain was prepared by dissolving 20g Ponceau S dye in lOOmL of dilute aqeous trichloroacetic acid (30g/ 100mL). Deionized water was used for the rinsing of destained cellulose acetate plates before visual examination. Method The protocol used in this study is that of the Helena Laboratories. The steps are slightly modified to adapt to the micro-cells. The basic directions are outlined below: - 1. The cellulose acetate plate was cut into strips for the minielectrophoretic cells. 2. Some of the reconstituted buffer was poured into a plastic boat from which some was retrieved to--soak the cotton in each of the outer compartments. A disposa- ble micro-pipet was used to drain the cotton swabs. 3. Two cut strips of Zip Zone Chamber wicks were soaked in the buffer and then draped over each support bridge so that contact wasmade with the buffer. The covered chamberwas now ready for electrophoresis. 4. The leads of the cell are connected to the Heath Power supply. By means of a switch then regulationwas kept at standby. At the same time the marked acetate strip was soaked in the buffer for 5 minutes prior to use. 5. The hemolysate containing globin chains under study was properly shaken; it was allowed to stand for a while and swirled just prior to use. 6. Using the microdispenser 5 microliter of the hemo- lysate was fed into the well plate. The maximum number of well plates filled was three, but initially one Sam- ple well plate was used. The hemolysate was added as a drop into the well plate. 7. The sample applicator tips are cleaned in deionized water and dry blotted prior to use. The applicator was loaded by depressing the tips into sample wells several times. The first loading was used to prime the applicator and hence was applied to a blotter pad. The second and more uniform loading was used. 8. The wetted cellulose acetate plate was removed from the buffer and firm blotted once. With the cellulos~ acetate side up, the applicator was depressed about 10 millimeters from the cathodic base. Additional superimposed application may be necessary for-.the en- hancement of minor fractions. 9. The strip was quickly placed over the cell with the acetate side down. The application site was nearest the cathode. The lid of the mini-cell was put in place and the voltage was turned on to 350V. The stop-clock was set for 2 minutes. 10. At the end of the time, power was cut off and the strip was removed from staining. 11. Thereafter, the stripwas stained for 3 minutes in Ponceau S. 12. The stained stripwas destained in three successive washes of 5 percent acetic acid for 2 minutes each or until the background turned white. The strip was now dried and inspected, the strip can be stored away for permanent record. The electrophoresis study was carried out in two phases, one in the form of a conventional electrophoresis as described above. The other was the coupling of the applied field strength of the NMR magnet. Once step #9 was reached, the prepared Sam- ple and cell are placed within the two faces of the magnets as illustrated in Figure 14, prior to turning on the power supply. All other steps remained the same. By comparison of the two runs, the effect of the magnetic susceptibility on the resolu- -. tion of the hemolysate was determined. Electrophoresis under the influence of the magnetic field was carried out in the X,Y, and Z -- directions. The polarity of the mini-cell was reversed to in- vestigate which direction, in relation to the polarity of the cell, was more favorable and reproducible. Several determina- - - tions were performed on each of these parameters mentioned above. support b Power Supply (40 d* Figure 14. The set-up for coupled magneto-electrophoresis. I t CHAPTER V Results and Discussion The results of this study are based on time-scheduled determinations for 2 minutes. The hemolysate migrated proper- ly at 25'~ and 350V without the presence or appearance of any artifacts. When the hemolysate was electrophoresed within the poles of a magnetic field in the X, Y, Z directional axes, the out- come was distinctly different for each run. In the Z-axis, the resolution was poor due to the clustering of the bands along an aligned angle. In the X-axis, the resolution produced was only slightly improved when compared to the conventional runs. Comparatively, the migration path length for both the X-axis and the conventional runs were approximately the same. However, electrophoresis performed in the Y-axis produced some distinct and noticeable differences when compared to the con- - - ventional electrophoresis technique. First, there was a splitting of the separated bands into two distinct peaks with intense coloration on staining with - - Ponceau S. he shades of color intensity of the two peaks correspond to HbA for the more intense band while the less 1 intense one corresponded the HbA2 fraction. Next, on comparing the migration distances of the conventional and magneto-elec- trophoresis runs, a striking result was that mobility distances - for magneto-electrophoresis diminished by about half the length for the conventional runs. Equal distances were obtained when magneto-electrophoresis was performed at a time schedule nearly 4 minutes instead of 2. The set up for the Y-axis runs is illustrated in Figure 14. When the polarity of the applied electric field was reversed, the results were similar to the conventional electrophoresis run. Magneto-electrophoresis runs had results (for Y-direc- tional axis) which are due to the coupled effect of the mag- netic susceptibility of the hemolysate and the applied voltage. Diamagnetic effect of the heme or globin chains produced a retarding force in the applied magnetic field. Thus, the com- bined effect was a greater separation of hemoglobin variant due to the magnetic field. This technique results in an en- hanced separation of hemoglobin A2 from HbA. This is shown in Figure 15. Further efforts will employ other variant specimens. The clustering effect produced in the Z-axis may be due to the multi-nucleate nature of the hemoglobin molecule. Para- magnetic susceptibility is well pronounced only in mononucle- ated molecules when subjected to a magnetic field in the Z- 20,21 - direction. In the X-axis, the applied magnetic field is ineffective since the electrophoresed sample appeared to be enveloped in the centre of the field. The circulating secondary fkld either hampered, or barely improved the resolution. Many runs in the X-axis produced diffused bands as well. Conclusions Based on this study, it is evident that the combination - of electrophoresis with an applied magnetic field is a useful technique for the separation and evaluation of hemoglobinopathies. Figure 15. Illustration of the effect of magneto-electrophor- esis on a normal Hb hemolysate. (a) conventional electrophor- esis of hemolysate, (b) the same hemolysate after magneto- electrophoretic separation. The time schedules for the same migration distance as shown above: 2 minutes for a, %-minutes for b. This technique produced more distinct band resolution over the conventional electrophoresis; it is relatively easy to perform and may provide time optimization for multiple samples, if a larger cell is employed. The effective net charge on mutant globin chains can be very prominent in establishing the various magneto-electrophoresis loci, since the migration path length happens to be shorter than the conventional electrophoresis. The net charge will either enhance or retard the magneto-electrophoretic migration, which will be related to the effective globin chain's magnetic suscep- tibility. One problem is that of the magnetic field strength; the mag- netic field used in this preliminary study is small (14.7 kilo- gauss). It is not high enough to penetrate each globin mass and demonstrate its effect on the iron (11) molecule. Each globin mass has one iron (11) molecule. The massive arrangement of - peptides partially shields the iron atom from the applied mag- netic field. For better effects, a magnetic field strength oT 20 about 25.3 kilogauss has been recommended in the literature. In addition to the above assertions, it was conclusively proven that paramagnetic materials such as ferrohemogl~bin do migrate substantially when placed in an applied magnetic field. In this study, hematin (ferrohemoglobin) was shown to migrate substantially, about 2.0 cm, in a cellulose acetate electro- phoresis cell placed in a magnetic field. No electric field voltage was involved. On the other hand, normal Hb that has - been treated with sodium thiosulfate migrated only slightly against gravity (about 0.40 cm) confirming that normal Hb is diamagnetic. It is hoped that in the future other graduate students will undertake the evaluation of the magneto-electrophoretic effect on some of the known globin mutants or variants; this will eventually culminate into the establishment of the diff- erent loci for these hemoglobinopathies. APPENDIX A The Gouy Technique The Gouy technique is often used to measure magnetic susceptibilities, and the instrument consists of a sensitive balance from which hangs the sample in the form of a narrow cylindrical tube as in Figure 15. The sample hangs between the poles of a magnet. If the sample is paramagnetic, its energy is apparently less within the magnetic field; hence, there is a drawing force into the field. Contrariwise, a diamagnetic substance has a lower ener- gy outside the field, and so there is a repelling force out of the field. This force is proportional to the susceptibility. Determining the balance point allows X, to be determined. The instrument is usually calibrated against a sample of known sus- 20 ceptibility . Figure 16. Gouy balance arrangement for measuring magnetic 21 - - susceptibility: APPENDIX B The Modified Gouy Technique The modified Gouy technique by Quinke is mainly applica- ble to liquids and solutions. In this version, the magnetic force acting on the capillary sample is measured in terms of the hydrostatic pressure. By experimentation, the change in capillary column,Ah, with the field on and off is determined by means of a cathetometer. Paramagnetic liquids are known to show the greatest in- crease in height; diamagnetic liquids show a decrease. Appli- ed field of 25 x lo3 Gauss are recommended for use, and results are comparable to the Gouy method. Since solutions exposed to air consist of a dynamic equi- librium mixture of air and its vapor, the relationship could be expressed by equation (8) wherepandlare densities of the liquid and the gas above the liquid respectively; k is the volume susceptibility oT the liquid and k is the volume susceptibility of the gas above the 0 meniscus; H is the applied field, and g is the acceleration due to gravity. The hydrostatic pressure, g (p-k )Ah is counterbalanced 2 - by the field strength, 1/2H (k-k ) to yield equation (8) above. 0 By rearrangement, the susceptibility per gram, X, is de- fined by equation (9) where x and are the gram susceptibility and density of gas 0 0 above the liquid. However, the term x0,& is negligible when compared to the larger diameter reservior to the capillary in addition to the small gas susceptibility, xo. Thus, a simpler definition is given by X =2gAh (10) F12 measures the liquid or solution susceptibility. Thus, suscept- ibility measurements are independent of density and tempera- ture. Under identical conditions, a reference sample is treated in like manner as the sample investigated. Then the relation holds; x and x are the susceptibilities of sample and refer- s r ence respectively. Illustration of the Quinke balance is shown in Figure 17. Reservior Capillary Figure 17. Principle of the Quinke balance based on Gouy 20 technique. 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