Electrophoresis R. D. Strickland, Research Service, Veterans Administration Hospital, Albuquerque, N. M.
I
is now used routinely in so many biological investigations that the subject matter of its literature parallels and is supplementary to that of simple electrophoresis. For this reason, this review will refer to immunoelectrophoretic findings under appropriate headings along with reports of work done by other electrophoretic techniques. The alternative of treating immunoelectrophoresis as a special category would require virtually duplicating in form and scope the biological portion of the review. It should be remembered that the goals of electrophoresis and immunoelectrophoresis differ. Electrophoresis is used to isolate the components of a mixture either as a preparative measure or for analytical purposes; immunoelectrophoresis accomplishes neither of these ends, but is used to detect as many as possible of the antigenic substances in a sample. The excellent resolving power of immunoelectrophoresis is achieved by supplementing the discriminatory effect of electromigration with those of differential diffusion and immunological specificity. The precipitation arcs in immunoelectrophoresis mark the lines along which the diffusion fronts of an antigen and its antibody come together. They signify the presence of components even though these may not be isolated in the electrophoretic sense and are, in any event, combined with antibody protein. The word electrophoresis is sometimes altered by rearrangement, by pruning, or by grafting to it sundry combining forms in order to indicate some modification or specialized application of the basic electrophoretic technique. Some examples are : electroimmunophoresis, immunoosmophoresis, stereoimmunoelectrophoresis, radioimmunoelectrophoresis, electroosmophoresis, chromatoelectrophoresis, enzymoelectrophoresis, and electrochemography. Authors should think cerefully before using some of these words in the titles of their papers. The prefix of a title keyword determines its position in the indexes of the abstract literature; i m m u n o - is likely to be found b y immunochemists, and electro- is a p t to come to the attention of persons interested in electrophoresis, but stereo-, even though it accurately describes a technique useful to both groups, directs itself to the attention of neither. MMUKOELECTROPHORESIS
The term microelectrophoresis has been used to denote electrophoresis on a small scale and also as a synonym for iontophoresis, which is a method for administering medication by electrical means. Another word that seems in danger of acquiring two meanings is plasmapheresis, which has long been a medical term for a therapeutic technique that has nothing to do with the electrophoresis of plasma. The sample pattern produced by electrophoresis has been given a surprising number of names: electrophoregram, electropherogram, electrophoreogram, electrophoretogram, electrochromatogram, electrophoretic pattern, phoregram, pherogram, proteinogram, proteinographic chart, zymogram, immunoelectropherogram. Of these, electrophoregram, with the third e pronounced as “uh,” indicated by the e of the International Phonetic Alphabet, conforms to the parent word and to sound Greek usage. This review refers to a few articles that were published in 1960, 1961, and 1962, but most citations date from the second half of 1963 through the early part of 1965; it continues the coverage of a previous review (2137). Once again in the bibliography, the Chemical Abstracts reference numbers have been appended wherever the article mentioned has appeared in a publication that is not readily accessible. BOOKS AND REVIEWS
I n addition to books on the general subject of electrophoresis (204, 232, 2091), two books describe electrophoresis in agar (1736, 2397), and one discusses the uses of starch as a supporting medium (425). There are books concerned with the application of electrophoresis to the specialized fields of physiology (lSO.43, neurology (1351), and immunoelectrophoresis (797). A number of books contain sections on the electrophoresis of specific materials such as gastric juice (762), histones (459), nucleic acids (575), pituitary hormones (640), carbohydrates (2378), radioactive natural compounds (1878), and metal complexes (227). One review (la@) is concerned with the effects upon fractionations of chemical and physical conditions within the electrophoretic bed; others discuss measuring proteins (1074) and calculating their mobilities and ion-binding
constants (2356). General methodology has been covered thoroughly (818, 1318, 1521, 2019, 24lS), as have disk electrophoresis (865, 1687), thinlayer electrophoresis (876, l5.47), and immunoelectrophoresis (796, 15‘86). Reviews covering diagnostic applications include the use of immunoelectrophoresis in hematology (998) and for analyzing serum (411, 1510, 1819) or serum and spinal fluid ( 7 4 l ) , and electrophoresis of bile (2526), spinal fluid (1751),serum and bone marrow (1699), and serum proteins (889, 1105, 1193, 1237, 1466, 1988, 2108, 2166) with attention to group-specific proteins ( g o ) , lipoproteins (180, 878, 879), and globulins (365, 2432). The uses of electrophoresis for separating polysaccharides (257),amino acids and peptides (215), cells (1472), nitrogenous components of molasses and yeast cultures ( I l O S ) , and isotopes (735, 20.49) have been surveyed. Discussions of supporting media include paper (2147), fibers (825), cellulose acetate (1154, 1536), starch gel (989, 1743, 2092), agar (1509), and polyacrylamide gel (946, 1649, 1550). FUNDAMENTAL DEVELOPMENTS
Among studies contributing to the theory of electrophoresis are analyses of the behavior of reversibly interacting systems (361-363, 1628) and of the mobility of complexes (2031). There are theoretical treatments of migration in stabilized media (288, 750, 2062), some of which employ a thermodynamic approach (564,2295). Thermodynamic methods have also been used to describe dye-binding by proteins (1075, 1737). A statistical method for evaluating the significance of changes in the electrophoretic patterns of serum proteins has been proposed (2120). The effects of variable conditions (e.g., pH, temperature, current intensity) upon sample behavior (266, 1196, 1199, 24423, as well as factors affecting the homogeneity of voltage gradients (849,1198,1201)and solution movements within the bed (12, 169, 1197, 1202, 1203, ,241) have been investigated, as have the optimum conditions for polyacrylamide electrophoresis (%$11), chromatographic phenomena (l200), protein adsorption on paper (1629), and gel filtration by starch (639). There are favorable reports of the effects of magnetic fields (1205, 1866) and of alternating current imVOL. 38, NO. 5, APRIL 1966
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posed a t right angles to the direction of electrophoretic migration (10). Gels with high solid content reduce sample mobility and tend to be distorted by electrokinetic liquid movements (812). The electrophoresis of particles is assuming increased importance; there are fundamental studies of the effects upon particle behavior of size (96), dialysis (1238-1240), and ionic content in the electrolyte (756, 971, 2148), as well as a theoretical discussion of particle mobility (1746). Accurate measurements of particle mobility can be made by observing their movements in an alternating electrical field (2039). APPARATUS
Electrophoresis apparatus have come to be classified according to whether they use low or high voltages. Among newly designed low-voltage apparatus are those using paper (161) or paper and other materials (1462, 2133), as well as cellulose acetate (406, 665), and thin layer materials which require special manipulative techniques (452, 803). There are many new apparatus for electrophoresis in gels (41.4, l 4 l 2 ) , including agar (181, 1698, 1770, 2042, 2069, 2130, 2297), starch gel (1325, 1416, 2372), and synthetic gels (1346, 1798, 1835, 1928). The beds of some apparatus are small columns of gel (1507, 1609, 1883, 2332); their use is called disk electrophoresis because of the shape of the separated fractions. One apparatus handles a number of samples a t once in multiple columns of acetylated cellulose powder (464), two use density gradients for stabilization (1144, 2082), and one employs countercurrent effects (722). There are apparatus for the electrophoresis of cells (885, 1590) and particulate material (1841, 1797, 1915). High-voltage apparatus employ gradients in excess of 30 volts per centimeter; there are several new designs for such apparatus (666, 672, 1172, 1328, 1691, 1720, 1979, 2122, 2304, 2398). Their advantage is that they permit fractionations in times so short that the action of diffusion and other distorting factors is minimized; a disadvantage is that they require provision for dissipating the increased heat that accompanies high power consumption. Although high-voltage apparatus are usually thought of as having highvoltage power supplies, it should be remembered that applying 90 volts to a 3-centimeter bed gives the same gradient as 900 volts in a 30-centimeter bed. It would be more exact to speak of lowand high-voltage gradient apparatus. There are numerous apparatus for preparative electrophoresis on the batch basis (98, 484, 660, 783, 925, 926, 1047, 1294, 1307, 1773, 1805, 1832, 2214). Many new continuous apparatus use
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twodimensional movement on a sheet of filter paper (163, 249, 1546, 1571) or particulate supporting media (841-843), but others depend upon maintaining a laminar flow of free solution (224, 536, 802, 863, 1055, 1163, 1164, 1319, 2392, 2396). One continuous apparatus makes use of a bed with concentric annular compartmentation (955) and another uses serpentine columns to stabilize against thermal convection (1165). An article discusses recent developments in preparative electrophoresis (1 772). There are a number of examples of apparatus developed for use in conjunction with electrophoresis, such as an incubator for measuring enzymes (1223), a fraction collector for low temperature columns (200),and devices for electrical destaining (1833, 2008). Commercial densitometers have been improved (813, lll4), spectrophotometers have been modified for densitometry (1573, 2185), and designs have been suggested for scanning (lo@), integrating (1347), and recording (1728) densitometers. A device permits ultraviolet photography of electrophoregrams (1081). Several ways to reduce the size of freesolution electrophoresis cells without loss of resolution have been developed (1840); most of these are patented (853, 1947, 19.48, 2259). STABILIZING MEDIA
There is growing evidence for the superiority of cellulose acetate over filter paper (444, 629, 826,828, 830, 1111, 1155, 1380, 1381, 1496, 2455). Thin layers of inorganic materials such as alumina, kieselguhr, and silica gel (455, 613) and organic materials like cellulose powder (488)or cellulose ion exchangers (2447) seem likely to find increasing use. Papers modified by incorporating ion exchange characteristics (606, 2447) or by impregnation with agar gel (2382) have been reported to be superior to ordinary paper; xirradiation of paper, however, increases trailing and diminishes sample movement (11). Inorganic compounds have been fractionated on the gelatin coating of photographic film (1254, 14%) and on poly(viny1 alcohol) fibers (1433). Agar can be improved by washing (f221),by freeing it from sulfate (952), or by processing it to agarose (338, 1662, 2146). Noble agar is best for separating lactic dehydrogenase isoenzymes (1220). Agar is as satisfactory as acrylamide gel for the immunoelectrophoresis of urinary colloids (1118). It is sometimes used with a supporting agent such as filter paper (2382), vegetable parchment (2463), Teflon-glass paper (2383), cellophane (&$?), or plastic films (369, 370). To enhance resolution by gel filtration, agar has been mixed with starch gel
(50, 874) or with Sephadex ($317); it can also be made t o give gel filtration effects by varying its concentration (2006). Sephadex, which has gel filtration properties (lO24), and diethylaminoethyl Sephadex, which has both ion exchange and gel filtration properties (1308), are comparatively recent additions to the list of supporting media. There is a method for preparing an improved and standard starch gel directly from potatoes (1145). Background staining of starch gel can be minimized by using starch that has been treated with protease (1876). Certain fast-moving hemoglobin components can be demonstrated on starch gel but not on polyacrylamide (892). Incorporating Pevikon into starch gel increases protein recovery (242). Coagulated egg albumen can be used as a supporting medium, but it has less resolving power than agar gel and creates a strong electroosmotic flow (241). Carboxycellulose gel (961, lo@), because of its ion exchange properties, is excellent for hemoglobin separation. There are reports favoring polyacrylamide gel for immunoelectrophoresis (1119), for separating histones (553), plant proteins (1927), and hemoglobin (6@), and for preparing bovine serum albumin (43); however, some proteins appear to polymerize in this medium (1693). The effect upon fractionation of varying the concentration of synthetic gel has been investigated (l834). Electrophoresis on a preparative scale can be accomplished in beds of particulate or porous material, including calcium sulfate blocks (247, 5 3 4 , starch powder (406), granules of agarose (918), ethanolyzed cellulose (2101), microscopic glass beads (707, 1442, 2@0), poly(viny1 chloride) (280), and Pevikon (238-240, 245), a copolymer of poly(vinyl chloride) and poly(viny1 acetate) which causes a smaller electroosmotic flow than does poly(viny1 chloride). At low ionic strengths, mobilities in density gradient electrophoresis are the same as in free solution (1657). BUFFERS
No descriptions of new buffering agents for proteins have been observed by this reviewer, but there have been a number of attempts to use known buffers in novel ways; for example, in discontinuous buffer systems (910,1614) or by establishing optimum ionic strengths (1449). The performances of a number of buffers for serum lipids (1110), of cacodylate with other buffers for hemoglobin (379), and of various buffers in polyacrylamide gel (642)have been compared. Separations of nucleotides and of phosphoric esters in carbonate (1837), of mucoproteins in McIlvaine’s buffer (2443), and of organic acids in . p H gradients (1011) have been recommended.
PROCEDURES
General Methodology. Descriptions are given of procedures for t h e electrophoresis of serum proteins (137, $412) and of hemoglobin (138, 800, 975, 992, 1378, 1910, 2200) on cellulose acetate; of serum (2400) and hemoglobin (296, 1458) in agar; of proteins (1443),tissue homogenates (485),hemoglobin (186, 1440), and serum isozymes (739) in starch gel; and of enzymes (1017) and serum proteins (482) in polyacrylamide gel. Techniques for immunoelectrophoresis on agar (18, 1280), starch gel (l779), and cellulose acetate (1621) have been detailed. Variant techniques for immunoelectrophoresis have been given descriptive names : stereoimmunoelectrophoresis (1763),immunoosmophoresis (258,259), and antigen-antibody crossed electrophoresis (1275). There are many procedures for separating and measuring amino acids (89, 298, 439, 671, 1020, 1076, 1926, 1943, 2366), some of which combine electrophoresis and chromatography (216, 761, 1021, 1655, 1706). Physiological cations can cause false ninhydrin reactions in electrophoregrams of amino acids (820). Other articles describe the separation of peptides (1317, 1870, 2396). There is a method for establishing the number of free sulfhydryl groups in peptides (1095), and one for measuring sodium, potassium, and phosphate in biological fluids (807, 808). Manipulative Techniques. Urine can be concentrated for electrophoresis by dialyzing it against polyglycol in Verona1 buffer (420) or against powdered sucrose (1461). Optimum conditions for dialyzing serum previous to free-solution electrophoresis have been established (1671). Hemolyzates for hemoglobin screening tests can be obobtained by freezing blood clots (222). Interfering salts can be removed from sugar sample spots by electrophoresis previous to paper chromatography (2446). Chromatographic action affects buffer composition if filter paper is moistened by capillary action rather than by dipping (1049). A template (469), a stamping device (111), and a comb that forms troughs (2298) have been found useful for shaping gel beds for immunoelectrophoresis. There are simple devices for applying hemolyzates t o starch blocks (126) and introducing serum into gels (951). Samples can be applied to the surface of a grl rather than embedded in it (790, 794). Capillary blood samples for haptoglobins and erythrocyte catalase can be taken up on filter paper strips and stored a t -20' C., then pressed on the surface of a gel for electrophoresis (147). An electrophoregram developed in starch can be sectioned and applied to an agar gel bed for immunoelectro-
phoresis (1186). Mixing serum samples with agar before it solidifies modifies the beta-globulins (2477). For disk electrophoresis it is better to introduce samples in high-density solutions of sucrose (1608), urea or sucrose (409),or Sephadex and sucrose (318) than by using a rigid gel. Amino acid mixtures can be transferred from electrophoregrams to chromatographic wedges by capillary action (1869). Proteins can be recovered from starch gel by electrodialysis (1151). Photographs of cellulose acetate electrophoregrams should be made with trans-, mitted light (410); contact prints on paper of haptoglobin typings in starch gel can be stored permanently (2374); starch gel electrophoregrams can be plasticized and dried for storage (148, 47.9) ; polyacrylamide electrophoregrams also can be dried (902). Detection and Measurement. Proteins on paper (2178) and in agar and starch gels (2265) and ribonucleic acids in agar (2262, 2263) can be measured by direct ultraviolet spectrophotometry. The progress of serum protein fractions can be observed by their fluorescence (1980) or by using a marker solution (1846). Metal ions associated with protein fractions can be detected by emission spectroscopy (l340). The reducing properties of proteins make it possible to measure them by polarography (1726, 2479) or by means of reagents containing molybdenum blue (1756), potassium permanganate (2346, 25-47), or potassium ferricyanide (541). Stains that have been used for measuring serum proteins include Amido Black 10-B (386, 895), Bromophenol Blue (1963, 2470), Lissamine Green (1272, 1939), Ponceau S (730, 1871), Sulfo Green (1784, 1785), and p-xylenolsulfonephthalein (540). The Coccine dyes can be used as markers in serum electrophoresis because they stain but do not affect mobility (1529). Sources of error in measuring stained proteins have been investigated (1448); elution is more accurate than densitometry for measuring electrophoregrams (1296); densitometers give erroneous values when samples are too small or too large (83). Erroneous electrophoregrams can be caused by blood-extending dextrans which stain with Amido Black (923). Drying electrophoregrams in a special oven (791) or freeze drying them (454) prevents spreading. Several papers compare electrophoresis with other methods of serum protein fractionation (71, 458, 645, 780, 1009, 1677, 2375). Quantitative or semiquantitative measurements of serum proteins can be made on the basis of the diameter (17), the area (1469),or the position (1917) of the precipitation arcs produced by immunoelectrophoresis. Fractions obtained by gel electrophoresis can be visualized by flooding the electro-
phoregram with immune serum (2417); visualization of immunoelectrophoretic patterns is enhanced by flooding the bed with cadmium sulfate solution (2433) Many methods have been developed for measuring specific proteins in human blood. These include detection or quantitation of abnormal hemoglobins (390, 1113, 1697, 2164), ways to distinguish between alpha and beta chain anomalies by forming hybrid hemoglobins (2284) or by enzymatic means (933), ways of measuring haptoglobins (858, 1889, 2300), fibrinogen (2l4, 470, 1739), ceruloplasmin (463, 201.4), and globulin subfractions (197, 418, 1357, 1780). One method for visualizing globulins uses electron microscopy (931). Macroglobulins can be detected by gel filtration during agar electrophoresis (1180) and can be distinguished from myeloma globulins by simple tests (349, 1931), b u t the methyl green-pyronineorange G stain for myeloma proteins has been called unsatisfactory (963). Staining lipoproteins with mixed Sudan dyes both before and after electrophoresis has been recommended (517-519). Sudan Black is superior to other dyes for lipoprotein staining (535, 1735). Measurements of betalipoproteins by dye are unsatisfactory (565, 1957). I n addition to a number of modified Schiff's tests for glycoproteins (928, 929, 1120, 2129, 2458), a new reaction for characterizing them (2293) has been developed. Spectrophotometric measurements of glycoprotein colors should be prompt to avoid fading (1914). There are procedures for measuring mucoproteins in serum (839,1236,1?'90) and in serum and ascitic fluid (1121); the staining properties of saliva mucoproteins have been studied (1669); acid mucopolysaccharides can be stained with Acridine Orange (1971); sulfated polysaccharides inhibit protein staining with Amido Black (1379). There is a simplified method for measuring casein (728) and a nephelometric method for quantitating betalactoglobulins (1344). Thyroglobulin can be estimated by immunoelectrophoresis (1036) and the thyroxine-binding capacity of serum protein fractions by using '3II-labeled thyroxine (337, 513, 838). Assays for human growth hormone involve immunoelectrophoresis against 1321-labeled antiserum (669, 973). An agent that inhibits the immunoelectrophoretic assay of insulin can be inactivated with (ethy1enediamine)tetraacetate (1644). Isoenzymes might be named by numbering them according to the order of their electrophoretic mobilities, the most rapid component to be number one (2373). General systems of detecting enzymes in electrophoregrams have been developed (14Q5, 2077, d.890, VOL. 38, NO. 5, APRIL 1966
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2291). There are also methods for detecting or measuring specific enzymes such as glutamine-oxalacetate transaminase (2352), glutamic oxalacetic transaminase (2015), lactic acid dehydrogenase (42,118,1674,1789,1802), glyceraldehyde-3-phosphate dehydrogenase (24l6), esterases (2198, 2361), catalases (145, 2274), amylases (60, 1769, 2287), beta-glycosidases (1345), the proteolytic activity of biological mixtures (2121), rennet (2004), aminopeptidases (2S54), and deoxyribonucleases (2349). BIOLOGICAL APPLICATIONS
Interactions. When substances interact, the products can often be detected electrophoretically. Examples of interacting proteins are complex formation between acidic and basic proteins (1303, 2242), cryomacroglobulins and serum proteins (1952, 1953), serum proteins and concanavalin A (1678),and Bence Jones protein with protein J from Jackbean meal (1600). Proteins also interact with nucleic acids (297), mucopolysaccharides (206), hyaluronic acid (481),and glucose (630). Cryoglobulin is a nucleic acid-protein complex (709). The complexes of streptokinase (125, 498) and of streptokinase and urokinase (1960) with plasminogen have been demonstrated, as have complexes of various enzymes with their inhibitors (1125, 1S'70), which include glutathione (284), dyes (24), and ribonucleic acid (947). The products of proteolytic enzyme action on lipovitellin (616), alphaz-macroglobulin ( l O O 4 ) , gamma-G globulins (1990), serum proteins (1000,1013,1514, 214O), myeloma proteins (1781), and hemoglobin (@) have been investigated. Electrophoresis has been used to study the reactions with proteins of radioactive ions (@3, l248), metal ions (64), chromium (419, 944, l l O g ) , mercury (1428), aluminum (1528), and plutonium (271), as well as quaternary ammonium salts (2037) and boric acid (1436). Low concentrations of alcohol in serum alter globulin distributions (2093, 2286). A miscellany of complexes, including metals with humic acid (1667), antibiotics with nucleic acid (534),bilirubin with EDTA (105S), and polyglucosamine with bivalent anions ( C S l ) , have been subjected to electrophoresis. A number of reports describe the effects upon proteins of ultrasound (1300, 2099, 2459), heat (956, 937, 997, 1116, 1559, 1655, 1560, 1561, 2379), ultraviolet light (609-511), x-rays (404, 608,1358), and gamma rays (6459). Biological Fluids. Milk and colostrum are of interest both as biological fluids and as food products. Because interest this year has centered around research aspects rather than
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matters of industrial interest, these fluids will be discussed in this section. Electrophoresis has been used to characterize proteins of colostrum from various sources (8, 325, 788, 1080, 1194, 1459, 1843) and of milk from humans (787, 1S72-1374, 1591), SOWS (1646), mares (12S1), buffaloes (1816), rats (1277), and domestic animals (516); electrophoretic work with the proteins of cow's milk has been extensive (78, 910, 1126, 1598, 1745), with special attention to casein (79, 254, 441, 591, 621, 729, 731, 736, 1171, 1367, 1399, 1461, 1582, 1615, 199'7, 2005, 2227, 2278, 24262427, 2481) and beta-globulins (591, 72SJ1554, 1702, 1787). Some research has been concerned with the effect of processing on the proteins of milk and milk products (92, 397, 601, 949, 990, 1553, 1905, 2B99), the effect of souring on the mcbility of fat globules (1813), and detecting milk proteins in food (866). Blood albumin can be detected in the milk of cows with mastitis (2OS2). I n addition to studies of urinary proteins in pathological states such as renal diseases (964, 2182, 2231), neoplastic states (964, 1625, 2577), burns (781), and cadmium poisoning (55), studies have been made of proteins (150, 637, 662, 701, 900, 1774, 2332) and glycoproteins (231, 530) in normal human urine. The urinary proteins of mice ( ~ 4 658), , rats (2S69), rabbits (563), dogs (1774),newborn ruminants (1460), and cows (2106) have also been investigated. Urine contains enzymes, examples of which are one with renninlike properties (321), alkaline phosphatase (343, 344), lactic dehydrogenase isozymes (1865), and esterases (2314). Metabolic products in urine that have been isolated by electrophoresis include urobilins (1565) , porphyrins (991, 9322), alpha-keto acids (193), alpha-aminoadipic acid (2195), phenolic acids (580, 681, 1483, 1818, 2l48),and creatine and creatinine (667). Antibodies have been demonstrated in human urine (868). A number of papers concerned with amino acids in urine are cited in the section on General Methodology. Methods for fractionating saliva proteins (416)on paper (607,664,932,2184, 2384) and in polyacrylamide gel (347, 15oo), including descriptions of normal patterns, and salivary protein patterns in mucoviscidosis ($36) and nephrosis (718) have appeared; there is even a study of rat saliva (867). Proteins in sputum from asthma patients (495) and patients with chronic bronchitis (267, 268, 270), as well as the components of bronchial mucus (884),have been examined. Gastric juice proteins from patients with various diseases (4, 311, 915, 1246, 1624), carcinoma (3, 1964, 1965, 2188), gastritis (1177, 185S), ulcers (1177, 2275), and cirrhosis (1420) have been
fractionated. The vitamin BIZbinding factor in gastric juice is reduced or absent in pernicious anemia (974, 6282, 2389). Carbohydrates in gastric juice (764) include the blood-group-specific substances (763). Gastric mucus contains proteases, carboxylesterases, acid phosphatases, and several other protein fractions (1821). The compositions of gastric and duodenal extracts have been compared with gastric juice (1734), and the protein fractions in the duodenal juice of dogs have been reported (984). The composition of normal human bile (1097, 2306) as well as that of various domestic animals (881) has been reported; specific substances in bile measured with the help of electrophoresis include enzymes and mucopolysaccharides ( C l S ) , cholesterol (1603, 1650), lipids in disease (2306), and urobilin (1366). Plasma proteins normally occur in feces and their distribution changes significantly in a wide variety of diseases (1434); normal meconium and that from patients with meconium ileus differ in protein content because of enzymolysis (1981). There is a study of variations in the sugar content of feces with relation t o age (832). Improvements in methods for cerebrospinal fluid include recommendations for agar over paper (2000) because of freedom from trailing, and for polyacrylamide gel (462) because the fluid need not be concentrated. There is a method for concentrating cerebrospinal fluid previous to electrophoresis (1582). Methods have been proposed for localizing enzyme activity in electrophoregrams (647-649). Storing cerebrospinal fluid a t -10' C. causes an extra gamma-globulin fraction to develop (927). A number of papers discuss the normal protein distribution (117, 422, 706, 1539), as well as lipoproteins (2097), and transferrins in neonates (1716). The protein composition of cerebrospinal fluid has been studied in various diseases (121, 286, 806, 847, 2257), tumors of the central nervous system (440,1329), encephalitis (1157-1159), brain abscesses ( l d O S ) , meningeal leukemia and mental deficiency (1903),multiple sclerosis (1270), and syphilis (1644). There are abnormalities in cerebrospinal fluid protein patterns in tardive dyskineasia (623),but not in any of the major categories of mental patients (9S0, 1622). The proteins of schizophrenics have high sulfhydryl content, as do those of post-convulsive epileptics (2285). d miscellany of reports describe the composition of fluids associated with the reproductive process. Except for one paper reporting neuraminic acid derivatives in human ovarian cysts (2060), interest has been confined to protein composition, which has been reported for seminal plasma in humans
(1289) and domestic animals (174, $40,
2054), human prostatic fluid in health and disease (2094), human cervical secretion (1532, 2012, 2258), uterine fluids from rabbits in estrns (2126), amniotic fluid from humans (328, 1182, 2117, 2136, 2139, 2431) and rabbits (1255),and rabbit fetal fluids (2406). Electrophoresis has been used for analyzing the proteins in human sweat (403), sodium chloride in the sweat of diabetics (625), and proteins ( 4 1 , 104.8) and sialic acid (346) in tears. The lipoproteins in lymph (1475, 1476), the proteins (208, %$IO), including glycoand lipoproteins (726, 985) and hyaluronate-proteins (861, 1949), of synovial fluid, as well as the proteins of the aqueous humor of the eye (87, 310, 1776) and of the inner ear perilymph ( 1 146, 1776), have been studied. Among fluids of pathological origin, the proteins in ascitic fluid from humans (445, 9 1 7 ) , rats (543), and mice ( 3 3 4 , blister fluid (306, 586, 587, 1316, 1563, 1778, 1933), edema fluid (445, l7l4), hydrocele fluid of filariasis (1816), and sheep hydatid fluid (396) have been fractionated. Blister fluid contains an antibody to staphylococcus (2079). Human Serum. Recent investigations of normal serum proteins in adults (1297) are usually concerned with distributions on media other than paper, such as cellulose acetate (2038), polyacrylamide gel (17%), urea-starch gel (1932), and anion-exchangers (779), or with special populations-e.g., racial groups (1721, 2027, 6251) and the aged (2423). Much interest has centered around special proteins such as the group specific proteins (149, 287, 914, 940, 1135, 1565, 1842), immune globulins (339, 504, 599, 867, 899, 913, 1880, l906,1908,1989,2l24, 2221,2256'), and previously unreported fractions such as normal double albumin fractions (1873, 2201), pink pigmented plasma proteins (1994), and a zone that appears in pregnancy (20, 435, 2@9), or fractions that inhibit bacterial growth (2001). There are studies of normal distributions of serum proteins (281), including immunoglobulins (635), in neonates and infants and in children (2435, 1613, 2283). Serum protein patterns in children more than two years old resemble those of adults except for lower alpha2and gamma-globulin fractions (2386). Specific proteins that have been investigated are transferrins in cord serum (1716), fetal fibrinogen (1366, 2132), a fetal beta-protein (2311), a fetal protein that disappears in the seventh month of pregnancy (724), and proteins during the fetal period (1023, 1230, 2209) and early infancy (356). Two statistical studies correlate characteristic serum protein patterns with diseases (291,2155). Diseases in which supernumerary protein fractions occur
have been listed (1338) and the significance of alpha-globulin changes in response to disease has been explored (196). Articles relate the distribution of serum proteins to clinical conditions, including cardiac insufficiency (348, 1298, 1322, 2087, 2235) , arteriosclerosis (598, 1453, 1678, 2337) with emphasis on lipoproteins (173, 775, 1376, 1653), cerebrovascular accidents (136), arterial hypotonia (61), periarteritis nodosa (36, 2308), polyarteritis nodosa (1640),myocardial infarction (657, 673, 1083, I l O Z ) , anemias (314, 1002, 1480, 1792, 2048), thrombocytopenia (1413), cold agglutinin disease (2393), blood grouping difficulties (1804), hematological disorders (324),leukemia (477, 612, 751, 996, 1264, 1405, 2033), various lung diseases (1641, 1649), tuberculosis (620, 1189, 1387, 2104, 2312), pulmonary sclerosis (14 ) , respiratory diseases in infants (715, 1168,2084),asbestosis ( l 4 ? O ) , silicosis (474, 694, 1057), pndumoconiosis (1463), pulmonary asparagonimiasis pergillosis (212, I$@), (1972, 1973), various hepatic disorders (1439, 1515, 1841), cirrhosis (786, 2208, 2256), cholecystitis (629, 1077), hepatitis (57, 123, 1123, 1166, 1764, 2149), obstructive jaundice (278), Fasciola distomatosis (3.54),amebic liver disease (1966), gastric and duodenal ulcers (1107, 1478, 1940, 1941, 2457), chronic oral inflammation (1648, 2435), gastrointestinal surgery ( l 5 2 5 ) , dysentery (2294, 2394), bacterial dysentery and amebiasis (993),cholera (1707), secondary pellagra caused by enterocolitis or cancer (88),the toxicosis of pregnancy (67, 770,1167,1244,2080,2310), various kidney diseases (1236, 2289, 2316), nephrosis (23.40, 240.2), renal tuberculosis (476), renal homotransplant rejection (1795), various skin diseases (305), bullous dermatosis (1069, 1315, 1564, 2468), scleroderma (677, 2112), chronic pyodermatitis (2276), neurodermatitis (2254), exudative diathesis (746), cutaneous amyloidosis (1775), leprosy (269), dermatomyositis ( e l l s ) , lichen myxedematosis (1465), psoriasis (299, 1218, 1273, 1274), abnormalities of skin pigmentation (584, 585, 1882), schizophrenia (644, 725), drug therapy of the mentally ill (623, 827), encephalitis (1226, 1885), encephalomalacia @bo),tuberculous meningitis (663, 1881), multiple sclerosis (2205), poliomyelitis (1012, 1767), mongolism (1078), diabetes (578, 1056, 1681, 2111), cystic fibrosis (804), thyrotoxicosis (811, 1925, 2472), rheumatic and rheumatoid diseases (40, 142, 619, 873, 953, 980, 1263, 1371, 1426, 1501, 1561, 1896, 2125, 2176, 2316), collagen diseases (2320), skeletal tuberculosis (1072), osteomyelitis (l717), muscular dystrophy (977, 1477), myasthenia gravis (1184), cataract (1938), ocular tuberculosis ( 11Q5),gynecological con-
ditions (1660), diphtheria (1396), scarlet fever ( I l Z Z ) , influenza (1583), tickbite fever (2313), brucellosis (552, 703, 720, 1026), trypanosomiasis ( I 75-1 78, 641, 1046), leptospirosis (1269), kalaazar (443), sarcoidosis (805, 1645), lupus erythematosus (493, 882, 1984), cranial trauma (1252), bone fractures (402, 747, 2240), hemorrhage (277), blood stasis ( d o l ) , burns (674), radiation dermatitis (1794),fatal mechanical injuries (51), and poisoning by lead (374, 740, 1576, l757), mercury (11Q1), carbon monoxide (1854), morphine (1519, 1520, 2248), chloroquine (1530), and mushrooms (246). Nuch attention has been given to serum protein changes that accompany chorionepitheliomas (777), cancers of the alimentary tract (230, 1052, ZO47), thyroid (866), uterus (97, 1904), and urinary bladder ( 5 ) , and various kinds of cancer ( 2 , 33, 562, 801, 1759, 1848, 2437, 2466); some reports are concerned with the effects of surgical (1156), radiation (551, 1174, 1227, 1634), and chemical (2464, 2465, 2467) therapy. Three tests for diagnosing cancer by electrophoresis have been proposed (1188,1537,2210). Studies of myeloma proteins of both the plasmacytoma (252, 679, 680, 1073, 1987, 1991) and the multiple myeloma types (93, 461, 582, 618, 840,862, 870, 1150, 1186, 1299, 1375, 1383, 1856, 1907, 2189, 2,207) have been numerous. It is possible to distinguish between macroglobulinemias and the myelomas by fractionation of the paraproteins (52, 461, 665, 869, 2222, 2345, 2399, 2480). d special stain to identify Bence Jones proteins has been developed (2452). Mammalian Serum. Studies of normal bovine serum protein distributions include fetuses (512), calves (2187), developing heifers (2333), cows (1016), variations in serum proteins with milk production (678), and transmission of immunities through colostrum (107, 1608, 1747). There are descriptions of bovine serum protein polymorphism (58, 80-82). Polymorphism also occurs in the serum proteins of reindeer (294). The serum betaglobulins of red deer have been fractionated (1350). Normal protein patterns of sheep ( 1 , 395, 1225, 1796) and comparative studies of fetal, lamb, and maternal variations (1466, 2281) have been published. There are developmental changes in the serum proteins of swine through the fetal and postnatal periods (1173), in growing pigs (475), and from birth to maturity (1282). Horses (897, 1336, 1966),dogs (15, 358, 2245), moles (466),laboratory rodents (1206), mice (426, 609), rats (65, 172, 251, 542, 606, 792, 793, 1626, 2158, 2249), guinea pigs (883, 1016, 1676, lQ85),and primates (56, 432, 450, 816) are among the mammals for which normal values of serum proteins have VOL. 38, NO. 5 , APRIL 1966
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been published; numerous comparative variant in humans (2018). There are studies of the effects upon lipoprotein? studies involve a wide variety of aniof vitamin D (1630),chorionic gonadomals (23, 22, 134, 608, 610, 614, 615, tropin (1008), prednisone (456), and 1029, 1216, 1580, 1589). Among the pathological conditions of heparin (822); serum lipoproteins also are affected by many diseases, including domestic animals that have been incoronary disease (2436),arteriosclerosis vestigated are brucellosis (1894), tuberculosis (833),schistosomiasis ( % % @ ) , (54, 1887, 1919, 2368), cerebral hypertension (1395), cancer (1061, 1362, paratyphoid fever (26), starvation 1604), placental insufficiency (1901), (2086), and numerous diseases (2007) skin reticulosis (2216),multiple sclerosis in cows; liver disease (2105) and starva(734),hepatitis ( l d O l ) , syphilis (1356), tion (2086) in horses; liver disease rheumatism (1923), lymphogranuloma( 152), eczema (1719 ) , and toxoplasmosis tosis (1060), and chronic enterocolitis (719) in dogs; poisoning (Z),arthritis (330). Lipoprotein distribution in the (1712 ) , plague (345), and parakeratosis serum of normal and hypercholesterol(1400) in swine; neurological disease emic dogs, rats, and guinea pigs (831) (2070), kidney stones (1697) and infectious polyarthritis (622)in sheep; and and vitamin C-deprived guinea pigs Aleutian disease in mink (1115 , 2145). (472), and in the brains of guinea pigs Serum proteins have been investi(1502) has been studied. gated in diverse experimentally proGlycoproteins. Serum glycoproteins have been studied in normal duced pathological conditions in animals, including liver damage by alhumans (1208, 1425, 1993, 1996), chiidren (457, 1633), premature infants lergies (1245), hepatectomy (2023), blood vessel ligations ( 9 , 427), liver, (1631),in relation to age and sex (1733), and in such conditions as venous stasis necrosis (308), atherosclerosis (1824, (659),myeloma (809),thyroidism (272), encephalopathy (217 7 ) , rabies (1481), diabetes (697, 2081), mongolism (1620), paratyphoid (487), tuberculosis (309, hemoblastosis (1233), asthma (1912), 982), arthritis induced by mycobacterial rheumatism (1389), tuberculosis (986), preparations (233, '795, 1247, 1349, amyloidosis (1918), and various condiand hepatitis (1699). The carbohydrate content of serum globulins has tions requiring hormone therapy (140, been measured (1417, 1646); there are 978, 1491, 2309) ; ionizing radiation many subfractions of alphal- and alpha2(122, 167, 388, 682, 1190, 1579, 1686, glycoproteins (1995, Z.241); the glyco1868, 2103) and irradiation by 3-cm. peptides of thyroglobulin are heteroelectromagnetic waves (2175); hemorgeneous (384). Serum glycoproteins rhage (2236), poisoning with lead (32), have been investigated in various mammercury (1636), alcohol (685, 1851), mals (669,570),including normal gerbils and silica (94, 1556, 1557), vitamin C (492), arteriosclerotic rabbits (1527, deprivation (189), starvation (2380, 1596), and cancerous rats (1209). 2381), and low atmospheric pressure Bovine submaxillary mucin is hetero(1619). Antibiotic therapy affects genedus (948). serum protein composition (1872,2325), Hemoglobin. By electrophoresis as does the inhalation of charged atmosin combination with other methods, pheric particles (2083). The changes many new hemoglobin variants have in serum proteins in rodents that occur been discovered (106, 675, 676, 714, as a result of carcinoma caused by 893, 959, 1041, 1117, 128'7, 1311, 1418, carcinogens (320, 661 , 2068), trans1419, 1581, 2002, 2041, 2044, 2074, plantation (260, 627, 890, 898, 2296), or 2165, 2342, 2371, 2444). Fetal hemospontaneous development (486, 1803, globin (383, 1139, 1516, 2046) and the 1825) are variable, depending upon the hemoglobins of Heinz-body anemia etiology and classification of the tumor. (467) and thalassemia (46, 72, 161, 711, Lipoproteins. Cholesterol on elec1170, 1411, dl42) have been investitrophoregrams can be measured and gated extensively. There are studies related to the distribution of lipoproteins of hemoglobin distribution according (743, 2M.2). Measuring lipoproteins to nationality (73, 2043, 2174). Minor by eluting the stained fractions gives concentrations of variant hemoglobin higher results than densitometry (1968). Staining lipoproteins after immunooccur even in normal blood (47, &, 255, 965, 2302); one individual has electrophoresis requires special techfour major hemoglobins (1818). Hemoniques (100, 1101); during immunoglobins H and Bart's are not bound by electrophoresis, alphal-lipoproteins may haptoglobins (1593). Much work has precipitate nonspecifically (2216). Low been done on the chemistry of hemo(844) and high density serum lipoproglobin, including isolating the chain teins (1302, 1978) from ultracentrifugasubunits (329, 939, 960, 962), amino tion have been examined electrophoretiacid analysis (389), and molecular cally. Variations of serum lipoproteins aging (1891). I n the presence of heavy in normal subjects and in various dismetal ions, canine and human hemoeases (386), as a function of age (@O), globins hybridize (597). Atropine in and as a result of senescence (424) have blood is bound mainly by hemoglobin been studied; a double beta-lipoprotein (576). has been reported to be a normal genetic
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ANALYTICAL CHEMISTRY
Electrophoretic examinations of animal hemoglobins include sheep (380, 1096, 1225, 1612, 2334), goats (SO,$), cows (1512), horses (2141), pigs (342), camels (119), armadillos (5 7 2 ) , elephant shrews (332), moles (466), mice (449, 891, 1814, 1866, 24Zl), elephants ( l l d l ) , elephants and hyraxes (333), primates (331, 1288, 2344), opossums (1696), domestic fowl (273, 341, 400, 698, 966, 1160, 1725, 1810, 1827, 1983), birds of various species (1929, 2151), fish (364, 776, 2307), amphibians (1406, 1407, 2217, 2218), and various animals (1810). The mobilities of whole red blood cells (544, '760, 2016, 2184, 2135) have been a matter of interest, as have the stroma proteins (74, 101, 392, 393, 712) and nonhemoglobin erythrocytic substances (283, 660, 880, 905, 921, 954, 1503,l6O4,1506,2238,2303). Specialized Blood Proteins. .\ number of methods for measuring and typing haptoglobins (146,223,829, 869, 1570, 1890) have been proposed, and haptoglobins have been classified by phenotypes (748, 1011) and their properties investigated in humans (66, 282, 1377, 1392, 1831) and in various animals (307, 904, 1330, 1694, 2323). Some serum protein hemoglobin and myoglobin binding cannot be attributed to haptoglobins (1278, 2390). Transferrins in man (426, 1179, 2364), in primates (150, 1262, 2005), and in cows (479, 1829, 1830), and ferritin from humans (717 ) ,humans and horses (656), horses (1176, 2167), horses, mules and donkeys (1861),and cell cultures (360) have been examined. A number of papers are concerned with clotting factors in plasma (202,557,887,1494,1652, 1654,1732,1992). Cell and Particle Electrophoresis. Electrophoresis of whole cells (686,1283, 2076, 2362, 2367, 2485), of formed cellular elements (480, 628, 919, 1760) or of other particles such as airborne antigens (782) not only is a way of classifying particles, but, particularly when combined with chemical treatment, supplies information concerning the nature and magnitude of surface charges. Tissues. Most kinds of tissue have been analyzed electrophoretically: brain for proteins (253, 646, 1414, 1643, 1610, 1700, 1750, 2020, 2280, 2341, 2460), lipoproteins (744),a metal-bound lipoprotein (854),phosphoproteins (628), basic proteins (943),and enzymes (133, 1362, 1354); ganglia for acetylcholine (708); skeletal muscle for proteins in disease (381, 382, 1337, 1353, 2288), after activity (2434), after tourniquet application (1602),from various animals (144, 1005, 1050, 105l,l@O, l 7 4 l ) , and for thyroxine-binding proteins (l4Og), myosin (1742),actin (363, 1099, laid), and myoglobins (1192, 1801) ; cardiac muscle for proteinq (367, 813, 814, 823, 896, 1210, 1950, 1951, 1964); skin
for soluble components (2473, 2478), proteins (190, 265, 668, 699, 995, 1441, 2159, 2474-2476), and allergens (191, 192); wool for proteins (2226); endocrine gland proteins from thymus (992, 1681, 1777), pituitary (115, 1038, 1306, 1526), thyroid (681, 1740, 1954), and corpus luteum (1292); secretory gland proteins from prostate (494,mammary gland (1709, 1718), submaxillary gland (1957), and kidney (525, 771); liver proteins from humans (312, 1966) and animals (243, 2.44, 546, 727, 784, 875, 1142, 1569, 2100), in disease (112, 785, 981, 1054, 11.49, 1509, 1312,1315, 1542, 1701, 2102), and as affected by diet (956, 2887); spleen for proteins (546, 799, 1388); eye lens for proteins (218220, 264, 527, 979, 1176, 1178, 1368, 1584-1586, 1471, 1659, 1817, 2454, 247l), products of enzymatic proteolysis (2448-2450), and amino acids (695); arteries for proteins (1844, lipoproteins (2252), glycoproteins (2021, 2022), and mucoproteins (75, 1888) ; gingivae for glycoproteins (2010); gastric mucosa for proteins (76, 497, 1424, 1820); bone marrow for proteins (1162, 1566, 2024); placenta for proteins (1688, 1921, 2195); dental pulp fluid proteins (856) ; connective tissue for antigens (850) and collagens (257, 506, 507,1591, 1606, 1607, 1752, 1859) ; amyloid tissue for proteins (170, 412); tumor tissue for proteins (19, 1515, 2273); assorted tissues for proteins (276, 293, 401, 567, 1355, 1961, 2051, 2152); and soluble proteins of free cells, including leukocytes (752), thrombocytes (1862), spermatozoa (969, 1310), cultured cells (1025, 1217), and cell nuclei (159, 1722). Electrophoresis is useful for studying the curing of pork (1727) and for identifying kinds of meats (1750). Lower Vertebrates. Protein fractions in chicken serum (815) have been characterized with attention to developing chicks (214 4 , sex differences (%%@), high cholesterol diets (946), molting (789),laying (2558), and disease (1211, 124.9, 2277); reports on specific blood proteins include a new globulin (1857, 1858), lipoproteins (2?9), interferon ( 2 4 3 , erythrocyte histones (398),hemoglobins and ferritin (478). Papers about the serums of fowl other than chickens deal with proteins of zinc-deficient quail (690), proteins and enzymes of ducks (1065, 1064, 1067, 1068), glycoproteins of domestic birds (568), and enzymes of 18 bird species (157). Articles on an assortment of avian substances too diverse to classify cover oviduct proteins (322, 1658), pigeon perilymph (1694), lenses (547, 845, 846, 1212), pancreatic enzymes (894),feather keratin (872),effect of ionizing radiation (2150) and cold storage (1616) on tissue proteins, and embryonic spermine and spermidine (1807), brain proteins (1982), and the mesodermal iaduction factor (2233). Other reports per-
2110, 2181, 2570), fungus-infected bean leaves (2118), pea root segments ( 1535), pea seedlings (%'la@,pea plants (689), soybeans (911 , 22 79), various legumes (772),vetch and alfalfa (1232),potatoes (1295, 1320), peanuts (2243), onion and garlic seeds (1228), cucumber seeds (5.4) , sesame seeds (1935),poppy seeds ( 1 124), pine seeds (1098),pollen (210, 851), and miscellaneous plant substances ( 1 492, 1708, 1930, 2061, 2171, 2228). Other plant substances that have been investigated are allergens from castor beans (1281, 1655, 2109) and from rye grass pollen (1030),corn enzymes (169),phytohemagglutinins from jackbeans (1601) and lilies (l749), amino acids (168, '1585), pigments from beets (1783) and cactus fruit (602,603),xylansfromgrains (558), polysaccharides from soybeans (1548), saponin from tea (877), lignin sulfonates (116, 2451), I4C-labeled photosynthesis products from algae (1884), hyoscyamine and scopolamine (1130) and solasonine and solamargine (1924) from nightshade, poisons from Amanita (286), products from herbaceous vines (1427), a uridylic acid-like substance from flowers (2244), chloro1665). Invertebrates. Substances of inplastin (1482), chlorophyll derivatives (lOl4), plant organelles (549, 821 , sect origin t h a t have been analyzed 8 6 4 , and heavy metals in plant mateelectrophoretically include hemoglobin rial (1062). Biochemical substances (SOO),hemolymph proteins (1259, 1260, 1548, 1988, 2085) and amino acids (1565), humic compounds (566), and nitrogenous material (2557) have been (2078), soluble body proteins (522, 957, isolated from soil. Humic acids are 1659, 21 14-21 l a ) , phosphatases (1 54, found in soft coal (1187). 156, 755) esterases (85, 155, 433, 679, Electrophoretic applications to fungi 1487,1964), and nitrogen compounds in include separating the proteins of corn royal jelly (994). Other studies involve smut (243) and the amino acids of crustacean blood proteins (471, 2403), hemocyanin (194),and digestiveenzymes actinomycetes (2072) and a system for identifying species of Pythium (407). (506); molluscan blood and egg (%$SO), Microorganisms. Substances isotissue (1@O), hemocyanin ( I 627), axolated from microbes include toxins plasm (967), and stile (987) proteins; and toxoids from clostridia (871, 2336), annelid hemoglobin (1879), roundworm proteins (901, 1675), and acid phosstaphylococci (lodo), corynebacteria (1766,2034,2035),and pertussis vaccine phatase in flukes (1361). Taxonomy. Electrophoresis, par(1134); Vi antigens (577, 1148, 1261, 2211) and antigens from streptococci ticularly immunoelectrophoresis, is (1647, 1748, 2418), mycobacteria (16, becoming increasingly important as a 1086, 1623, 1811 ) , Pasteurella (538, tool in taxonomy. Both relation2239), Rickettsia (1806), and Erysipeloships and unsuspected differences thriz (2261); allergens from Brucella may appear in electrophoregrams. (254); hemolysins from streptococci Groups that have been studied in this (757), staphylococci (1108, 1135), and way include sea cucumbers (1,404, Listeria (758); proteins (559, 651, 1825, mollusks and crustaceans (496), mol2519, 2565) and colicins (1408) from lusks (387), fish (1593, 1946, 2271), coliform organisms; nucleotide-bound amphibia (886),cerebral proteins of fish, peptides (118), cell wall components frog, reptile, rat, and ox (2025), serum proteins of fish, crustacea, and insects (1006), and enzymes (350) from myco(1664), and lens proteins of various bacteria; and antibodies from Serratia animals (260,1397,2269). (1 791). ,Methods have been suggested for differentiating the pleuropneumoniaPlants. Among plant proteins t h a t like organisms by electrophoresis (688); have been fractionated are those from wheat (171, 4~6,592,632,757,758,1039, the effects on cell mobility of ionizing 11 12, 115~,1~85,1286,1485,1675,16S5, radiation and p H (72l), of antibody binding (908), and of drugs (713) have 2090,2138,2428), rye (1161, 1229, 1761 , 1762, 2076, 2358, 2359), barley (77, 695, been studied. Viruses (59, 1540, 1915, 798, 909, 1084,1085, 11 58,1786, 6559), 2229, 2424) and bacteriophages (301, rice (102, 950, 2071), oats (2560), corn 302) have been studied electrophoretgerm (S5), corn and sorghum (2268), ically. beans (179,1005,1207,1689,1799,1977, Extracts of paramecia (749), leptotain to chicken egg proteins from yolks (1555, 1554, 1822,2073) and whites (162, 1292, 1475, 1541, 1598, l715), and the effects upon them of storage (958, 1498, 1970). Egg proteins from birds of different species have been compared (114, 408, 2067, 2253). Only one example of electrophoretic studies on the serum of reptiles, Iranian snakes ( l 2 7 l ) ,could be located; investigations of snake venoms are mentioned elsewhere. Amphibia are better represented with studies on serum (428,2588), muscle (20%) , hemoglobin (105, 860), and embryonic tissue (2052) of frogs, serum (195,371) and hemoglobin (1828) of toads, and serum of salamanders (732). There is a comparative study of pteridine distribution in the skin, of amphibia (108). The blood proteins of fish (120, 129, 683, 1129, 1359, 1754), including perch hemoglobin (399), eel serum (554, 653, 654, 2225), and salmon serum (1216, 1656), have been investigated, as have fish muscle proteins (115, 1704, 1705, 2870), lamprey collagen (175S), salmon pituitary hormones (1999), and fish egg proteins (999, 1037,
VOL. 38, NO. 5 , APRIL 1966
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spira (1864), and trypanosomes (1900) have been analyzed for proteins, and free amino acids have been determined in ciliates (2331). There are studies of the electrophoretic mobility of trypanosomes (935) and Entamoeba invadens
(2223). Enzymes. The enormous volume of literature concerned with enzyme electrophoresis cannot be treated adequately in this review. iis an alternative to omitting valuable material, citations are grouped without comment under appropriate key phrases: Amylases in human blood (857, 2234, 2365, 2408), human blood and urine (491), human blood and tissue (2353), human and animal serum (185), rabbit serum (184), human saliva (1587), saliva and pancreatic juice (1643), saliva and gastric juice (916); electrophoresis increases activity of amylase (2224); aspergillus amylase, dextrase, and protease (1684) ; aspergillus (1446) and streptomyces (596) cellulase; pancreatic glycosidase and proteases (2292), apricot glycosidase (2045),E . coli betagalactosidase (68), bovine liver betaglucuronidase (1785); beef tissue hetaglucuronidase, hyaluronidase, and uricase (1497); influenza virus sialidase (2028); lysozyme (1518) ; hexokinase (1911); phosphohexose isomerase (906); fish muscle enolase (2272); isocitrate lyase (1027) ; Pepsinogen (124, 378, 941, 942, 1245, 1489, 1922, 2088), streptokinase (520), trypsin (531, 976), chymotrypsin (604); trypsin-inhibiting substances in serum (601, 888, 1276, 2321) and in soybeans (2180) ; rennin purification (1729) and mode of action (1250,2267,2445),antirenninfactorin swine serum (1661-1663), action of pancreatin on casein (566), isolation of elastases (141 5 ) , aspergillus protease (162, 183), B. subtilis protease (1867), bromelin (368) ; leucine amidopeptidase in human serum (645) and tissue (1695), cattle leucine aminopeptidase (1219), rat aminopeptidases (1538, 1724), human serum angiotensinase ( l l 3 7 ) , human placenta peptidase (24829, E . coli mucopeptidases (1738), asparaginase in rat livers (1961, 2196), locating asparaginase during continuous electrophoresis (2328); Esterases in human serum (2360), human liver (573), human organs ( 6 ) , human cell cultures (1169), mammals (448, 611), rats (447, 600, 1874), mice (38, 704, 1771), duck serum (1065), and houseflies (84); human brain esterases and phosphatases in multiple sclerosis (132), human gastrointestinal esterases, phosphatases, and lactic dehydrogenase in normals and in sprue (2385), fungus esterases and phosphatases (14.99); esterase electrophoresis discussed (972); demonstrating esterase inhibition by organic pesticides (1836); carboxylic esterases in cirrhosis (2017) and in horse, donkey, and mule (1066); cholinester-
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ANALYlICAL CHEMISTRY
ases in man (1267) and in horses (1670, 2173); arylesterases in man (2407), in mammals and man (91), move with prealbumin (2331); atropine esterase is a sialoprotein (1409); phosphatases in and human lymph (1109),organs (E@), intestine (1558, 1559), calf intestine (164) and rat embryo (1028); alkaline phosphatases in yeast (460) and in serum of sheep (18/t5), chickens (1071), and diseased humans (824, 852, 1016, 1611,2205); acid phosphatase detection in prostatic fluid (2058), differentiation between seminal and vaginal (63),associated with liver mitochondria and lysosomes (37), and in Tetrahymena (39); insect inorganic pyrophosphatase (1256) ; creatine kinases (335, 503, 1892), pyruvate kinases (633, 634), nucleases (209, 359, 1312, 1593, 2069), glucose-ATP phosphotrnnsferase (1loo), and phosphoglucomutase (2107); Lactic acid dehydrogenase in serum (907, 1222, 1253, 1944), erythrocytes (616, 526, 1605, 2119), and tissues (362, 594, 1847, 2324, 2405), in muscle disease (317,693,1279),tumors (972,745,2095), sprue (105),myocardial infarction (661), rheumatic disease (417 ), various diseases (1059), and experimental hepatic lesions (373),in mammals (262,263,912, 1152, 1438, 1808, 1892) and goldfish (924), and compared among vertebrates (261); method for lactic acid dehydrogenase (774) and problems of measurement (1849); lactic acid and malic acid dehydrogenases (773,1079, 1946, 2404) ; malate dehydrogenase in tissues (1058, 1974), neonates (514), and sea urchins (207); glutamate dehydrogenases from various sources (2192) and in higher plants (2230); glucose-6-phosphate dehydrogenases (660, 1128, 15.42, 2036, 2053, 2456) and alcohol dehydrogenase (810, 1464, 1465, 1711); uricase (2327); drosophila xanthine oxidase (765); Straub’s diaphorase (2123), homogentisic acid oxidase (2365), mushroom tyrosinase ( l o % ) , potato phenolase (1723), galactose oxidase and neuramidase (1877);catalases from animals (938, 1140,1637) and plants (160),peroxidases and catalases from animal and vegetable sources (2330), horseradish peroxidase (394, 1136), and ceruloplasmins from various animals (141 2) ; Glutamic-oxalacetic transaminase in tissue (1181, 1886), reticulocytes and erythrocytes (6138), and rheumatic heart disease (188) ; glutamic-aspartic transaminase in tissues (438, 1423) and in disease (1447); ornithine carbamoyltransferase (2329); Carbonic anhydrase (652, 1505, 1863, 2202) ; thiaminase (571); and various kinds of isoenzymes in human cell cultures (158). Hormones. The fractionating power of electrophoresis in conjunction with the specificity of immunological methods and the sensitive measurements possible with radio-
isotope labeling have made accurate assays of the protein hormones practical a t concentrations normally associated with biological material. This achievement should signal a new era in endocrinology. Pituitary hormones, including growth hormone (7, 481, 670, 1258,1265,1266,1305,1800,1860,2361), prolactin and growth hormone (366), prolactin (1267), adrenocorticotrophic hormone (8439), follicle stimulating hormone (1467, 1976), luteinizing hormone (502), and pituitary (and chorionic) gonadotropins (983), have been studied. It has been suggested that electrophoretically plural fractions of pituitary hormones may be artifacts (2089). There are papers describing the electrophoretic behavior of insulin (361,632, 733, 1703, 1782, 2212, 2213) as studied by I3lI tagging (468, l o o f , 1213, 1488, 1522, 2438) and as affected by immunities (326, 327). Tris buffer causes insulin to dissociate from globulins (700). From 1 3 1 1 studies, glucagon does not appear to be associated with any serum protein (1487). Both thyroxine (44, 86, 1429, 1860, 2250) and triiodothyronine (303, 316 , 1523, 1788) are bound to proteins; thyroxine and triiodothyronine can be separated from each other (213) and from iodinated organic compounds used for x-ray contrast (2279). Detergents cause thyroid hormones to dissociate from serum proteins (l624). Parathyroid hormone-like activity has been detected in prealbumin (1291); bovine parathyroid hormone is antigenic to rabbits (2414); hot 0.251HC1 affects the mobility but not the activity of parathyroid hormone (2203). Steroid hormones migrate best a t a high p H (225); free and conjugated steroid hormones can be separated on paper and the fractions located with tetrazolium (366, 367) ; conjugated steroids can be fractionated with high-voltage electrophoresis using paper, and the fractions located by ultraviolet light and spot tests (1183). There is an electrophoretic way to measure the corticosteroid-binding capacity of globulin (1690). Niscellaneous hormones that have been investigated include placental hormones (45, 1045, 1360, 1672, 1962), adrenaline (25), catecholamines (626, 1482), urogastrone (437), and nerve growth factor (1986). Biochemicals. There has been a marked increase in work relating to the separation and preparation of the nucleic acids (548, 588, 1673), deoxyribonucleic acid (430, 574, 1106, 1584, 1897-1899, 2030, 2183) and ribonucleic acid (104, 903, 968, 1323, 1324, lS32, 1354, 1859, 1860, 2246, 2416, 2462), as well as nucleotides (276, 415, 1710) and purines and pyrimidines (1082). A number of papers report fractionations
of histones (165, 166, 533, 1147, 1369, 1454, 161 8 , 1674, 1683). Electrophoresis has proved useful in examining the biochemistry of vitamins Be (SO) and ljI2 (187, 1087-1094, 2186, 2194), thiamine (1909), biotin (2301), and the flarinoids (70, 4 3 0 , and for analyzing vitamin mixtures (376). Studies of miscellaneous biochemical substances include polysaccharides (1268, 1651), sugars (211,638,1363,1468,1592,1685), amine sugars (1127,1666--1668),heparin (127, 1010), amino acids (690, 1007, l36& 1517, 1668, 1968, 1969, 2469), and peptides (817, 1826, 1875, 2009). h method for preparing radioactive phosphate esters from plant tissues has been suggested (203). Applications to Pharmacology and Toxicology. Drugs can be introduced into limited body sites by electrophoretic action, a process t h a t is called iontophoresis (135,235,236,292, 465, 489, 617, 1022, 1224, 1744, 2190, 2159). Drug substances investigated by electrophoresis include alkaloids (141, 42S, 988, 1394, I d l o , 1511, 1617, 1713, 1959,2015,2419),antibiotics (521,lSl4, 1768, 1793, 1942), hypnotics (2247), cardenolide glycosides (2484),the coloring matter of ergot (%), and various drugs (499). There has been an unusual number of papers on venoms from snakes (69, 295, 377, 483, 555, 1018, 1019, 1284, 1301, 1437, 1474, 1552, 2376), toads (2391), salamanders (256), hymenoptera (848,1895),and arachnids (525, 710, 1679, S4Ol). Toxicological applications include methods for detecting organophosphorus insecticides (323),identifying arsenicals (1695), detecting polyphosphates (2429), determining inorganic poisons (636, 637), and tests to establish time of death (716, 1290, 2098). References to the effects of poisoning upon the proteins of serum have been cited in the section on Human Serum. GENERAL CHEMICAL APPLICATIONS
Apparatus for and biological applications of particle electrophoresis have already been described. The attention of the reader is directed especially to the last paragraph of the section on Fundamental Developments. This rapidly developing new method has been used to study the behavior of suspensions of asphaltenes (583), polystyrene particles (1444, 1976),styrene-acrylic copolymers (1445), poly(sodium acrylate) (1646), ion exchange resin ( r o d ) , polyethylene (1131), octadecanol (934),and inorganic colloids or suspensions (198, 199, 970, 1070, 1241, 2237). A general system for identifying organic compounds by electrophoresis is being developed (692, 693) ; organic compounds that have been fractionated include caprolactam polymers (1902), dyes (62, 453, 1327, 1632, 2219,2220),acyl hydrazides (1926),keto acid 2,4-dinitrohydrazones (1765), car-
bamates (691), diphenylamino acids (15?7), and caramels (2260). Detailed studies of substances used in photographic preparations are available (2160-21?2, 2326). Several systems have been proposed for separating inorganic ions (819, 1531, 2191, 2343), including alkali metals (181, lZ42, 2029, 2064, 2065), calcium and strontium (2066), copper and cadmium (205), selenium and tellurium (2266), molybdenum and rhenium (24.55),rare earths (23, 778, 835, 836, 1132, 1149), bismuth from other cations (1572), auric ions from colloidal gold-198 (168), thorium, protactinium, and uranium (1490), and decay products of tellurium-132 (2057). X method for measuring cobalt has been described (2031). Electrophoresis has been used t o investigate the chemistry of organometallic compounds (759), germaiiiuni compounds with the brown material of soft coal (2096), polythionates (767), and of metallic complexes, including those of chromium with acetate (2060), lead-210, bismuth-210, and polonium-210 in solution (2055), technetium and rhenium (2063), gallium and indium ( I l O ) , chromium (l234), mercury (2056), uranium (1251), germanium (687), iron (1432), iron and cobalt (2338),cobalt (226, 1908), and rhenium, osmium, iridium, and platinum (228, 229). Complexing agents are useful for cation separations (375); electrophoretic properties of complexing agents have been investigated (217,lO32-lO34, 1467). Csing ion exchange substances as supporting media for the electrophoresis of ions yields very sharp sample zones (7b3, 754, 1452), a phenomenon that is often described as “ion focusing.” Fused salts have been used as the liquid phase for separating isotopes (1479), cations ( S l ) , uranyl salts (103), and thiocyanate complexes (109). Molten sulfur can be freed from carbon impurities by electrophoresis (631). Industrial uses of electrophoresis are increasing in scope. Some examples are the application of coatings (96, 97, 274, 315, 329, 539, 589, 684,696, 742, 766, 768, 769, 1688, 1756, 1852, $040, ZWOd), deposition of strontium titanate (2336), dyeing (2197), printing (1692), condensation of pastes (1390), purification of water (289, 624, 2440), and analyses for quality control (21, 28, 29, 221, 500,
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16.579f.
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