University of Southern California Dissertations and Theses

Chemical, physicochemical, and biological studies on the mucoproteins of plasma and serum

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Chemical, physicochemical, and biological studies on the mucoproteins of plasma and serum

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Content CHEMICAL, PHYSICOCHEMICAL, AND BIOLOGICAL STUDIES ON THE MUCOPROTEINS OP PLASMA AND SERUM A Dissertation Presented to the Faculty of the Department of Biochemistry and Nutrition, the University of Southern California In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy by Henry Eben Weimer June 1950 This dissertation, written by _...... under the guidance of h.%3... F a cu lty Com m ittee on Studies, and approved by a ll its members, has been presented to and accepted by the C o u n cil on Graduate Study and Research, in p a rtia l fu l fillm e n t of requirements fo r the degree of D O C T O R O F P H I L O S O P H Y Date^^yL^Ai ^ . .. ....... Committee on Studies ACKNOWLEDGMENTS I wish to express my gratitude to Dr. Eloise Jameson Tor the most generous use of her electrophoresis apparatus and to Hyland Laboratories, Los Angeles, Califor nia, for the donation of human plasma. TABLE OF CONTENTS CHAPTER PAGE I. INTRODUCTION . . . . . . . . . . . . . . . . 1 II. REVIEW OF THE LITERATURE.................... 5 Current nomenclature of carbohydrate-rich proteins occurring In normal plasma . . . 3 Investigations related to the isolation and characterization of carbohydrate-rich proteins from plasma or serum ...... 9 Studies dealing with serum polysaccharide levels In normal and pathological states 20 Theories concerning the physiological source and significance of the mueo- proteins of plasma • »••••••••• 25 III. MATERIALS AND METHODS........................ 33 Plasma and serum ••••••••••••• 33 Preparations studied ••••••••••• 34 Mucoprotelns •••.•••••••••• 34 Seromucold • • •••••• ............. 36 Mucoidahnllche Substanz ••••...«• 36 Chemical analyses ............... 37 Carbohydrate determinations .. ......... 37 Nitrogen determinations............ 37 Lipid determination • •«.•••••• 37 Ill CHAPTER PAGE Hexosamine determination ••••*••• 37 Tyrosine determination • ••*••••• 38 Determination of hexuronic acids • • • • 38 Moisture determinations • •••••••• 38 Determination of ash •••••••••• 38 Amide nitrogen determinations.......... 38 Total protein determinations • ••••• 39 Phosphorus determinations 39 Sulfur determinations...........* . • • 39 Amino acid analysis................... 39 Electrophoretic studies • .............. • • 39 Turnover studies » « • • • ............... 41 IV. EXPERIMENTAL................................ 42 Characterization of a homogeneous mucoprotein from human plasma ......... 42 Isolation............. 42 Chemical composition ••••••»•.• 44 Amino acid composition ............. 46 Molecular weight from amino acid content 49 Physicochemical characterization • • • • 53 Electrophoretic studies ••..«••• 53 Determination of partial specific volume 58 Sedimentation and diffusion constants • 60 iv CHAPTER PAGE Determination of molecular weight * . 60 Viscosity studies............. . . . 63 Frictional coefficient **••*•*, 66 Approximate dimensions of molecule • • 69 Rate of turnover studies............... 71 Comparative studies * • 74 A comparative study of carbohydrate-rich protein fractions isolated from human plasma by different methods ......... 75 A comparative study of carbohydrate-rich protein fractions isolated from the sera of five animal species • •»*•*•• 86 Demonstration of mucoprotein in normal and malignant tissue • ................. . . 94 Electrophoretic demonstration of an acidic protein in two strains of tumor-bearing rats...........* • • • • * • .......... 100 Electrophoretic demonstration of human plasma mucoprotein, MP-1, in human um bilical cord serum • 104 Effect of buffer anion upon electrophoretic mobility in the pH range, 2*0 - 4*0 . . 107 Studies pertaining to the MP-2 component * 109 V CHAPTER PAGE Experiment 1 . . . . . . . . . . . . . . 112 Experiment I I . . . . . . . . . . . . . . 113 Experiment III . . . . . . . . . . . . . 113 V. DISCUSSION................................... 119 VI. SUMMARY AND CONCLUSIONS...................... 137 BIBLIOGRAPHY......................................... 142 LIST OP TABLES TABLE PAGE I# Chemical Composition of Human Plasma B&ico- protein (MP-1) • ••••••••••,• 45 II. Amino Acid Composition of Human Plasma Mucoprotein (MP-1) and Human Plasma Albumin • 47 III, Molecular weight of Human Plasma Mucoprotein (MP-1) From Amino Acid Content • • • • • 51 IV, Summation of an Electrophoretic Investigation of Human Plasma Mucoprotein MP-1 , , • • 54 V, Summation of a Partial Specific Volume Deter mination of Human Plasma Mucoprotein (MP-1)............................ 61 VI, Relative Viscosity of Human Plasma Muco protein (MP-1) ••,•••••••«•• 64 % VII, Summation of Data Employed In the Determina tion of the Intrinsic Viscosity of Human Plasma Mucoprotein (MP-1) •••••••• 67 VIII, Summary of a Rate of Turnover Study of Serum Protein Fractions of Rats Administered C^4 Methyl-labeled Glycine •*,,••• 73 IX. Comparative Study of Carbohydrate-rich Protein Fractions Isolated From Human Plasma by vii Six Different Procedures............. 76 X. Summary of an Electrophoretic Investigation of Carbohydrate-Rich Protein Fractions Isolated from Human Plasma By Six Different Procedures * . ......... • •••••• 77 XI* Comparative Study of Mucoproteins Isolated from the Sera of Five Animal Species • 88 XII* Summary of an Electrophoretic Investigation of Mucoproteins Isolated from the Sera of Five Animal Species •••••••• 89 XIII. Comparative Study of Carbohydrate-Rich Protein Fractions Isolated From Tissues 97 XIV* Summary of an Electrophoretic Investigation of Carbohydrate-Rich Protein Fractions Isolated From Tissues ••••*•••• 98 XV* Comparative Electrophoretic Mobilities of Acidic Protein Components in Rat Serum and Human Plasma ••••••••••• 103 XVI. Effect of Buffer Anion Upon the Electro phoretic Mobility of Human Plasma Muco protein (MP-1) in the pH Range 2.0-4.0 108 XVII. Summation of Electrophoretic Studies Per taining to Human Plasma Mucoprotein MP-2 . ................................ 116 viii TABLE PAGE XVIII. Protein Components of Normal Human Plasma Characterized by Physicochemical Methods 121 XIX. A Comparison of Human Plasma Mucoprotein, MP-1, with Ovomucoid •••«.•••• 124 XX. Amino Acid Composition of Human Plasma Proteins •••••••• ........... . 126 XXI. A Comparative Study of Carbohydrate-Kich Fractions Isolated from Plasma and Serum ••••••••••«••••.• 151 XXII. Comparison of Two Human Plasma Mucoprotein Preparations Isolated by Procedure B • 133 LIST OP FIGURES FIGURE PAGE 1# Isolation Procedure for Human Plasma Muco protein (MP-1) ••••••••••••• 43 2# Electrophoretic Patterns of Human Plasma Mucoprotein (MP-1) at Various pH values * 56 3* Mobility of Human Plasma Mucoprotein (MP-1) as a Function of pH •••••••••• 57 4* Electrophoretic Patterns of Descending Boundaries of Normal Human Plasma, and of Normal Human Plasma with Added Mucoprotein at pH 8.6 ••••••••••••••• 59 5. Plot of m versus •••••••••«•• 62 6• Plot of n/no versus C.* • • • • • • « • • • 65 7. Graphical Determination of the Intrinsic Viscosity of Human Plasma Mucoprotein (MP-1)................................. 68 8. Electrophoretic Patterns of Mucoprotein (MP-1) and Mucoidahnliche Substans • • • 80 9. Electrophoretic Patterns of Mucoprotein Preparations (PCA-AS) and (PCA-PTA) • • • 81 10* Electrophoretic Patterns of Seromucoid and Mucoproteins (PCA-D-PTA) . * ........... 82 X FIGURE PAGE 11* Electrophoretic Patterns of Human Plasma Mucoprotein and Guinea Pig Serum Mucoprotein 90 12* Electrophoretic Patterns of Beef and Rat Serum Mucoprotein • ••••••«••••• 91 15. Electrophoretic Patterns of Carbohydrate- Rich Protein Fractions Isolated From Normal and Malignant Tissues • •••«•••••• 99 14. Electrophoretic Patterns of the Serum of Two Strains of Tumor-Bearing Rats at pH 4.0 • • 102 15. Electrophoretic Patterns of Human Umbilical Cord Serum at pH 4.5 and pH 7.4. • • • » • 106 16. Electrophoretic Patterns of Mucoprotein Preparations Isolated In Experiment I . . • 111 17. Electrophoretic Patterns of Mucoproteln Preparations Isolated in Experiments II and III . .................................. 114 CHAPTER I INTRODUCTION The presence of carbohydrate-rich proteins in plasma and serum has been demonstrated by many investigators who have studied the plasma proteins* These conjugated proteins are characterized by a relatively high content of hexose and of hexosamine and a relatively low nitrogen content* They are more soluble than the major protein constituents of plasma when "salted out11, and In general do not coagulate on being heated in aqueous solution* The isolated proteins have been designated as polypeptides, peptones, proteose, seronrucoid, seroglycoid, mucoidahnliche Substanz, Tiergummi, and muco protein by various workers and may be considered as represen tatives of the general class, glycoproteins* They have been isolated from human, horse, beef, rat, chicken, dog, rabbit, and guinea pig plasma and serum* Chemical analysis and solubility in various solvents are the principal criteria that have been employed to charac terize these materials in the past* Data obtained by these methods provide no Indication of the homogeneity of the isolated material since a contaminant may be present in constant amounts* The employment of electrophoretic or ultracentrifugal analysis affords a procedure for the deter mination of the degree of homogeneity of an isolated glyco- 2 protein. Data obtained from chemical and physicochemical studies of a homogeneous material is obviously of greater significance than that obtained from studies carried out on a mixture of proteins * Recent interest in these compounds has arisen from the fact that the serum mucoprotein level rises markedly in patients with cancer, with myocardial Infarctions, and with the pyogenle Infections, pneumonia and tuberculosis. This investigation was undertaken for the purpose of further elucidating the nature of mucoprotelns by chemical, physlcochemleal and biological studies. CHAPTER II REVIEW OF THE LITERATURE Due to the wide scope of the present Investigation this section of the dissertation will be presented under the following headings: Current nomenclature of carbohydrate- rich proteins occurring in normal plasma; Investigations related to the isolation and characterization of carbohydrate- rich proteins from plasma or serum; Studies dealing with serum polysaccharide levels in normal and pathological states Theories concerning the physiological source and significance of the mucoprotelns of plasma* CURRENT NOMENCLATURE OF CARBOHYDRATE-RICH PROTEINS OCCURRING IN NORMAL PLASMA The classlfication of proteins that is most generally accepted was originally formulated and recommended by the American Physiological Society and the American Society of Biochemists (Committee on Protein Nomenclature, 1908)* In this classification the carbohydrate-rich proteins of plasma would be Included In the subclasalficatlon, glycoproteins, under the general class, conjugated proteins* Glycoproteins are, by definition, Compounds of the protein molecule with a substance or substances containing a carbohydrate group <*• 4 other than a nucleic acid*n At the present time no further definition exists for this class of compounds and there is no accepted nomenclature* As Meyer (1945) stated, WA re viewer of mucopolysaccharides, mucoids, and glycoproteins is faced with the problem of giving hie own definitions and classlfications,since in this field, unlike the general field of proteins, there is no accepted terminology*w Levene (1925) considered mucoprotelns to be conjugated with a carbohydrate prosthetic group* He stated that, t tthe carbohydrate group of all mucoprotelns is conjugated with sulfuric acid* The group is built of four components in equlmolecular proportions* The components are sulfuric acid, acetic acid, hexosamine and glucuronic acid*” The muco protelns isolated from various tissue sources were thought to differ only in respect to the protein moiety* Blix (1940) proposed the following chemical classifi cation of glycoproteins* 1* Neutro glycoproteins. Glycoproteins containing a neutral polysaccharide, e*g*, ovomucoid and seromucold* 2. Acldo glycoproteins* Glycoproteins containing an acid polysaccharide* a* Chondroproteins* Contain chondroitin sul furic acid, e.g., chondromucoid. 5 b* Hyaloproteins* Contain hyaluronic acid* c* Sialoproteina. Contain the acid polysaccharide present in submaxlllary mucin* d* Mucoprotelns* This term should be reserved for compounds between mucoitin sulfuric acid and protein* A wide variety of nomenclature has been employed in designating the carbohydrate-rich protein fractions Isolated from serum and plasma* The first term to find wide acceptance was "seromucold*” This name was employed by Zannetti (1897, 1905} to designate a carbohydrate-rich protein Isolated from beef serum* His choice of terminology was based on the similarity of his Isolated material to ovomucoid with respect to carbohydrate content, to solubility, and to method of iso lation* The term wseromucoidt t has also been employed by Bywaters (1909), Rlmlngton (1940), Rimington and Van Den Ende (1940), Jayle and Judas (1946), Staub and Rlmlngton (1948), McCrea (1948), and Jacobs (1949) in referring to similar fractions* Hewitt (1957b, 1939) referred to a carbohydrate-rich protein fraction isolated from the sera of several animal species as "seroglycoid** The chemical data reported for his "seroglycoid* preparations are very similar to those reported by Rimington (1940) for ffseromucoid•f , 6 Mayer (1942) Isolated two carbohydrate-rich protein fractions with similar properties from human and horse serum. He designated these materials as "raucoidfihnliche Substanz" because of the high sulfur, carbohydrate, and glucosamine contents. Winzler and Burk (1944) employed the term "proteose1 1 to designate non-heat-coagulable, sulfosalicylic acid-soluble, non-dialyzable, protein precipitable by saturated ammonium sulfate that they Isolated from rat blood. Their "proteose" fraction had a relatively high carbohydrate content and con tained polarographically demonstrable cystine. The term "proteose" was also used by Vassal, Partridge, and Crossley (1947) in referring to a similar fraction isolated from dog serum. Meyer (1945) in his review entitled "Mucoids and Glycoproteins" limited his discussion to compounds containing hexosamine. He classified hexosamine-containing compounds into three main groups, the mucopolysaccharides, the mucoids, and the glycoproteins. The following definitions were given, "As mucopolysaccharides we define polysaccharides which contain hexosamine as one component, whether they occur free or whether they be obtained by chemical manipulation from substances of higher molecular weight." "As mucoids we define substances which contain a mucopolysaccharide in firm chemical union with a peptide where the hexosamine content 7 is greater than 4 per cent* The group of proteins which contain less than 4 per cent hexosamine, classified as glyco proteins, embraces many proteins listed as albumins and globulins in the accepted classification of proteins." Meyer emphasizes that, "The distinction between mucoids and glycoproteins based on a hexosamine content of 4 per cent or over is arbitrary. This figure is chosen since compounds with such a hexosamine content possess the solubility pro perties of mucoids and remain soluble after precipitation by alcohol.M Stacey (1946) defined mucoprotelns as, "protein- carbohydrate compounds with relatively high protein or peptide content, the chemical reactions of which are pre dominantly protein.f t He adds, win general they do not coagulate on being heated In aqueous solution. All muco- proteins contain a hexosamine constituent.w Stacey points out that, "the most radical change of classification in this group is the abandonment of Levene’s (1925) definition that the carbohydrate group of all mucoprotelns is conjugated with sulfuric acid.1 * The recent work of Weimer, Mehl and Winzler (1950) indicates that Levene's (1925) assumption may have been correct, at least with respect to plasma mucoprotein* Since the publication of Stacey's review the term , , mucoprotein, , has been employed by several Investigators in the field to compounds with the properties described by Stacey. Winzler and Mehl and their collaborators, (1948, 1949, 1950), Simkin, Bergmann, and Prinzmetal (1949) have designated carbohydrate-rich protein fractions Isolated from human plasma as "mucoprotelns." Fredericq and Deutsch (1949) refer to a purified ovomucoid preparation as a "mucoprotein." Surgenor and coworkers (1949) have designated an oc fraction which does not meet the solubility requirements of Stacey’s definition, as a mucoprotein* They state, "Muco protelns have been characterized by euglobulln properties, high viscosity in solution and a tacky behavior when pre cipitated* Glycoproteins have often appeared to be pseudo globulins resembling the albumins in their solubility proper ties*" Cohn, et al, (1950) have announced the isolation of three carbohydrate-containing proteins from human plasma which they have designated as <£ g-mucoprotein, cfg-glyeo- protein, and OCi-lipid-free glycoprotein. No information has been presented as to their choice of nomenclature other than electrophoretic mobility* It is obvious from the foregoing discussion that the terminology pertaining to the carbohydrate-rich proteins of serum is quite arbitrary at the present time* The term "glycoprotein" seems quite adequate as a generic name for this type of conjugated protein or for undefined mixtures* However, with the Isolation and characterization of discrete 9 chemical entitles many of the designations applied In the past appear to he Inadequate, If not misleading* For example, the use of the term t t mucoidw would Imply mucin-like proper ties Including a high viscosity when in solution* This is not confirmed by the data of Fredericq and Deutsch (1949) for ovomucoid or by the data of Smith, et al*(195Q) for human plasma mucoprotein* The time seems to be propitious for the appointment of a new committee to consider the revision of protein nomenclature* The rapid development in isolation techniques and the availability of modern physicochemical methods for the characterization of proteins emphasize the need for a system of nomenclature more definitive than one based on solubility and chemical analysis* INVESTIGATIONS RELATED TO THE ISOLATION AND CHARACTERI ZATION OF CARBOHYDRATE-RICH PROTEINS FROM PLASMA AND SERUM Serum mucoprotein was first described by Freund (1892) who, however, failed to detect nitrogen in his preparation which he named t , Tiergummi*w It was later investigated by Zannettl (1897, 1903) who gave it the name ^seromucoid*, considering it to be similar to ovomucoid* He found a vari able carbohydrate content of from 3 to 34 per cent in his 10 preparations• Bywaters (1909) studied the material more thoroughly with the purpose of determining whether it served as a means of transport of carbohydrate from the small Intestine to the tissues. The yield of purified material was 0.3 to 0.9 gram from a liter of serum, varying according to the nutri- tlonal state of the animal. The mucoprotein contained 24.3 percent carbohydrate, and a pentabenzoyl glucosamine deriva tive was obtained from an acid hydrolysate. All of the above early workers Isolated mucoprotein by alcohol precipitation from the filtrates of serum from which proteins had been removed by heat coagulation. Lustig and Haas (1931) isolated 11 fractions from beef serum by ammonium sulfate fractionation and fractional ex traction with different solvents. Carbohydrate determinations were made employing the orclnol reaction. Subfraetlons of euglobulin and pseudoglobulin were found to have the highest carbohydrate content, 6.4 - 8.5 per cent. Albumin subfrac tions were lowest in carbohydrate, 0.47 - 0.65 per cent. Ozakl (1936) has described the preparation of a muco protein by a method similar to that used by Bywaters, which contained 13.4 per cent reducing sugar, calculated as glucose, after hydrolysis. Nilsson (1937) determined the glucosamine content of 11 the globulin, albumin, and seromucold fractions of healthy Individuals and of patients with pneumonia. Average values of 2*IB per cent, 0.52 per cent, and 13 mg. per cent respec tively were obtained with healthy Individuals and 1.38 per cent, 3*33 per cent, and 8 mg. per cent respectively were obtained with pneumonia patients. Hewitt (1937a, 1937b, 1939) precipitated a glyco protein which he called "seroglycoid* from an acidified filtrate of serum by the addition of acetone, after having first removed successive fractions of globulin and albumin by precipitation with ammonium sulfate. Following a brief treatment of "seroglycoid* with pepsin, he isolated a partial breakdown product with the same properties as those ascribed to ftserum mucoid.* He suggested that this latter product, which he could not detect in normal serum, was produced by disaggregation of "seroglycoid.* A complete review of the problem was undertaken by Rimington (1940), who developed an Improved method for iso lation leading to a main fraction of *seroraucoidn with uniform properties. Working with ox serum, Rimington (1940) removed albumins and globulins by reducing the pH of the serum to 4.7 with acetic acid and heating on a steam bath. The fil trate was concentrated in vacuo at 50<>c. and "seromucold* was precipitated by the addition of 10 volumes of aleohol. 12 Els main fraction contained 10.7 per cent hexose calculated as galactose-mannose, 5.61 per cent glucosamine, and 13.6 per cent nitrogen. Rimington and Van Den Ende (1940) compared the globo- glycoid, crystalbumin, seroglycoid, and seromucoid fractions of normal horse serum chemically and imraunologically by the Schultz-Dale reaction. They concluded that: 1. Crystalbumin and globoglycoid are identical or closely similar. 2. Seroglycoid differs quantitatively from crystal- bumin in its carbohydrate and amino acid content. 3. Seroglycoid and seromucoid, although both rich in bound carbohydrate, differ from one another when compared by immunological methods. Blix, Tiselius and Svensson (1941) determined the lipid and polysaccharide content of electrophoretically separated blood serum proteins. All fractions were found to contain carbohydrate with the cC - and j3 -globulins having the highest percentage. Mayer (1942) obtained mucoprotein from dialyzed sulfo- salicylic acid filtrates of horse serum by fractionation with alcohol. He referred to his preparation as a wmucoidahnliche Substanz1 * and found the isoelectric point (minimum solubility) to be at pH 3.4. 13 Winzler and Burk (1944) Isolated a similar material from rat blood by lyophilizlng or precipitating with alcohol the non-dlalyzable residue from sulfosallcyllc acid filtrates. They found their preparations to be polarographically active and concluded that the Isolated material was Identical with that giving ”the index of polypeptidemia*1 of earlier French workers (Crlstol and Puech, 1926} as well as the ”polaro- graphic filtrate wave” of Brdleka, Novak and Klumpar (1939). Jayle and Judas (1946) in an investigation of the glycoproteins of human plasma employed an equlmolar mixture of potassium acid phosphate and potassium basic phosphate as a fractionating agent* They Isolated three carbohydrate- rich protein fractions* The first fraction which precipitated at a concentration of 0*75 molar was designated as mucoid F* They postulated that this fraction was detached from the fibrinogen molecule at the moment of coagulation* The second fraction which they considered to be part of the {3 -globulin, was precipitated between 1*7 and 2*45 molar* They believe that this fraction may migrate to the albumin in pathological states> and consider it one of the reasons for the increase of the sugar albumln/sugar globulin ratio in pathology* The third fraction which precipitated between 2*45 molar and 3*0 molar was designated as seromucold. They found its concen tration to be quite constant in normal subjects and to be considerably increased in patients with tuberculosis* 14 Jayle and Abdellatlf (1946) reported the carbohydrate content of a serum fraction designated as haptoglobin to be 10 per cent* The carbohydrate moiety was composed of galac tose, mannose, and N-acetylglucosamine. Vassal, Partridge, and Crossley (1947) fractionated the serum from dogs, before and during Type I pneumococcal pneumonia* They Isolated material with the properties of mucoproteins, which they referred to as proteoses, by ammonium sulfate saturation of the supernatant from the seroglycoid precipitation (Hewitt, 1937a) at pH 6.8. An Increase in "proteose” concentration was found during infection. Slight decreases were found in cysteine and cystine concentrations in the infected animals. Winzler, Devor, Mehl, and Smyth (1946) Isolated muco proteins from human plasma by saturating sulfosallcyllc and perchloric add filtrates of plasma with ammonium sulfate at pH 4.0. The composition of the product obtained was found to be nearly the same regardless of which acid was used as a protein precipitant. Time of storage of plasma was noted to be a factor in the yield obtained, the yield increasing with time of 8torage. Staub and Rimington (1948) carried out constant solubility studies on a "seromucold" fraction obtained from ox serum (Rimington, 1940). They obtained a fraction satis fying the criteria of constant solubility and composition. 15 This fraction was electrophoretically homogeneous at pH 8.0* By successive treatments of "seromucold* with a phenol-alco hol mixture, chloroform, and amyl alcohol, they obtained a fraction richer in carbohydrate than the original preparation* Petermann, Karnovsky and Hogness (1948), by electro phoretic analysis of human plasma at pH 4*0, demonstrated the presence of an acidic protein component with a mobility similar to that of Isolated mucoprotein prepared by the method of Winzler, Devor, Mehl, and Smyth (1948) • They found an Increase in the concentration of this component in patients with cancer and also in patients having certain non-neoplastic diseases* Winzler and Smyth (1948) demonstrated by chemical analysis marked increases in the plasma mucoprotein levels of cancer patients* McCrea (1948) identified the serum inhibitor ("Francis inhibitor") of heated LEE influenza virus as a component of the heat-stable seromucold fraction of rabbit and human sera* Hirst (1949) has objected to this view on the grounds that only 15 per cent of the inhibitory activity of serum could be accounted for by this fraction, and that seromucold was an ill-defined mixture. Cohn in his review (1948) mentions two glycoproteins contained in Fraction IV-6 (Cohn e£ al*, 1946): 16 1. oC -2 glycopseudoglobulln with an Isoelectric point of approximately 4*9. 2• CC -2 mucoid globulin with an isoelectric point of approximately 4.9• These materials were estimated to occur in plasma to the ex- tent of 0*7 gram and 0*5 gram per 100 grams of plasma respec tively. Jacobs (1949) studied the variations in the bound glucosamine of serum mucoid in a wide variety of pathological conditions with special emphasis upon diabetes mellitus* He found the glucosamine levels to be higher for diabetic than for non-diabetic subjects. Of great Interest was finding that the serum mucoid glucosamine levels followed the blood sugar levels and that the glucosamine level fell with the administration of insulin* Garst and Friedgood (1949) Isolated a protein from urine containing 10 per cent nitrogen* The Isolated material gave a positive Mollsch reaction and was considered to be a glycoprotein* It was shown to be effective in stabilizing ether-water emulsions. Mehl, Humphrey, and Winzler (1949) characterized electrophoretically mucoprotein preparations obtained from pooled, normal, human plasma by saturating the perchloric acid filtrate of plasma with ammonium sulfate at pH 4*0* Their preparations consisted of three components designated 17 as MP-1, MP-2, and MP-3* Those components were round to be Isoelectric at pH values of 2*3, 3*4, and 4*3 respectively* Me hi, Golden, and Winzler (1949) were able to demon strate by electrophoresis the presence of two proteins in serum that were negatively charged at pH 4*5* These com ponents which were designated as M-l and M-2 were found to Increase in concentration in the serum of patients with cancer or pneumonia* The electrophoretic mobility of M-l corres ponded to that of MP-1 but the mobility of M-2 did not correspond to that of either MP-2 or MP-3* Glick and coworkers In collaboration with Winzler and Mehl (1949) demonstrated a lack of identity between the hyaluronldase inhibitor of serum and certain mucoprotelns of serum Isolated both by salt fractionation and electrophoretic methods* Employing the procedure developed by Winzler and Smyth (1948), Slmkln, Bergman, and Prinzmetal (1949) investigated the quantitative changes in serum mucoprotein following the occurrence of myocardial infarction* In all but 2 of 23 patients with a proven diagnosis of myocardial infarction there was a definite rise in the serum mucoprotein level* It was found that serial determinations offered more Information than the absolute level at any time after the onset of the patients attack* The Increase in serum mucoprotein above the patients* normal value varied from 22 to 160 mg* per cent* 18 A definite elevation always occurred by the third day after the onset of the attack, and was maintained until the ninth day; thereafter the serum mucoprotein concentration gradually declined approaching normal values at the end of the month# Although the Increase In serum mucoprotein levels Is not specific for this condition, elevated levels being found In patients with pneumonia, with bronchogenic carcinoma, and with postoperative patients, the procedure proved helpful as a diagnostic aid under certain circumstances# Surgenor and coworkers (1949) announced the Isolation of a mucoprotein from Fraction IV-4 by the low temperature, ethanol fractionation procedure (Cohn et al«, 1946)# The product which was 82 per cent <£ -2 globulin showed 3 poorly resolved components In the ultracentrifuge# The principal component, comprising 65 to 75 per cent of the material sedlmented with S * 6 to 9 depending upon the protein eoncen- tration* This mucoprotein was found to be extremely labile under conditions of acid pH and therefore would not seem to be related to the mucoprotelns isolated by other investi gators# (c.f# Winzler, Devor, Mehl, and Smyth, 1948; Welmer Mehl, and Winzler, 1950)# Chemical analysis gave the follow ing values: Hltrogen, 14#5 per cent; hexose, 4*35 per cent; hexosamlne, 2#84 per cent# According to the terminology of Meyer (1945) this material would be classified as a glyco protein due to Its low hexosamlne content# 19 Seibert, Pfaff, and Seibert (1948) determined the polysaccharide content of the fractions isolated from human plasma by the low temperature, ethanol procedure (Cohn at al., 1946)* Fractions IV-1 ( -1 (60 per cent) plus lipides), IV-4 ( OC 2- plus /3 ), and IV-6 ( (X -2 (95 per cent)) yielded the highest values, containing 3*39, 3.73, and 6.83 per cent respectively. Fraction V (95 per cent albumin) yielded the lowest value, 0.30 per cent. Dische and Osnos (1950) isolated neutral mucopoly saccharide fractions from rat brain, kidney, muscle, heart and pancreas, from mouse kidney and sarcoma 180, and from human serum. Carbohydrate determinations showed the presence of galactose, mannose, glucosamine, and methyl pentose. They suggested that their findings indicate the presence in most animal tissues of polysaccharides of the type found in serum and blood group substances. Good at al. (1950) found increased serum mucoprotein levels in animals after chronic chilling, hyperimmunization, and injection of adrenalin. Cohn and coworkers (1950) reported the isolation of a very soluble plasma protein which they designated as OC 1- small acid protein and as CC 1-lipid-free glycoprotein. This protein, which forms Precipitate VI-2 was found to comprise 0.5 per cent of the total plasma proteins. The following 20 approximate values were given for certain physicochemical constants; Sgo** 2*0; pHj, 3.0, It seems probable in view of the solubility and approximate physical constants that this protein may be identical to the mucoprotein studied by the author. STUDIES DEALING WITH SERUM POLYSACCHARIDE LEVELS IN NORMAL AND PATHOLOGICAL STATES If by the usual blood sugar determination methods, the reducing power of blood or serum is determined before and after acid hydrolysis, there is an Increase In reducing sub stances following hydrolysis. This fact has been known since 1855 (Grevenstuk and Laquer, 1925) and has formed the ration ale of many studies of serum in normal and pathological states. These increases were traced by most authors to the presence of carbohydrates In serum proteins and therefore designated as Eiweisszucker, du sucre proteidique, and serum polysaccha ride. In the case of healthy individuals there has been demonstrated a definite content of hydrolyzable sugar, which is constant within certain limits. Deviations from this nor mal range have been observed in many pathological conditions. The determination of the carbohydrate constituents of the protein molecule is a problem that as yet has not been resolved. The classical methods are usually Inapplicable due to the small amounts Involved. Numerous colorimetric methods have been employed for the detection of small amounts of sugars. All of these methods lack specificity and are subject to interference by other substances that may be present. Many questions have been raised regarding their use. However, when employed under rigidly controlled conditions they have yielded valuable information regarding the fluctuations of serum polysaccharide levels In normal and pathological states. Recent investigations dealing with serum polysaccharides have relied exclusively upon colorimetric methods (Shetlar et al.j 1949a, b; Friedman, 1949; Werner, 1949)* The present evidence indicates that the serum poly saccharides which are associated with protein, contain galaetose, mannose, and glucosamine. Bierry (1928) isolated mannose as the phenyl hydrazone and galactose (1929) as the osazone from the plasma proteins of the horse* Rimington (1929) Isolated glucosamine as the hydrochloride and mannose as the phenylhdrazone from carbohydrate fractions obtained after the barium hydroxide digestion of serum albumin and serum globulin. Winzler and coworkers (1948) reported the Isolation of a galactose derivative, mucic acid, from a mixture of mucoprotelns. Friedman (1949) in a critical study of colorimetric reactions for the determination of poly saccharides in biological proteins concluded that the orclnol 22 reaction was superior for their quantitative determination. Employing the orcinol, carbazole, and skatole methods, the hexose component of the serum proteins of normal and immunized horses was analyzed and the results were found to be con sistent with an equimolar ratio of mannose and galactose* In spite of the fact that there is a considerable concentration of polysaccharide in association with the proteins of serum, which is as great or greater than that of glucose, comparatively few attempts have been made to evaluate it quantitatively with a view to determine its significance* Lustig and Ernst (1937) determined the polysaccharide content of the serum proteins by the orcinol reaction for representatives of several animal orders and species* They found mammals to have the highest serum protein values and, with the exception of crustaceans, the lowest values for protein sugar* For man, the average protein-bound sugar value was found to be 144 mg* per cent* The protein content of sera was found to be lower during growth but the poly saccharide content of the protein was similar to that of the adult animal* Seibert and Atno (1946) determined human serum poly saccharide levels directly on untreated sera by a modifica tion of the carbazole method* They reported an average value of 102 mg* per cent* Slight differences, 13 mg* per cent, were noted between the concentrations during fasting and 23 after the Ingestion of meals containing large amounts of carbohydrates• Seibert and coworkers (1947) found that the carbohydrate content of human serum proteins Increased in cases of cancer and of tuberculosis. It was suggested that, in diseases accompanied by tissue destruction or pathological activity, an Increase may occur In serum polysaccharide. Seibert, Pfaff, and Seibert (1948) correlated the Increased polysaccharide content of serum in cancer and in tuberculosis with electrophoretic analysis. In cases where the serum polysaccharide was increased, the 0C-2 globulin as determined by electrophoresis, was also increased. They postulated that an increase in <X -2 globulin occurs in con ditions characterized by tissue destruction or pathological activity* Shetlar, Everett and coworkers (1949a) determined the serum polysaccharide levels in malignancy and in other patho logical conditions. They found Increased levels in cancer, arthritis, cholelithiasis, ulcerative colitis, nephritis, pemphigus, and In most infections. No differences were noted between carcinoma patients and those having other types of malignancy. The suggestion was made that elevations of serum polysaccharide are involved in some way with tissue proli feration. 24 Shetlar at al, (1949b) determined serum polysaccharide levels in dogs following the production of sterile turpentine abscesses, of bacterial abscesses, and of talc granulomas, the injection of turpentine intrapleurally, the following experimental surgical operations. In all cases the poly saccharide level rose and the maximal level depended some what on the type of inflammation. Elevation occurred both in the presence and absence of fever. The maximal elevation appeared within 3 to 6 days after the initial Injury. The suggestion was made that this phenomenon Is correlated in some way with tissue proliferation or repair. Werner (1940) determined the changes in the serum polysaccharide and in the serum proteins during the regenera tion of serum proteins in normal and intoxicated rabbits. In normal rabbits he found an increase in the oC - and /3 - globulins and a decrease in the albumin and 2P -globulin after bleeding. After bleedings, single or repeated for long periods, an Increase in the glucosamine content of serum was found. Since this increase did not parallel any of the main fractions of the serum proteins he postulated that there must be a subfraction present more rich in carbo hydrate than the 0C -2-globulin. 2 5 THEORIES CONCERNING THE PHYSIOLOGICAL SOURCE AND SIGNI FICANCE OP THE MUCOPROTEINS OP PLASMA At the present time there Is little definitive evidence regarding the source and function of the plasma mucoprotelns* There are, however, a large number of Independent Investi gations on different normal and pathological states which appear to be related: 1* The demonstration, polarographically, of high molecular weight compounds containing cystine In sulfosalicylic acid filtrates of serum (Albers, 1940; Brdicka, Novak, and Klumpar, 1939; Muller and Davis, 1947; Schmidt, 1940; Waldschmldt-Leitz and Mayer, 1939; Winzler and Burk, 1944)* 2* The demonstration of polypeptlde-llke substances in serum deproteinated with trichloracetic acid (Cristol and Puech, 1926; Golffon and Spaey, 1934; Hahn,1921). 3* The demonstration of increased levels of serum polysaccharides In certain malignant and patho logical states (Godfried, 1939; Seibert et al* > 1947, 1948; Shetlar, e£ al** 1949a, b; Werner, 1949). 4* The demonstration of Increased serum mucoprotein and serum mucoid levels In certain malignant and 26 pathological states (Winzler and Smyth, 1948; Jacobs, 1949; Slmkln et al«, 1949)* 5* The isolation of carbohydrate-rich, non-heat- coagulable, non-dialyzable, polarographically active proteins from the sera of several species of animals (Mayer, 1942; Winzler and Burk, 1944; Welmer, Mehl and Winzler, 1950). The investigations cited above may be summarized as follows: There occurs normally in animal sera relatively small amounts of carbohydrate-rich, non-heat-coagulable, non- dialyzable, sulfosallcyllc acid-soluble, polarographically active proteins which are precipitated by phosphotungstic acid, by high concentrations of ethanol, and by saturation with ammonium sulfate* The amounts of such materials are markedly Increased in pathological conditions such as cancer, pneumonia, tuberculosis, myocardial infarction, arthritis and in experimental inflammations* Investigations in which Increases in serum polysaccha rides or serum mucoprotein levels were correlated with changes in electrophoretic patterns indicate that more than one species of carbohydrate-rich protein may be involved. Mehl, Golden, and Winzler (1949) correlated Increased serum muco protein levels in cases of carcinoma and of pneumonia with 27 electrophoretic studies* In electrophoretic analyses con* ducted at pH 4*5 they found Increased levels of two acidic components designated as M-l and M-2* The M-l component which had been previously Isolated in a homogeneous state (Welmer, Mehl, and Winzler, 1950) was shown to migrate with the cC 1-globulins at pH 8*6* Seibert, Pfaff, and Seibert (1948) correlated the Increased polysaccharide content of serum In cancer and in tuberculosis with electrophoretic analyses* In cases where the serum polysaccharide was in creased, the oC 2-globulins as determined by electrophoresis were also Increased* Shedlovsky and Scudder (1942) noted that a rise in oC 2-globulin is associated with inflammation or tissue destruction, irrespective of cause* Additional substantiating evidence for the hypothesis that more than one species of carbohydrate-rich protein may be involved are the recent findings of Cohn and coworkers (1950)* They report the isolation of three carbohydrate-rich proteins designated as oC 2-glycoprotein, OC 2-mucoprotein, and oC 1-lipid-free glycoprotein (also designated as ofl- small acid protein) respectively* Several theories have been advanced as to the source of mucoprotelns in blood. Piesslnger (1934) and Crlstol (1935) believed that hyperpolypeptidemia might result (1) from a failure of the liver to metabolize the normal amounts of 28 polypeptides formed, (2) from a failure of the kidneys to excrete them normally, or (3) to an Increase In their forma* tlon In certain tissues. Cancer, trauma, and many Infections were thought to cause hyperpolypeptldemla through excessive protein decomposition In the affected tissues. A similar view (3) has also been advanced by Seibert and her colleagues (1947, 1948). They suggested that Increased serum poly saccharide levels with a concommittant Increase in materials migrating with the £ 2-globulins as found In carcinoma and In tuberculosis are due to Increased tissue destruction# Godfrled (1939) considers Increased polypeptide levels to be due to (1) Increased parental destruction of proteins, (2) diseases of the liver, (3) diseases of the kidney. Brdicka (1939) has suggested that the material giving the polarographlc filtrate test Is formed by the degradation of serum proteins, possibly through the action of the protective enzyme, "Abwehrferment,* of Abderhalden. That such enzymes may occur due to the presence In the blood stream of a foreign protein seems probable from the work of Merten (1948)# Crossley, Kienle, Vessel, and Christopher (1941) concluded from kinetic studies that the serum proteins of dogs Infected with pneumococcus were partially degraded In vivo to molecules of lower molecular weight# Winzler and Burk (1944) considered the possibility that the proteoses in the blood of tumor- 29 bearing animals originate largely, even if not wholly, from serum albumin rather than from tumor nitrogen* Sixnkin, Bergman, and Prinzmetal (1949) postulated a breakdown of inf are ted heart muscle in determining serum mucoprotein levels of patients with myocardial infarction. Roberts and White (1949) Incubated surviving rat liver minces with the homologous serum and noted Increases in the oC 1- globulin fraction after incubation. In all experiments, a decrease of albumin in the serum medium accompanied the in crease of CC 1-globulin as determined by electrophoresis. In contrast to the above theories which are postulated primarily on tissue destruction and protein degradation, Shetlar, Everett, and coworkers (1949a, b) have suggested that Increased serum polysaccharides are due to increased tissue proliferation or repalr. Their suggestion is based on the evidence that maximum Increases in serum polysaccharide levels occur after maximum tissue destruction has occurred and when the repair processes of the organism would be ex pected to be functioning at their greatest rate. Werner (1949) studied changes in the serum polysaccha ride and in the serum proteins in rabbits following intoxi cation in an attempt to ascertain the source of the serum polysaccharide. In animals given benzene or phosphorous perorally, resulting in liver poisoning, an increase in 30 cC - and j3 -globulins was observed but no increase in the glucosamine content of serum was found. Poisons of the bone marrow such as lead and benzene given parenterally only slightly affected the regeneration pattern of the serum pro teins and a normal Increase In the glucosamine content of serum following bleeding was observed. Werner concludes that, "the results of the poisoning experiments Indicate that the liver is the site of the serum polysaccharide (protein-bound carbohydrate.w It seems to the author that the question as to the source of the plasma mucoprotelns Is but part of the general problem, "What is the site of origin of the plasma proteins?” While it has been generally accepted that the liver is the chief site of formation of certain of the plasma proteins, notably, albumin, prothrombin, and fibrinogen (Madden and Whipple, 1940), little definitive evidence is available regarding the site of formation of the plasma globulins. Sabin (1939) has suggested that both normal serum globulin and antibody globulin arise by a process of shedding of surface films of cytoplasmic material from monocytes or macrophages. White and Dougherty (1946) have presented evi dence which suggests that retlculo-endothelial cells other than those of the liver may be the source of certain of the serum globulins. The function of the plasma mucoprotelns is unknown at the present time* Most of the Investigators and reviewers in this field have avoided this question entirely or have confined their remarks to suggestions and speculations* Bywaters (1909) suggested that they might serve as a means of transport for carbohydrates and presented data indicating that there was less mucoprotein in the serum of an animal in inanition than in the serum of one in a good nutritional state* In view of recent work on the /3 1-lipoprotein as a means of transport for lipids (Oncley, Gurd and Melin, 1950), Bywater1s investigations might well merit repeating* Rimington (1940) remarked ffThe physiological significance of the protein-bound carbohydrates in serum is not known." Meyer in his review (1945) devotes one line to this topic, "Little known about the metabolism of these substances and little about their function." Stacey in his review (1946) stated, "The physiological significance of mucoprotein in serum is unknown but it is tempting to speculate that they may be intermediate stages in the synthesis of albumins and globulins*" The suggestion of Shetlar, Everett and coworkers (1949a, b) that serum polysaccharides are involved in tissue proliferation implies a function which is certainly non specific* 32 Werner (1949) states, "The physiological role played hy the serum polysaccharide (protein-hound carbohydrate) is unknown* It may however be suggested that they participate in protein metabolism. The fact that the polysaccharide content rapidly increases in conditions of protein loss might indicate a role in the formation of the serum proteins. Possibly the carbohydrate-rich fractions of the (£- and /3 - globulins are Intermediary stages in the synthesis of other serum proteins. It is also conceivable that part of the carbohydrate may be transformed into amino acids. The rapid appearance of the protein rich in carbohydrate may be related to the fact that carbohydrate material Is more easily avail able to the body than are proteins and amino acids.* CHAPTER III MATERIALS AND METHODS PLASMA AND SERUM Human plasma used in this investigation was pooled, normal plasma donated by the Hyland Laboratories of Los Angeles and out-dated, lyophilized plasma donated by Dr* A* G. Ware, Chemist, Los Angeles County General Hospital* Rat serum was colleeted from albino rats, University of Southern California strain, by exsanguinatlon, from the ab dominal aorta after ether anesthesia, the blood being allowed to clot at room temperature and centrifuged* Guinea pig serum was collected by cardiac puncture following ether anesthesia, the blood being allowed to clot at room tempera ture and centrifuged* Beef serum, donated by the Swift Packing Company, was collected from the jugular vein after the animal had been stunned by a blow on the head* The serum was decanted after clotting had occurred* Horse serum, donated by the Victory Packing Company, was obtained In a similar manner* Umbilical cord serum was donated by Dr* Elolse Jameson, Research Associate, University of Southern California Medleal School* PREPARATIONS STUDIED 34 Mucoprotelns* Miieoproteins were isolated from plasma and serum by the following methods: 1* Winzler, Devor, Mehl, and Smyth (1948). One volume of plasma (1800 ml.) was diluted with an equal volume of water, and one volume (1800 ml*) of 1*8 molar perchloric acid was added while stirring* Filtration through a double thickness of Whatman No* 1 filter paper was started 30 minutes after protein precipitation* The filtrate was dlalyzed against running tap water until nearly free of acid, and the muco- proteins were precipitated by saturating the dialysate with ammonium sulfate at pH 4*0* The materials were exhaustively dlalyzed against distilled water aid dried by lyophlllzatlon* 2* Weimer, Mehl, and Winzler (1950). One volume, usually 1 to 3 liters, of pooled, normal, human plasma was diluted with 0*1 volume of 1 molar sodium acetate and 0*9 volume of distilled water* Ammonium sulfate was added with stirring to a concentration of 2*73 molar* The mixture was allowed to stand for a minimum of 16 hours at 4<>c* and then filtered through a double thickness of Whatman No* 1 filter paper* The pH of the filtrate was reduced to 4*9 with 1 N hydrochloric acid, and the resulting suspension was allowed to stand at 4°C* for a minimum of 16 hours* The precipitate was removed by filtration through a double thickness of 35 Whatman No* 1 filter paper as before* The pH of the filtrate was then reduced to 3.7 with 1 N hydrochloric acid, the mixture was again allowed to stand at 4<>C* tor a minimum of 16 hours, and the precipitated proteins were filtered as above* The mucoprotein was then precipitated by saturating the filtrate with ammonium sulfate at 4°C* The mixture was allowed to stand for a minimum of 72 hours at 40C* to Insure complete precipitation of the mucoprotein which was collected by fil tration through a double thickness of Whatman No. 1 filter paper* It was then dissolved in a small volume of water, dlalyzed against distilled water until free of ammonium sulfate, and dried by lyophllizatlon* 3* A macroadaptation of the micromethod of Winzler and Smyth (1948)• One volume of plasma (1800 ml.) was diluted with an equal volume of water, and one volume (1800 ml*} of 1*8 molar perchloric acid was added while stirring* The mixture was allowed to stand at 4<>G* for 4 hours, then fil- tered through a double thickness of Whatman no* 1 filter paper* The volume of the filtrate was measured and 1/5 volume of 5 per cent phosphotungstlc acid in 2 N hydrochloric acid was added while stirring* Filtration through a double thickness of Whatman No* 1 filter paper was begun 30 minutes after the mucoprotein precipitation* The materials were ex haustively dialyzed against distilled water and dried by lyophili zation. 36 Seromucold* Rimington (1940). One volume of plasma was diluted with 3 volumes of distilled water# The pH of the diluted plasma was reduced to 4*7 with 10 per cent acetic acid. The aeidified, dilute plasma was placed in a boiling water bath until coagulation was complete and filtered through a double thickness of Whatman Ho# 1 filter paper# The fil trate was placed in dialysis tubing, a few drops of toluene added, and a current of air was blown over the tubing for several days to reduce the volume# The temperature of the concentrated filtrate was reduced to -2°C# in a brine bath and 10 volumes of 95 per cent ethanol at the same temperature was added slowly with stirring# The mixture was allowed to stand overnight, then filtered through a double thickness of Whatman No# 1 filter paper# The precipitate was dialyzed against distilled water and lyophlllzed# Mucoidflhnliche Substanz* Mayer, (1942)# One volume (2 liters) of plasma was diluted with an equal volume of water# The diluted plasma was heated for 10 minutes in a boiling water bath# Two volumes (4 liters) of 20 per cent sulfosalicyllc add were added while stirring# The precipi tate was filtered through a double thickness of Whatman Jfo* 1 filter paper* The filtrate was dialyzed overnight against running tap water and then against distilled water until 37 free of the precipitant* The filtrate was concentrated at 4°C* by blowing a current of air across the dialysis tubing* Mucoldfihnllehe Substanz, was precipitated by the addition of 10 volumes of 95 per cent ethanol while stirring* The pre cipitate was dialyzed against distilled water and dried by lyophlllzatlon* CHEMICAL ANALYSES Carbohydrate determinations * Carbohydrate determina tions were based on the orcinol reaction of Sflrensen and Haugaard (1933) as modified by Hewitt (1937a)* An equlmolar mixture of galactose and mannose was used as a standard* Nitrogen determinations. The micro-Kjeldahl, direct nesslerizatlon method of Koch and McMeekln (1924) was em ployed* Ammonium sulfate and human plasma albumin were used as standards* Lipid determinations* Lipid determinations were carried out by hot 95 per cent ethanol extraction for 12 hours in a Soxhlet apparatus* Hexosamlne determinations * The method of Rimington (1940) employing Elson's and Morgan's acetylaeetone reagent and Ehrlich1 s reagent (0*8 gram p-dlmethylamlnobenzaldehyde 38 dissolved in 30 nil* of absolute ethanol and 30 ml* of con centrated hydrochloric acid) was followed* Glucosamine hydro chloride was used as a standard* Tyrosine determinations * A method employing Folinfs phenol reagent (Folin and Clocalteu, 1927) as adapted by Winzler, Devor, Mehl and Smyth (1948) was followed* Although the values obtained are reported as "tyrosine,* it is recog nized that this reagent is not specific for tyrosine groups in proteins* Tyrosine was used as a standard* Determination of hexuronic acids* The method of Dlsche (1947) was used* Glucuronic acid was employed as a standard* Moisture determinations * Moisture was determined by heating the material overnight in a vacuum oven at 60<>C* Determination of ash* Samples were ashed by heating in a muffle furnace at 550°C* for 12 hours* Amide nitrogen determinations ♦ The mucoprotein sample was hydrolyzed under reflux for 4 hours in 20 per cent hydro chloric acid* Aliquots were placed in Conway microdiffusion cells, made alkaline with sodium hydroxide, and allowed to stand for 40 hours* Ammonia was determined by direct ness- lerization of an aliquot* Ammonia sulfate was employed as a standard* 59 Total protein determlnations* Total protein was determined by the biuret method* Human plasma albumin was used as a standard* Phosphorus determinations* The procedure of Fiske and Subbarow (1925) was employed* Monopotassium phosphate was used as a standard* Sulfur determinations♦ The sulfur analysis was per formed by Elek Laboratories, Los Angeles, utilizing a gravi metric procedure* AMINO AG ID ANALYSIS The amino acid analysis was performed by Shankman Laboratories, Los Angeles, utilizing microbiological pro cedures* ELECTROPHORETIC STUDIES The electrophoresis experiments were carried out in a modified Tiselius apparatus by the method of Longsworth (1942). Barbiturate, acetate, trichloroacetate, phosphate, and perchlorate buffers were employed* The buffer systems were 0*02 molar with respect to the sodium salt of the buffer anion and 0.08 molar sodium chloride* One per cent solutions of protein in buffer were prepared, and 40 were dlalyzed for 48 hours against two changes of buffer* The protein solution was centrifuged if necessary to clarify it prior to filling the cell* Boundaries were established by either removing 1.0 to 2*0 ml. of buffer from one of the electrode vessels or by adding similar volumes* Electro phoresis was carried out at 4 to 6 volts/cm* for 3 to 6 hours* A constant temperature of 2<>C* was maintained* The conduc tivity of the final dlalysate was measured by means of a Wheatstone bridge assembly, with the conductance cell mounted in the electrophoresis bath* The distance a component moved from the starting boundary was measured from enlargements of known magnification, obtained by tracing on coordinate paper the outlines of the patterns projected from the film in a photographic enlarger* The electrophoretic mobility of the descending boundary (Longsworth, 1942} was employed in plotting the pH- mobility curve* Mobilities were deter mined by the equation of Longsworth e£ al* (1959)* in which: XT * electrophoretic mobility, expressed in cm*/sec./ volt/cm* h = distance that boundary has moved from the starting position in centimeters* 41 Kp « specific conductance of the final dlalysate, assumed to be Identical with the protein solution* Expressed in reciprocal ohms (mho)* I » current strength in amperes* t a time in seconds A * cross sectional area of the cell in square centi meters • Relative concentration of components was determined by the method of Tlselius and Kabat (1938)* TURNOVER STUDIES Turnover studies were made with methyl labeled glycine* Counting was carried out in a Geiger-Muller counter, equipped with a gas chamber* Relative rates of turnover were expressed as specific activities, i*e., counts/min./mg* protein. CHAPTER IV EXPERIMENTAL CHARACTERIZATION OP A HOMOGENEOUS MUCOPROTEIN FROM HUMAN PLASMA Isolation* It has been possible to Isolate the major component of normal plasma mucoproteln In an electrophoreti- cally homogeneous state by a series of ammonium sulfate precipitations (Welmer, Mehl, and Winzler, 1950)* This ihuco- protein has been designated as MP-1 by Mehl, Humphrey, and Winzler (1949)• Their terminology will be employed. Figure 1 is a schematic outline of this procedure. Sixteen fraction ations of normal, pooled, human plasma by this method have yielded an average of 0.5 0.05 gram of an electrophoretlcally homogeneous mucoproteln per liter of plasma when volumes of plasma up to 3 liters were employed. The fractionation of larger volumes has yielded a slightly lnhomogeneous product which could be rendered homogeneous by refractlonating. The somewhat hygroscopic preparations are readily soluble in aqueous, salt, acid, and alkaline solutions to produce very slightly turbid solutions. Reprecipitated pre parations give clear solutions. Aqueous solutions are not coagulated by boiling even when acidified with acetic acid. The mucoproteln is not precipitated by 0.6 M perchloric acid, 43 FIGURE 1 ISOLATION PROCEDURE FOR HUMAN PLASMA MUCOPROTEIN (MP-U Plasma Filtrate A pH reduced to 4*9 Filtrate B pH reduced to 3.7 Filtrate Cl To one volume of plasma were added one-tenth volume of molar sodium acetate and 0.9 volume of distilled water. Ammonium sulfate was added to a final con centration of 2.73 molar. Precipitate A Precipitate B Precipitate C Ammonium sulfate added to saturation at 4oC. Filtrate D| Precipitate D Mucoproteln 44 by 0*2 M sulfosalicylic acid, or by 0*3 M trichloracetic acid, but Is precipitated by phosphotungstlc acid, by 75 per cent ethanol at pE 3.5, and by saturation with ammonium sulfate at all pH values employed (pH 1,8 to pH 7,4). Chemical composition# The chemical composition of MP-1, given in Table I, is a tabulation of average values obtained from preparations that were found to be homogenous by electrophoretic and ultracentrifugal analysis# Table 1 shows the characteristically high carbohydrate and hexosamine and low nitrogen content for this material# This protein would accordingly be placed in the category of mucoprotelns according to the classification of Meyer (1945) and of Stacey (1946)• It was thought that the high hexosamine content might only be apparent and due to the reaction of carbohydrate with amino acids# However, no positive test for this reaction was obtained by the procedure of Vasseur and Immers (1949)# Amide nitrogen is present in approximately the same ratio to the content of aspartic and glutamic acids in MP~1 as Is found in plasma albumin (Table XX)# Whether the lipid present is an integral part of the molecule or an adsorbed contaminant is not known# It seems probable that it is an Integral part since only a small TABLE I CHEMICAL COMPOSITION OP HUMAN PLASMA MUCOPROTEIN (MP-1) Component gm./lOO gnu* Hexose 16.4 Hexosamine 11.9 Ni trogen 10.1 Amide nitrogen 0*7 Total protein 63*0 Lipid 3.6 Ash 1*6 Sulfur 1.02 Phosphorous 0.03 Hexuronlc aoid neg. \Ka=sssxsssxssmssssaBBsasarwBsaaBt Moisture-free basis* 46 amount of the total lipid could be extracted with boiling ether. The small amount of phosphorous present might occur as a phosphoric acid ester of the hydroxy amino acids or as a constituent of phospholipids. It should be noted that the sulfur content is in ex cess of methionine or cystine sulfur (Table II). The signi ficance of this will be discussed later* Amino acid composition. It was suspected that some insight into the source of the plasma mucoproteln might be obtained from an examination of its amino acid composition* Accordingly the protein was subjected to amino acid analysis using microbiological procedures. The content of amino adds shown In column 2 of Table II does not correspond to that of any of the plasma proteins studied in detail (Edsall, 1947)* The differences between the amino acid composition of MF-1 and plasma albumin become less significant when the amino acid contents of MP-1 are multiplied by the factor 105.9/58*24 to compare comparable amounts of protein (columns 5 and 4)* This factor is the ratio of the per cent of the known amino acids in plasma albumin (105.9) and MP-1 (58.2). However, significant differences between the proteins persist, especially for the amino acids cystine, isoleucine, and TABLE II AMINO ACID COMPOSITION OP HUMAN PLASMA MUCOPROTEIN (MP-1) AND HUMAN PLASMA ALBUMIN Component Mucoproteln gm./lOO gnu* Mucoproteln gm./l82 gm. Albumin gm./lOO gm. Arginine 3.65 6.7 6.2 Aspartic acid 7.44 13.5 10.4 Cystine and cysteine 0.60 1.1 5.6 Glycine 0.82 1.5 1.6 Glutamic acid 10.73 19.6 17.4 Histidine 1*31 2.4 3.5 Isoleucine 3.15 5.7 1.7 Leucine 5.21 9.5 11.0 Lysine 5.03 9.2 12.3 Methionine 0.65 1.2 1.3 Phenylalanine 3.91 7.1 7.8 Proline 2.37 4.3 5.1 Threonine 4*80 8.7 5.0 Tryptophan 1.25 2.3 0.2 Tyrosine 1.99 3.6 4.7 Serine 2.51 4.6 3.7 Valine 2.82 5.1 7.7 Total 58.24 106.1 105.9 ** Grains of amino acid liberated by hydrolysis of 100 gms. of MP-1, moisture-free, ash-free basis. 48 tryptophan. It therefore seems unlikely that plasma albumin Is a direct precursor of MP-1* The cystine and methionine values account for 0.22 per eent sulfur In comparison to the 1.02 per cent actually found (Table I)* This may be explained by the presence of sulfuric acid esters which would also aceount for the low Isoelectric point exhibited by the muco- protein. This possibility was strengthened by the observa tion that, after mild acid hydrolysis of the mucoproteln, a precipitate was produced by the addition of barium chloride or by benzidene In acetic acid. If the nitrogen contained in the amino acids In Table II and the hexosamine and amide nitrogen shown in Table I are summated, they account for 90 per cent of the nitrogen actually found In MP-1, This value does not Include any alanine which may be present. It Is probable that this lack of agreement Is In part due to the loss by destruction of amino acids during hydrolysis. Block (1945) has noted that, Mthe amount of destruction during the conventional acid hydrolysis of a protein Is Increased by the presence of non protein substances, especially carbohydrates and lipids.w Both of these substances are found In appreciable amounts In MP-1. Ninety-eight per cent of the total weight of MP-1 Is accounted for by the Indicated hexose, hexosamine, lipid, 49 ash, sulfur, amide nitrogen and total protein values* How ever, only 86 per cent of the total weigjht of MP-1 is accounted for if the amino acids (corrected for hydrolysis water) are substituted for the total protein value in the above summa tion* This also suggests that there may have been a loss of amino acids by destruction during acid hydrolysis, or that the amino acid analysis is incomplete* Molecular weight from amino acid content* One of the Important methods of obtaining the molecular weight of a protein is based on the amino acid content. If a given amino acid is a constituent of a protein, it is evident that at least one molecule of this amino acid must be present in each molecule of protein* Consequently the molecular weight cannot be less than the mass which contains one mole equiva lent of the amino acid* The protein may contain more than one mole of the amino acid, in which case the molecular weight is an integral multiple of the value calculated on the assumption that only one mole Is present* These relations are given in the following equation* Molecular weight of = n Molecular weight of amino acid x ^qq protein Per cent of amino acid in protein where n represents the number of moles of the amino acid present* TABLE III MOLECULAR WEIGHT OF HUMAN PLASMA MUCOPROTEIN (MP-1) FROM AMINO ACID CONTENT Amino acid Content* Minimal Number of Molecular molecular amino acid weight weight residues Cystine 0.60 40,000 1 40,000 Methionine 0.65 22,900 2 45,800 Glycine 0.82 9,150 5 45,750 Tryptophan 1.25 16,300 3 48,900 Histidine 1.31 11,800 4 47,200 Tyrosine 1.99 9,100 5 45,500 AVERAGE 45,525 Proline 2*37 4,850 9 43,650 Serine 2.51 4,180 11 45,980 Valine 2.82 4,150 11 45,650 Isoleucine 3.15 4,150 ia 45,650 Arginine 3.65 4,760 10 47,600 Phenylalanine 3.91 4,210 n 46,310 Threonine 4.80 2,480 18 44,640 52 TABLE III (continued) MOLECULAR WEIGHT OP HUMAN PLASMA MUCOPROTEIN (MP-1) PROM AMINO ACID CONTENT Amino acid Content* Minimal molecular weight Number of amino acid residues Molecular weight Lysine 5.03 2,900 19 46,400 Leucine 5.21 2,520 18 45,360 Aspartic acid 7.44 1,790 25 44,750 Glutamic acid 10.73 1,370 33 45,210 AVERAGE OP 17 AMINO ACIDS 45,545 * Grams/lOO grams of protein* Moisture-free, ash-free basis* 55 figures are not of quantitative significance and this degree of accuracy in the analytical analysis is not implied. Physicochemical characterization. In order to more completely characterize MP-1, the following studies were made. Electrophoretic studies. The mucoprotein (MP-1) was found to he electrophoretically homogeneous at pH values from pH 1.5 to 8.5. Table IV is a summation of the data. Figure 2 shows the descending boundaries of representative electrophoretic patterns at the indicated pH values. These patterns demonstrate that the mucoprotein isolated by the procedure appears electrophoretically homogeneous over the entire pH range studied. That is, of course, not an absolute criterion of the purity of the protein, since small amounts of impurities are not detected by this method. However, the homogeneity of the material over the wide range of pH values used in this investigation limits the possibility that the preparations contain more than one major component. Figure 3 shows the pH-mobility curve obtained by plotting the mobility of the descending boundary against the pH of the protein solution. The isoelectric point of the mucoprotein shown in the pH-mobility of Figure 3, is at pH 1.8. This is an unexpectedly low value for a protein occurring in plasma. Evidence indicating that this may be due to the presence of sulfuric acid esters has been pre sented (see page 46). In addition It should be noted that 54 TABLE XV SUMMATION OP AN ELECTROPHORETIC INVESTIGATION OF HUMAN PLASMA MUCOPROTEIN (MP-1) Buffer system pH Mobility x 10"^ Cm./volt/sec Ascending Descending boundary boundary Sodium diethyl barbiturate — sodium chloride 8*5 -7.2 -6*5 8.45 -6.5 -6.5 8.4 -7.3 -6.1 Sodium acid phos phate - sodium chloride 7.45 -7.1 -6.5 6.8 -6.4 -5.8 5.6 —5.9 —5.3 Sodium acetate — sodium chloride 4.7 -4.7 -4.5 4.7 * -4.6 4.55 -4.5 -4.2 3.9 —3.3 -2.8 3.8 -2.9 -2.5 Sodium triehlor- acetate — sodium chloride 2.9 -» -1*7 Sodium perchlorate - sodium chloride 2.3 -1.4 -1.1 Sodium trichlor- acetate -- sodium chloride 2.0 -1.3 -1.2 Sodium perchlorate - sodium chloride 1.85 -0.28 -0.26 55 TABLE IV (continued) SUMMATION OP AN ELECTROPHORETIC INVESTIGATION OP HUMAN PLASMA MUCOPROTEIN (MP-1) Buffer system PH Mobility x 10"5 Ascending boundary Cm .9Vol t /s e c • Descending boundary Sodium trichlor- acetate — sodium chloride 1.8 0.0 0.0 Sodium perchlorate - sodium chloride 1.76 1.0 0.9 Sodium triohlor- acetate — sodium chloride 1.6 1.3 1.3 # Not possible to determine# FIGURE 2 ELECTROPHORETIC PATTERNS OF HUMAN PLASMA MUCOPROTEIN (MP-1) AT VARIOUS pH VALUES Vertical line indicates position of starting boundary* FIGURE 3 MOBILITY OF HUMAN PLASMA MUCOPROTEIN (MP-1) AS A FUNCTION OF PH C D C D C D Small open circles - barbiturate buffer Double circles - phosphate buffer Small dots - acetate buffer Dotted circles - trichloracetate buffer Large open circles - perchlorate buffer 58 the pKg of sulfuric acid Is pH 1*75* A buffer system containing sodium diethylbarbiturate at pH 8*6 Is employed In most current electrophoretic analyses of sera* Under these conditions there Is the most effective resolution of components (Longsworth, 1942)* It was of value then to determine with which component mucoprotein migrated under these conditions. In order that changes in mucoproteln levels in different normal and pathological states might he correlated with changes in concentration of the other com ponents of serum* Three ml* of freshly drawn, pooled, normal, human serum and an equal volume of serum from the same pool plus 50 mg* of a mucoprotein preparation were diluted to 15 ml* with sodium diethylbarbiturate-sodium chloride buffer at pH 8*6 and subjected to electrophoretic analysis* Figure 4 shows photographs of the descending boundaries of the pat terns after electrophoresis* An examination of the two photographs indicates quite clearly that under these condi tions mucoproteins migrate with the oC 1-globulins of serum* Determination of partial specific volume* The partial specific volume was determined pycnometrlcally in aqueous solution* The protein concentration was determined by drying aliquots of the solutions to constant weight in a drying oven at 105©C* A plot of m vs* w^ was used in deter mining dav/dw^* The partial specific volume was then determined from the formula: 59 FIGURE 4 ELECTROPHORETIC PATTERNS OF DESCENDING BOUNDARIES OF NORMAL HUMAN PLASMA, AND OF NORMAL HUMAN PLASMA WITH ADDED MUCO PROTEIN AT pH 8.6 Normal Human Plasma Normal human plasma with added -h -«■ 60 * V (X- (~ Wl ) ) (Svedberg and Pederson, 1940} In which? V * specif! c volume W1 * weight fraction of protein (0*01 times per cent) nx * weight of solution Am/Aw^ = slope of line determined from the plot of 0 78* W^* Table V is a summation of data obtained in the Investi- gatlon* Figure 5 is a plot of m versus w^ for the determina tion of dn/dwi which was found to be 5*2• The average value of the partial specific volume for the 4 highest protein concentrations was found to be 0*675* The linear relation ship obtained in Figure 5 indicates that the partial specific volume is substantially Independent of concentration over the range employed* Sedimentation and diffusion constants* This data was supplied by Dr* Emil L* Smith, School of Medicine, Uni versity of Utah, and has been reported elsewhere (Smith, Brown, Weimer, and Winzler, 1950)* It is Included here for use in the following section* The average sedimentation constant <S20w) of MP-1 is 3*11 x 10*^ cm*/sec« and the average diffusion constant (J>20w) is 5*27 x 10*? cm*2/sec* Determination of molecular weight* Substituting the values obtained for the sedimentation and diffusion 61 TABLE V SUMMATION OF A PARTIAL SPECIFIC VOLUME DETERMINATION OF HUMAN PLASMA MUCOPROTEIN (MP-1) Concen tration g./ml. W1 Weight* Weight Pycnometer solution plus m solution (grams) (grams) dm/dwi (from Figure 5) Specific volume V Partial specific volume Vl 0.0 19.303 9.628 3.2 1.00000 0.0638 19.509 9.834 3.2 0.97905 0.678 0.0386 19.430 9.755 3.2 0.98708 0.676 0.0300 19.400 9.725 3.2 0.99003 0.674 0.0211 19.370 9.695 3.2 0.99309 0.672 0.01275 19.344 9.669 3.2 0.99576 0.671 0.00745 19.330 9.655 3.2 0.99720 0.670 0.0045 19.315 9.640 3.2 0.99870 0.669 0.0031 19.312 9.637 3.2 0.9906 0.669 0.0018 19.307 9.632 3.2 0.99950 0.669 * Weight Pycnometer: 9*675 grams* 62 FIGURE 5 PLOT OF m VERSUS 9.850 - 9.825 / 9.800 / 9.775 9.750 / n i (grams) / 9.725 / 9.700 / 9.675 - / 9.650 - / 9.625 9 600 1 1 1 1 1 1 1 0.0 0 .0 1 0.02 0.03 0.04 0.05 0.06 0.07 W i (grams/ml.) 63 constants and partial specific volume in the equation: RTs M * g" (l-vp) (Svedberg and Pedersen, 1940) in which: M = molecular weight R = gas constant » 8*313 x 107 ergs/degree/mole. T » absolute temperature * 293° s = sedimentation constant * 3*11 x 10-13 cm,/sec, D * diffusion constant * 5*27 cm, 2/sec, V * partial specific volume * 0,675 p - density of solvent * 1,0 the molecular weight of MP-1 was found to be 44,100, Viscosity studies, A mucoprotein preparation which was 90 per cent electrophoretically homogeneous was employed in viscosity studies. The results of viscosity measurements in aqueous solution are presented in Table VI, Figure 6 is a plot of e/hq vs* will be seen that a linear relationship exists between the relative viscosity and the protein concentration for dilute solutions. At higher concentrations there is a departure from linearity. The intrinsic viscosity was determined by plotting the function H * (In n/n0)/C. vs, C, and extrapolating to zero protein concentration. The values obtained for the four highest protein concentrations were employed in this 64 TABLE VI RELATIVE VISCOSITY OP HUMAN PLASMA MUCOPROTEIN (MP-1) Cone entrat ion C. Solvent Temperature t Relative Viscosity n/no grama per loO ml* 6*58 Water 25 1.605 5*86 Wat er 25 1.518 5.00 Water 25 1.257 2.18 Water 25 1.167 1*275 Water 25 1.110 0*745 Water 25 1*066 0.45 Water 25 1.051 0.51 Water 25 1.055 0.18 Water 25 1*019 65 PIGTJRE 6 PLOT OF a/11*) VERSUS C* 66 determination* Table VII is a summation of the data and Figure 7 is the plot of H vs. C* The intrinsic viscosity, Ho# was found to he 0*069* The value of the Einstein vis cosity coefficient (viscosity Increment), calculated from the intrinsic viscosity, Ho, and the partial specific volume, V^, using the equation F * 100 Hq/V^ (Oncley, Scat chard, and Brown, 1947) was 10*2* Frictional coefficient* An estimate of f/f0 (ratio of experimentally determined molar frictional constant to molar frictional constant calculated for an unsolvated spherical particle of the same mass) can be made from the value of the viscosity increment (page 63)* This estimate Involves the Important assumptions that the molecule behaves as a rigid ellipsoid of revolution obeying the Simha equation for viscosity (Mehl, Oncley, and Simha, 1940; Simha, 1940) and the Perrin equation for f/f0 (Perrin, 1936). The numeri cal relations between the viscosity Increment and the axial ratio of an ellipsoid of revolution have been calculated by Mehl, Oncley, and Simha (1940)* Their tabulation gives an axial ratio of 8*0 for an elongated ellipsoid and 13*5 for a flattened ellipsoid with viscosity increments of 10.2 From these axial ratios the value of the frictional coefficients may be determined from the tabulation of Svedberg and Pederson (1940)* An axial ratio of 8*0 corresponds to an f/fc value 67 TABLE VII SUMMATION OP DATA EMPLOYED IN THE DETERMINATION OP THE INTRINSIC VISCOSITY OP HUMAN PLASMA MUCOPROTEIN (MP-1) Cone entra tlon C. Relative Viscosity n/*o ln(n/no) H grams /I Oo ml. 6.38 1.605 0.4731 0.0742 3.86 1.318 0.2761 0.0715 3.00 1.237 0.2127 0.0709 2.18 1.167 0.1544 0.0708 FIGURE 7 GRAPHICAL DETERMINATION OF THE INTRINSIC VISCOSITY OF HUMAN PLASMA MUCOPROTEIN (MP-1) 0.076 0.075 0.074 0.073 0.072 0.07/ 0.070 0.069 0.068 0.0 2 .0 6.0 8.0 C ( grams/ioo m l.) 69 of 1.43 for an elongated ellipsoid and an axial ratio of 13*5 corresponds to an f/f0 value of 1*58 for a flattened ellipsoid* These values are for the anhydrous protein molecule* If it is assumed that 0*2 gram of water is bound by 1*0 gram of protein (Oncley, Seatchard, and Brown, 1947) the same esti mations may be made for the hydrated molecule* In this case 6 9 the viscosity increment is 7*9 ( 0*675 ^*072 ) corresponding to axial ratios of 6*5 and 10*0 for an elongated ellipsoid and a flattened ellipsoid respectively* These values yield frictional coefficients of 1.34 and 1.46 for the hydrated elongated ellipsoid and flattened ellipsoid respectively. It seems probable from the above values that the mucoprotein (MP-1) molecule is asymmetric in shape and that considerably more water of hydration would be required for it to approach a spherical shape. Smith et al* (1950) determined the frictional coefficient of MP-1 from sedimentation and dif fusion studies. They found f/fc values of 1.78 and 1.63 for the anhydrous and hydrated elongated ellipsoids respectively. Their values indicate a more asymmetric molecule than was estimated from viscosity measurements. Approximate dimensions of molecule. An estimate of the approximate dimensions of the hydrated mucoprotein molecule may be obtained by assuming that it is a rigid ellip soid of revolution and that 0.2 gram of water is bound by 70 1*0 gram of mucoprotein* Substituting the values for the molecular weight and partial specific volume In the expression: Hydrated molar volume » + 0*2 M (1) * 44,100 (0*675) + 44,100 (0*2) « 38,700 cm.3 A value of 38,700 cm.3 is found. Each molecule would occupy a volume of — S§J?9S. . . ., = 0.65 x 10-1® cm.® 6.06 x 1023 = 6.5 x 104 i 3 The volume of an ellipsoid of revolution is given by the expression: V * 4/3- r r a2b (2) in which a * 1/2 equatorial axis b * 1/2 axis of revolution The axial ratio for the hydrated mucoprotein molecule determined from the viscosity Increment was 6*5 (page 66)* Then: b « 6* 5a Substituting in equation (2) and solving for f f at t yielded a value of 15*4 &• Thus, the approximate dimensions of the 71 hydrated molecule would be - length = 174 % width = 27 % Rate of turnover studies# These studies were carried out with the serum of 2 male albino rats that have been given C^4 methyl-labeled glycine by stomach tube# Rat number 1 weighing 100 grams was given 600 mg* of glycine with a specific activity of 2000 cts./min./mg* (counted as barium carbonate)* Rat number 2 weighing 144 grams was administered 627 mg* of glycine with a specific activity of 2000 cts*/ min./mg* The animals were sacrificed after 17 hours by ex- sangulnation from the dorsal aorta under nembutal anesthesia* The blood was allowed to clot at room temperature, centri fuged, and the serum fractloned by the following procedure* 1* To one volume of serum was added an equal volume of saturated ammonium sulfate, a precipitate forming* The mixture was allowed to stand over night in the refrigerator* The precipitate was removed by centrifugation (Globulin fraction). 2* To the supernatant was added 1*0 ml, of N hydro chloric acid, a precipitate forming. After standing overnight in the refrigerator the pre cipitate was removed by centrlguatlon (Albumin fraction)• 72 3* The supernatant from (2) (Mucoprotein fraction) and the Globulin and Albumin fractions were dialyzed against distilled water until free of the precipitant, then lyophllized# Although the component by which the fraction is desig nated forms the major portion of each fraction* none of these fractions can be considered homogeneous In any sense of the word# Samples were prepared for reading in the Q-gas chamber of a Gelger-M&ller counter by making up aqueous solutions of the three fractions to a concentration of 1 mg./ml* and drying a 2 ml# aliquot on a planchet under an Infra red lamp# One drop of detergent was added to the planchet preparatory to drying. A summary of the results obtained In this Investigation is presented in Table VIII# A comparison of the specific activities of the three fractions would seem to indicate that the mucoprotein frac tion has a greater rate of turnover than the albumin or globulin fractions# This may well be more apparent than real# It Is a well established fact that glycine Is a precursor of carbohydrate# The mucoprotein fraction has a much higher carbohydrate content than the other two (Winzler, unpublished results)# Whether the radio-activity of any or all of these TABLE VIII SUMMARY OP A RATE OP TURNOVER STUDY ON SERUM PROTEIN FRACTIONS OP RATS ADMINISTERED C14 METHYL-LABELED GLYCINE w t m t - Bat Serum number fraction Total Counting Counts counts interval per in minute minutes Back- Corrected Average Specific ground counts activity per minute cts./' min./mg. Globulin 2490 5 498 91 407 Globulin 2435 5 487 91 396 Globulin 2434 5 487 91 396 400 200 Albumin 1921 5 384 91 293 Albumin 1898 5 380 91 289 291 146 Mucoprotein 2892 5 578 91 487 Mucoprotein 2860 5 572 91 481 484 242 Globulin 1876 5 375 115 260 260 130 Albumin 1871 5 374 115 259 259 150 Mucoprotein 3295 5 659 115 544 544 272 # An accident during lyophilization caused the loss of the bulk of the protein frac tion from this animal* 74 Tractions resides in the carbohydrate or in the protein moieties or both is not known* A similar investigation in volving hydrolysis and starch chromatography of the Isolated fractions might be expected to be more conclusive* COMPARATIVE STUDIES A review of the literature (Chapter II) emphasized the need for fundamental comparative investigations involving methods of Isolation of carbohydrate-rich, non-heat-coagul- able, non-dialyzable proteins of serum and species differences in these materials* Carbohydrate-rich proteins have been isolated from human (Wlnzler et al*, 1948), beef (Rimington, 1940), horse (Mayer, 1942), rat (Winzler and Burk, 1944), chicken (Hewitt, 1957b), dog (Vassel et al*, 1947), and rabbit (Hewitt, 1957b) sera* The isolated materials have been designated as mucoprotein (Winzler e£ al** 1948), sero- mucoid (Rimington, 1940), seroglycold (Hewitt, 1957b), pro teose (Winzler and Burk, 1944), and xnucoid&hnliche Substanz (Mayer, 1942) by various investigators* With the exception of the investigation of Mehl, Humphrey, and Winzler (1949), characterization of these substances has been largely based on the results of chemical analysis and solubility properties* It is of considerable Interest to know whether the variations 75 In chemical composition noted in the above investigations are due to (1) species differences (2) different isolation pro cedures (3) varying degrees of purity or (4) isolation of different proteins. The following comparative studies were undertaken in an attempt to provide some Information on these questions. A comparative study of carbohydrate-rich protein frac tions isolated from human plasma by different methods. In this series of experiments the only variable factor was the method of isolation. All of the preparations were isolated from pooled, normal, human plasma and all analyses were carried out under the same conditions. The carbohydrate- rich protein fractions are designated by the nomenclature employed by the original investigators. Mucoproteins were prepared by the procedures of Winzler, Devor, Mehl, and Smyth (1948), Weimer, Mehl, and Winzler (1950), and a macroadaptation of the micromethod of Winzler and Smyth (1948)« Seromucold was prepared by the method of Rimington (1940). Mucoldahnllche Substanz was Isolated by a procedure similar to that followed by Mayer (1942). Table IX is a summation of the results of chemical analyses of the various preparations. Table X is a summary of the results of the electrophoretic analyses. Where the preparation contained more than one TABLE IX COMPARATIVE STUDY OP CARBOHYDRATE-RICH PROTEIN FRACTIONS ISOLATED PROM HUMAN PLASMA BY SIX DIFFERENT PROCEDURES Material Yield in gms ♦/liter Chemical Analysis* Hexose Hexos- amlne Nitro gen Tyro sine total proteins Ash Mucoprotein PCA-AS 0.345 f 14.9 % 11.2 % 11.9 % 4.3 $ 69.0 % 7.5 Mucoprotein MP-1 0*500 16.5 12.0 10.1 4.0 62.0 1.8 Mucoprotein PCA-PTA 1*20 14.3 8.3 14.0 4.7 60.0 59.6 Mucoprotein PCA-D-PTA 0*340 6.9 3.9 3.9 3.4 29.0 57.0 Seromueold 0.250 9.6 6.3 12.1 3.5 82.0 «* Mucoidahnllche Substanz 0.345 9.9 9.4 10.2 3.9 50.0 6.9 * Moisture-free basis ** Insufficient material for determination -a o> TABLE X SUMMARY OP AH ELECTROPHORETIC INVESTIGATION OP CARBOHYDRATE-RICH PROTEIN FRACTIONS ISOLATED PROM HOMAN PLASMA BY SIX DIFFERENT PROCEDURES Preparation pH Number of components Mobility x 10*5 cm, Ascending boundary ,2/volt/s ec. Descending boundary Mucoprotein, PCA-AS Component 1 -5.1 -3.8 (Winzler, Devor, 4*5 3 Component 2 -3.5 *2.9 Mehl and Smyth, Component 3 -0.7 -0.7 (1948)) Component 1 -8.2 -6. 6 8*5 3 Component 2 -7.3 -5.3 Component 3 -0.9 -0.9 Mucoprotein, MP-1 4.5 1 -4.7 -4.2 (Weimer, Winzler, 8.5 1 -7.2 -6.5 and Mehl, 1950) Mucoprotein, PCA-PTA Component 1 -5.7 -4.5 (Winzler, and 4.5 2 Component 2 -5.1 -3.4 Smyth, 1948) 8.5 2 Component 1 -6.4 -5.6 < 1 TABLE X (continued) SUMMARY OP AN ELECTROPHORETIC INVESTIGATION OP CARBOHSDRATE-RICH PROTEIN FRACTIONS ISOLATED PROM HUMAN PLASMA BY SIX DIFFERENT PROCEDURES Preparation PH Number of components Mobility x 10“^ cm Ascending boundary .2/volt/sec. Descending boundary Seromueoid 4.6 1* -1*0 -1.0 (Rimington, 1940) 8.5 2 Component 1 -6.7 -6.6 Component 2 -1.0 -1.5 2.7 2 Component 1 -0.7 -0.4 Component 2 2.5 2.0 Mucoid&hnliche Substana (Mayer, 4.5 1 -3.9 -3.4 1942) 8.5 1 -6.9 -6.1 Mucoprotein PCA-D- 4.5 2 Component 1 -5.2 -5.2 PTA Component 2 -4.7 -4.2 Component 1 -5.8 -5.7 8.4 3 Component 2 -5.2 -5.2 Component 3 -2.0 -2.0 * Spreading boundary indioates heterogeneity. CD 79 component, the components are designated in order of their rate of migration* Figures 8, 9, and 10, are photographs of the ascending and descending boundaries after electro* phoresis at the indicated pH values* In these tables and figures, mucoprotein preparations are designated by the following symbols: MP-1 - isolated by ammonium sulfate fractionation of plasma (Welmer, Mehl, and Winzler, 1950} PCA-AS- isolated by ammonium sulfate precipitation from perchloric acid filtrates of plasma (Winzler, Devor, Mehl, and Smyth, 1948)* PCA-PTA - isolated by phosphotungstic acid precipitation from perchloric acid filtrates of plasma (Winzler and Smyth, 1948). PCA-D-PTA - Isolated by phosphotungstic acid precipitation from dialyzed perchloric acid filtrates of plasma* An examination of Tables IX and X indicates that the properties of carbohydrate-rich protein fractions isolated from human plasma are affected by their mode of isolation* The yield, composition and electrophoretic properties have been found to vary with the isolation procedure employed* The yields varied from 0*25 gram per liter for sero- mucold to 1*2 grams per liter of plasma for PCA-PTA* The 8 0 FIGURE 8 ELECTROPHORETIC PATTERNS OF MUCOPROTEIN (MP-1) AND MUCOID- Ihnliche SUBSTANZ (MP-1) Mucoldahnliche Substanz -I - + + ^ 1- pH 8.5 -I 4 pH 8.5 pH 4.5 pH 4.5 81 FIGURE 9 ELECTROPHORETIC PATTERNS OF MUCOPROTEIN PREPARATIONS (PCA-AS) AND (PCA-PTA) (PCA-AS) (PCA-PTA) J . L . pH 8«5 pH 8*5 "Mi 1 I j l pH 4*5 D 4 4 pH 4*5 82 FIGURE 10 ELECTROPHORETIC PATTERNS OF SEROMUCOID AND MUCOPROTEIN (PCA- D-PTA) Seromucoid Serozsucold Sercjnucoid (PCA-D-PTA) pH 2.7 pH 8*4 83 very high yield of the latter preparation Is apparently due to the extremely high ash content (59*6 per cent) and the precipitation of polypeptides with an appreciable carbohydrate content* This is indicated by the high carbohydrate and nitrogen contents of preparation PCA-PTA In contrast to that of preparation PCA-D-PTA which was exhaustively dlalyzed after the initial preparation with perchloric acid. It was con firmed by the finding that a concentrated dlalysate of a perchloric acid-filtrate of plasma yielded a precipitate upon the addition of phosphotungstic acid* The reagent control was negative* It is well established that phosphotungstic acid is a precipitant for serum polypeptides (Hahn, 1921; Godfrled, 1939)* That these polypeptides are rich In carbo hydrate is indicated by the work of Rimington (1940)* If the yields are corrected for ash content, the highest yield is that of the mucoprotein preparation, MP-1 (0*49 gram/liter)* The greater yield of this material Is due to less coprecipl- tatlon in the method of isolation (cf* page 109)* The fractionation procedure employed (Weimer, Mehl, and Winzler, 1950) is the most satisfactory of those attempted for the isolation of carbohydrate-rich protein fractions from human plasma with the properties of mucoprotelns* In addition to greater yields, the product is electrophoretlcally homo geneous, possesses the greatest hexose and hexosamlne and 84 lowest nitrogen and ash contents, and might be expected to better approximate the native protein since a milder method of fractionation was employed in its Isolation* The hexose contents of the preparations varied from 6*9 per cent for PCA-D-PTA to 16*5 per cent for MP-1. Iso lation procedures involving the use of heat or strong acids resulted in preparations with lower hexose contents than the one utilizing salt fractionation, MP-1. This suggests that the more drastic Isolation methods might result in the dis aggregation of the native mucoprotein molecule. The hexosamlne content varied from 3*9 per cent for PCA-D-PTA to 12*0 per cent for MP-1. Hexose/hexosamine ratios varied from 1.05 for mucoidfihnliche Substanz to 1.76 for PCA-D-PTA. No constant relationship was apparent among the preparations studied. With the exception of PCA-PTA, nitrogen values were appreciably lower than those for other plasma proteins (Edsall, 1947). The high nitrogen content of PCA-PTA is probably due to its polypeptide content. Some evidence bearing on this has already been presented. Additional support for this statement is its relatively low total protein value (60.0 per cent) in relation to the nitrogen content (14.0 per cent). 85 Tyrosine values showed little variation (3.4 to 4.7 per cent) and in general paralleled the nitrogen contents of the preparations. Total protein content as measured by the biuret reaction varied considerably (29.0 to 82.0 per cent). This large distribution can be explained by the variable carbo hydrate, polypeptide and ash contents of the preparations* Values for total ash varied from 1.8 per cent for MP-1 to 59.6 per cent for PCA-PTA. Isolation procedures utilizing phosphotungstic acid resulted in preparations with the highest ash content (PCA-PTA, PCA-D-PTA). Electrophoretic analyses showed differences in the number of components and in mobilities. Although in some preparations its electrostatic charge has been altered by the method of isolation (ef. page 109), it is believed that the major component present in all preparations is the muco- protein MP-1. This is indicated by the following evidence: 1. All the Isolated preparations are negatively charged when subjected to electrophoresis at pH 4.5. Mehl, Golden, and Winzler (1949) and Mehl and Golden (1950) have demonstrated the presence of two components, negatively charged at pH 4.5. The faster moving component, M-l, was found to be identical with the Isolated mucoprotein MP-1. With the exception of seromucoid the mobility of all prepara- 86 tlons at pH 4*5 approximates that of MP-1, -4*2 x 10*5 cm*V volt/sec. 2. With the exception of the preparation isolated hy the employment of phosphotungstic aeld, the mobility of the major component of all preparations Is similar to that of MP-1, at pH 8*5, -6*6 x 10*5 cm.2/volt/sec* 3. All preparations contain a greater carbohydrate content than any other known protein component of normal, human plasma (cf* Cohn et al., 1946)* 4* Solubility* None of the preparations are coagulated by boiling in aqueous solution* The results obtained in the above series of experiments emphasizes that the mode of isolation of any naturally occurring high polymer Influences its composition and proper ties* A comparative study of carbohydrate-rich protein frac tions isolated from the sera of five animal species* In this series of experiments the only variable factor was the species involved* Human, rat, horse, beef and guinea pig sera were fractionated by the method of Weimer, Mehl and Winzler (1950)* The Isolated fractions were subjected to chemical and electro phoretic analyses under similar conditions* Table XI pre sents the results of chemical analyses* Table XII is a 87 summary or the findings of the electrophoretic analyses* Figures 11 and 12 are photographs of the ascending and de scending boundaries after electrophoresis at the indicated pH values* All of the animal sera were hemolyzed and it was necessary to refractionate the final product at least once in order to remove the contaminating hemoglobin* This pro cedure was not successful in the case of horse serum muco- protein. After three refractionations the carbohydrate-rich protein fraction still contained 4 electrophoretic components* Due to the necessity of refractionation with a concommittant loss of material, no accurate estimate can be made regarding the relative amounts of mucoprotein present in the sera of the various species* With the exception of the horse preparation, none of the mucoprotein preparations were coagu lated by boiling in aqueous solution* An examination of Tables XI and XII indicates a marked species difference in the mucoprotein fraction of serum* Disregarding the carbohydrate-rich protein fraction from horse serum and confining our observations to those prepara tions which are substantially homogeous, it is apparent that pronounced differences exist in composition and in electro phoretic mobility* TABLE XI COMPARATIVE STUDY OP MUCOPROTEINS ISOLATED PROM THE SERA OP FIVE ANIMAL SPECIES Species Chemical Analysis*___________ Hexose Hexos amine Nitrogen Tyrosine Total protein % % % % %----- Human 16.5 12.0 10.1 4.0 63.0 Horae**- 6.7 4.6 15.8 5.5 wTwWW Rat 7.1 5.2 11.4 2.5 tf Mil m n c Beef 10.7 7.9 11.5 2.3 79.0 Guinea pig 9.1 6.6 11.7 2.0 80.0 * Moisture-free basis 4* Material contained 4 electrophoretic components ■**** Color of solution interfered with test TABLE XII SUMMARY OP AN ELECTROPHORETIC INVESTIGATION OF MUCOPROTEINS ISOLATED PROM THE SERA OF FIVE ANIMAL SPECIES Species PH Number of Mobility x 10"5 cm.2/volt/sec components Ascending Descending boundary boundary Human 4.5 1 -4.7 -4.2 8.5 1 -7.2 -6.5 Beef 4.5 1 -3.2 -2.5 8.6 1 -6.2 -5.6 Rat 4.5 1 -0.8 • 0 1 8.6 1 -6.2 -5.6 Guinea pig 4.6 1 -0.8 -0.8 8.5 1 -5.8 -5.0 Horse 4.5 4 Component 1 -2.8 -1.3 Component 2 i O • -a -0.5 Component 3 0.5 0.8 Component 4 3.0 1.3 90 FIGURE II ELECTROPHORETIC PATTERNS OF HUMAN PLASMA MUCOPROTEIN AND GUINEA PIG SERUM MUCOPROTEIN Human Guinea pig + pH 8.5 D 4* 4~ " + ■ — j — pH 4.5 4- 4- pH 8.5 -*-4- + — | - D A pH 4.6 FIGURE 12 ELECTROPHORETIC PATTERNS OF BEEF AND RAT SERUM MUCOPROTEIN Baef Rat 92 The hexose contents varied from 7*1 per cent for rat serum mucoprotein to 16.5 per cent for human serum mucoprotein. The hexose contents of all animal species was substantially lower than that of human* The hexosamine content of all preparations was suffi ciently high to place them in the category of mucoproteins according to the definitions of Meyer (1945) and Stacey (1946) • The hexosamine content of all preparations was found to be directly proportional to the hexose content* The following hexose/hexosamine ratios were determined: Human* 1*57; rat* 1*56; beef* 1.55; guinea pig* 1*58* This finding indicates a ratio of 4 moles of hexose to 5 moles of hexosamine in the serum mucoprotein molecules of the above species* Nitrogen values varied from 10*1 per cent for human serum mucoprotein to 11*7 per cent for guinea pig serum muco protein* 3he nitrogen content of all preparations was signi ficantly lower than those determined for the major protein constituents of human plasma (Cohn et al,, 1946)* Tyrosine values ranged from 2*0 per cent for guinea pig serum mucoprotein to 4*0 per cent for human serum muco protein* The animal serum mucoprotein showed little vari ation* 2*0 to 2.5 per cent* The most striking difference was between the human and animal preparations* 93 Total protein values exhibited an almost direct relationship to the nitrogen content or the preparations the ratio showing but a slight variation, 6,24 to 7*0. With the exception of the fraction from horse serum, all the preparations were substantially homogeneous when subjected to electrophoresis. The slight boundary spreading in the beef and in the rat serum mucoprotelns at pH 4,5 is believed due to the concentration of the solution. Due to the small amount of material available it was necessary to used 0,6 per cent solutions of the respective proteins* At this dilution, diffusion is much greater than at higher protein concentrations. The skewness in the ascending boundary is due to the delta boundary. All preparations were negatively charged at pH 4,5, This has been found to be a unique characteristic of a muco protein Isolated from human serum by ammonium sulfate frac tionation (see page 63) and is apparently true for the muco protelns of other species Isolated by the same procedure. Marked differences are apparent in electric mobility. These differences are most pronounced at pH 4,5, The rat and guinea pig preparations possessed a very low mobility at this pH, -0,7 and -0,8 x 10~5 cm,2/volt/sec, respectively. This might suggest that the Isoelectric point for these materials is higher than the serum mucoprotelns of the other 94 species* At pH 8.5 less variation in mobility was found, -5*0 to -6.5 x 10*^cm.^/volt/sec* Human serum mucoprotein exhibits a greater electrostatic charge at the pH values employed than the serum mucoprotelns of the other species investigated* Further chemical analyses are indicated to ascertain the reasons for this difference in electrophoretic behavior. DEMONSTRATION OF MUCOPROTEIN IN NORMAL AND MALIGNANT TISSUE The presence of glycoproteins in tissues has been demonstrated by several investigators employing cytochemical procedures (Harter, 1948; Gersh and Catchpole, 1949). None of these workers attempted to Isolate and characterize the material that was stained by periodic acid and leucofuchsln. This stain was given chemical significance by Hotchkiss (1948) who showed it to be fairly specific for water and/or alcohol-Insoluble polysaccharides or polysaccharide-containing protein complexes. Elevated serum mucoprotein levels have been found in patients with cancer and with pneumonia (Wlnzler and Smyth, 1948; Mehl and Golden, 1950) and in laboratory animals with cancer (Wlnzler and Burk, 1944). It was therefore decided 95 to investigate rat fibrosarcomas and beef lung tissues to ascertain if they contained mucoprotein* It was postulated that such a material in tissues should have the same solu- bility properties as serum mucoprotein* Isolated tissue was coarsely minced with shears then extracted with 10 volumes of distilled water for one week at 4<>c* The suspended tissue was filtered and the filtrate fractionated by the method of Welmer* Mehl, and Wlnzler (1950). The isolated fractions were then subjected to electrophoretic analysis at pH values of 4*6 and 8*5 and to chemical analysis* Table XIII gives the results of the chemical analyses* Table XIV is a summation of the data obtained in the electrophoretic studies* Figure 15 shows the electrophoretic patterns obtained* For comparative purposes chemical and electrophoretic data of rat and beef serum mucoprotein are included in Tables XIII and XIV. The results of chemical analysis demonstrate that all the isolated fractions contain carbohydrate-rich proteins* The chemical composition insofar as it was investigated does not differ greatly from the serum mucoprotelns of the same species* Hie differences in chemical composition of the frac tions Isolated from the two rat fibrosarcomas is interesting 96 and may provide some clue as to the source of serum naico- proteins when correlated with the areas of visible necrosis. Well developed rat fibrosarcomas, AX-C9935, usually contain from 50 to 80 per cent of their mass as necrotic tissue in contrast to well developed rat fibrosarcomas, E 2730, which consist of only 10 to 30 per cent of necrotic tissue. Whether the greater area of necrosis is related to the in creased carbohydrate content of the fraction is not known but it is a point that merits further investigation. The fraction isolated from beef lung was unique in the fact that the hexosamine content exceeded the hexose content. The nitrogen content was very high in relation to the total protein content. With the exception of values for total protein, the composition of the fraction isolated from rat fetuses (albino rats, Southern California strain}, does not differ significantly from the material isolated from the rat fibro sarcoma, E 2730. Electrophoretic analyses showed that the Isolated tissue protein fractions are mixtures containing at least three components. Due to the small amount of material avail able, refraetionatlon was not attempted. It is significant that all the preparations analyzed contained at least one negatively charged component at pH 4.5. The mobilities of 97 TABLE XIII COMPARATIVE STUDY OF CARBOHYDRATE-RICH PROTEIN FRACTIONS ISO LATED FROM TISSUES* Tissue Hexose Hexos- Nitrogen Tyro- Total amine sine protein ■ ■ y ■ ■ - - y a r . J o J o J o J o J o Rat fibrosarcoma (AX-C9935) 11.6 7.3 10.6 3.3 72.0 Rat fibrosarcoma (E 2730) 7*4 5.5 11.6 3.1 75.0 Rat fetus** 7.4 5.0 9.9 3.2 59.0 Rat serum muco protein 7.1 5.2 11.4 2.5 Beef lung 8.0 8.3 15.1 2.8 68.0 Beef serum muco protein 10.7 7.9 11.3 2.3 79.0 * Moisture-free basis ** Sufficient material for chemical analysis was Isolated from rat fetuses by the same procedure. TABLE XIV SUMMARY OP AN ELECTROPHORETIC INVESTIGATION OP CARBOHYDRATE- RICH PROTEIN FRACTIONS ISOLATED PROM TISSUES Tissue PH Number of Mobility x 10“5 components Ascending boundary cm.2/volt/s ec« Descending boundary Beef lung 4*6 3 Component 1 -3.3 -3.5 Component 2 0.0 0.0 Component 3 1.5 1.0 8.5 2 Component 1 -6.2 -5.2 Component 2 -2.5 -2.7 Beef serum muco 4.5 1 -3.2 -2.5 protein 8.6 1 -6.2 -5.6 Eat fibrosarcoma 4.6 1* O • O to -1.0 0.0 to -1.0 (AX-C9935) 8.5 3 Component 1 -6.3 -5.4* Component 2 -5.2 Component 3 -3.5 Hat fibrosarcoma 4.5 1* o • o to —1.0 0.0 to -1.0 (E 2730) Eat serum muco 4.5 1 -0.8 -0.7 protein 8*6 1 -6.2 -5.6 Diffuse boundary indicates heterogeneity. 99 FIGURE 15 ELECTROPHORETIC PATTERNS OF CARBOHYDRATE-RICH PROTEIN FRACTIONS ISOLATED FROM NORMAL AND MALIGNANT TISSUES Rat fibrosarcoma AX-C 9935 Beef lung D A pE 8.5 pH 8.5 pH 4.6 pH 4*6 100 this component are within the range of v&luea found for the homogeneous serum mucoprotein of the same species* It Is well established that the electric mobility of a protein may vary considerably with variations in the types and quantities of other proteins and other substances in the solution (cf* Edsail, 1947)# The similarity of mobilities of serum mucoprotelns and one component of the carbohydrate- rich protein fractions from tissues is more marked at pH 8*5* On the basis of chemical composition, solubility and electrophoretic mobility It seems probable that one com ponent of the Isolated tissue fractions Is either Identical to or closely related to the serum mucoprotein of that animal species* ELECTROPHORETIC DEMONSTRATION OP AN ACIDIC PROTEIN IN TWO STRAINS OP TUMOR-BEARING RATS Peterman, Karnovsky, and Hogness (1948) demonstrated the presence of an acidic protein component In the plasma of patients with gastric cancer by electrophoresis at pH 4*0. This component has been shown to be identical with the muco protein, MP-1, isolated from normal human plasma by Mehl, Golden, and Wlnzler (1949)* Two strains of tumor-bearing rats, Marshall and Irish, maintained in this laboratory afforded an opportunity for 101 determining whether electrophoresis at pH 4.0 could be employed as a procedure for determining the presence of a similar component in rat serum* Blood was obtained from adult rats with well developed fibrosarcomas by cardiac puncture under ether anesthesia* The serum was diluted to a protein concen tration of 1*0 per cent with buffer* Dialysis and electro phoresis were carried out as described in Chapter II (Materials and Methods)• Figure 14 shows photographs of the ascending and de scending boundaries after electrophoresis at pH 4*0* The patterns of both strains are similar and show a negative component migrating toward the anode* Better resolution of this component is obtained in the ascending boundaries where the albumin and globulins are ascending and the negatively charged component is descending* In the descending boundary the acidic component is apparent as a shoulder on the globu lin boundary* The electrophoretic mobilities found for the negative component are given in Table XV. For comparison the data of Peterman at al* (1948) for the mobilities of the most acidic component in human plasma and the mobilities of iso lated rat and human serum mucoprotein determined in the present investigation are included* 102 FIGURE 14 ELECTROPHORETIC PATTERNS OF THE SERUM OF TWO STRAINS OF TUMOR- BEARING RATS AT pH 4*0 Irish strain 103 TABLE XV COMPARATIVE ELECTROPHORETIC MOBILITIES OF ACIDIC PROTEIN COMPONENTS IN RAT SERTJM AND HUMAN PLASMA Material pE Mobility x 10~5 cm.g/volt/sec Ascending Descending boundary boundary Serum Prom Marshall 520 strain tumor-bear ing rats 4*0 Serum from Irish strain tumor-bearing rats 4*0 Plasma from patient with gastric cancer (Peterman et al., 1948) 4.0 Isolated human plasma mucoprotein (MP-1) 4.0 Isolated rat serum mucoprotein 4*5 -0.9 -0.4 •3.3 •0.8 -0.6 -0.3 -2.7 -2.8 -0.7 104 The mobilities of the negative component in serum from tumor-bearing rats differ substantially from that reported by Peterman, Karnovsky, and Hogness (1948) for the most acidic component in human plasma. This species difference has been noted for Isolated mucoprotelns (page 86). It is probable in view of the similar rate of electrophoretic migration that this acidic component which can be demonstrated in the sera from tumor-bearing rats is identical with the Isolated mucoprotein from normal rat serum. This could be ascertained by the procedure of Mehl et al. (1949), adding isolated rat mucoprotein to normal serum and to serum from tumor-bearing rats and subjecting the sera to electrophoretic analyses at pH 4.0. ELECTROPHORETIC DEMONSTRATION OP HUMAN PLASMA MUCO PROTEIN, MP-1, IN HUMAN UMBILICAL CORD SERUM The electrophoretic mobility of a protein can be utilized as a means of identification. Mehl and his associ ates (1949, 1950) have employed electrophoresis in acetate buffer at pH 4.5 as a method for the demonstration and identi fication of mucoprotein in serum. Since it had been suggested that increased serum poly saccharide levels (Shetlar et al., 1949a, b) and Increased 105 serum mucoprotein levels (Wlnzler, personal communication) are due to tissue proliferation, It seemed worthwhile to subject serum from a human umbilical cord to electrophoretic analysis under the conditions employed by Mehl, Golden, and Wlnzler (1949). Figure 15 shows the patterns of the ascending and descending boundaries after electrophoresis. At pH 4.5, Figure 15 shows one negatively charged component, comprising 16 per cent of the total protein concentration. This com ponent migrated toward the anode with mobilities of -2.4 x 10~5 cm*2/volt/sec. and -3.6 x 10*5 cm.^/volt/sec. in the descending and ascending mucoprotein boundaries respectively. These mobilities are similar to those found by Mehl and co- workers (1949) for the component in serum which they desig nated as M-l and found to be identical with the Isolated mucoprotein MP-1. The relative concentration of MP-1 In the umbilical cord serum, 16 per cent, is approximately 20 times as great as that found in the serum of normal subjects (Mehl, personal communication). This finding Increases the possi bility that increased serum mucoprotein levels are in some manner Involved with tissue proliferation* The apparent absence of the M-2 component in the descending boundary Is interesting. This component has been found to be present by Mehl, Golden, and Wlnzler (1949) in 106 FIGURE 15 ELECTROPHORETIC PATTERNS OF HUMAN UMBILICAL CORD SERUM AT pH 4.5 AND pH 7.4 + -— | —► - — < I » + D A pH 4.5 ~ I + 4 * <- -1 — 1 n a l D pH 7.4 107 normal serum and In Increased amounts in the sera of patients with a wide variety of pathological conditions (Mehl and Golden, 1950)• The lack of resolution of any of the globulin components of normal serum in either the ascending or descending boun- daries at pH 4.5 suggests a protein - protein interaction, since they were found to be present by electrophoresis at pH 7.4 (Figure 15). EFFECT OF BUFFER ANION UPON ELECTROPHORETIC MOBILITY IN THE pH RANGE, 2.0 - 4.0 In the determination of the pH- mobility curve of human plasma mucoprotein (MP-1) the selection of suitable buffer solutions below pH 4.0 presented the greatest diffi culty. At the suggestion of Dr. John W. Mehl, four different buffer systems were employed. The preparations of the buffer systems, electrophoretic analyses, and mobility deter minations were carried out as previously described (Chapter II). Table XVI is a summation of data obtained in this investigation. It is evident from an inspection of Table XVI that, apart from pH, the species of buffer anion also exerts a marked influence upon the rate of electrophoretic migration. 108 TABLE XVI EFFECT OF BUFFER ANION UPON THE ELECTROPHORETIC MOBILITY OF HUMAN PLASMA MUCOPROTEIN (MP-1) IN THE pH RANGE 2,0 - 4.0 Buffer system PH Mobility x 10“5 Ascending boundary cm.2/volt/sec Descending boundary Sodium acetate - sodium chloride 3.9 -3.3 -2.8 Sodium acetate - sodium chloride 3.8 -2.9 -2.5 Sodium acetate - sodium chloride 3.5 C* . H 1 . H a Sodium monocitrate - sodium chloride 3.1 -0.8 « 0 1 Sodium perchlorate - sodium chloride 2.9 -3.1 -2.8 Sodium trichloracetate - sodium chloride 2.9 * -1.7 Sodium perchlorate - sodium chloride 2.7 -2.7 -2.0 Sodium perchlorate - sodium chloride 2.3 -1.4 -1.1 Sodium trichloracetate - sodium chloride 2.0 —1. 3 -1.2 Not possible to ascertain* 109 This effect has been related to the activity of the electro lyte (Cohn and Edsall, 1943)* It is especially pronounced in the pH range 2*9 - 3*9. The citrate buffer system pro duced a marked reduction in electric mobility. This finding confirms an earlier observation of Tiselius (Dr* £* Jameson, personal communication) * The perchlorate anion on the other hand apparently Increases the rate of migration* A marked decrease in mobility in acetate buffer was observed as soon as the pH was reduced to a point outside the effective buffer range for acetic acid and its conjugate base* The slight difference in mobilities reported at pH 2*0 and pH 2*3 Is well within the experimental error of the method* In the region of the isoelectric point (pH 1*8), the perchlorate and trichloracetate anions do not seem to have any detectable difference upon electric mobility* Although both of these anions are protein preclpitants, the great solubility of mucoprotein allowed them to be used* STUDIES PERTAINING TO THE MP-2 COMPONENT Wlnzler, Devor, Mehl, and Smyth (1948) showed that mucoprotein preparations obtained by saturating perchloric acid filtrates of human plasma with ammonium sulfate were mixtures, containing at least three electrophoretic com- 110 ponents. Mehl, Humphrey, and Wlnzler (1949) designated these components as MP-1, MP-2, and MP-3 in the order or their isoelectric points which were found to be approximately 2*3, 3.4, and 4.3 respectively. The proportion of components was found to vary considerably from preparation to prepara tion and the MP-2 component was not well resolved at pH values above 6.0. In Figure 16 c and d are shown electrophoretic patterns at pH 8.5 and pH 4.5 respectively of material Isolated by the method employed by the above authors. At pH 8.5 the pattern is almost Identical to that reported by Winzler et al. (1948), but at pH 4.5 the material shows evidence of only a trace of the MP-2 component. In contrast the electrophoretic patterns reported by Winzler ^t al. (1948) and Mehl, Humphrey, and Winzler (1949) show substantial amounts of the MP-2 component with decreased amounts of the MP-1 component at pH 4.5. The preparations employed by the latter authors had been exposed to acid solutions for varying lengths of time at room temperature. The above data suggested that the MP-2 component might arise from a modification of the MP-1 component by exposure to strongly acid solutions. To test this hypothesis the following experiments were performed. Ill FIGURE 16 ELECTROPHORETIC PATTERNS OF MUCOPROTEIN PREPARATIONS ISOLATED IN EXPERIMENT I - j -v 1 D pH 3#5 pH 8.5 pH 8.5 pH 4.5 112 Experiment 1^. One gram of a homogeneous mucoprotein, MP-1, Isolated by the method of Weimer, Mehl, and Winzler (1950) was dissolved in 50 ml* of distilled water and added to 700 ml* of pooled, normal, human plasma. The plasma was fractionated by the procedure of Winzler, Devor, Mehl, and Smyth (1948) and the Isolated material was subjected to electrophoresis at pH 8.5. Figure 16, a, shows photographs of the ascending and descending boundaries after electro phoresis. For comparison electrophoretic patterns of the added mucoprotein, MP-1 (Figure 16, b) and plasma fractionated by the above method without the addition of MP-1 (Figure 16 c, d) are included. Figures 16, a, and 16, c, are quite similar and no relative increase in the amount of the MP-2 component can be noted even though the plasma fractionated contained an Increased amount of the MP-1 component. This indicates that the MP-1 component is not dissociated into the MP-2 component by strongly acid solutions at room temperatures for short periods of time (30 to 60 minutes). A coneommittant aspect of this experiment was the determination of the extent of coprecipitation of muco protein (MP-1) with the perchloric acid-precipitated proteins of human plasma. The average yield of mucoprotein isolated by this method was 0.345 graav^liter of plasma or 0.242 gram/ 700 ml. In the above experiment, in which 1.0 gram of 113 mucoprotein (MP-1) was added to 700 ml* of plasma prior to fractionation of 0*504 gram material was isolated. The difference, 0.262 gram (0.504 - 0.242) represents the amount of added mucoprotein that was isolated. Thus, there was a 26.2 per cent recovery of the added mucoprotein. The next experiment attempted was more direct and more productive of results. Experiment II. Two hundred mg. of an MP-1 preparation was dissolved in 10 ml. of distilled water and 5 ml. of 1.8 M perchloric acid were added. The solution was allowed to stand at room temperature (25°C.) for 6 hours. The solution was dialyzed against running tap water for 4 hours and against distilled water for 36 hours at 4°C. It was then dialyzed against two changes of acetate huffer, pH 4.5, for 48 hours and subjected to electrophoretic analysis. Figure 17, a and b, show the electrophoretic patterns obtained for material and for an untreated control respectively. It is evident that the above treatment has resulted In the forma tion of a new electrophoretic component comprising 32 per cent of the total protein. Experiment III. Of interest in relation to the pre ceding experiments was the effect of mild acid hydrolysis upon the electrophoretic mobility of human plasma mucoprotein, 114 FIGURE 17 ELECTROPHORETIC PATTERNS OF MUCOPROTEIN PREPARATIONS ISOLATED IN EXPERIMENTS II AND III D + -t * “ A pH 4*5 — -----> - V - * — - 4- -f -«- - - - - - - - - D A pH 4*5 d I — pH 4*5 - t - + <---1 — D A pH 4*5 115 MP-1. Two hundred mg* of an MP-1 preparation was dissolved in 20 ml • of distilled water and the pH of the solution ad justed to pH 4.7 with dilute acetic acid* The solution was placed in a boiling water bath for 30 minutes, cooled and filtered* Aliquots of the filtrate were tested qualitatively for the sulfate ion with benzidene and barium chloride re agents and a positive test was obtained in each case* The filtrate was dlalyzed against acetate buffer at pH 4*6 for 4 days and subjected to electrophoretic analysis. Figure 17 c and d show the electrophoretic patterns for the material subjected to hydrolysis and an untreated control respectively. Table XVII is a summary of the electrophoretic studies for this section. It is apparent that there has been no loss of homo geneity as a result of hydrolysis. However, the mobility of the material has been markedly reduced. The mobilities for the descending boundary for the treated material and the control are -3.1 x 10"^ cm.^/volt/sec. and -4.3 x 10*^ cm.2/volt/sec. respectively. The value for the material subjected to mild acid hydrolysis corresponds very closely to that of the MP-2 component obtained by treatment of MP-1 with perchloric acid, -3.4 x 10~5 cm.2/volt/sec. and approaches the maximum value reported for the mobility of the MP-2 com ponent at pH 4.5, -2.8 x 10~5 cmm2/volt/sGCm (Mehl, Humphrey, TABLE XVII SUMMATION OF ELECTROPHORETIC STUDIES PERTAINING TO HUMAN PLASMA MUCOPROTEIN, MP-2 Preparation* Figure Number of Mobility x 10"5 cm.2/volt/sec. reference pH components Aseending Descending boundary boundary PCA-AS (added a 8.5 MP-1) MP-1 (Control) b 8.5 PCA-AS (Control) c 8.5 PCA-AS (Control) d 4.5 MP-1 (Perchloric a 4.5 acid - treated) MP-1 (Control) b 4.5 3 Component 1 -7.7 -6.3 Component 2 -6.5 -4.7 Component 3 -0.9 -0.8 1 -7.3 -6.1 3 Component 1 -8.2 -6.6 Component 2 -7.3 -5.3 Component 3 -0.9 -0.9 3 Component 1 -5.1 -3.8 Component 2 -3.5 -2.9 Component 3 -0.7 -0.7 2 Component 1 -4.9 -4.3 Component 2 -4.0 -3.4 1 -4.3 -3.8 116 TABLE XVII (continued) SUMMATION OP ELECTROPHORETIC STUDIES PERTAINING TO HUMAN PLASMA MUCOPROTEIN, MP-2 Preparation* Figure Number of Mobility x 10“® cm.^/volt/sec. reference pH components Ascending Descending boundary boundary MP-1 (Heated) c 4.6 1 -3.1 —3.1 MP-1 (Control) d 4.6 1 -4.9 -4.3 * PCA-AS - Material isolated by procedure of Wihzler, Devor, Mehl and Smyth (1948), MP-1 - Material Isolated by procedure of Welmer, Mehl, and Winzler (1950). H H -a 118 and Winzler, 1949)* Results from this series of experiments suggests that exposure to solutions of strong acids at room temperature or hydrolysis in weak acid solution at 100°C* are capable of modifying the MP-1 molecule, resulting in the formation a new electrophoretic component of mucoproteln* The mobility of the modified mucoproteln is apparently influenced by the nature and severity of the chemical treatment to which the MP-1 molecule has been exposed* CHAPTER V DISCUSSION A more complete knowledge of both the chemical and physicochemical characteristics of the plasma proteins is one of the essentials to our understanding of their function* The fractionation of pooled, normal, human plasma with ammonium sulfate as described in this thesis has resulted in the isolation of a homogeneous mucoproteln. This mucoproteln, which has been designated MP-1, has satisfied three of the four criteria usually applied for the purity of proteins, constancy of chemical analysis, homogeneity by electro phoresis, monodlsperse in the ultracentrifuge. Although constant solubility studies were not carried out, the satis faction of the above criteria indicates that the material consists of but one major component. The amino acid composition, chemical composition, and physicochemical characteristics of MP-1 have been determined as completely as for any of the other protein components of human plasma with the possible exception of albumin. The physicochemical properties of the various plasma components have been summarized In Table XVIII, together with an esti mate of the amount present in plasma. Data for the globulins, albumin, and fibrinogen were obtained from Oncley, Scatchard, 120 and Brown (1947) and Cohn et al* (19 50)• The variations in these physicochemical properties of the plasma proteins are great* The wide range of these physicochemical properties does not seem so unusual, however, if the wide range of functions of these proteins and the unusual composition of many of the molecules Is considered* It is apparent that the MP-1 molecule resembles many of the other proteins in one or two constants, but is dis similar in all others* The sedimentation constant of MP-1, 3*11, is similar to that of one of the 1-globulins, 2*9; the partial specific volume, 0*675, approximates that of OC 2-globulin, 0*693; the intrinsic viscosity, 6*9, is close to that of oC 1-globulin; the frictional ratio, 1*43, Is almost identical to those of gC I-globulin, 1*38, /5 1- globulin, 1.37, and 2T-globulin, 1.38* In certain constants plasma mucoproteln, MP-1, is an unusual protein in comparison with the other components of plasma* It has the smallest molecular weight, 44,100, the lowest Isoelectric point, pH 1.8,end the smallest partial specific volume, 0*675, of any of the Isolated components of human plasma. In respect to these constants it resembles fetuin which Pedersen (1947) has isolated from the fetal sera of the cow by ammonium sulfate fractionation* He reported the following physico chemical constants for this material: S2q = 3*09, D20 * 5.0, 121 TABLE XVIII PROTEIN COMPONENTS OF NORMAL HUMAN PLASMA CHARACTERIZED BY PHYSICOCHEMICAL METHODS Electrophoreti c Component Approxi- mate amount in plasma Sedimen tation Constant s20* w Diffu sion Con stant D20* w Partial specifle volume Intrin sic vis cosity H0 x 10: gms ./liter Albumin 32 4.6 6.1 0.733 4.2 dC 1-globulin 2 5.0 0.841 6.6 oC 2-globulin 1 9.0 0.693 9.2 /3i-globulin 2 5.5 0.725 5.5 /31-globulin 2 7.0 0.740 /SI-globulin 1 20.0 0.740 < 61-globulin 2 2.9 0.950 4.1 / 62-globulin 2 7.0 7T-globulin 5 7.2 0.739 6.0 Tf-globulin 1 10.0 Fibrinogen 2 9.0 25.0 Mueoprotein 1 3.11 5.27 0.675 6.9 122 TABLE XVIII (continued) PROTEIN COMPONENTS OF NORMAL HUMAN PLASMA CHARACTERIZED BY PHYSICOCHEMICAL METHODS Electrophoretic component Frie- Molecular Approximate Approximate tional weight Dimenaions isoelec- ratio ten- Dia- trie point f/f© M gth meter pHj ft ft Albumin 1*28 69,000 150 38 4.7 (£ 1-globulin 1.38 200,000 300 50 5.2 2-globulin 1.68 (300.000) 4.9 (G 1-globulin 1.37 90.000 190 37 5.0 /3 1-globulin (150,000) 5.4 /$ 1-globulin (500,000) -1,000,000 /61-globulin 1.7 1,300,000 185 185 P 2-globulin (150,000) 6.3 7T -globulin 1.38 156,000 235 44 6.3 Y-globulin (300,000) 7.3 Fibrinogen 1.98 400,000 700 38 5.3 Mucoproteln 1.43 44,100 174 27 1.8 123 V20 ** 0*692, Molecular weight * 48,700, pHj * 3.5. Petuin also had a relatively low nitrogen content, 12*3 per cent* It does not seem probable that the fetuin described by Pedersen (1947) is beef mucoproteln, due to the difference in "salting out" characteristics* Pedersen precipitated fetuin at ammonium sulfate concentrations of 45 per cent of saturation and washed the precipitate with 0*50 saturated ammonium sulfate solutions* Beef mucoproteln is soluble under these conditions and is not precipitated until an ammonium sulfate concentration approaching saturation Is attained* Zannettl (1897, 1903} designated a carbohydrate-rich fraction from serum as seromucold due to Its similarity to ovomucoid* Recently, Fredericq and Deutseh (1949) have referred to an ovomucoid preparation, which they characterized, as a mucoproteln* It is of interest to compare the charac teristics of an electrophoretically homogeneous mucoproteln isolated from egg white, ovomucoid, to those of an electro phoretically homogeneous mucoproteln isolated from human plasma, MP-1* Table XIX summarizes the data reported by Predericq and Deutseh (1949) and gives the corresponding values for MP-1* The similarity in composition between these two muco- proteins is striking* The total carbohydrate contents are 124 TABLE XIX A COMPARISON OP HUMAN PLASMA MUCOFROTEIN, MP-1, WITH OVOMUCOID Chemical composition Ovomucoid MP-1 Hexose (per cent) Hexosamine (per cent) Nitrogen (per cent) Ash Physicochemical constants Molecular weight Sedimentation constant Diffusion constant Partial specific volume Frictional ratio Intrinsic viscosity Axial ratio Isoelectric point 9*7 17.0 13.2 0.05 27,000 2.8 8.0 0.685 1.35 5.5 6.5 3.9 16.4 11.9 10.1 1.8 ,100 3.11 5.27 0.675 1.43 6.9 8.0 1.8 125 almost Identical although an Inverse relationship exists between the hexose and hexosamine contents of the two pre parations. The higher nitrogen value of ovomucoid Is pro bably due to its greater content of hexosamine. In respect to physicochemical constants, the similarity persists. Both are small proteins, asymmetric In shape with low Iso electric points. An outstanding characteristic is the remarkably low partial specific volume of these materials. The values reported are the lowest appearing in the litera ture (cf. Svedberg and Pedersen, 1940). It seems reasonable to assume that they are related to their high carbohydrate contents. Table XX presents the amino acid composition of the major protein components of human plasma (Edsall, 1947) for comparison with that of MP-1. The amino acid composition of MP-1 Is qualitatively identical to that of the other proteins insofar as they have been determined. However, great quantitative differences exist. The differences become less significant when the amino acid contents of MP-1 are multiplied by the factor 105.9/58.24 (column 5) to com pare comparable amounts of protein. This factor is the ratio of the average per cent of known amino acids in the major components (105.9) and MP-1 (58.24). However, certain marked differences still persist between the amino acid TABLE XX AMINO ACID COMPOSITION OP HOMAN PLASMA PROTEINS* (1) (2) (3) (4) (5) (6) (7) (8) Component MP-1 MP-1** Alb. dC nP F. Total nitrogen 10*1 15.95 15.24 16.03 16.09 Total sulfur 1.02 1.96 1.32 1.02 Amide nitrogen 0*7 0.88 1.11 Arginine 3.65 6.7 6.2 7.7 6.8 4.8 7.9 Aspartic acid 7.44 13.5 10.4 9.0 9.8 8.8 13.6 Cystine and Cysteine 0.60 1.1 6.3 1.5 3.5 3.1 2.7 Glycine 0.62 1.5 1.6 3.1 5.6 4.2 5.6 Glutamic acid 10.73 19.6 17.4 21.6 14.5 11.8 14.3 Histidine 1.31 2.4 3.5 2.8 2.8 2.5 2.8 Isoleucine 3.15 5.7 1.7 1.7 5.0 2.7 4.8 Leuelne 5.21 9.5 11.0 14.2 7.9 9.3 7.1 Lysine 5.03 9.2 12.3 8.9 6.6 8.1 8.3 H to 0> TABLE XX (continued) AMINO ACID COMPOSITION OF HUMAN PLASMA PROTEINS* (1) Component (2) MP-1 (3) MP-1** (4) Alb. (5) CC (6) /$ (7) T (8) F. Methionine 0.65 1.2 1.3 1.4 1.7 1.1 2.5 Phenylalanine 3.91 7.1 7.8 4.6 4.7 4.6 4.2 Proline 2.37 4.3 5.1 4.7 7.1 8.1 5.7 Threonine 4.80 8.7 5.0 4.9 6.1 8.4 6.6 Tryptophan 1.25 2.3 0.2 1.9 2.0 2.9 3.3 Tyrosine 1.99 3.6 4.7 4.5 6.0 6.8 5.8 Serine 2.51 4.6 3.7 5.0 7.1 11.4 9.2 Valine 2.82 5.1 7.7 5.2 7.0 9.7 4.4 Total 58.24 106.1 105.9 102.7 104.2 108.3 108.8 * Grams/lOO grams, moisture-free, ash-free basis, ** Grams/182 grams, moisture-free, ash-free basis. 127 128 composition of MP-1 and the major components of human plasma* The albumin content of cystine, isoleuclne, and tryptophan, the oc-globulin content of aspartic acid, leucine, and iso leucine, the ^-globulin content of glycine and glutamic acid, the 'ZP-globulin content of aspartic acid, glutamic acid, serine, and valine, the fibrinogen content of glycine, glutamic acid, and serine differ greatly from the corresponding amino acid contents of MP-1* It should also be noted that the total sulfur content of the major components of human plasma is accounted for by the sulfur contents of the sulfur containing amino acids* The cystine, cysteine, and methio nine sulfur account for only 22 per cent of the total sulfur content of MP-1* It therefore seems unlikely that any of the major protein components of human plasma are direct precursors of MP-1* (The following symbols are employed in Table XX: Alb. indicates albumin, oc Indicates oC -globulin, /3 indicates /S -globulin, IT indicates "ZP -globulin, F* indicates fibrinogen*) The results obtained from chemical and electrophoretic analyses of carbohydrate-rich protein fractions isolated from pooled, normal, human plasma by six different procedures demonstrate that the method of isolation affects the proper ties of the material* 129 Analysis of serum mucoproteins isolated by the same procedure from several species of animals showed pronounced species differences* It is believed that the two factors (1) method of isolation, and (2) species, account for the major part of the wide range of analytical results reported in the litera ture for carbohydrate-rich fractions Isolated from plasma and serum* Evidence pertaining to this has been presented in Chapter IV, page 74* Table XXI is a summary of the re sults of chemical analyses of preparations isolated from plasma and serum in the current investigation compared with analytical results reported in the literature* In Table XXI the following symbols are used to indicate isolation pro cedures : A - isolated by ethanol precipitation from dialyzed, concentrated sulfosallcylie acid filtrates of serum and plasma (Mayer, 1942; Winzler and Burk, 1944)* B - isolated by precipitation with ammonium sulfate from perchloric acid filtrates of plasma (Winzler, Devor, Mehl, and Smyth, 1948)* C - isolated by a series of ammonium sulfate precipi tations (Weimer, Mehl, and Winzler, 1950). 130 D - isolated by ethanol precipitation from concentrated heat-coagulated filtrates of plasma and serum (Rimington, 1940). E - isolated by phosphotungstic acid precipitation from perchloric acid filtrates of plasma (Winzler and Smyth, 1948). El - Isolated by phosphotungstic acid precipitation from dialyzed perchloric acid filtrates of plasma (Winzler and Smyth, 1948). It is unfortunate that so little physicochemical data has been reported thus limiting comparison to partial chemical composition. The results presented in Table XXI confirm the authorfs thesis that the properties of carbohydrate-rich protein fractions Isolated from plasma and serum are profoundly affected by the species and by the method of isolation. The differences among the preparations Isolated by the author have already been discussed (Chapter IV, page 74). The present discussion will be confined to a comparison of the authorfs results with those reported in the literature. Fractions (mucoldahnliche Substanz) Isolated from human plasma and serum by Procedure A are in reasonably good agreement with the exception of the value for hexose* The difference in the hexose values reported by Mayer (1942) TABLE XXI A COMPARATIVE STUDY OP CARBOHYDRATE-RICH PROTEIN FRACTIONS ISOLATED PROM PLASMA AND SERUM* Species Isolation procedure Reference Hexose Hexos- amine Nitro gen Tyro sine Sul* fur Ash % % % % % $ Hwaan A Mayer (1942) 18.9 10.6 10.4 4.0 A Author 9.9 9.4 10.2 3.9 6.9 B Winzler e£ al»(1948) 15.1 11.9 7.9 4.2 1.3 2.8 B Author 14.9 11.2 11.9 4.3 7.5 C Author 16.5 12.0 10.1 4.0 1.0 1.8 D Author 9.6 6.3 12.1 3.5 £ Author 14.3 8*3 14.0 4.7 59.6 £1 Author 6.9 3.9 3.9 3.4 57.0 Rat A Winzler and Burk (1944) 9.0 9.2 5.8 2.9 C Author 7.1 5.2 11.4 2.5 Beef D Rimington (}940) 10.7 5.6 13.6 3.0 1.6 0.4 C Author 10.7 7.9 11.3 2.3 Horae A Mayer (1942) 18.3 10.3 10.6 3.7 D Bywaters (1909) 13.6 10.7 11.6 1.8 C Hewitt (1936) 8.5 2.7 13.0 5.4 C Author 6.7 4.6 13.8 3.5 (17.4) (12.0) Guinea pig c Author 9.1 6.6 11.7 2.0 * Moisture-free basis. 131 132 and the author Is the greatest round in Table XXI* This difference Is difficult to reconcile since the other values show but slight variation. No explanation is proffered. The chemical composition of the fraction isolated from human plasma by Procedure B is similar to that reported by Winzler, Devor, Mehl and Smyth (1948). Greatest differences are In nitrogen and ash values. The nitrogen content of their preparation seems unusually low for a protein. The similarity of these preparations was borne out by electrophoretic analy ses. Both preparations contain at least three components at pH 4.5 and at pH 8.5. The average mobilities reported at these pH values (Mehl, Humphrey, and Winzler, 1949) are in good agreement with those determined in the present investi gation. Table XXII summarizes the data. For comparison with the mucoproteln Isolated from rat serum by the author, the results of Winzler and Burk (1944) are the only ones available. It should be noted that two different methods of isolation were employed. Winzler and Burk reported a higher hexose content and a lower nitrogen content than found by the author. It seems significant that both hexose values are lower than those found for human plasma mucoproteln MP-1. The analytical data reported by Rimington (1940) for beef seromucoid is in the same range as that for beef muco- TABLE XXII COMPARISON OP TWO HUMAN PLASMA MTJCOPROTEIN PREPARATIONS ISO LATED BY PROCEDURE B* Component Mobility x 10*5 cm# 2/volt/sec. Mehl, Humphrey and WLnzler (1949) pH 8.5 pH 4.5 Author pH 8.5 pH 4.5 MP-1 -6.5 -3.6 -6.6 -3.8 MP-2 -5.0 -2.3 -5.3 -2.9 MP-3 -0.7 -0.9 -0.7 Procedure B - ammonium sulfate saturation of perchloric acid filtrates of plasma. ** Not reported. 134 protein. However, the data reported by the author is for an electrophoretically homogeneous preparation* Staub and Rimington (1948) presented evidence that the beef seromucold of Rimington (1940) consisted of three components* Evidence that seromucold preparations isolated from human plasma are mixtures has been presented. The chemical composition of human plasma seromucold approximates that of beef serum seromucold. This similarity of composition between sero mucold preparations of two species suggests that the some what drastic method of Isolation (Procedure D) alters the native protein* A marked species difference was found be tween human plasma mucoproteln and beef serum mucoproteln isolated by milder chemical treatment (Procedure C)* The results of several investigations on the carbohy drate-rich protein fractions of horse serum are recorded in Table XXI. The hexose value reported by Mayer (1942) is - appreciably higher than those reported by other investigators* Mayer also determined the isoelectrle point of his mucoid- ahnliche Substanz preparation* Prom minimum solubility measurements he found it to be at pH 3*4* The preparation Isolated in the present investigation contained at least 4 electrophoretic components* Its chemical composition most closely approximates the seroglycoid preparation of Hewitt (1936)* If it is assumed that the carbohydrate content of 135 of the author’s preparation resides entirely In the components that are negatively charged at pH 4*5 and the hexose and hexosamine contents are corrected on this basis, the values obtained are similar to those reported by Mayer (1942), All of the preparations listed in Table XXI are glyco proteins and with the exception of the seroglycoid prepara tion of Hewitt (1936) can be classified as mucoprotelns by current definitions (Meyer, 1945; Stacey, 1946)* A survey of the results of the current investigation and of the data reported in the literature permits the fol lowing general summary to be made regarding the nature of serum mucoprotelns* Mucoprotelns are low-molecular weight glycoproteins In which the protein moiety Is conjugated with a polysaccharide* They are characterized by relatively high hexose and hexosamine contents and a relatively low nitrogen content in comparison with the major protein constituents of plasma* The amino acid composition Is similar to that of other plasma proteins Insofar as they have been determined* Certain quantitative differences exist which suggest that the major protein constituents of plasma are not direct pre cursors of plasma mucoproteln* An excess of sulfur over that accounted for by the sulfur content of the amino acids cystine, cysteine, and methionine is present. At least part of the excess sulfur Is present as sulfuric acid esters* 136 The presence of sulfuric acid esters as an Integral part of the mucoproteln molecule accounts for the extremely low Iso electric points of these materials, ttSalting out* properties indicate that mucoprotelns are the most soluble proteins of plasma. Certain species differences exist both as regard to chemical composition and physicochemical properties. Apart from species differences the properties of a mucoproteln preparation are determined by the isolation procedure em- polyed. Although the physiological function of plasma muco protelns Is unknown, present evidence indicates that they are present In Increased amounts when there is a demand for protein. CHAPTER VI SUMMARY AND CONCLUSIONS Prom the results of the present investigation the following summary can be made* 1* A mucoproteln, MP-1, isolated from pooled, normal, human plasma by a series of ammonium sulfate precipitations, has been characterized by the determination of its chemical composition, amino acid composition, and physicochemical cons tants• a« The chemical composition of MP-1 showed the rela tively high hexose and hexosamine contents and rela tively low nitrogen content characteristic of proteins of this type* An excess of sulfur was found over that accounted for by the sulfur content of the amino acids methionine, cystine, and cysteine* Evidence was presented indicating that at least part of the excess sulfur was present as sulfuric acid esters* b. The amino acid composition was qualitatively iden tical to that of other human plasma proteins Insofar as they have been determined* Certain quantitative differences were found which suggest that other plasma proteins are not direct precursors of plasma rauco- protein MP-1* 138 c. The physicochemical constants of human plasma mucoproteln MP-1 were found to differ significantly from those of the major protein constituents of human plasma. The most striking differences were in the values for the molecular weight, partial specific volume, isoelectric point, and estimated size of the molecule. MP-1 is the smallest protein to be isolated from normal human plasma and has the most acid iso electric point. ^Salting out* characteristics suggest that it is the most soluble protein of normal human plasma. MP-1 was found to be electrophoretically homogeneous at pH values from 1.5 to 8.6. At pH 8.6 it was found to migrate with the OC 1-globulins of human serum. 2. A rate of turnover study employing C*^ methyl- labeled glycine indicated that the mucoproteln fraction of rat serum had a greater rate of turnover than the albumin or globulin fractions. It was suggested that this finding may be more apparent than real. A method of investigation was proposed for determining whether an apparent or real differ ence exists. 3. Carbohydrate-rich protein fractions isolated from pooled, normal, human plasma by six different procedures were compared by chemical and electrophoretic analyses. Marked 139 differences in chemical composition, in the number of electro phoretic components, and in electrophoretic mobilities were found* The various fractions were similar in that all were negatively charged when subjected to electrophoresis at pH 4*5 and that all satisfied current definitions for a muco- protein* It was suggested that the mode of isolation may affect the properties of mucoprotelns* 4* Electrophoretlcally homogeneous mucoprotelns isolated by the same procedure from human plasma and the serum of several animal species were compared by chemical and electrophoretic analyses* Significant species differ ences were found in chemical composition and in electrophoretic mobilities* The differences were most pronounced between human plasma and the animal serum mucoprotelns* A constant hexose/hexosamlne ratio of 1*36 was found in all preparations* The mucoprotelns of all species were negatively charged when subjected to electrophoresis at pH 4*5* All preparations exhibited the solubility properties and chemical composition characteristic of mucoprotelns* 5* Carbohydrate-rich protein fractions Isolated from normal and malignant tissues were characterized by chemical and electrophoretic analyses* The chemical composition and electrophoretic mobilities were found to be similar to the serum mucoprotein of the species of origin* It was suggested 140 that these preparations, which were mixtures, contain at least one component either identical with or similar to the serum mucoproteln of the same species* A difference In chemical composition was found in materials isolated from two different strains of rat fibrosarcoma. 6. An acidic component was found to be present In the serum from two strains of rats with well developed fibro sarcomas by electrophoresis at pH 4*0. The mobility of this component was found to be similar to that of isolated rat serum mucoproteln* 7. An increased level of human serum mucoproteln MP-1 was found to be present in the serum from a human umbilical cord. The bearing of this finding on current theories regarding the source of serum mucoprotelns was diseussed. 8. In the determination of the pH- mobility curve of human plasma mucoproteln MP-1, the species of buffer anion was found to exert a marked effect upon the mobility in the pH range 2.0 to 4.0* 9. The source of the MP-2 component of human plasma mucoprotelns prepared by ammonium sulfate saturation of perchloric acid-filtrates of plasma was investigated* It was found to be formed by the prolonged exposure of MP-1 to a strong acid solution at room temperatures. 141 10* Although significant progress has been made in the isolation and characterization of mucoprotelns from serum and other tissues, a better understanding of these proteins Is dependent upon further chemical, physicochemical, biochemi cal, and physiological studies# The two major problems ares a* What is the function of plasma mucoprotelns? b# What is the source of plasma mucoprotelns? Correlated with a solution of these are the following: c# Elucidation of the nature of the polysaccharide component of mucoproteln# d. Isolation of the slower acidic electrophoretic component of human serum, M-2# e. Constant solubility studies# f# Enzyme inhibition studies# g. 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Creator Weimer, Henry Eben (author)

Core Title Chemical, physicochemical, and biological studies on the mucoproteins of plasma and serum

School Department of Biochemistry and Nutrition

Degree Doctor of Philosophy

Degree Program Biochemistry

Degree Conferral Date 1950-06

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor [illegible] (committee chair), Bartholomew, J.W. (committee member), Deuel, H.J. (committee member), Visser, Donald (committee member), Wehl, John W. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-254

Unique identifier UC11345320

Identifier DP21543.pdf (filename),usctheses-c17-254 (legacy record id)

Legacy Identifier DP21543.pdf

Dmrecord 254

Document Type Dissertation

Rights Weimer, Henry Eben

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

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