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A comparative study of thre blood proteins of five species of Oncorhynchus using starch-gel electrophoresis Jones, Helen 1963

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A COMPARATIVE STUDY OF THE BLOOD PROTEINS OF FIVE SPECIES OF ONCORHYNCHUS USING STARCH-GEL ELECTROPHORESIS by HELEN OONES B . S c , The University of B r i t i B h Columbia, 1961 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of ZOOLOGY We accept this thesis as conforming to the required standard THE UNI VERS ITY^O"F~BRITISH COLUMBIA January, 1963 In presenting this thesis in pa r t i a l fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It i s understood that copying or publication of this thesis for financial gain shall not be allo\\red without my written permission. - i i -i ABSTRACT The blood proteins of f i v e species of Oncbrhynchus: chum, coho, pink, sockeye and spring salmon, have been separated by starch-gel electrophoresis. Comparisons the protein fractions obtained from representatives of fresh water downstream migrants, maturing upstream migrantsi^jmd mature adults, have yielded indications of i n t r a s p e c i f i c differences associated with physiological states. I n t e r s p e c i f i c comparisons of the protein fractions obtained from salmon i n comparable physiological states have yielded further indications of blood protein differences associated with species differences. A taxonomic grouping of the f i v e species has been suggested based on the constant characteristics of t h e i r respective protein patterns. The use of a standard protein has provided an » e f f i c i e n t means of removing i n t e r - g e l variations In protein mobility. ACKNOWLEDGMENTS I wish to express my sincere gratitude to Dr. W. S. Hoar for his time and many valuable suggestions which he gave freely throughout this research. I am indebted to Dr. H. Tsuyuki of the Fisheries Research Board of Canada who provided me with many blood samples and introduced the starch-gel electrophoretic techniques for their study. Special thanks are due Mr. Harold Harvey of the International SaiLmon Commission who obtained blood samples for my research from many locations in the Province of British Columbia. . I wish to acknowledge the financial support provided by a National Research Council Studentship. - i i i -TABLE OF CONTENTS I. INTRODUCTION . 1 A. PROTEINS AS TAXONOMIC CHARACTERS .' B. PROTEIN STUDIES IN PACIFIC SALMON 2 II. MATERIALS AND METHODS 5 A. BLOOD SAMPLES 5 1. Source of Blood Samples 5 2. Sampling Technique and Sample Storage 5 B. ELECTROPHORETIC TECHNIQUE, APPARATUS, AND MATERIALS 1. Introduction 7 2. General Outline of Procedure 8 8. Detailed Description of Procedure 9 a) Preparation of Gel 9 b) Dialysis of the Sample 11 c) Procedure Prior to Electrophoresis 11 d) The Electric Field IS e) Treatment of the Gel Following Electrophoresis 13 f) Gel Photography 14 C. A METHOD FOR THE QUANTITATIVE DESCRIPTION AND COMPARISON OF THE MOBILITIES OF PROTEIN FRACTIONS SEPARATED BY STARCH-GEL ELECTROPHORESIS 15 1. Rf Values of Chromatography 15 2, Electrophoretic Mobility 15 - i v -3. A Standard Protein and the % Value 16 4. Assumptions of the. Value 18 5. Incomplete Standards 18 III. RESULTS 20 Y A. DATA PRESENTATION 20 B. STANDARD « 20 C. THE EFFECT OF FREEZING, DIALYSIS, AND HEPARIN ON THE BLOOD PROTEINS OF A FEMALE SOCKEYE SALMON 25 D. SERUM AND PLASMA PROTEIN FRACTIONS COMMONLY OBSERVED WITHIN SPECIES GROUPS 26 1. Spring Salmon 26 2. Chum Salmon 27 3. Pink Salmon 28 4. Sbckeye Salmon 29 5. Coho Salmon 32 D. AN INTERSPECIFIC COMPARISON OF SERUM AND PLASMA PROTEINS 33 1. Intra-Gel Species Comparisons 33 a) Chum-Sockeye 33 b) Pink-Spring 34 c) Pink-Sockeye 34 2. Inter-Gel Species Comparisons 35 E. SEX 38 1. Pink Salmon 38 2. Chum Salmon 38 8. Coho Salmon 38 4. Soc^eye Salmon 39 5. Spring Salmon 39 F. AGE 40 1. Pink Salmon 40 2. Coho Salmon 40 3. Chum Salmon 40 4. Sockeye Salmon , 41': G. GEOGRAPHICAL LOCATION 42 IV. DISCUSSION 57 A. FREEZING, STORAGE, AND DIALYSIS 57 CONTENTS OF THE STAINED FRACTION- 57 C. LABILITY OF BLOOD PROTEINS 59 D. INTERSPECIFIC COMPARISONS OF BLOOD PROTEINS: WHEN ARE THEY VALID? 62 E. SPECIES RELATIONSHIPS WITHIN ONCORHYNCHUS 63 V. BIBLIOGRAPHY 67 i - v i -LIST OF FIGURES Figure 1. Diagram of electrophoretic and dialysis apparatus 2 . Plasma proteins of a mature male spring salmon Plasma proteins of a mature male pink salmon 3. Plasma proteins of a mature male spring salmon Plasma proteins of a mature female spring salmon 4. Serum proteins of immature and maturing chum salmon Plasma proteins of a maturing female sockeye salmon 5. Serum proteins of fresh water downstream migrating chum salmon 6. Plasma proteins of a mature male pink salmon Plasma proteins of a mature female pink salmon 7. Plasma proteins of a mature female pink salmon Serum proteins of a maturing female sockeye salmon 8. Plasma proteins of fresh water downstream migrating pink salmon 9. Plasma proteins of a mature male sockeye salmon Plasma proteins of a maturing male sockeye salmon 10. Plasma proteins of a maturing male sockeye salmon taken from Great Central Lake, British Columbia Plasma proteins of a maturing male sockeye salmon taken from Cultus Lake, British Columbia 11. The effect of freezing and dialysis on the serum proteins of a maturing female sockeye salmon - v i i -12. Serum proteins of a maturing female sockeye salmon Plasma proteins of fresh water downstream migrating sockeye salmon S3 13. A comparison of the serum and plasma proteins of a maturing female sockeye salmon (the back surface of the gel h a l f shown i n Fig. 14) 54 14. A comparison of the serum and plasma proteins of a s maturing female sockeye salmon (the cut surface of the gel h a l f shown i n Fig. 13) 55 15. The plasma proteins of a mature male coho salmon The plasma proteins of a mature female coho salmon The plasma proteins of young coho sampled i n June of Ifheir f i r s t year 56 16. A diagrammatic summary of the R m zones consistently occupied by blood protein fractions within species groups 32 - v i i i -LIST OF TABLES TABLE \ I. A.summary of sampling details including the sample reference number (BRN), species, weight, length sex, approximate physiological condition, location, and dafee of sampling II. A summary of the treatment of blood samples Including the sample reference number (SRN), i f heparinized, thermal history, and duration of storage III. A summary of some suggestions as to the phylogenetic relationships of five species of Oncorhyiachus I. INTRODUCTION A. PROTEINS AS TAXONOMIC CHARACTERS The c h a r a c t e r i s t i c s and physiological status of the protein molecule make i t one of the more promising taxonomic characters. Sibley (1960) defines such characters as "any attributes of an organism which are diagnostic and, i d e a l l y , ..... furnish clues to phylogenetic r e l a t i o n -ships" . The enoranous- p o s s i b i l i t i e s f o r v a r i a t i o n i n protein molecular structure provide a sensitive index to the genetic information which has organized that structure. The structure of s p e c i f i c proteins together with t h e i r immediate dependency on the genetic materials which bridge animal groups should provide conservative, diagnostic information on the relationships of those groups. A number of methods u t i l i z i n g differences i n protein structure, or i functions of that structure, have been employed as taxonomic tools for the evaluation of the relationship of animal groups. The p r e c i p i t i n reaction which characterizes the interaction of antigen and antibody i n immune sera has been shown to be s p e c i f i c and i s the basis of many serological ,fkeys'' to animal groups. The fact that the s p e c i f i c i t y of the p r e c i p i t i n reaction i s heritable i n accordance with Mendelian p r i n c i p l e s has provided the basis of Immunogenetics, a second method of evaluating phylogenetic relationships. A review of the l i t e r a t u r e on immunogenetics i s provided by Sibley (1960) and by Cushing (1962). -2-Other methods f i n d t h e i r basis i n the structure of proteins of the red blood c e l l . Jacobs, Glassman, and Parpart (1950) studied the rates of penetration of four non-electrolytes into red blood c e l l s , rates which are dependent, i n part, on the s p e c i f i c structure of the proteins of the c e l l membrane. Their r e s u l t s were interpreted as indications of the zoological relationships of the animals they examined. Sibley notes that studies on the crystallography of hemoglobins were i n t e r -preted by Reichert and Brown (1909) as having taxonomic implications. The chemical and physical properties of the whole protein molecule, i i t s s i z e , weight,, net charge, shape, s o l u b i l i t i e s , etc. as demonstrated by the techniques of chromatography and electrophoresis have also been used as taxonomic characters, although not extensively i n the case of chromatography (Sibley, 1960). B. PROTEIN STUDIES IN PACIFIC SALMON A number of studies on the body proteins of P a c i f i c salmon have been conducted using s e r o l o g i c a l , column chromatographic, and paper and starch-gel electrophoretic techniques. Some of these have cast l i g h t on the systematic relationships within t h i s group. Hourston (1949) studied the serological relationships of the Salmonoids and concluded that the coho, spring and sockeye salmon (indistinguishable from kokanee) form a closely related group, with chum and pink salmon being more d i s t a n t l y related both to t h i s group and to each other, Ridgway and his co-workers have published a series of papers on the serology of -3-Pacific salmon. They were able to differentiate populations of sockeye salmon serologically (Ridgway, Gushing, and Durall, 1958) and to demonstrate 14 antigenic components in the sera of the Alaskan red salmon (Oncorhynchus nerka), two of which were missing in a large per-centage of the red salmon taken in Asian waters. They concluded that two stocks of red salmon exist, Asian and Alaska, which intermingle in the North Pacific and Bering Sea (Ridgway, JCLontz and Matsumoto, 1962). The existence of blood groups in the red salmon has been established by Ridgway (1962) and Ridgway and Klontz (1960). The myogen (muscle proteins soluble in low ionic strength salt solutions) of the spring salmon (0. tshawytscha), have been studied by Tsuyuki and Roberts (1961) using column chromatography and starch-gel electrophoresis; that of coho, spring, sockeye and chum salmon was studied by T.suyukl, Roberts, and Gadd (1962,11) using column chromatography; and the myogen of pink salmon and of the previously mentioned four species were studied again by Tsuyuki, Roberts and Gadd (1962,111) using starch-gel electrophoresis. The results of these studies have been taken as an indication that the chum, pink, and sockeye salmon are more closely related to one another than to the spring and coho salmon and that the latter species are in turn more closely related to the genus Salmo than are the former. No published reports of starch-gel electrophoretic comparisons of the blood proteins of different species of Pacific salmon have been located. Vanstone and Chung-Wai Ho (1960) have studied the plasma proteins of coho salmon by paper electrophoresis and were able to show differences in the fractions associated with smolting, sex, and state of maturity. -4-In the present study, the serum and plasma proteins of the five species of West Coast Pacific salmon were investigated by starch-gel electrqphoretic techniques. - 5 -I I . MATERIALS AND METHODS A. BLOOD SAMPLES 1. Source of Blood Samples Five species of P a c i f i c salmon were sampled: Oncorhynchus  gorbuscha (Walbaum), pink; 0. keta (Walbaum), chum; 0. kisutch (Walbaum), coho; 0. nerka (Walbaum), sockeye; and 0. tshawytscha (Walbaum), chinook. Table I summarizes the sampling information, tabulating the species of salmon sampled, i t s location when sampled, i t s weight, length, sex, i t s approximate physiological condition at the time of sampling, and the date on which sampling occurred. I t w i l l be noted that the specimens are representative of a fresh water downstream migrating f i s h , an upstream migrant, green on the spawning ground, or ripe on the spawning ground. Blood from different sexes of the same species was also obtained. Fish located i n various regions of the State of Washington, the Province of B r i t i s h Columbia, and the offshore waters of that province were sampled. 2. Sampling Technique and Sample Storage The blood of f i s h less than 30 cm i n length was sampled by removal of the caudal peduncle with a p a i r of sharp, clean scissors. Thereafter the anterior portion of the body was elevated and a heparinized c a p i l l a r y tube placed d i r e c t l y adjacent the severed caudal artery. L i t t l e r e s i s t -ance 'was offered to the body undulations of the f i s h during sampling, and -6-I t was noted that death almost always postdated the sampling. When the blood flow ceased or the tube had been f i l l e d , one end of the c a p i l l a r y tube was sealed with plasticene. The rube and contents were immediately centrifuged f o r f i v e minutes at approximately 3100 r.p.m. (arm radius = 13.2 cm). The hematocrit was then recorded, the open end of the tube sealed with plasticene, and the tubes wfire stored v e r t i c a l l y , a c e l l u l a r portion uppermost, i n sealed l a b e l l e d corrugated cardboard squares at -5°C. At a l l times, the id e n t i t y of in d i v i d u a l blood samples was maintained; none was pooled. The f i s h was then weighed. The fork-length was recorded p r i o r to the severance of the caudal peduncle. The sex and state of maturity of adult f i s h was recorded. Almost without exception, f i s h greater than 30 cm were sampled i n the f i e l d . Here the caudal peduncle was severed with a clean knife and the pressurized blood stream directed into a clean funnel and centrifuge tube sometimes heparinized, as indicated i n Table I I . The tube was stoppered and sealed with Parafilm, and, i n cases where a centrifuge was not immediately available, c h i l l e d i n ice water u n t i l one could be reached. This delay was usually about s i x hours, though occasionally as long as a day. After centrifugatioh, the plasma (or serum) was removed to a clean container, stoppered, sealed with Parafilm, and placed at temperatures below freezing. The thermal h i s t o r i e s of the stored samples are not i d e n t i c a l ; some samples were placed at -20°C only after i n i t i a l storage at -6°C, others were placed at -20°C d i r e c t l y . A l l samples, however, were stored i n a frozen state. The duration of storage varied from a year and two months down to less than a day, with the majority of storage -7-times being less than six months. Details of the temperature and duration of storage are given in Table II. B. ELECTROPHORETIC TECHNIQUE, APPARATUS, AND MATERIALS 1. Introduction Extensive discussions on the methods and general theory of electrophoresis are presented in the texts of Block, Durrum, and Zweig (1955) and Bier (1959). Electrophoresis may be defined as "the migration of charged particles in an electrolyte solution which occurs when an electric current is passed through the solution" (Harper, 1961). Zone and moving boundary electrophoretic techniques were f irst des-cribed in 1937 by) Kbnig and Tiselius, respectively. The electrolyte may be contained in a tube as was done in moving boundary electro-phoresis or i t may be distributed evenly in a supporting matrix such as paper, agar-gel, or starch-gel as In zone electrophoresis. The migration of the charged particle in the electrolyte has been shown to vary with the physical and chemical characteristics of the particle (charge, size, shape, amphoteric properties and dissociation constant) and with the nature of its environment such as the electrolyte concentration, ionic strength, pH, temperature and the intensity and distribution of the applied field (Block, Durrum, and Zweig, 1955). The migrations may be i classified as one-dimensional, two-dimensional, or continuous; they may also be classified according to the matrix used (none, paper, cellulose, resins, gels, etc.); Block et aL classify electrophoretic techniques in -8-terms of the degree of evaporation permitted. The variety of classifica-tions possible provides dome indication of the immense diversity in electrophoretic techniques. One of the more recent advances in electrophoretic techniques which was employed in this study is the use of a starch-gel supporting media first described by Smithies (1955 ,b). The properties of this matrix permitted a considerable increase in the resolving power of the technique. Poulick and Smithies (1958) were able to demonstrate more than 30 com-ponents in human sera by two-dimensional starch-gel electrophoresis. A review of the literature of starch-gel electrophoresis is provided by Smithies (1959^b). Perhaps equally varied as electrophoretic techniques are the applications of electrophoresis. Immunoelectrophoresis, taxonomy, pathology, the isolation and purification of compounds, classification of the compounds of body fluids, are just a few of the applications which led Bier to write "The contribution of electrophoresis to our knowledge of proteins Is second to no other method. Its impact is felt in biochemistry, physiology, and medicine" (1959). 2. General Outline of Procedure;: With slight modifications, the procedure followed and the o materials employed were those described by Smithies (1955,b) in his original description of zone electrophoresis In starch-gels and the improved procedure for starch-gel electrophoresis in his later paper (Smithies, 1959£). The apparatus is diagrammed in detail by Smithies (1959$ and is I -9-illustrated as used here in Fig. 1. The modifications consist mainly of the placing of the gel tray in a horizontal position during migration, rather than vertical, and the substitution of the suggested buffer trays and electrodes by those designed by the E-C Apparatus Company (538 Walnut Lane, Swarthmore, Pa.) for zone electrophoresis. Starch~gel electrophoretic techniques are relatively new. A brief outline of the general procedure follows. A starch-gel is prepared with an appropriate buffer and a commercially obtainable starch. This is poured into a plastic tray and covered with a plastic sheet with eight downward projecting tabs extending the width of the gel (Fig. 1). When the hot starch sol cools to its gel phase, the cover is removed, and the samples injected into each of the slots formed by the cover. They are sealed with mineral o i l , and strips of f i l ter paper soaked in buffer are clamped against the gel ends so that the remainder of the strips dip into two chambers at eigher end of the gel containing the same buffer. A current of the desired voltage Is passed through the gel for a given time and then turned off. The gel is then placed in a slicing tray and sliced horizontally into two halves. The halves are stained selectively with an appropriate dye and then rinsed with a dye solvent until the stained fractions are resolved maximally. The gel halves are removed, protected from desiccation, and placed in cold storage until further use. Following is a detailed description of the procedure used in these experiments. 3. Detailed Description of Procedure a) Preparation of Gel A quantity of hydrolyzed starch (Connaught Medical Research -10-C«.Uo \o\& — sample. Seal -buf fer 4* power supply upper cooling plote O i l e d f \ o » \ l C &heet qe.1 t r a y , •f{\ter p a p e r l o w e r C O O l i n a p la te . buffer Fig. 1. a. Dialysis method for small samples b. Gel cover with slot formers c. Electrophoretic apparatus assembled -11-Laboratories, Toronto) was weighed according to i ts batch specifications (about 59 gra) and combined with 500 ml of borate buffer which was 0.0230 M H3B03 and .0092 M NaOH at pH 8.5. The suspension was heated and stirred until f luid, clear, and boiling. The flame was extinguished and a vacuum applied to the flask until a l l bubbles had been removed. The gel was then poured into a gel tray coated with mineral o i l and covered with an oiled plastic sheet with eight transversely oriented slot formers (manufactured by Otto Hil ler , Madison, Wisconsin). The cover was clamped on tightly with plywood sections and the entire prep-aration stored at 5°C for a minimum of 4i\ hours. b) Dialysis of the Sample The majority of plasma and serum protein samples were dialyzed before electrophoresis for at least 24 hours in borate buffer at pH 8.5. The dialysis of small volumes necessitated the development of a suitable method of dialysis which would eliminate the loss of a large portion of the sample due to capillarity in the collapsed dialysis tubing supplied commercially (Fig. 1). A 2.5 cm section of collapsed celluloid dialysis tubing with a 6 mm diameter was pinched and rolled at one end on an axis perpendicular to that on^which i t had been flattened. The rolled end was then dipped In hot parafin several times and allowed to cool. When used, a fine wire hook thcough the open end suspended the capsule with i ts contents in buffer solution. Very small quantities ( i .e . 30 ul) were thus reclaimable from the dialysis chamber. c) Procedure Prior to Electrophoresis A l l electrophoretic equipment was housed in a constant temperature environment room at 10°C - 1°C. Additional cooling of the -12-gel was accomplished by maintaining a water bath at 3.5°C within the environment room, and circulating water from this bath through two plastic plates between which the gel tray was placed. The gel was taken from the refrigerator, undamped, and the cover carefully removed in the environment room. The gel was then temporarily covered with an oiled plastic sheet to prevent desiccation. The ends of the gel tr.ay were removed and two buffer saturated squares of f i l ter paper (Whatman's #17, 6\ux5 ") clamped in place at the gel ends completing the electrolyte bridge, including both buffer chambers (each containing two l iters of buffer), the f i l ter paper wicks, and the gel (Fig. 1). The f i l ter papers were separated from the electrodes by two large sponges which acted as baffles for convention currents developed in the buffer with the passage of current. Samples of 40 u l quantities were placed in the gel slots with a 50 u l microsyringe (#80701, Hamilton Company, Inc.. , Whittier, Cal i f . ) . In rare cases much smaller volumes were used e.g. when young fish yielded very small blood volumes. The slot capacity is approximately 50 jul 1 5 pi. Where several types of samples were applied to one gel, the syringe was flushed thoroughly with distilled water between applications. After samples had been applied to each slot, a droplet of mineral o i l was placed over them preventing their desiccation and their movement onto the gel surface. Smithies (1959a) suggested hot vaseline for this purpose mainly for use with vertical electrophoresis which would require a seal of a more solid nature. In horizontal electrophoresis, however, mineral o i l Is probably an Improvement since there is no risk of harming the sample contents with the excess heat of liquid vaseline. -13-The edges of the gel tray were also oiled improving the seal with the oiled plastic sheet when i t was replaced after sample application. The upper cooling cover was then placed directly on the gel tray so that no space intervened. Both cooling plates had been precooled with water at 3.5°C prior to use. d) The Electric Field The jjower supply was turned on and the voltage regulated to 800 v with a current of 2 milliamps yielding a voltage gradient of 30 volts/cm in the gel which was 27 cm long. Since Smithies (1959^ ) used only 135 v or a 5 v/cm gradient with a gel of identical dimensions, i t seems reasonable to assume that the apparatus used in these experiments offered a greater resistance. Longer f i l ter paper wicks were used in these experiments than in Smithies' which may have Increased the resistance of the system. Smithies quoted a voltage gradient of 5 v/cm in a gel 27 cm long with an applied voltage of 135 v. This indicates that he assumed a resistance of zero for the f i l ter paper wicks, an assumption which might produce a large error once the wicks were lengthened. Although a high voltage was applied In these experiments, a rather small current of 2 milliamps was measurable. The power supply used in this work was a DC model 1331 obtained from the E-C Apparatus Company. The time at which the power supply was turned on was noted, as was the temperature of the room and the water bath. The current was passed in a l l cases for a period of 16 hours. e ) Treatment of the Gel Following Electrophoresis After the power supply had been turned off, the upper -14-coollng cover was removed and the f i l ter paper wicks discarded. The cutting tray (Otto Hil ler , Item #5) was placed over the gel and the latter, inverted into i t . The entire gel was sliced horizontally into two haives with' the microtome-knife blade supplied with the tray. The cut surfaces of the halves were then placed face up in a porcelain tray and stained with Amido Black 10B (2.5 gm Amido Black/450 ml absolute metyl alcohol/ 50 ml glacial acetic acid) for five minutes. This dye is specific for proteins. The stain was drained off and saved for future use, and the gel halves were sl id into the washing chamber of an 'Automatic Gel-Wash-ing Machine' designed by Pert et al.(1959) and supplied by Otto Hil ler , Madison, Wisconsin. The washer contained a methanol dy§ solvent (5 parts abs. methyl alcohol/5 parts dist. water/l part glacial acetic acid) which cycled continuously through activated charcoal; washing continued for about eight hours. If oontinued for days stained fractions wil l be removed as well as the dye held in the gel matrix. When the rinsing was completed, the gel halves were wrapped in Saran wrap, labelled and stored at 3°C until photographed. Storage times ranged from several days to four months. No visible effects on the stained traces resulting from prolonged storage were noted. f) Gel Photography For photographic records of stained gel surfaces, the best results were obtained by placing two lamps at 45° angles to the gel surface and photographing from above. > Commercial films have a blue ahiliation factor and can be used without a f i l ter at approximately one second at f 16 on standard lenses. For an alternate method, which may be used i f residual stain is not too dense and the gel is even in thickness, -15-expose the gel with a back-light system at approximately four seconds at o f 16 with standard lenses. This has the advantage of recording the stain held by the protein throughout the depth of the gel. The photographs presented here were taken with the f irst method. A metric scale was included with each. C. A METHOD FOR THE QUANTITATIVE DESCRIPTION AND COMPARISON OF THE  MOBILITIES OF PROTEIN FRACTIONS SEPARATED BY STARCH-GEL  ELECTROPHORESIS 1. _Rf Values of Chromatography The analysis of paper chromatograms have long been dependent on the use of R^  values as a means of comparing the mobilities of the spots obtained on one chromatogram and between chromatograms (Block, Durrum, Zweig, 1955). The R^  value is obtained by dividing the distance the spot travelled from the origin by the distance the solvent front travelled from the origin. The quotient is useful as an expression of the mobilities of different spots in relative terms; that i s , where a correction has been e applied for the influence of the mobility of the solvent on the mobility of the spot. 2. Electrophoretic Mobility Similar types of comparisons of the mobility of fractions separated by electrophoresis have been made possible by the calculation of the Mobility (u) of any given fraction, where the Mobility has been defined as the distance in eentimeters the fraction has moved in one -16-t second for a field strength of one volt/cm (Raymond, 1955). The mobility of any given fraction wil l remain a constant i f the pH, temperature, com-position of the medium, and other factors are fixed. Experimentally, this constancy of mobility was not achieved, although every effort was made to apply the same ensironmental parameters to each electrophoretic separation. Discrepancies were noted in the mobility of a given fraction across the width of a single gel as well as between gels. 3. A Standard Pfotein and the R^ , Value To assist in the estimation and correction of the error in the apparent mobilities between gels, a standard protein was included in every electrophoretic separation. The standard protein used was a com-mercially prepared "family" of proteins in powdered egg albumen (Nutritional Biochemical Corp., Cleveland, Ohio). This was dissolved in distilled water (0.4 gm egg albumen/10 ml distilled H 20), stored at 3°C, and freshly prepared at frequent intervals. When applied, a 40 ;ul sample of the soluble portion of this preparation was placed inhone of the eight slots present in the gel, usually second from one edge and labelled as slot #2. The conspicuous appearance and known location of the standard trace greatly decreased the likelihood of ever confusing the order in which the other seven samples had been applied. The characteristics of the fractions obtained from the standard protein were fairly constant between gels and may be seen In Fig. 4. They consist of a very definite end point of the fastest moving fractirin, a darkened constric-tion in the width of the trace occurring at about 0.36 of the distance from the origin to the end point of the fastest fraction, and several narrow bands distributed between that constriction and the origin at -17-about 0.12, 0.1'6, 0.20 and sometimes 0.27 the distance from the origin to the end of the fastest fraction. It was the characteristics of the standard protein, and more particularly, their distances from the origin which were used to calculate the relative mobilities of each of the other protein fractions which accompanied the standard. These may subsequently be compared with the relative mobilities of fractions on other gels. This calculation was accomplished in the following manner. The distances from the side of each slot nearest the cathode (the electrode toward which a l l fractions migrated) to the leading edges of each visible frac-tion originating from that slot were measured in centimeters. Measure-ments of a l l fractions from a l l eight slots on every gel were taken. It was noted that in every instance the leading edge of the fastest moving fraction of the standard had travelled farther from its slot than that of any other fraction on the gel. The distances travelled by a l l blood protein fractions were then expressed as a decimal of the distance between the leading edge of the fastest moving fraction of the standard and the cathodal margin of its slot (origin). Thus each visible blood protein fraction was assigned a value describing its mobility relative to the standard, a value which for convenience may be referred to as an value. For example, a fraction which had travelled 4.3 cm from its origin on a gel waece the leading edge of the standard protein was 16.1 cm from its respective origin would have an value of 4.3/16.1 or 0.26. By the use of the value, i t was hoped that the variation between gels might be eliminated leaving only the variation within gels, a much smaller error. -18-4. Assumptions of the Value It should be noted that the successful removal of the variation between gels by the application of the R - value is based on two assump-tions: f i rs t , that the factors influencing the absolute mobility of fractions on separate gels influence a l l the visible fractions; second, that they do so in a multiplicative fashion, not In an additive fashion. If the mobilities of fractions on separate gels are changed, i t must be a change proportional to their distance from the origin, not a change resulting from the addition or subtraction of a constant from that dis-tance. If the effects were additive, then the value for the same protein on two separate gels would not remain constant i f the mobility of the standard on both gels were not identical. For example, i f the leading edge of the standard on the f irst gel was three arbitrary units of measure from its origin and the test fraction was one unit, and on the second gel the standard and the test fraction were both one unit farther from their origin, then the Rm value for the test fraction would have changed from 0.33 to 0.50 and Its usefulness diminished. Data from these experiments Is later presented which indicate a multiplicative response to experimental changes. 5. Incomplete Standards Variation in the mobilities of fractions between gels sometimes resulted in the loss of the fastest moving fraction of the standard i f i t migrated off the gel field into the end zone-filter paper junction. Knowing the relative positions of other fractions in the standard to the fastest moving fraction of the standard in gels where the entire trace was present, the slower moving fractions may then be used to calculate the -19-Rn, values In gels where the fastest f r a c t i o n of the standard has been l o s t . The leading edges of v i s i b l e fractions were taken as the posi t i o n of those fractions when measuring distance from the o r i g i n . Where a f r a c t i o n appeared broad or di f f u s e , an additional measurement of the t r a i l i n g edge was included. -20-III. RESULTS A. DATA PRESENTATION Description of the starch-gel electrophoretic separations of Pacific salmon blood proteins are presented in theotext and in the photographs of stained protein fractions seen in Figures 2-15. Each photograph is presented with an overlying transparent grid of lines which are parallel to the origin and represent 0.1 intervals of R m values, where 0.0 coincides with the cathodal margin of the slot (origin) and 1,0 coincides with the leading edge of the fastest fraction of the standard. Since each Rf„ value is expressed in two decimal places, the position of a fraction referred to by its Rm value may be obtained by visual extra-polation between grid lines. Each blood sample used is referred to by a sample number. Refer to the appropriate sample number in Tables I and II for further details on the nature and history of, each sample. The relative position of the fractions as well as their general appearance must be considered together to gain an impression of the whole. Figure 16 summarizes the zones which were consistently occupied by blood protein fractions within species groups. B. STANDARD Although care was taken to maintain constancy in experimental TABLE I A SUMMARY OF SAMPLING DETAILS Sample Reference Number Species Weight gm Length ' cm Sex .Approximate Physiological Condition - Location Date 1-a (Fig.2 ,3) Spring Male Mature Spring • River'-, Washington State ;Sept.! 16, 1962 1-b (Fig. 3) Sp ring Female Mature •. • -p r - . • - ' 2-a . (Fig.2,6) Pink Male Mature Beia:'G'oola',' British Columbia Oct. 1, 1962 2-b (Fig. 6) Pink Female Mature 3-a 3-c 3-b (Fig. 4) Chum Chunr Chum 21.6, • 15.1- • 23,. 8 14;: 6 -13:0 -14.3 -Immature: •. in spring"of'2nd year ... Collected from-;' Apr.:; 2 , 1962 Cultus" L a k e , " " ' British Columbia; Held, invfreshwater • -,. at the University — -of British Columbia 4-a 4-b 4-c (Fig. 4) Chum Chum Chum 86.3 61.5 77. 7 21.5 19.2 21.5 Male Male Female Maturing: in f a l l of 2nd year; eggs visible^ (as above) Oct. 24, 1962 5-a Sockeye (Fig.4,13,14) 5-b Sockeye (Fig. 7,11,12) Female Female Maturing:, ^ approx-imately one month away from spawning Cultus Lake, British Columbia Oct. 24, 1962 Oct-.- 17, 1962 6-a,b,c,d Chum (Fig. 5) approx. 2.,0 approx. ,6. . Fresh water downstream migrants Cheakamus River,, British Columbia; Aug. t, 1962 7-a,b,c (Fig. 8) ,d,e,f Pink approx. 1.0 approx. -5.0 Fresh' water -J downstream - -migrants. Collected MayoS, 1962- from-Cultus -Lake, British Columbia May 31, 1962 8 (Fig. 9) Sockeye 2600 60.5 Male Mature Horsefly •.River y British Columbia Aug. 26 1961 9 (Fig. 9) Sockeye 1130 •• 47.4 Male Maturing: "six to eight weeks from spawning Cultus Lake, British Columbia Sept. "27", 1961 10 (Fig. 10) Sockeye 2260 59. Male Maturing Great Central-LakeBr i t i sh Columbia Aug: 11, 1962 l l -a ,b ,c (Fig. 12) Sockeye 3.78 4.9.2 . 4.'48 ' ^8.0 .8.8 8.7 '' Fresh water . downstream , migrants .. Collected.May 11, 1962j'from Cultus Lake, British Columbia May 16., 1962 12-a 12-b (Fig. 15) Coho" approx. 1800 "approx. 60 Male Female Mature Samish River, Washington State Nov . 27, 1962 13-a,b (Fig.-'IS) Coho approx. 1.0 approx. 4.7 Freshwater ' • • residents in June of their f irst year Obtained as fry from the Samish Hatchery, Washington State ofiune 19, 1962 -22-TABLE II THE TREATMENT OF BLOOD SAMPLES Sample Reference Number Heparinized Thermal history and Storage 1-a (Fig. 2,3) 1-b (Fig. 3) , Yes Yes stored 1 month at -5°C 2—a (Fig. 2,6) 2-b (Fig. 6J Yes Yes stored 2 weeks at -5°C 3-a ' 3-b 3-c (Fig. 4) No then sfcfcred stored 5^ months at -20°C, 1 month at -5°C 4-a 4-b 4-c (Fig. 4) No stored 2 days at 3PC 5-a ,(Fig. 4,13, K b 1 4 J (Fig.7,11,1 k yes; 4; No stored 1 day at 3°C 2) . No i I 2-stored stored for 1 or 3 days at -5°C for 1 or 3 days at 3°C 6-a,b.c.d (Fig. 5) No then Stored 1 month at -20°C stored 5 weeks at -5°C 7-a,b,c,d,e Q(Fig. 8) ,f Yes then stored stored Z\ menths at -20°C, 1 month at -5°C 8 (Fig. 9) Yes then stored 3 weeks at ~20°C, stored 1 week at -5°C 9 ( F i g , 9) Yes then stored stored 1 year at -20°C, 1 week at -5°C 10 (Fig.10) Yes then stored stored 1 month at -20°C, 1 week at -5°C l l - a , b , c , (Fig. 12) Yes then stored stored 4 months at -20°C, 1 month at -5°C 12-a 12-b (Fig. 15) Yes stored 1 month at -5°C l3-a,b (Fig. 15) Yes then stored stored 4 months at -20^0, 3 months at -5°C -23-conditions, changes in the mobilities of fractions between runs were con-siderable. The distances which the end point of the standard travelled in nineteen runs ranged from 13.2 cm to 23.4 cm (an estimated value) with a mean distance of 18.33 cm.and a standard deviation of - 3.5 cm. Thus In this experiment, the use of a standard protein, or some comparable means of monitoring variation between gels, is desirable. As mentioned previously, the 1^  values were based on the position of the fastest moving fraction of the standard. In cases where that fraction migrated off the gel f ield, i t was necessary to calculate its position in order to obtain Rp, values for the remaining gel fractions. The position of the end point was obtained by multiplying the distance from the origin to the dark constriction in the standard by a factor of 2.789. The con-stant relative position at 0.36 made possible its use in this capacity. The rel iabil i ty of estimating the position of the leading edge of the standard on the basis of the position of this region was examined in nine traces where the entire standard was visible. These standards migrated varying distances from the origin, ranging from 10.1 cm to 18.0 cm. The average error in predicting the actual location of the leading edge was 0.48 cm. The errors in prediction ranged from 1.0 cm to 0.2 cm and had a standard deviation of 0.27 cm. This relatively good agreement indicates that the practice of estimating the position of the end point of the standard was a reliable one. This is based on the assumption that the error in estimation is not related to the distance the standard travelled. Out of 23 runs using the standard, 11 possessed incomplete standards. Either a reduction in the migration time or the voltage would be an -24-appropriate modification for further studies. y Errors in estimating incomplete standards would inevitably cause errors In the % values. Another source of error in the R^ values results from variations in mobilities within a single gel. Air pockets or other faults in the gelJs electric field wil l sometimes cause the same protein to migrate at different rates in different parts of the gel. An example of this type of variation may be seen in Fig. 11. In this gel, the fastest fraction of the blood proteins of spring salmon ranges in distance from the origin from 14.8 cm to 15.5 cm, with a corresponding RJJ, range of 0.77 to 0.81. Although the range in variation within gels is usually less than this, the fact that i t is occasionally present places lower limits on the accuracy necessary in the estimation of incomplete standards. For example, In Fig. 1 1 a variation within the gel of 0.7 cm in the distance traveled by the leading fraction introduces an error of 0.04 in the % values of those fractions. Correspondingly, an error of approximately 1.0 cm in the estimation of the end point of the standard In that gel would be necessary to produce the same error In ^ values; an estimation error more than twice the average noted in the test group and just on the upper limit of two standard deviations of that group. Thus, as a general conclusion, the position of the leading edge of the fastest fraction in incomplete standards could be estimated with satis-factory accuracy considering the degree of variation within gels occasionally observed in these experiments. The estimation of the end points of Incomplete standards is seen to be reliable in practical application when the R^ values of the same blood protein fractions are compared on two separate gels, one with a complete standard and one with -25-an Incomplete standard. Figures 7 and 13 represent such a pair of gels. The serum proteins of a female sockeye are see In Fig. 7 which has an incomplete standard and are also seen in Fig. 13 which has a complete standard. Comparison of the 1^  values of the serum protein fractions corresponding to the appropriate slots in both gels reveals that they are in close agreement. A large error in the estimation of the end point of the incomplete standard would have prevented this agreement. C. THE EFFECT OF FREEZING, DIALYSIS, AND HEPARIN ON THE BLOOD PROTEINS  OF A FEMALE SOCKEYE SALMON A portion of the serum taken from a female sockeye salmon approxi-mately one month away from spawning condition was frozen and the remaining portion was placed at 3°C. Part of the serum placed at 3°C was dialyzed for one day in borate buffer at pH 8.5. The protein fractions of the three treatment categories can be seen in Fig. 11. There appear to be no differences in any of the traces which can be correlated with either freezing or dialysis. Certain differences do appear in the traces but none which occur in both members of a treatment category. Thus, the plasma proteins of a green female sockeye, at least, do not appear to be affected by dialysis or freezing. Another female sockeye, in the same state of sexual maturity as the previous individual, was sampled and heparin was added to a portion of that sample. The serum and plasma proteins of this i n d i v i d u a l are compared In Figures 13 and 14, which are two sides of the same gel. In 26-Fig . 1 3 the traces of serum and plasma exhibit no differences, but on the cut surface 6f the gel half , F ig . 1 4 , the traces di f fer in the region 0.0 to 0.2. Each of the traces of plasma proteins has an additional thin fraction in the range 0.10 to 0.12 and lacks the dark t r a i l s in the region 0.09 to 0.18 which occur in the traces of serum proteins. The additional protein fraction in the plasma traces may be one of the proteins involved In the c lott ing mechanism in salmon blood. D. SERUM AND PLASMA PROTEIN FRACTIONS COMMONLY OBSERVED WITHIN  SPECIES GROUPS 1. Spring Salmon Photographs of fractions obtained by starch-gel Electrophoresis of the plasma of spring salmon are presented in Figs. 2 and 3. In F ig . 2 the plasma of a male spring salmon In spawning condition Is shown, while in F ig . 3 the plasma of the same animal i s compared with the plasma of a female spring salmon of the same population in spawning condition. The Rm values of fractions common to male and female plasma in these traces are l i s t e d as fellows. A narrow but prominent band l i e s between RJJJ values 0.23 and 0.25; that i s , the band may be found within those l imits In either photograph, but i s not necessarily 0.02 Rm units long. More diffuse and longer i s a band lying between 0.28 and 0.34 followed by a band between 0.37 and 0.44. A fourth band common to both sexes In varying densities Is that found in the range 0.45 and 0.52. The 1^ values of the terminal or fastest fraction differ somewhat in Figs. 2 and 3. Because -27-the migration In Fig. 2 was obtained under unusual experimental conditions (brief power failure), preference wil l be given to that of Fig. 3.where the fastest fraction is seen to range between 1^ values 0.58 and 0.66. Additional fractions are apparent in Fig. 2 which are slower moving than those already mentioned. They possess values of 0.22, 0.20, 0.08, 0.06, and 0.04. The resolution of these fractions in Fig. 2 as well as the presence of fractions on the anodal side of the origin, the only ones obtained in these experiments, might be related to the unusual experimen-tal conditions mentioned above, since they are not present in Fig. 3. In conclusion, the. plasma^proteins of spring salmon have appeared consistently in five zones. 2. Chum Salmon Photographs of the protein fractions resolved from the sera of chum salmon are presented in Figs. 4 and 5. Fig. 4 shows the serum proteins of six individuals collected from the same location and sub-sequently held in fresh water, but sampled six months apart. Three traces are of sera taken one year after emergence as fry and three traces (two males and one female) are of sera taken one year and six months after emergence. Figure 5 presents the serum proteins of fresh water donn-stream migrants. Fractions common to both figures possess the following Rgj values. Two bands at 0.21 and 0.24 in Fig. 5 are similarly placed as a diffuse band in Fig. 4 In the range 0.18 - 0.23. Both figures show a short dark band with the same value of 0.27. Two well defined bands in the blood corresponding to slots 6, 7, and 8 of Fig. 4 having values of 0.34 and 0.36 are apparently represented in the sera of the younger -28-individuals in Figs. 4 and 5 by a diffuse band ranging from 0.33 to 0.38. The next fraction common to both ages of Fig. 4 lies between 0.44 and 0.53, noting that a slower fraction of the range 0.39 to 0.44 present in the older individuals is lacking in the younger. In this respect, the younger chum of Fig. 4 and those of Fig. 5 are similar in that a rather diffuse band in the corresponding region in the latter begins at about 0.43 and with an upper margin at approximately 0.55. Two final bands follow these in each of the three,ages tested having the respective ranges of 0.60 fco 0.68 and 0.70 to 0.74 in Fig. 4 but moving slightly faster in Fig. 5 with ranges of 0.61 - 0.70 and 0.71 to 0.78. Thus a minimum of six fractions appear in each of the three ages of chum salmon which possess comparable values. 3. Pink Salmon Photographs of the plasma protein fractions obtained from the blood of pink salmon are presented in Figs. 6, 2,. 7 and 8. In Fig. 6 the plasma proteins of mature male and female salmon in spawning condition are compared. Figure 2 presents the protein fractions of the plasma of a male pink salmon in spawning condition and of a male spring salmon in spawning condition; the blood of the pink salmon was taken from the same individual as was the male plasma in Fig. 6. Figure 7, another species comparison, presents the plasma protein fractions of a mature female pink salmon in spawning condition toggther with the serum protein fractions of a green female sockeye salmon judged to be about one month away from spawning. The plasma protein fractions of young pink saSimon caught as downstream migrants and sampled twenty days later are seen in Fig. 8 -29-representing the sampling of six individuals. Because only small samples in the order of 10 ul were obtainable, two of the traces are too faint to see. The four traces obtained show no obvious signs of individual variability. For comparison, the plasma protein fractions of a mature female pink salmon in spawning condition may also be seen in Fig. 8. There are a number of protein bands which consistently appeared In the plasma of each pink salmon tested which can be listed. Starting with the slowest moving, the f irst band is a narrow one seen in each figure between overall limits of 0.25 and 0.28. This band is seen closely associated with another at 0.25 or 0.26 in Figs. 7 and 8. Next, and perhaps most reliable In appearance with this species, is a series of narrow but prominent bands between 0.84- and 0.41. Appearing as a dark pair of bands at 0.37 and 0.39 or 0.40, respectively, they are sometimes flanked by additional bands at 0.35 (Figs. 6, 7, and 8) and at 0.42 as in Fig. 8. This series is then followed by two rather diffuse bands lying between 0.44 and 0.53 and between 0.55 and 0.65. The trace is completed with a terminal band between 0.70 and 0.75 seen in Figs. 7 and 8, though poorly in Fig. 2 and not at a l l in Fig. 6 due to excessively high mobilities In the latter two runs and the subsequent loss of the terminal fraction from the gel fields. Thus, plasma protein fractions of pink salmon have appeared consistently at at least six gel locations, each with its own relatively constant Rm address. 4. Sockeye Salmon Photographs of the protein fractions obtained from the serum and plasma of sockeye salmbn'are' presented in Figs. 9, 10, 11, 7, 12, 14, 13, and 4. Figure 9 presents a comparison of the plasma proteins of a -30-mature male sockeye In spawning condition and a maturing male sockeye judged to be six to eight weeks: away from spawning. The plasma proteins of two green male sockeye from widely separated spawning areas are com-pared in Fig. 10. The plasma of a male taken from Great Central Lake on Vancouver Island, British Columbia, is compared with that of a male taken from a weir at Cultus Lake, British Columbia. A l l the serum proteins visible in Fig. 11 are those of a female sockeye salmon judged to be about one month from spawning condition. Figure 7 presents the serum proteins of a maturing female sockeye approximately one month from spawning condition for comparison with the serum proteins of a mature female pink salmon in spawning condition. The serum of the female sockeye salmon seen in Fig. 7 is also shown In Fig. 12 where i t is accompanied by the plasma proteins of young sockeye collected as fresh water downstream migrants and sampled five days later. Figures 13 and 14 are two sides of the same gel half showing the plasma and serum protein fractions of a female sockeye salmon also judged to be about one month from spawning condition, but differing in identity from that pre-viously mentioned. Photographs of both sides of the gel half, the cut surface (Fig. 14) and the back surface (Fig. 13), were included as an illustration of the manner in which the appearance of the trace of the same sample may alter within the thickness of a gel. Only one slot in Fig. 4 contained the blood proteins of sockeye salmon, the remaining slots held those of chum salmon; the f irst slot corresponds to the plasma proteins of a green female sockeye salmon, three traces represent the serum proteins of young chum salmon in'their second year, and three more traces represent the serum of maturing chum six months older than the i - 3 1 -previous group. There are a number of 1^ , values or ranges at which protein fractions obtained from the blood of sockeye salmon frequently, i f not consistently, appear. Beginning with the slowest fractions, the f irst is found in the RJJJ range 0.18 to 0.21. This fraction may be seen in each of the figures with traces of sockeye blood proteins, though In some i t is quite faintly stained. In the bloods examined, this fraction is invariably followed by a much darker band In the R,,, range 0.23 to 0.28 being most frequently found in the upper portion of that range. Another fraction which fre-quently appeared is located between 0.35 and 0.37. It can be seen clearly in Figs. 1 1 , 7, 1 3 , and 4 and faintly in Figs. 12 and 14. These bands are typically followed by a broad darkly staining band with a well defined trailing margin and a diffuse leading margin occupying the range 0.38 to 0.51. This-band existed as such in a l l the figures presented except Fig. 1 0 where i t appears to have been resolved into two bands in the ranges 0.38 to 0.44 and 0.45 to 0.50. This broad band is followed by another darkly staining band which conversely shows a diffuse trailing margin and a well defined leading margin and, in most cases, lies between 0.60 and 0.75. Figure 10 does display a band in this region but is unusual in showing an additional band in the region of 0.50 to 0.60, a region void of fractions in a l l the other figures. Since the plasma of the green male sockeye in Fig. 1 0 may be seen In Fig. 9 to exhibit the more usual fraction distribution, the R„, values of fractions seen in F ig .10 may be considered somewhat unreliable due to the poor trace of the standard which accompanied them. This factor however does not reduce the usefulness of the gel for purposes of Intra-gel comparisons of protein fractions. - 3 2 -That i s , valid comparisons of the protein fractions appearing within this gel can s t i l l be made since they were obtained under identical experimental conditions. The fractions already mentioned are next preceded by a shorter darkly staining band which terminates the trace. This fraction i s located in the RJJJ range 0.73 to 0.80 in most figures though i t may l i e nearer to the origin at 0.70 to 0.73 as in Figs. 1 3 , 1 4 , and 4. This band i s seldom longer than 0.04 1^ units. No such fraction i s seen in Fig. 1 0 , since the appropriate Rjj, gone l i e s beyond the gel f i e l d . In conclusion, serum and plasma protein fractions of sockeye salmon have appeared frequently, i f not consistently, in at least six relatively constant zones. 5. Coho Salmon. A photograph of the plasma proteins obtained from coho salmon is presented in Fig. 15. Here the plasma protein fractions obtained from a mature female coho in spawning condition are presented with the plasma protein fractions of a mature male coho in spawning condition, and with the plasma protein fractions of young coho sampled in June of their f i r s t year. There are at least four zones in this gel which are consistently occupied by protein fractions in each of the eight traces obtained. The slowest moving of these fractions may be seen in the zone 0.26 to 0.28 as a short darkly staining band. The next band Is stained less darkly and i s located between 0.33 and 0.36. These are followed by two broad heavily stained regions occupying the zones 0.41 to 0.54 and 0.64 to 0.80. 1 - 3 3 -The latter band shows some Indication of being divisible into two in the traces of the female and young coho with the fastest band of the pair between 0.71 and 0.78 and no greater In length than 0.03 1^  units. OMier traces of coho plasma proteins were obtained but the standards accompany-ing them were not usable. They did not, however, indicate any individual differences within sexes; three individuals of each sex were tested. D. AN INTERSPECIFIC COMPARISON OF SERUM AND PLASMA PROTEINS This comparison is based on two sources of information. A direct source may be used based on intra-gel comparisons of protein fractions of two species appearing on the same gel, or an indirect source may be used which Is based on inter-gel comparisons of RJJ, zones of protein fractions of two or more species appearing on separate gels. A summary of the zones occupied by protein fractions in each species is presented in Fig. 16. 1. Intra-Gel Species Comparisons a) Chum-Sockeye As mentioned previously, Fig. 4 presents one trace of the plasma proteins of a maturing female sockeye and six traces of the serum proteins of two age groups of chum salmon. In general appearance, the blood proteins of both species are quite similar; only minor differences are apparent. The protein fraction of the sockeye in R„j zone 0.38 to -34-0.50 differs from the younger chum serum proteins which display a fraction only in the upper region of that zone starting at 0.45. The serum frac-tions of the older chum salmon are, however, very similar to those of the sockeye. The sockeye is seen to lack the paired fractions at 0.34 and 0.36 of the older chum but is in this region no different than"-:the younger chum salmon, Generally speaking, the differences between sockeye and chum fractions in this gel seem to be no greater than those existing between individual chum. Thus no real interspecific differences are implied here. b) Pink-Spring Figures 2 and 7 show comparisons of serum proteins of pink salmon with the plasma proteins of spring salmon and the serum proteins of sockeye salmon respectively. In Fig. 2 both species were mailie and in Fig. 7 both species were female. The fractions of pink salmon differ most markedly from those of spring salmon, in Fig. 2, at Rm values 0.37 and 0.39 where paired bands characterize pink blood proteins. Bands present in the interval 0.0 to 0.1 in traces of spring blood proteins are absent in those of pink. Absent in the 0.55 to 0.60 regions of spring traces are the bands present in pink traces at the same level. Another gap in the spring tasaces at 0.45 is f i l led in the traces of pink salmon blood. Both species exhibit short bands at approximately 0.25. c) Pink-Sockeye Short bands at 0.25 are also present in the traces of sockeye and pink seen in Fig. 7. Darkly staining areas, in the traces of sockeye serum proteins in the regions 0.38 to 0.50, 0.62 to 0.72, and 0.73 to 0.77 are absent or reduced in the traces of pink serum proteins. -35-Lacking in the sockeye traces are the series of short bands at 0.34, 0.39, 0.41 present in the pink traces with the exception of the band at 0.37 which appears in both. The terminal or fastest fractions of the sockeye traces are situated in higher ^ zones than are those of the pink. In conclusion,' the Intra-gel comparisons of chum and sockeye salmon reveal no differences larger than the variation within individual chum; whereas, the comparisons of pink and spring, and of pink and sockeye reveal marked trace differences. The pink traces differ from both the spring and sockeye traces by the series of bands at 0.37 and 0.39 and 0.34. 2. Inter-Gel Species Comparisons A graphic summary of the R,,, zones in which the blood proteins of each species are commonly located is presented In Fig. 16. These zones are sometimes longer than the fractions which occupy them since they represent eye-fltfed brackets of variability about the position of any given fraction. They also represent a minimum of the number of fractions seen in the trace of any given species since only those zones were recorded which were occupied by a fraction in a l l the traces of that species. Thus, any given trace may differ from its species counterpart in Fig. 16 by having more fractions than Indicated, but i t is not likely to differ by having less. Since almost every zone is seen to differ in range from any other zone at the corresponding level, comment wil l be restricted to those zone characteristics which appeared very consistently and are easily recognized. The f irst of these to be mentioned is the pair of fractions which l ie at S P E C I E S R m V a l u e s O.I 0 . 2 0 .3 0 4 . 0.5 0.6 07 O.S CHUM n COHO D PINK D[ -SOCKEyE SPRING U Fig. 16. A diagrammatic summary of the Rp, zones consistently occupied by blood protein fractions within species groups. -37-0.37 and 0.40 in the traces obtained from the blood of pink salmon. The traces of the bloods of other species sometimes showed one fraction in either of these positions (as did sockeye traces in Fig. 7), but none of the traces obtained for any other species presented a pair. The traces of pink salmon blood are also unusual In lacking a very darkly stained fraction between 0.40 and 0.50 as is present In the traces of sockeye, certain chum, and coho. Pink and spring both show fractions in this region but they are not stained as darkly as those of the other species. Stained proteins In this region of the traces of spring salmon blood are divisible into two fractions which is not obviously tame of the traces of any other species. The traces of chum and sockeye salmon are unique In their possession of a fraction In the range 0.18 to 0.22. In the traces obtained, there Is at least one Rm zone which is con-sistently occupied by a protein fraction in a l l of the five species. A short dark band in the range 0.24 to 0.27 can be seen in each of the figures presented. This protein or group of proteins may be present In the blood of a l l of the Pacific salmon. In general appearance and distribution of protein fractions, the serum and plasma proteins of chum, coho, and sockeye salmon appear to be more like one another than like those of spring and pink salmon. Each member of the former group exhibits two very darkly staining fractions in,the ranges 0.40 to 0.50 and 0.60 to 0.7 , while the latter species do not. Pink and spring blood proteins, however, do not appear to be very similar in their appearance and distribution, differing in the presence of paired band at 0.37 and 0.39 and at 0.55 in the pink traces. -38-E. SEX i 1. Pink Salmon Three runs were made In which the blood proteins of male and female pink salmon were compared. One of these is presented in Fig. 6 where four traces of the plasma proteins of a mature male pink salmon are presented with three traces of the plasma proteins of a mature female;: pink salmon. Both fish were sampled on October 1, 1962. Iksnone of the three gels were any differences noted which might be correlated with sex. A comparison of the 1^  values of protein fractions of pink salmon In Figs. 2 and 7, which were taken from a mature male and a mature female respectively, also does not indicate any obvious sex differences. 2. Chum Salmon The blood proteins of maturing male and female chum salmon sampled on October 24, 1962, may be seen In Fig. 4. No differences in these traces are apparent. None of these individuals weee sexually mature; eggs in the female were the sifce of a pin head. Differences in the plasma proteins of sexes may become more obvious as maturity is approached. 3. Coho Salmon The terminal fractions of mature male coho plasma proteins in Fig. 15 appear more heavily stained and longer than those of the mature female coho. Those of the male range between 0.61 and 0.80 while those of the female are found between 0.64 and 0.76, and show some Indication -39-of being divisible into two fractions. These fish were both sampled on November 27, 1962. 4. Sockeye Salmon A comparison of the serum and plasma proteins of a maturing female sockeye (sampled October 24, 1962) in Figs. 13 and 14 with the plasma proteins of the maturing and mature male sockeye seen in Fig. 9 yield some Indication of positional differences. The terminal fraction In the female traces is found between 0.67 and 0.73 while those of the immature male l i e between 0.71 and 0.77. This difference in position, however, is not borne out in the mature male which appears to be similar to the female in this region, but in addition, displays a very faint fraction at about 0.80. The mature and maturing male sockeye were sampled in late August and late September, respectively, 5. Spring Salmon The plasma proteins of a mature male and a mature female spring salmon are compared in Fig. 3. These fish were both sampled on September 16, 1962. The female trace displays a darker band than the male in the region 0.37 to 0.42. The male, in turn, shows a darker band than the female in the region 0.45 to 0.49. No other differences are apparent. In conclusion, indications of sex differences exist for coho, spring, and possibly sockeye salmon, but not for chum and pink salmon. For spring and sockeye salmon the differences are in the nature of a darkened region in the female trace at about 0.40. Coho differences are the reverse with female bands staining less heavily and with an additional band present in the terminal region. -40-F. AGE 1. Pink Salmon In Fig. 8 the plasma of young pink salmon caught as fresh water downstream migrants is compared with the plasma of a mature female. The traces obtained are very similar with the exception of the lack of a terminal fraction between 0.72 and 0.75 in the female trace. The traces of the young pink show few signs of individual variability. 2. Coho Salmon The plasma proteins of the young coho in.Fig..IS differ from the traces of the mature male coho in the same manner as do the females, which has been presented previously. The young differ additionally from the female traces in their display of fractions at 0.38 and possibly 0.30. 3. Chum Salmon The serum proteins of young chum taken as fresh water downstream migrants are presented in Fig. 5. The serum proteins of two ages of chum salmon held in fresh water which were sampled in the spring of their second year and in the f a l l of their second year are presented in Fig. 4. The serum of the oldest chum differs from both the younger age groups in the display of a fraction between 0.18 to 0.22, a pair of fractions seen at 0.33 and 0.35, and a dark band between 0.40 and 0.44. Both of the younger age groups are alike in lacking these fractions, but differ slightly in the terminal zone which in Fig. 5 l ies at a higher Rn, value than that of either of the age groups in Fig. 4. I -41-4. Sockeye Salmon Comparisons of different ages of sockeye salmon can be seen In Figs. 9 and 12. The plasma proteins of young sockeye taken as fresh water downstream migrants are presented in Fig. 12 together with the serum proteins of a green female sockeye. The blood proteins of both ages are very similar in appearance; the 1^  zone 0.40 to 0.50 Is less darkly stained in the young than in the adult, but this may be a result of the small samples applied. The young sockeye show two fractions at 0.30 and 0.33 which are not apparent in the trace of the adult. The plasma proteins of two adult male sockeye salmon in different stages of s^Ktial maturity are presented in Fig. 9. The plasma of a male sockeye taken'from Cultus Lake, British Columbia, ;six to eight weeks prior to spawning is compared with that of a ripe male sockeye taken from the Horsefly River, British Columbia. The plasma proteins of the green male •iffer from those of the ripe male in the display of a terminal band In the range 0.74 to 0.78 and in the greater fetensity of the fraction irLwthe region 0.60 to 0.70. In conclusion, the traces of pre-smolting coho, downstream migrating spring, and downstream migrating pink salmon a l l differ from the adult salmon with which they were compared by the possession of additional fractions between 0.30 and 0.38, in the case of cbho and spring, and between 0.73 and 0.75 in the case of coho and pink. Contrarily, downstream migrating chum lacked fractions seen in the traces of the adults between 0.18 and 0.35. G. GEOGRAPHICAL LOCATION A comparison o f the plasma p r o t e i n s o f two i n d i v i d u a l s o f the same s p e c i e s , s e x , and s t a t e o f s e x u a l m a t u r i t y , d i f f e r i n g o n l y in the l o c a t i o n o f t h e i r spawning ground are p r e s e n t e d i n Fig. 10. Two male sockeye , both ftbout s i x weeks away from spawning c o n d i t i o n were c o l l e c t e d , one from Cultus Lake, British Columbia, and one from Great Central Lake on Vancouver Island, British Columbia. The plasma p r o t e i n s o f these two i n d i v i d u a l s a r e Remarkably s i m i l a r in appearance and p o s i t i o n . This is an Indication,-at l e a s t , t h a t t h e plasma p r o t e i n p a t t e r n s w i t h i n s p e c i e s remain r e l a t i v e l y constant i n i n d i v i d u a l s o f the same sex and p h y s i o l o g i c a l c o n d i t i o n . -43-Fig. 2. A comparison of the plasma protein fractions of a mature male spring salmon in spawning condition seen corresponding to slots 1, 3, 5, and 7 (Sample Reference Number* (SRN): 1-a) with the plasma protein fractions of a mature male pink salmon in spawning condition seen corresponding to slots 4, 6, and 8 (SRN: 2-a). The standard corresponds to slot 2. The grid of parallel lines over the photograph repreeBBt 0.1 Rn, intervals, where 0.0 coincides with the origin and 1.0 the end point of the standard. * For further details on the sources and treatment of blood samples refer to the appropriate Sample Reference Number in Tables I and I I . i -44-. 3 . A comparison of the plasma proteins of male and female spring salmon, with the traces of a mature male i n spawning condition corresponding to slot s 4, 6 and 8 (SRN: 1-a) and a mature female spring salmon from the same population i n spawning condition corresponding to s l o t s 1, 3, 5, and 7 (SRN: 1-b). The standard corresponds to s l o t 2. Fig. 4. The blood protein fractions of two age groups of chum salmon and ft! eockeye salmon are compared here. Traces of the serum of three chum salmon sampled in the spring of their second year In fresh water correspond to slots 3,4, and 5 (SRN: 3-a,b,c) the serum protein fractions of maturing chum salmon of the same population sampled in the f a l l of their second year correspond to slots 6 (male), 7 (female), and 8 (male)(SRN: 4-a,b,c); the plasma proteins of a maturing female sockeye correspond to slot 1 (SRN: 5-2). The standard corresponds to slot 2. Fig. 5. The serum protein fractions of young chum salmon taken as fresh water downstream migrants correspond to slots 1, 3, 4, 5, 6, and 7 (SRN: 6-a,b,c,d,e,f). The standard corresponds to slot 2. -47-.1 .s 1 \ I I M I I M I M i l I l l I I I I M I I I I I I I I I I I I I I I I I l l l l l l l l l l l l l l l l l l l . i l Fig. 6. A comparison of male and female pink salmon, where the plasma protein fractions of a mature male pink salmon in spawning condition correspond to slots 1, 3, 5, and 7 (SRN: 2-a) , and the plasma protein fractions of a mature female pink salmon from the same spawning population correspond to slots 4, 6, and 8 (SRN: 2-b). The standard corresponds to slot 2. ?6 LC s IIIIIIIHIIIIIIIIIII I ! Illlllllllllllllll lllllllllllll Ill IIIIIIIHIIIIIIIIIII HHHHHHIIIHHI IIIIIHIIIIIIIHIIIIIIIHIIIIIIIIIIHIIII II S •1 lllllll IIIIHIIIIIIIIIIIHI HIIHIIIIIIIHIIIIII 111 Fig. 7. A comparison of the plasma protein fractions of a mature female pink salmon in spawning condition corresponding to slots 4, 6, and 8 (SRN: 2-b), with the serum protein fractions of a maturing female sockeye salmon corresponding to slots 1, 3, 5, and 7 (SRN: 5-6). The standard corresponds to slot 2. -49-Fig. 8. A comparison of young and adult pink salmon, where the plasma protein fractions of young pink salmon taken as fresh water downstream migrants correspond to slots 1, 3, 5, 6, 7, and 8 (SRN: 7-a,b,c,d,e,f), and the plasma protein fractions of a mature female pink salmon in spawning condition correspond to slot 4 (SRN: 2-b). The standard corresponds to slot 2. -50-Fig. 9. A comparison of the plasma protein fractions of a mature male sockeye in spawning condition corresponding to slots 1, 3, 5, and 7 (SRN: 8) with the plasma protein fractions of a maturing male sockeye judged to be six to eight weeks from spawning corresponding to slots 4, 6, 8 (SRN: 9). The standard corresponds to slot 2. Fig. 10. A comparison of two individual sockeye salmon from widely separated spawning populations. The plasma protein fractions of a maturing male sockeye taken from Great Central Lake, British Columbia, correspond to slots 1, 3, 5, and 7 (SRN: 10); the plasma protein fractions of a maturing male sockeye taken from Cultus Lake, British Columbia, correspond to slots 4 and 6 (SRN: 9). The standard corresponds to slot 2. -52-Fig. 11. The effect of freezing and dialysis on the serum protein fractions of a maturing female sockeye salmon (SRN: 5-b), where unfrozen, undialyzed serum proteins correspond to slots 1 and 5; unfrozen serum dlalyzed for 24 hours corresponds to slots 2 and 6; and frozen, undialyzed serum protein fractions correspond to slots 4 and 8. The standard corresponds to slot 3. -53-Fig. 12. A comparison of young and adult sockeye salmon, where the serum protein fractions of a maturing female sockeye salmon (SRN: 5-b) correspond to slots 5, 7, and 8; and the plasma protein fractions of young sockeye salmon taken as fresh water downstream migrants correspond to slots 1, 3, and 4 (SRN: ll-a,b,c). -54-rtg. .13. A comparison of the serum and plasma protein fractions of a maturing female sockeye salmon (SRN: 5-a), where the serum protein fractions correspond to slots 4. 6, and 8; and the plasma protein fractions (heparinized portion of fehe sample) correspond to slots 1, 3, 5, and 7. The standard corresponds to slot 2. This i s the uncut surface of the lower gel half; the cut surface Is presented In Fig. 14. -55-Fig. 14. A comparison of the serum and plasma protein fractions of a maturing female sockeye salmon (SRN: 5-a) , where the serum protein fractions correspond to slots 4, 6, and 8; and the plasma protein fractions (heparinized portion of the sample) correspond to slots 1, 3, 5, and 7. The standard corresponds to slot 2. This i s the cut surface of the lower gel half; the uncut surface i s presented In Fig. 13. Fig. 15. A comparison of the plasma proteins of a mature male coho salmon in spawning condition corresponding to slots 3, 5, and 8 (SRN: 12-a), with the plasma protein fractions of a mature female coho salmon in spawning condition correspond-ing to slots 1 and 7 (SRN: 12-b), with the plasma protein fractions of young coho salmon taken i n June of their f i r s t year as fresh water non-migrants corresponding to slots 4 and 6 (SRN: 13-a ,b). The standard corresponds to slot 2. -57-IV. DISCUSSION A. FREEZING, STORAGE, AND DIALYSIS The effect on blood proteins of storage at subzero temperatures has been studied by Hughes (1959) and Tsuyuki et al.(1962). Hughes found that one month of storage at - 25°C did not qualitatively affect the pattern of human sarcoplasmic muscle obtained by starch-gel electrophoresis. Tsuyuki demonstrated no significant differences between the protein patterns of fresh salmon and those stored at - 30°C for up to four months. In the present study, the electrophoretic pattern of frozen sockeye serum failed to differ qualitatively from that of an unfrozen portion of the same sample (Fig. 11). The serum proteins of additional portions of the same sample, one of which was dialyzed 24 hours at pH 8.5, also failed to differ significantly (Fig. 11). Brown (1957) noted that the pH of fish bloods range from 6 to 8 and quoted a pH of 7.41 for Salmo gairdnerii unexercised venous blood. The similarity of the undialyzed and dialyzed sera, which most probably differed in pH when applied to the gel slots, indicates that any changes in the net charges of the undialyzed serum proteins must have occurred prior to, or soon after, the onset of migration. B. CONTENTS OF THE STAINED FRACTION As Tsuyuki et al.(1962, III) noted, what Is referred to in this -58-dlscussion as a protein band is not necessarily a simple protein but may instead be a conjugated protein possessing nonprotein, or prosthetic, groups. It may be any proteinaceous substance with an affinity for Amirio Black 10B. Furthermore, since the separation of proteins by electro-phoresis depends solely on their mobility in an electric f ie ld , and since plasma proteins which differ in size, shape, composition, and physiological role may sometimes have the same mobility, protein bands are l ikely com-posed of ^'families" of proteins rather than single protein components (Harper, 1961). The question of the actual identification of the proteins obtained by electrophoretic separations of the components of animal bloods has been resolved In only a very few cases, some of which are cited by Cowan and Johnston (1962) as Brandt et al.(1951), mbr ick and Blonstein (1948), and Tiselius (1937). In many publications, the assumption of similarity Is made and the bands obtained are referred to In order of decreasing mobility as albumen, alpha-one globulin, alpha-two globulin, beta-globulin, and gamma-globulin even though their characterization was not accomplished. More cautious reporting may refer to the blood protein fractions of one species by a number. This eliminates interspecific extrapolations on previous characterizations but necessarily contains in i t the implication that bands obtained from different Individuals contain identical protein components when they are referred to by the same number. In this study, the use of a standard protein has permitted reference to a range in which a specific fraction appeared, a range expressed as a percentage of the length of the standard f^td which may differ from one trace to another. Thus the Identities of stained bands are not fused before i t is warranted. -59-iDf the bands are subsequently assumed or shown to be Identical, this may be stated and limits of variability may be placed about the range. Sibley (1960) considers this assumption to be reasonable in the case of closely related species. He states "....because protein structure is genetically determined tt seems reasonable to assume that two proteins are extremely similar i f they are derived from closely related species and have identical electrophoretic characteristics. For example, i t seems reasonable to assume that electrophoretically identical peaks in different species of the same genus result from proteins nearly identical in basic structure". There is perhaps some'merit, however, in using a reference system which allows for the possibility of individual and species differences rather than implying the absence of such differences by the manner in which the data is presented. C. LABILITY OF BLOOD PROTEINS The merit undoubtedly l ies in a second property of protein structure (the f i rs t being that i t is genetically determined) which is tremendous diversity associated with equally diverse physiological roles. West and Todd (1961) state "There is greater diversity in the chemical composition of proteins than in that of any other group of feibgically important compounds This difference in proteins extends to the composition of different tissues within the organism", to the composition of "the major organic structures of the protoplasmic machine", hormones, enzymes, and disease defense mechanisms. In short, the regulatory mechanisms of the -60-body are of protelnaceous origins, and thus are the physiological states within the individual. Meisner and Hickman (1962) briefly review studies which they conclude "have demonstrated the lability• of serum proteins to physiological change" with reference to the findings that migration in teleosts is associated with a decrease In the albumen-globulin ratio, and that starvation and exposure to industrial wastes may Influence the electrophoretic patterns of certain fish. Their own studies demonstrated the lab i l i ty of serum proteins of the Rainbow trout to external stimuli by inducing a higher albumen-globulin ratio at the lowest of two temperatures. Vanstone (1960) and Ridgway, Klontz, and Matsumoto (1962) have demonstrated intraspecific differences in the blood proteins of Oncorhynchus associated with physio-logical change. Ridgway et al. observed an antigen (or antigens) which occurred only in the blood of mature or maturing female red salmon (0. nerka); Vanstone was able to demonstrate several changes in the plasma protein fractions obtained by paper electrophoresis of coho salmon blood (0. kisutch) which were associated with smolt transformation, with the Immature sea salmon, and with the onset i*n' maturation of the female. In the present study, intraspecific blood protein differences were also demonstrated which were associated with sex differences and age differences. Intraspecific comparisons of the plasma and serum proteins of mature and maturing male and female salmon gave indications of isex differences In the coho, spring, and possibly sockeye salmon but not in chum and pink salmon. As Vanstone demonstrated for the maturing female coho salmon, the mature female blood proteins of the spring and sockeye salmon also appear to stain more darkly (at about R^ j 0.40 in both species) than those -61-of the mature male, but this was not demonstrated for the coho salmon. The mature female coho blood proteins appeared lighter than those of the male, but showed an additional terminal fraction. Vanstone concluded that the additional band in the female trace which also contained l ip id stain-ing components was associated with egg production and was likely "a mixture of yolk constituents Including serum vlte l l in ." Using paper electrophoresis, the intraspecific differences in the plasma proteins of coho salmon associated with smolt transformation and immature sea salmon which Vanstone (1960) was able to demonstrate were in the nature of the loss of the fifth and fastest fraction In the transform-ation from pre-smolt to smolt stages and Its subsequent appearance in the 2-year-old salmon In salt water. He Indicated Carlson had demonstrated a similar change in the plasma proteins of smolting and newly marine dwelling sockeye salmon. The traces obtained by Vanstone for the pre-smolting yearling coho in fcesh water, the Immature 2-year-oldecoho in salt water, the maturing male coho in fresh water, and the spawned-out male coho were almost identical. In'"the present study, the traces of pre-smolting coho, sampled In June of their f irst year, and of mature male coho were not identical, but differed in the pre-smolts possession of additional fractions at Rm 0.34, 0.38, and between 0.75 and 0.78. Similarly, young spring salmon collected as fresh water downstream migrants possessed fractions at 0.30 and 0.33 not present in the maturing female spring salmon traces. Young pink salmon collected as fresh water downstream migrants also differed in their traces from a mature female pink salmon by the addition of a fraction, though in a terminal position at 0.73. In the traces of ifchum salmon blood, i t is -62-the maturing males and female which display fractions at R 0.18, 0.33, m and 0.35 not seen in the traces of chumr;,taken as fresh water downstream migrants. It is evident that the trend indicated for coho.and sockeye by Vanstone of a disappearance of the terminal fraction during downstream migration normally present in a l l other conditions is not bom out in the" results for sockeye, chum and pink salmon. In fact, for the latter species the reverse was indicated. The blood of coho downstream migrants was not examined. An explanation offered by Vanstone for the appearance of additional slow moving fractions (low R|g values) associated with an increase in age as noted in chum salmon traces was an increased antibody production corresponding to a greater exposure time to antigenic sub-stances. Sibley (1960) not^ ed a positive correlation in globulin peaks with age in birds. ' D. INTERSPECIFIC COMPARISONS OF BLOOD PROTEINS: WHEN ARE THEY VALID? Having considered certain examples of the labi l i ty of the blood proteins of Salmonidae to external stimuli and internal physiological change, the question of when valid Interspecific comparison can be made introduces itself. Sibley (1960) considered this problem In his inter-specific electrophoretic comparisons of Avian proteins and concluded that "if reliable quantitative measurements of avian sera are desired the sample size must be large, the birds must be healthy and they must be segregated by age and sex". In the present studies the sample sizes were not ideal but at least four individuals of each species were examined and for most species the traces of about ten individuals were obtained -63-and frequently duplicated in almost f i f t y electrophoretic separations. When placing the blood of different species on the same gel, care was taken to select individuals of the same sex, approximate age and state of maturity i f these were available. Having done this, the only other major sources of variation l i k e l y to influence the species comparison are environmental factors, such as diet or thermal history, and the range of individual variation. Since most samples were taken directly from f i e l d animals, l i t t l e control could be exercised over environmental factors. These factors, however, could be expected to augment variation betwen individuals; several traces were obtained from Individuals of the same sex ( i f adult), age, and state of maturity which indicated no greater differences than those seen between traces of the same sample. For example, Fig. 8 shows the plasma proteins of four young pink salmon col-lected at the same time as fresh water downstream migrants which appear very similar; Fig. 10 shows the plasma proteins of two maturing male sockeye taken from separated spawning grounds (Cultus and Great Central Lakes, British Columbia) which also appear very similar; Fig. 4 demon* strates the same uniformity for the serum proteins of chum salmon, within age groups. The finding of uniformity among each of the animal groups examined i s taken as an indication that individual var i a b i l i t y i s not l i k e l y to obscure species differences i f they occur, providing the precaution of matching physiological states i s taken. E. SPECIES RELATIONSHIPS WITHIN 0NC0RHYNCHUS Many studies on the biology of the species of Oncorhynchus have yielded suggestions as to the nature of the systematic relationships -64-between them. These suggestions have been summarized in Table I I I . Tsuyuki (1962) noted that hybridizing studies on Pacific salmon by Foerster (1935) revealed that the sockeye, pink, and chum salmon were more readily hybridized than coho salmon. Other studies discussed by Tsuyuki segregated the genus into species groups of sockeye and pink salmon, then coho and spring salmon, with chum occupying an intermediate position on the basis of feeding habits; or into species groups of sockeye, chum and pink salmon, and of coho and spring salmon on the basis of the behavior of juvenile salmon, where the f i r s t group exhibited a stronger schooling tendency and the latter were more t e r r i t o r i a l (Hoar, 1958). Neave (1958) noted that pink, spring, and chum salmon had been shown capable of l i v i n g in strongly saline water at an earlier stage of develop-ment than coho and sockeye. Tsuyuki's studies on the muscle proteins of Pacific salmon led him to conlcude that the chum, pink, and sockeye salmon are more closely related to each other than to the coho and spring, but that the protein pattern of the chum salmon does have certain features common to both groups. In the present study, the traces of the blood proteins of the pink salmon were found to differ from those of spring salmon and from those of chum, sockeye, and coho salmon which resemble one another. The similarities and differences in the traces of blood proteins and the species groupings which have resulted are taken as an indication of the systematic relationships of these five species. Although this grouping does not agree entirely with any of those already described, there are many aspects of i t which are in par t i a l agreement. In each of the groupings.: described, the pink and coho salmon were con-sidered to differ significantly; in most of the groupings, the chum and TABLE III A SUMMARY OF SUGGESTIONS ON THE NATURE OF THE TAXONOMIC RELATIONSHIPS BETWEEN THE FIVE WEST COAST SPECIES OF ONCORHYNCHUS Bases for Suggestions Suggested Relationships Hybridizing studies (Foerster, 1935) Pink Sockeye Chum Coho Feeding behaviour of juveniles (Hoar, 1958) Pink Sockeye Chum Coho Schooling behaviour of juveniles (Hoar, 1958) Pink Sockeye Chum Coho -Spring Abili ty of juveniles to withstand high salinities (Neave, 1958) Pink Spring Chum Coho Sockeye Muscle proteins (Tsuyuki, 1962,111) Pink ~ Sockeye Chum Coho Spring Blood proteins (present study) Pink /Sockeye Chum Coho Spring * The order of l i s t ing is not significant. -66-sockeye were' regarded as closely related, while the spring salmon was considered to differ significantly from both the pink and the sockeye salmon. These generalizations are each supported by the results of this study. Unusual are the findings that the coho blood proteins resembled those of the sockeye but not those of the spring salmon, and that the blood proteins of the pink salmon do not resemble those of the chum and sockeye salmon. \ Regarding the degree lof differentiation shown by'ithe species of Oncorhynchus, Neave (1958) has written "the species are evidently closely related and frequently require careful examination for taxonomic separa-tion". In stressing the physiological and ecological similarities between the species, Neave also noted that they differ l i t t l e in their preferred temperature in fresh water and salt water, in lethal temperature, in salinity requirements and show much overlap in feeding habits. It is ^iot altogether surprising that suggestions as to the systematic relationships between the five West Coast species of Oncorhynchus based on their physiological and ecological characteristics should differ and sometimes prove contradictory. -67-V. BIBLIOGRAPHY Bier, M. (Editor). 1959. Electrophoresis theory, methods, and applications. Academic Press: New York. Block', R. J. , Durrum, E. L. and Zweig, G. 1955. A manual of paper chromatography and paper electrophoresis. Academic Press: New York. Brandt, L. W., Clegg, R. E. and Andrews, A. C. 1951. The effect of age , . and degree of maturity on the serum proteins of the chicken. J. Biol. Chem, 191: 105-111. Brown, M. E. (Editor). 1957. The physiology of fishes. Vol. 1. Metabolism. Academic Press: New York. Cowan, I. McT. and Johnston, P. A. 1962. Blood serum variations at the species and subspecies level in deer of the genus Odocoileus. Systematic Zool., 11: 131-138. Cushing, J. E. (Chairman). 1962. Symposium on immunogenetic concepts In marine population research. Amer. Nat. , 96: 193-246. Foerster, B. E. 1935. Inter-specific cross-breeding of Pacific salmon. Trans. Roy. Soc. Canada, Series 111, 29, Sec. V: 21-33. Harper, H. A. 1961. Review of physiological chemistry. 8th ed. Lange Medical Publications: Los Altos, California. Hoar, W. S. 1958. The evolution of migratory behaviour among juvenile salmon of the genus Oncorhynchus. J. Fish. Res. Bd. Canada, 15(3): 391-428. Hourston, W. R. 1949. The serological relationships of some Pacific coast Salmonoid fishes. M.Sc. Thesis: University of British Columbia. -68-Hughes, B. P. 1959. The electrophoresis of some human muscle proteins on starch gels. Biochem. and Biophys. Res. Commun. 1.(4): 194-198. Jacobs, M. H. , Glassman, H. N. and Parpart, A. K. 1950. Hemolysis and zoological relationship. Comparative studies with four penetrating non-electrolytes. J. Exp. Zool. 113: 277-300. Kibrick, A. C. and Blohstein, M. 1948. Fractionation of serum into albumen and a-, -, and -globulin by sodium sulfate. J. Biol. Chem. 176: 983-987; Konig, P. 1937. Actas e trabalhos do Terceiro Congresso Sud-Americano de Chimica, Rio de Janeiro e Sao Paulo. 2, 334. Meisner, H. M. and Hickman, C. P. Jr. 1962. Effect of temperature and photoperiod on the serum proteins of the rainbow trout, Salmo  gairdnerl. Can. J. Zool. Vol. 40: 127-130. Neave, F. 1958. The origin and speciation of Oncorhynchus. Trans. Roy. Soc. Canada. 52(3): 25-39. Pert, J. H., Engle, R. E. J r . , Woods, K. R. and Sleisenger, M. H. 1959. Preliminary studies on quantitative zone electrophoresis in starch-gel. J. Lab. and Clin. Med. 54: 572-584. Poulick, M. D. and Smithies, 0. 1958. Comparison and combination of the starch-gel and filter-pjaper electrophoretic methods applied to human sera: two-dimensional electrophoresis. Biochem. J. 68: 636-643. Raymond, S. 1955. Paper electrophoresis. 3rd Edition. E-C Apparatus Company: Swarthmore, Pa. Reichert, E. T. and Brown, A. P. 1909. The differentiation and specificity of corresponding proteins and other v i t a l substances in relation to biological classification and organic evolution: the crystallography of hemoglobins. Carnegie Inst. Wash. Publ. 116. -69-Ridgway, G. J . 1962. Demonstration of blood groups in front and salmon by Isoimmunization. Ann. N. Y. Acad. Sci. 97:111-115. Ridgway, G. J . , Cushing, J . E. and Durall, G. L. 1958. Serological differentiation of populations of sockeye salmon. Special Scientific Report. Fisheris No. 257: 1-9. ; >"•. Ridgway, G. J . , and Klontz, G. W. 1960. Blood types in Pacific salmon. U.S. Fish and Wildlife Service, Special Scientific Report. r Fisheries No. 324: 1-9. Ridgway, G. J . , KLontz, G. W. and Matsumoto, C. 1962. Intraspecific differences in serum antigens of red salmon demonstrated by immunochemical methods. International North Pacific Fisheries Commission. Bulletin Number 8: 1-13. Sibley, C. G. 1960. The electrophoretic patterns of avian egg-white proteins as taxonomic characters. Ibis, Vol. 102(2): 215-284. Smithies, 0. 1955b. Zone electrophoresis in starch gels: group variations in the serum proteins of normal human adults. Biochem. J . 61: 629-641. Smithies, 0. 1959a. An improved procedure for starch-gel electrophoresis: further variations in the serum proteins of normcQ; individuals. Biochem. J . 71: 585-587. Smithies, 0. l'9S9b'.'"Zone?e^ecir6pnd2Ssil^.iiAb8tarcK' gels1 andr:i±s .application to Istudies^of §erumrpr.oteinsC Advances'..inYProtein Chemistry. 14:65-113. Tieelius, A. 1937. A new apparatus for electrophoretic analysis of colloidal mixtures. Trans. Faraday Soc. 33: 524-531. Tsuyuki, H. and Roberts, E. 1961. Muscle proteins of Pacific salmon (Oncoi-hynchus). I. A note on the separation of muscle proteins soluble in low ionic strength salt solutions. J . Fish. Res. Bd. Canada, -7.0-18(4): 637-640. Tsuyuki, H., Roberts, E. and Gadd, R. E. A. 1962. Musclefproteins of Pacific salmon (Oncorhynchus). I I . An Investigation of muscle protein and other isubstances soluble in salt solutions of low ionic strength by column chromatography. I I I . The separation of muscle proteins soluble in, low ionic strength salt solutions by starch-gel electrophoresis. Can. J. Biochem. and Physiol. 40: 929-936. Vanstone, W.- E. and Chung-Wai Ho, F. 1961. Plasma proteins of coho salmon, Oncorhynchus kisutch, as separated by zone -electrophoresis. J. Fish. Res. Bd. Canada, 18(3): 393-399. West,. E. S. and Todd, W. R. 1961. Textbook of biochemistry. 3rd Edition. The Macmillan Company: New York. 

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