Serum proteins of the Japanese quail, Coturnix coturnix Japonica

Comp. Bioch~m. PhysioL, 1971, Vol. 39B,pp. 789 to 796. Pergamon Press. Printed in Great Britain

SERUM PROTEINS OF THE JAPANESE QUAIL, COTURNIX COTURNIX JAPONICA* A L F R E D C. S C H R A M and C H R I S T O P H E R P. C H R I S T E N S O N Department of Chemistry and the KiUgore Research Center, West Texas State University, Canyon, Texas 79015 (Received 8 December 1970)

Abstract--1. Fractionation of quail serum indicated two major protein components, one of which was an albumin fraction, and the other, a large molecular weight lipoprotein fraction. 2. Quaff serum albumin was physically and immunologieally similar to chicken serum albumin. 3. A large portion of the serum proteins of the hying female was found in the lipoprotein fraction, which, however, was only a minor component of the serum of the male. INTRODUCTION THE JAPANESE quail, Coturnix coturnix japonica and its European subspecies, C. c. coturnix are migratory galliformes. Perhaps as a result of migratory habits, their introduction as game birds in Eastern United States has been unsuccessful. T h e Japanese quail is, however, raised in large quantities as a laboratory animal for toxicity and genetic studies. After the brooding stage, the quails are very hardy, stand crowded conditions and changes in temperatures, and reproduce at a rapid rate, hatching after 17 days of incubation and reaching full maturity 7 weeks after hatching. Quails were hatched in an incubator from eggs collected in a cage containing two males and three females of about 1 year of age. T h e parents' stocks was not genetically homogeneous; occasionally, albinos were hatched. T h e newly hatched chicks were raised for 5 weeks in a brooder and fed Purina Game Bird Startena. Thereafter, they were kept in cages in an air-conditioned building, their feed being gradually changed to Purina Game Bird Layena. MATERIALS AND METHODS At 10 weeks of age, the birds were exsanguinated by heart puncture, after a 12-hr fast in some eases. The serum was stored at - 15°C until use. After thawing at 37°C, the serum was centrifuged for 15 min at 2000 rev/min. A 0"3-ml sample was mixed with about 1/zg of l*SI-labelled bovine serum albumin Q25I-BSA) (Day et al., 1967) as a marker and fractionated * Supported in part by Grant AE-189 from the Robert A. Welch Foundation, Houston, Texas, and by a grant from the Committee on Organized Research, West Texas State University. This investigation was carried out in the Killgore Research Center, West Texas State University, Canyon, Texas. 27

789

790

ALFRED C. SCHRAM AND CHRISTOPHER P. CHRISTENSON

at room temperature (21 _+I°C) on a 0"7 x 150 cm column of Sephadex G-200 (Pharmacia Co.) equilibrated with 0"15 M NaC1 in 0"02 M Tris-HC1 buffer p H 8-6, containing 0"02% NaN3 as a preservative. T h e effluent was collected mechanically in fractions of approximately 1 ml (55 drops) under 10-15 cm of hydrostatic pressure, at a rate of 3 ml/hr. T h e radioactivity of the fractions (count/min) was determined in a sodium iodide crystal detector; the absorbance at 280 m/x (O.D.2s0), the Lowry protein determination (Lowry et al., 1951) on 0-1 ml and the anthrone carbohydrate determination (Morris, 1948) on 0"5 ml of each fraction were also used to obtain the elution profiles. Double-gel diffusions (Ouchterlony, 1958) were carried out at room temperature in a moist atmosphere, in 1% agar in 0'15 M NaC1 with 0"02 M Tris-HC1 p H 8"6 and 0"02°,"0 NaN3 using a commercial rabbit antiserum against chicken serum (Nutritional Biochemicals Corp.), or in 1°/ agar in 13% NaC1 with 0"02 M Tris-HC1 p H 8"6, using a chicken antiserum against quail serum. This chicken antiserum was obtained from a 1-year-old white crested black Polish hen, 1 week after the last of four subcutaneous injections of 0-1 ml of pooled quail serum given at 4-weekly intervals, the first one in Freund's complete adjuvant, the following three in Freund's incomplete adjuvant. T h e data plotted on the graphs are expressed as the percentage of the total amounts eluted from the column, in order to use a single ordinate scale. RESULTS T h e e l u t i o n profiles o f q u a i l s e r u m (Figs. 1-3) are q u i t e d i f f e r e n t f r o m t h a t o f c h i c k e n s e r u m (Fig. 4) or of silver p h e a s a n t ( S c h r a m , 1970). A t first glance, the sera a p p e a r e d s i m p l e r in p r o t e i n classes c o m p o s i t i o n t h a n chicken s e r u m . M a l e a n d f e m a l e q u a i l sera differed m a r k e d l y b y t h e a m o u n t s o f a large m o l e c u l a r w e i g h t c o m p o n e n t w h i c h was o b s e r v e d in t h e c o l u m n ' s v o i d v o l u m e ( F r a c t i o n s 15-25,

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with 1/~g z=5I-BSA; fraction size 1 ml. Each fraction was analyzed by absorbance at 280 m/~ ( ); total radioactivity ( . . . . ); Lowry protein determinations on 0"1 ml (. . . . ); and anthrone carbohydrate determination on 0-5 ml ( - ' - " ). The results are expressed as a percentage of the total amount eluted.

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FIG. 2. Sephadex G-200 fractionation of 0"3 ml of fasted female quail s e r u m with 1 p g I " I - B S A . Conditions and analyses similar to those for Fig. 1.

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FIG. 4. Sephadex G-200 fractionation of 0"3 ml of fasted white crested black Polish hen (8 months old) serum with 1/~g I~SI-BSA. Conditions and analyses similar to those for Fig. 1. Figs. 2, 3). This component represented only a minor portion of the male's serum proteins. For both male and female serum, this component decreased following a 12-hr fast, at the rate of which all serum proteins decreased: it was 33.6 and 36.7 per cent of the total serum proteins in fasted and non-fasted females (Table 1). The first fractions containing this component (Fractions 16-20, Fig. 2; Fractions 15-20, Fig. 3) were always turbid, suggesting the presence of lipid material. These fractions were smaller in volume, apparently because of smaller drop volume, resulting from lower surface tension. This variation in fraction volume required the addition of the labelled BSA as a reference in each elution profile. The only other clearly separated serum protein was an albumin fraction, with an average molecular weight similar to that of BSA, as indicated by the coincidence of its maximum with that of a trace of labelled BSA added to the serum. There was no discrete globulin fraction, in line with the observation of Kubo & Benedict (1969) that quails have only a small amount of IgG immunoglobulins. The lack of a clear globulin fraction was apparently not a deficiency of the fractionation conditions, since under the same conditions, a chicken serum was separated into a macroglobulin, a globulin and an albumin fraction (Fig. 4). The sera always contained free hexose and a variable amount of free amino acids and other small molecular weight substances absorbing at 280 m/z. Gel diffusion contradicts the simplicity of the elution profiles. Both rabbit anti-chicken serum (Fig. 5b) and chicken anti-quail serum (Fig. 5a) show the complexity of the individual fractions• The chicken antiserum gave better resolution of the high molecular weight fractions, while the rabbit antiserum gave better resolution of the low molecular weight fractions. For comparison, the chicken

37 (albumin) 30 (globulin) 14-28 (macroglobulin) 29-50 (globulin + albumin) 47-58 (free hexose) Total

37 (albumin) 30 (globulin) 14-27 (macroglobulin) 28-48 (globulin+albumin) 46-57 (free hexose) Total

34 (albumin) 27 (globulin) 14-24 (macroglobulin) 25-30 (globulin) 31-41 (albumin) 47-55 (free hexose) Total

Quail (female, fasted) (Fig. 2)

Quaff (female, not fasted) (Fig. 3)

Hen (fasted) (Fig.4)

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36"7 63"3

33"6 66"4

3"4 96.6

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1.94 1"07 3"53 6-54

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mg Hexose/ml serum

* Based on the Lowry test with BSA as standard. t Protein content too low. The data were obtained by integration of the appropriate portions of the area under the curves in Figs. 1-4.

34 (albumin) 27 (globulin) 14-21 (macroglobulin) 22-49 (globulin + albumin) 44-59 (free hexose) Total

Fraction

Quail (male, not fasted) (Fig. 1)

Bird

TABLE 1--COMPOSITION OF JAPANESE QUAIL SERUM

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ALFRED C. SCHRAM AND CHRISTOPHER P. CHRISTENSON

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s._o @ @ @ @,c, Fx~. 5. Tracings of double-gel diffusions in 1% agar in 0"02 M Tris pH 8"6 with 13% NaC1 in (a) and 0"9% NaC1 in (b) and (c). Center wells: (a), chicken antiserum against pooled quail serum; (b) and (c), rabbit antiserum against chicken serum. Outer wells: (a) and (b), fractions of female quail serum recorded in Fig. 2; (c) fractions of hen serum recorded in Fig. 4. serum fractions (Fig. 4) were also analyzed by gel diffusion against the rabbit anti-chicken serum (Fig. 5c). DISCUSSION The high molecular weight fraction in the serum of the female quail is very probably related to egg laying. Both females were laying regularly prior to the exsanguination. In the Japanese quail, the egg weight is approximately 7 per cent of the body weight, and the females are prolific layers (at least 300 eggs per year). To compensate for this loss of weight, the female must have an efficient mechanism of egg proteins synthesis. The high molecular weight component's concentration, like egg-laying, decreases during fasting. The turbidity of some of the fractions is responsible for the high absorbance at 280 m/~. Thus it was necessary to use another test (Lowry) to measure protein. The low levels of protein found in the fractions prevented the use of the biuret test (Dittebrandt, 1948). The peak based on absorbance at 280 mt~ does not correspond to that based on the Lowry protein determinations, which suggested that the first fractions might be made of chylomicrons. However, the chicken antiserum precipitated a component found in the fractions with highest turbidity (Fractions 15-18, Fig. 5a), which eliminated the identification as triglyceride globules. This component was different from another component (Fractions 17-18, Fig. 5a-c) corresponding to the macroglobulins of the chicken. The component in quail fractions 15-18 is thus probably a very high molecular weight lipoprotein, with relatively high carbohydrate content

SERUMpROTEINS OF THE ~ A P ~

QUAIL

795

(Figs. 1-3). This component must not be present in the chicken serum, since the rabbit antiserum did not detect it, while the immunized chicken elaborated antibodies against it. It is interesting to notice that Fraction 37 (Fig. 2) which contains the peak of the albumin fraction does not react with the chicken antiserum, although the rabbit antiserum distinguishes in it at least three different antigenic components (Fig. 5b). This albumin fraction obviously contains proteins which are sufficiently similar in chicken and in quail sera to prevent the chicken from recognizing them as antigens. The ratio of absorbance at 280 mix to the amount of colour developed by the Folin reagent in this sample is not the same in the case of the chicken and of the quail (Table 1). Although there is a small difference between the ratios for male and female quails, there is a much larger difference between the ratios for quail and chicken. These fractions are thus different in chemical composition, but must contain many identical determinants. The molecular weight is similar to that of BSA in both quail and chicken serum. In the fractions (25-30) which would contain any globulin, the ratio of absorbance at 280 mix to that at 750 mix (in the Lowry test) was similar for all birds, indicating similarity in composition. Indeed, the rabbit antiserum precipitated components in this region, but the chicken antiserum also precipitated some components. The macroglobulin fractions were also precipitated by both chicken and rabbit antisera. Here again, we find similarity in hexose content per 100 mg of protein in quail and chicken macroglobulins. Thus for the globulins and the macroglobulins, with similarity in general composition, we find differences in immunogenie composition. The total amount of carbohydrate does not vary widely between male and female, or between fasted and non-fasted state in the female. However there are differences in the free and bound carbohydrate (Table 1). The male has a higher concentration of free hexose (4.77 rag/m1 of serum) than the female (3.57 and 3.35 mg/ml). In the female, the increase in total carbohydrate is due to the increased amount of bound carbohydrate in the globulin and especially in the macroglobulin fractions. In conclusion, the male Japanese quail serum contained only a small portion (3.4 per cent) of macroglobulins, while the laying female serum contained approximately ten times as much (33.6 and 36.7 per cent). The laying female serum also appeared to contain 50 per cent more protein than the male serum. A globulin fraction was not separated by gel filtration. The albumin fraction contained components which were antigenicaUy very similar to the components of hen serum albumin. Ack~.towledgement--We wish to thank Linda McCrary for her expert assistance. REFERENCES DAY E. D., LASStTER S. & FRITZ R. B. (1967) Radioiodination of antibodies adsorbed to insoluble antigens. 3t. Immun. 98, 67-71.

796

ALFRED C. SCHRAM AND CHRISTOPHER P. CHRISTENSON

DITTEBRANDT M. (1948) In Experimental Immunochemistry (Edited by KABAT E. A. & MAYER M. M.), pp. 559-560. Thomas, Springfield, Illinois, 1967. KUBO R. T. & BENEDICTA. A. (1969) Comparison of various avian and mammalian IgG immunoglobulins for salt-induced aggregation. J. Immun. 103, 1022-1028. LOWRY O. H., ROSEBROUGHN. J., EARR A. L. & RANDALLR. J. (1951) Protein measurements with the Folin phenol reagent. 9~. biol. Chem. 193, 265-275. MORRIS D. L. (1948) Quantitative determination of carbohydrates with Dreywood's anthrone reagent. Science 107, 254-255. OUCHTERLONY O. (1958) Diffusion-in-gel methods for immunological analysis. Prog. Allergy 5, 1-78. SCI-IRAMA. C. (1970) Serum proteins of the silver pheasant Gennaeus nyctheremus. Comp. Biochem. Physiol. 36, 481-492.

Key Word Index---Serum proteins in birds; quail; Coturnix coturnix japonica; lipoprotein in birds; macroglobulin in birds.