THE JOURNAL OF BIOLOGICAL CHEM~~Y Vd. 242, No. 17, Issue of September 10, pp. 3951-3956, 1967

Printed in U.S.A.

Quantitative Immunological Comparison of Bird Lysozymes*

(Received for publication, April 24, 1967)

NORMAI\: ARNHEIM, JR., AND ALLAN C. WILSON

From the Departments of Biochemistry and Genetics, University of California, Berkeley, California 94720

SUMMARY

Rabbit antisera were prepared against crystalline chicken lysozyme and characterized by immunodiffusion, immuno- electrophoresis, inhibition of enzyme activity, and quantita- tive micro-complement fixation.

Lysozyme was purified by Sephadex G-75 chromatography from the egg whites of 17 species of birds in the order Galli- formes and was tested for reactivity with anti-chicken lyso- zyme. The chicken and its closest relative, the jungle fowl, have lysozymes that appear to be indistinguishable from each other. The lysozymes of all the other species tested (e.g. partridges, quails, and pheasants) could be distinguished from the chicken enzyme by quantitative micro-complement tiation. Immunodiffusion failed to detect most of these differences in antigenic structure.

A particularly close relationship was observed between the lysozymes of the chicken, partridges, and American quails. This suggests that these lysozymes may show very small sequence differences. This finding may also have taxo- nomic significance.

The comparison of the structure of homologous proteins from different species can contribute towards an understanding of the relationship between protein structure and function as well as the nature of evolutionary processes and taxonomic relationships. Data on the amino acid sequence of cytochrome c (l), hemoglobin (2, 3), and insulin (4) have been analyzed from these points of view. By comparison, little information is available concerning the structural variations of lysozyme (muramidase, EC 3.2.1.17) among species (5-12). The only known lysozyme sequences are those of the Tq phage and the chicken (7, 13, 14). Since the three-dimensional structure of chicken lysozyme is known (15), information on structural variation among species is especially valuable. In the present article the lysozymes of 16 species of birds are compared with that of the chicken. Immunological methods were employed, with particular emphasis on the quanti- tative micro-complement fixation method (16). This method has

* This research was supported in part by Grant GB-3839 from the National Science Foundation and by Postdoctoral Traineeship GM-367 from the United States Public Health Service.

been shown to be sensitive to small differences in amino acid se- quence (17) and capable of providing an approximate measure of the degree of structural relatedness between homologous proteins (18, 19).

EXPERIMENTAL PROCEDURE

Materials

Eggs-Table I indicates the species and source of supply of all the egg whites utilized in this investigation. Most eggs were re- frigerated within 24 hours after laying. The egg whites were sep- arated from the yolks and stored frozen at -8”.

Proteins-Commercially prepared, crystalline chicken egg white lysozyme was obtained from Reheis Chemical Corporation (Division of Armour, control No. B18309), Worthington (crys- tallized twice, LY644A), and Pentex (crystallized three times, EZ1962). Ovalbumin (crystallized five times, No. 5614) and conalbumin (No. 7168) were purchased from Nutritional Bio- chemicals. Crystalline ovalbumin, further purified by chroma- tography on carboxymethyl cellulose, was supplied by I). Wachter of this laboratory.

Antisera-Antibodies to chicken lysozyme were obtained by injecting commercially prepared lysozyme into male New Zea- land white rabbits. Initially, 2 mg of any one of the three com- mercial lysozyme preparations were injected into each rabbit in- tradermally on the back. The lysozyme was suspended in Freund’s complete adjuvant (Difco) together with an additional 4 mg per ml of lyophilized, phenol-killed BCG (supplied by the Department of Bacteriology and Immunology). One month after the initial immunization, each rabbit was given an intrave- nous injection of lysozyme (2 mg per ml in saline). Sera were collected 11 days later. Two months later each rabbit was given an intravenous injection (2 mg of lysozyme per ml of saline) every other day for a total of three injections. Seven days after the final injection, sera were collected. Each antiserum (A) is identified by 2 digits; for example, A24 indicates the antiserum obtained from rabbit 2 after four intravenous injections. Rab- bits 2 and 3 were immunized with Worthington lysozyme, 4 and 5 with Pentex lysozyme, and 6 and 7 with the Almour prepara- tion.

Methods

Lysozyme assays-Lysozyme was assayed by the lytic activity against Micrococcus lysodeikticus (20). The assay mixture con-

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Species Diferences in Lysozymes Vol. 242, No. 17

I I I

75 150

EFFLUENT VOLUME (ml)

225

FIG-. 1. Amberlite chromatography of crystalline Armour chicken lysozyme. See “Methods” for further details.

tained 0.25 mg of M. lysode&icus cells (Worthington ML36) per ml in 0.05 M NaCl and 0.066 M phosphate buffer, pH 6.2. We have defined 1 unit of lysozyme activity as that amount which causes a change of 1 y0 transmittance per min at 540 mp. Under our conditions, Armour crystallized lysozyme has a specific activity of 660 units per mg. Assays were carried out in a Zeiss spectrophotometer. Routine assays were made at room temper- ature (23 f 1’). For the determination of specific activities of purified lysozymes, the temperature was maintained at 25 f 0.10”.

A semi-quantitative assay for lysozyme activity in the eluate from chromatographic columns was carried out by adding ali- quots from each fraction collected to test tubes containing the standard assay mixture. The change in the percentage of trans- mittance was recorded after 1 hour of incubation at 23’ with a Spectronic 20 spectrophotometer.

Protein was determined by measuring the absorbance at 280 ml with a Zeiss spectrophotometer. The extinction coeffi- cient (E?$,J for chicken lysozyme at pH 5.4 is 26.3 (21).

Immunological Methods-Immunodiffusion tests were con- ducted at room temperature by means of Ouchterlony’s double diffusion technique. The gel medium was a modification of that described by Fujio et al. (8). A 1% solution of Ionagar No. 2 (0x0 Ltd., London) was prepared in a 0.02 M sodium phosphate buffer (pH 7.1) containing 0.01% merthiolate and 0.14 M NaCl. Antigen and antiserum wells, each 3 mm in diameter, were placed 3 mm apart. In most cases, 10 ~1 of solution containing 250 pg of lysozyme per ml were added to each antigen well. Each antiserum well received 10 ~1 of undiluted antiserum.

Immunoelectrophoresis was performed in agar gels on glass slides (1 X 3 inch) (22). The gels contained 1 y0 Ionagar No. 2, NaCl (0.14 M), sodium phosphate (0.0064 M), and citric acid

(0.0008 M), the final pH being 6.9. Electrophoresis was carried out at room temperature with a voltage gradient of 3 volts per cm for 6 hours. Antiserum was then added. Twenty-four hours later the slides were examined for the presence of precipitin arcs, before and after staining with Amido black.

Micro-complement fixation experiments were performed ac- cording to the technique of Wasserman and Levine (16). Seven- milliliter reaction volumes were used. Hemolysin and guinea pig complement were obtained from Baltimore Biological Labo- ratory or Hyland Laboratories, Los Angeles. Sheep red cells were supplied by the Bennett Ranch, Woodland, California. All reagents were diluted in a buffer containing 0.14 M NaCl, 0.01 M

Tris-hydrochloride, 5 x 10m4 M MgS04, and 1.5 x 10m4 M CaClz at a final pH of 7.45. The reaction time at 5” was standardized at 18 hours.

Ultracentrifugation-Sedimentation velocity analyses were made with a Spinco model E analytical ultracentrifuge. Lyso- zyme solutions of 0.8% were centrifuged at 59,780 rpm in a buffer containing 0.02 M sodium acetate, pH 5.3, and 0.15 M

KC1 at 21”. Xephadex Chromatography-Lysozyme was purified from the

egg whites of 17 species by gel filtration. Columns of Sephadex G-75 (Lot T07118) 40 or 50 cm in length and 1.5 cm in diameter were equilibrated with 0.14 M NaCl. In most cases, 2 ml of a 1: 10 dilution of egg white were applied to the column. Flow rates in any one experiment were constant and were between 0.25 and 1.5 ml per min. All fractionations were carried out at 23”.

Amberlite Chromatography-Chromatography of crystalline lysozyme was carried out according to the method of Tallan and Stein (23). Bio-Rex 70 (200 to 400 mesh) resin was employed. A 0.2 M sodium phosphate buffer, pH 7.18, was used as an eluant. Chromatography was carried out at 23” on a column, 18 x 2.5 cm, with a flow rate of 1.5 ml per min.

Starch Gel Electrophoresis-Horizontal starch gel electrophore- sis was carried out in a phosphate-citrate buffer, pH 7.0 (24), for 18 hours with a voltage gradient of 6 volts per cm at 4’. The position of the protein was determined by staining with Amido black.

RESULTS

Purity of Immunizing Antigens-Sedimentation velocity ultra- centrifugation of all three commercial lysozyme preparations gave single symmetrical peaks having an average sso+, of 1.82, which is in agreement with the reported behavior of chicken lyso- zyme under the experimental conditions that were employed (21).

The three commercial preparations of chicken lysozyme were subjected to starch gel electrophoresis. All showed minor, ano- dally migrating, protein impurities in addition to the cathodally migrating chicken lysozyme. A comparison with the electro- phoretic separation of whole chicken egg white revealed that the Armour lysozyme preparation was contaminated with egg white albumins, globulins, and conalbumin. When similar amounts of the Pentex and Worthington preparations (approximately 1 mg) were applied to the gel, the globulin fraction was the only con- taminant observed.

Amberlite chromatography of 90 mg of Armour lysozyme re- vealed one major protein peak. Two minor peaks, one eluting at the effluent volume characteristic for ovalbumin and another eluting just before the major protein peak were also observed. Together, the minor peaks accounted for 4 % of the total absorb- ance at 280 rnp (Fig. 1).

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Issue of September 10, 1967 N. Amheim, Jr., and A. C. Wilson 3953

Electrophoretic and chromatographic behavior therefore indi- cate the presence of minor impurities in the commercial prepara- tions of chicken egg white lysozyme that had been utilized as im- munizing antigens.

Homologous Immunological Reaction-Six antisera were tested for the presence of antibodies directed against chicken lysozyme. Immunodiiusion revealed that every antiserum gave rise to a single precipitin line when tested against Armour crystalline lyso- zyme that had been further purified by Amberlite chromatog- raphy.

One of the antisera, A54, was tested and found to inhibit the enzymatic activity of chicken lysozyme. The antiserum did not inhibit human salivary lysozyme. It is known that antiserum directed against human lysozyme does not inhibit the enzymatic activity of chicken lysozyme (6).

Each antiserum was tested against chicken egg white by im- munoelectrophoresis. In every case, a strong precipitin arc was observed at a cathodal position, which was determined by paral- lel experiments with crystalline enzyme to be characteristic for lysozyme. Antisera A24, A31, and A54 also produced a second precipitin arc with egg white. Since an arc of identical mobility was produced upon immunoelectrophoresis of crystalline oval- bumin, these antisera must contain antibodies to ovalbumin as

well as to lysozyme. The remaining three antisera (prepared against Armour lysozyme) were shown by analogous methods to contain antibodies to both ovalbumin and conalbumin as well as to lysozyme.

Micro-complement fixation tests were conducted with antisera A24 and A54. In both cases it was demonstrated that the degree of complement fixation is independent of the purity of lysozyme. As shown in Fig. 2, the height and shape of the complement fixa- tion curves and the lysozyme concentration required for peak fixation are identical whether egg white or chromatographically purified crystalline lysozyme is used. Hence, the antibodies to ovalbumin in antisera A24 and A54 do not interfere with our com- plement fixation analysis of lysozyme. This conclusion was con- firmed by directly measuring the concentration of antibodies against ovalbumin in these antisera. Micro-complement fixation experiments conducted with chromatographically purified, crys- talline ovalbumin demonstrated that the concentration of anti- bodies against ovalbumin was more than 10 times lower than that

0 I 10 20 40 80

mpgm Lysozyme Added

FIG. 2. Reactivity of antiserum A24 with chicken egg white (0) and chromatographically purified crystalline chicken lysozyme ( l ), as determined by the micro-complement fixation technique. The reaction mixtures contained 1 ml of a 1:11,060 dilution of antiserum.

TABLE I

Species from which egg whites were obtained”

Common name

Fowl Domestic chicken Burmese red jungle fowl

Partridges Sharp’s francolin Chukar partridge

American quail Bobwhite quail California quail

Other gallinaceous birds Domestic guinea fowl Ruff ed grouse Blue-eared pheasant Lady Amherst’s pheasant Blue peafowl Golden pheasant Domestic turkey Swinhoe pheasant Japanese quail Reeves’s pheasant Ring-necked pheasant

Scientific name hpplier”

Gullus gallus A Gallus gallus B

Francolinus clappertoni Alectoris graeca

C B

Colinus virginianus Lophortyx californica

D E

Numida meleagris Bonasa umbellus Crossoptilon au&us Chrysolophus amherstiae Pavo cristatus Chrysolophus pictus Meleagris gallopavo Lophura swinhoei Coturnix coturnix Syrmaticus reevesii Phasianus colchicus

F G C F F F H F I C J

a In general, one egg was examined from each species. b A, Local supermarket; B, J. L. Duroy, Los Angeles, Cali-

fornia; C, Dr. R. Feeney, University of California, Davis, Cali- fornia; D, D. Dearer, Richmond, California; E, A. C. Wilson, Walnut Creek, California; F, Poisal’s Rare Bird Farm, Pleasanton, California; G, Dr. C. Sibley, Yale University; H, Armour Starr, Turlock, California; I, Dr. H. Abplanalp, University of California, Davis, California; J, State Game Farm, Vacaville, California.

Effluent Volume (ml)

FIG. 3. Sephadex G-75 chromatography of chicken egg white. Two milliliters of a 1:9 dilution of egg white were applied to a column, 52 X 1.5 cm. Absorbance at 280 rnp (O), lysozyme ac- tivity (A). See “Methods” for further details.

against lysozyme in both A24 and A54. The sharp dependence of complement fixation on antibody concentration (see Reference 19 and below) insured that our micro-complement fixation analy- sis of lysozyme is free from any interference by antibodies against other egg white proteins.

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3954 Species Diferences in Lysozymes Vol. 242, No. 17

Immunological Cross-reactions-In order to measure the reac- tivity of these antisera with the lysozymes of other species, lyso- zyme was first partially purified from the egg whites of the species listed in Table I. Since avian lysozymes are small molecules (mol u-t m-14,500), compared with other egg white proteins (8-10, 25), chromatography on Sephadex G-75 was employed for this purpose. Fig. 3 shows the result of an experiment in which chicken egg white was subjected to this procedure. One major protein peak was eluted beginning at the void volume of the col- umn. This peak was devoid of lysozyme activity. At an effluent volume of 60 ml, a second peak was observed which con- tained 94c/, of the initial lysozyme activity. The specific activ- ity of the lysozyme in the peak tube was 615. Chromatographi- tally purified, crystalline lysozyme has a specific activity of 660 (see “Methods” for definition of specific activity). When lyso- zyme purified from chicken egg white by Sephadex G-75 chroma- tography was tested against antisera A24 and A54 by immuno- electrophoresis, a single precipitin arc was observed at a position characteristic for lysozyme. This shows that Sephadex chroma- tography effectively separated ovalbumin from lysozyme.

1.6~160

Effluent Volume (ml)

FIG. 4. Sephadex G-75 chromatography of egg white from the Japanese quail. Two milliliters of a 1:9 dilution of egg white were applied to a column, 53 X 1.5 cm. Absorbance at 280 rnE.1 (O), lysozyme activity (A). See “Methods” for further details.

100 , I I I

10 20 40 80

mpgm LYSOZYME ADDED

FIG. 5. Reactivity of antiserum A54 with lysozymes purified by Sephadex chromatography from the egg whites of chicken (O), turkey (a), and ring-necked pheasant (A). The reaction mix- tures contained 1 ml of a 1:8800 dilution of antiserum.

80

60

’ 2o 1 phe(y Chicken 1

10/3000 1/10,000 1/30,000

ANTISERUM CONCENTRATION

FIG. 6. The dependence of complement fixation on antiserum concentration. Sephadex preparations of chicken (e) and ring- necked pheasant (0) lysozymes were used as antigens. Serial dilutions of each antigen were tested against several concentra- tions of antiserum A24. Each point represents the peak height of a complement fixation curve for a particular antiserum con- centration.

When other egg whites were subjected to Sephadex G-75 chro- matography, similar elution profiles resulted. As an example, Fig. 4 shows the results of an experiment with egg white from the Japanese quail.

The degree to which lysozyme from each species was purified by the Sephadex procedure was estimated by comparing the spe- cific activity of the peak lysozyme tube with that of chromatogra- phically purified crystalline chicken lysozyme (i.e. 660). Almost all of the preparations had specific activities greater than 500 and w-ere thus more than 75% pure according to this criterion.’

Our first cross-reaction experiments were done with turkey and ring-necked pheasant lysozymes. Fig. 5 shows the results of an experiment w-ith antiserum A54 used at a concentration of 1: 8800. At this antiserum concentration the homologous reaction with chicken lysozyme gave the greatest degree of complement fixation, measured at the peak of the curve. The reaction with turkey lysozyme was weaker, while essentially no reaction was detected with ring-necked pheasant lysozyme.

In another experiment with turkey lysozyme, the antiserum concentration was raised by a factor of 1.16. At this concentra- tion of antiserum, turkey lysozyme fixed as much complement as the chicken enzyme had at the original antiserum concentration. In a reaction with ring-necked pheasant lysozyme, the peak height could be made to equal that of chicken lysozyme if the an- tiserum concentration was raised by a factor of 1.70.

Experiments with both chicken and ring-necked pheasant lyso- zyme have shown that the height of the complement fixation

1 The lysozyme preparations that had lower specific activities may have resulted from either one of two phenomena. It is pos- sible, first of all, that some species have an unusual abundance of low molecular weight material which exhibits absorbance at 280 rnp and is eluted with lysozyme. On the other hand, the turnover number of lysozyme in some of the species may differ from that of the chicken. Although crystallized turkey lysozyme has been shown to have the same specific activity as crystallized chicken lysozyme (25), no other close relatives of the chicken have been examined with respect to the turnover number of lysozyme.

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Issue of September 10, 1967 N. Amheim, Jr., and A. 6. Wilson 3955

TABLE II Cross-reactions of lysozymes from various species with anti-chicken

1 ysozyme

Species

Fowl Domestic chicken ................. Burmese red jungle fowl. .......

Partridges Sharp’s francolin .............. Chukar. ............... .........

American quails Bobwhite quail. ............... California quail ...................

Other gallinaceous birds Domestic guinea fowl ............ Ruff ed grouse ............ ....... Blue-eared pheasant. ............ Lady Amherst’s pheasant. ........ Blue peafowl. .............. Golden pheasant ............ Domestic turkeyc. ............... Swinhoe pheasant ................ Japanese quail. .................. Reeves’s pheasant. ............... Ring-necked pheasant. ..........

-

-

--

-

Index of dissimilarity5

Antiserum A24 Antiserum A54

1.00 1.00

1.00 1.00

1.026 1.07 1.13” 1.10

1.04b 1.01 1.086 1.09

1.33 1.45 1.35 1.32 1.37 1.60 1.44 1.60 1.52 1.31 1.63 1.48 1.66 1.16 1.81 1.87 1.86 1.81 1.88 1.70 2.20 1.70

a Average for at least two experiments. b Experiments with a third antiserum confirm that these lyso-

zymes are immunologically distinct from chicken lysozyme, the index of dissimilarity for all four species being between 1.1 and 1.2.

c Experiments with three other antisera gave the following values for turkey lysozyme: 1.33, 1.37, and 1.63.

peak is linearly related to the log of the antiserum concentration (Fig. 6). The slopes of these two lines are identical, allowing us to use as a measure of the cross-reactivity of any lysozyme the factor by which the antiserum concentration must be raised in order that a peak height equal to that given by chicken lysozyme is obtained. This factor is called the index of dissimilarity (18, 19). This procedure for measuring cross-reactions has been used before in investigations of species differences in other pro- teins (18, 19).

Table II presents the results of cross-reaction experiments with lysozymes from 16 species. Two antisera were used, A24 and A54. In all cases the peak of the complement fixation curve oc- curred in the reaction tube containing 0.03 enzyme units of lyso- zyme (about 0.05 pg). With the exception of the red jungle fowl, all the species have a lysozyme that can be distinguished from that of the chicken by at least one of the antisera. These species fall into two categories. The first includes the partridges and American quails. These species have a lysozyme that is barely distinguishable from that of the chicken. Experiments with an- other antiserum confirm this conclusion. The remaining species have a lysozyme that is more easily distinguishable from that of the chicken by micro-complement fixation.

Immunodiffusion proved to be a far less discriminating techni- que. The lysozymes from 15 of the 16 species gave lines of iden- tity with chicken lysozyme in experiments with antisera A24 and A54. Ring-necked pheasant lysozyme, however, .was excep- tional in that a weak spur was formed with chicken lysozyme

when tested with antiserum A24 (Fig. 7a). With antiserum A54, ring-necked pheasant and chicken lysozyme gave lines of iden- tity (Fig. 7b).

nISCUSSION

All of the species ‘tested, with the exception of the chicken’s closest relative, the red jungle fowl, have an egg white lysozyme w-hich differs in structure from that of the chicken as judged by the micro-complement fixation technique. As judged by im- munodiffusion, however, all the species except the ring-necked pheasant have a lysozyme which is identical with that of the chicken. The relative insensitivity of immunodiffusion to small differences in protein structure has been shown before in studies on hemoglobin (17), serum albumin (19), and growth hormone (26).

Wetter, Cohn, and Ceutsch (11) reported that differences be- tween chicken lysozyme and turkey, guinea fowl, and pheasant lysozymes can he detected by quantitative precipitation with an-

FIG. 7. Photographs of immunodiffusion patterns obtained by reacting anti-chicken lysozyme sera (A) with Sephadex prepara- tions of chicken (C) and ring-necked pheasant (P) lysozyme. The antisera used were a, A24 and b, A54. See “Methods” for further details.

TABLE III Conventional zoological classijkation of species whose lysozymes

were investigated

A. Family Phasianidae Pheasants

Domestic chicken Burmese red jungle fowl Ring-necked pheasant Reeves’s pheasant Golden pheasant Lady Amherst’s pheasant Swinhoe pheasant Blue-eared pheasant Blue peafowl

Partridges Sharp’s francolin Chukar

Old World Quail Japanese quail

American Quail California quail Bobwhite quail

B. Other Families Domestic guinea fowl Ruff ed grouse Domestic turkey

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3956 Species Di$erences in Lysozymes Vol. 242, No. 17

tisera to chicken lysozyme. Other workers, however, state that chicken lysozyme and turkey lysozyme “can be regarded as hav- ing almost identical behavior in the quantitative precipitin test and in gel diffusion experiments” even though these lysozymes differ in amino acid composition (10). All of our antisera clearly distinguish turkey from chicken lysozyme in the micro- complement fixation test.

Aclcmwledgments We thank those persons listed in Table I for assistance in obtaining egg whites and the Misses J. Griebrok, L. Ferguson, and D. Midgarten for technical assistance.

REFERENCES 1.

2.

The fact that the immunological reactivity of partridge and American quail lysozymes is nearly equal to that of the chicken suggests that these enzymes differ only slightly from each other in chemical structure. Hemoglobins S and C, which diier from hemoglobin Ai by a single amino acid residue, are distinguishable from A1 by means of micro-complement fixation tests conducted with antisera prepared against hemoglobin A1 (17). The index of dissimilaril y was observed to be 1.3 in each case (18). As a smaller index of dissimilarity (1.1) was observed in the compari- son of partridge and quail lysozymes with chicken lysozyme, it is possible tha t these enzymes diier by not more than 1 amino acid residue from chicken lysozyme.

MARGOLIASH, E., AND SCHEJTER, A., Advance. Protein Chem., 21, 113 (1966).

ZUCKERKANDL, E., AND PAULING, L., in 5’. BRYSON AND H. J. VOGEL (Editors), Evolving genes and proteins, Academic Press, New York, 1965, p. 97.

3. BUETT~ER-JANUS&, J., AND HILL, R. L., in ?‘. BRYSON AND H. J. VOGEL (Editors). Evolvina genes and proteins, Aca- demic Press, kew York: 1965, p.“16j. .

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SMITH, L. F., Amer. J. Med., 40; 662 (1966). GLYNN. A. A.. AND PARPMAN. R.. Immunoloau. 7, 724 (1964). OSSER~AN, E: F., AND LAWL~R, 6. P., J. Eii: Med., i24, 921

(1966). INOUYE, M., AND TSUGITA, A., J. Mol. Biol., 22, 193 (1966). FUJIO, H., SAIKI, Y., IMANISHI, M., SHINKA, S., AND AMANO,

T., iike& J., 5,‘201’/1962). CANFIELD. R. E.. AND MCMURRY. S.. Biochem. BioPhus. Res.

Commuh., 26, $8 (1967). ’ ’ . .

It is of interest that over 100 additional species of partridges and American quails exist (27). Such a large number of lyso- zymes, possibly differing slightly from each other in amino acid sequence, would be of value in relating protein structure to cata- lytic and immunological activity.

IMANISHI, %I., &&CA, S., MIYAGAWA, N., AMANO, T., AND TSUGITA. A.. Biken J.. 9. 107 (1966).

WETTER, L’. R.‘, COHN, M’. END D&JTs&, H. F., J. Immunol., 70, 507 (1953).

Our cross-reaction data may also have taxonomic implications. All the species studied here are considered by zoologists to be closely related to the chicken. Many of them have been re- ported to be capable of hybridizing with the chicken (28). Ac- cording to classical zoological criteria, they belong to four related families in the order Galliformes2 (27, 29-35). Further details regarding the zoological classification of these species are given in Table III. It will be observed, for example, that the chicken and jungle fowl are grouped among the pheasants. Our data on lyso- zyme suggest instead a close relationship among the chicken, jungle fowl, partridges, and American quails.3

JOLLIES, P., CHARLEMAGNE, D., PETIT, J. F., MAIRE, A. C., AND JOLLBS, J., Bull. Sot. Chim. Biol., 47, 2241 (1965).

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PHILLIPS, D. C., AND SARMA, V. R., Nature, 206, 757 (1965). WASSERMAN, E., AND LEVINE, L., J. Zmmunol., 87, 290 (1961). REICHLIN, M., HAY, M., AND LEVINE, L., Immunochemistry, 1,

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LIN, M., AND ALLISON, W. S., Fed. Proc., 23, 1258 (1964). SARI~H, V. M., AND WI&ON, A. C., Science, 164, 1563 (1966). PARRY. R. M.. JR.. CHANDAN. R. C.. AND SHAHANI, K. M.,

Proc: Sot. E&p. iiol. Med., i19, 384’(1965).

Clearly, one of these alternatives is incorrect. Either the chicken is more closely related to pheasants, as suggested by conventional zoological criteria, or it is more closely related to partridges, as our lysozyme experiments indicate. Unpublished experiments which we have performed with the lactic dehydro- genases of these species agree m,ith the relationships suggested by the lysozyme data. Further studies on ovalbumin and serum albumin are in progress. Information concerning several pro- teins m;ill aid in the evaluation of the taxonomic significance of data on protein structure and may determine whether the conven- tional taxonomic scheme is in need of revision.

SOPHIANOPOUL~S, A. J., RHODES, C. K.; HOLCOMB, D. N., AND VAN HOLDE. K. E.. J. Biol. Chem.. 237. 1107 (1962).

SCHEIDEGGER,‘J. J., int. Arch. Allergy Appl. Ir/lmu&l., 7, 103 (1955).

TALLAN, H. H., AND STEIN, W. H., J. Biol. Chem., 200, 507 (1953).

FINE, I. H., AND COSTELLO, L., in S. P. COLOWICK AND N. 0. KAPLAN (Editors), Methods in enzymology, Vol. 6, Academic Press, New York, 1963, p. 958.

Micro-complement fixation may receive increasing use by biochemists interested in protein structure. The technique is exceedingly economical of materials. Cross-reactivity experi- ments are conveniently carried out with millimicrogram quanti- ties of protein antigens and microliter quantities of antiserum. Furthermore, extremely small differences in protein structure can be detected by this procedure. Micro-complement fixation could be especially useful in studies of chemically modified pro- teins as well as proteins from different species.

25.

26.

27.

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Norman Arnheim, Jr. and Allan C. WilsonQuantitative Immunological Comparison of Bird Lysozymes

1967, 242:3951-3956.J. Biol. Chem. 

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