Multiple Lysozymes of Duck Egg White

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Multiple Lysozymes of Duck Egg White Received for publication August 31 1970 ELLEN tl PRAGER AND ALLAN C WILSON From the Department of Biochemistry University of California Berkeley California 9 X20 SUMMARY Lysozyme was purified from a pooled sample of Peking

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524 Duck Lysozymes Vol 246 No 2, complement fixation and described previously 4 Antisera to as a base line for the other amino acids in line with glycine values. each of the two duck lysozymes found in greatest amount A given for duck lysozymes previously described 1 3 Values. and B were produced as follows The lysozyme 2 mg was close to integers were then obtained for the other amino acids. suspended in Freund s complete adjuvant supplemented by an Tryptophan and half cystine as well as the amides asparagine. additional 3 mg per ml of lyophilized phenol killed BCG sup and glutamine were not analyzed for Furthermore proline. plied by the Department of Bacteriology and Immunology and was also not determined because it was impossible to obtain a. injected intradermally into three spots on the back of each male reliable value on a lysozyme sample in which half cystine was. New Zealand white rabbit Four rabbits Nos 432 to 435 were neither carboxymethylated nor otherwise modified. injected with lysozyme A and four rabbits Nos 436 to 439. Starch Gel Electrophoresis, with lysozyme B Sera first bleeding were collected 24 days. later Thirty eight days after the primary immunization Horizontal starch gel electrophoresis at pH 5 3 7 0 and 11 9. intravenous boosts of 1 mg of lysozyme in isotris buffer were was performed at 4 as has been described 5 However. given every other day for a total of three injections One week starch was used at a concentration of 11 The three duck. after the last injection antisera second bleeding were collected lysozymes were most clearly distinguishable at pH 5 3 In. Antisera were stored at 10 those used for micro complement order to separate them cleanly a voltage gradient of 10 volts. fixation were heat treated at 60 for 20 min to inactivate the per cm for 18 hours was used This high voltage and the con. rabbit complement present and then stored at 10 comitant high current necessitated cooling the gel by directing a. stream of cold air from an Oster hair dryer over the gel during the. Lysozyme Assays run Under these conditions the current was 50 ma or less per. Lysis of i crococcus luteus formerly named Micrococcus gel. Downloaded from http www jbc org by guest on November 28 2019. lysodeikticus 11 was measured by recording the change in Following electrophoresis protein was located by staining with. percentage of transmittance at 540 rnp in a Zeiss spectrophotom 0 647 Amido black dissolved in a solution containing methanol. eter at 23 water acetic acid in the ratio 45 45 10 Excess Amido black. was removed by thorough rinsing of the gel in the same solvent. Densitometry, The absorbance at 280 rnp was used to measure protein. Lyophilized protein was weighed Photographs of the Amido black stained starch gels were made. with Polaroid type 46 L film The lysozyme bands of those eggs. Immunological Methods containing two lysozymes were then scanned on a Joyce Loebl. double beam recording microdensitometer using a narrow slit. Immunoelectrophoresis Ouchterlony double diffusion and. The amount of protein represented by each peak was determined. micro complement fixation were performed as described previ. by cutting out the peak from the densitometric tracing and weigh. ously 4 The degree of antigenic difference between pairs of. ing the paper, lysozymes is expressed as the index of dissimilarity This index. is defined as the factor by which the antiserum concentration Purification of Lysozyme. must be raised in order for a heterologous lysozyme to produce The purification procedure utilized was based upon published. a complement fixation curve the peak of which is equal in height methods 1 5 20. to that produced by the homologous lysozyme 4 Pooled Preparation Pooled Peking duck egg white 1 liter. was diluted 1 5 in ammonium acetate buffer pH 9 0 0 1 M. Ultracentrifugation, acetate homogenized for 1 to 2 set in a Waring Blendor and.
Sedimentation velocity studies were carried out with a Spinco filtered through tissue paper KimWipes About 42 g of car. model E analytical ultracentrifuge Lysozyme solutions of 0 6 boxymethyl cellulose equilibrated in the same buffer were added. to 0 7 To protein were centrifuged at 67 770 rpm in a buffer 12 of and the slurry was stirred at 4 overnight The rest of the. 0 02 M sodium acetate pH 5 3 and 0 15 M KC1 at 23 purification was carried out at room temperature The resin. with the lysozyme bound to it was allowed to settle and the. Amino Acid Composition, supernatant containing the vast majority of the egg white. Lysozyme samples were hydrolyzed under vacuum in 6 N HCl proteins was decanted The carboxymethyl cellulose was then. at 105 110 for 24 42 or 72 hours The hydrolysates were poured onto a Buchner funnel lined with eight layers of KimWipes. evaporated to dryness and subsequently dissolved in 0 2 M citrate and thoroughly washed with the ammonium acetate buffer. buffer at pH 2 2 Amino acid analyses were then carried out The lysozyme was eluted with 0 4 M ammonium carbonate pH. with either a Beckman 121 or a Beckman 120B autoanalyzer as 9 2 and lyophilized It was then subjected to gel chromatog. previously described 5 13 Values for serine and threonine raphy on a Sephadex G 50 column 4 5 X 53 cm equilibrated. were extrapolated to zero time of hydrolysis 14 whereas those with the ammonium acetate buffer As Fig 1 shows one large. for valine and isoleucine were taken from the 72 hour hydrolysate protein peak emerged as well as two small peaks The material. 3 14 To calculate the number of residues of each amino acid in the large peak which included all of the lysozyme activity. it was assumed that each lysozyme has 129 amino acids as has was lyophilized The residue dissolved in 0 05 M ammonium. been the case for all those bird lysozymes which have been se carbonate was then put onto a carboxymethyl Sephadex column. quenced i e chicken 15 16 Japanese quail 17 Duck II 18 2 4 x 14 5 cm equilibrated with the same buffer A linear. and turkey 19 This assumption has also been made for other gradient produced by utilizing 255 ml each of 0 05 M and 0 4 M. bird lysozymes such as those of bobwhite quail and ring necked ammonium carbonate eluted two lysozymes designated A and B. pheasant 5 In addition glycine was allowed to equal 12 00 Fig 2 Then 0 8 M ammonium carbonate was passed over the. Issue of January 25 1971 E M Prayer and A C Wilson 525. 15 LYSOZYME,O 100 200 300 400 500 600 700 800 9oc,EFFLUENT VOLUME ml C. 1 Gel filtration of partially,FIG purified duck lysozyme on a P. column of Sephadex G 50 L, Downloaded from http www jbc org by guest on November 28 2019. column eluting the lysoeyme designated C Fig 2 A further. increase in buffer concentration to 1 0 M ammonium carbonate I I I I. failed to elute any more lysozyme After lyophilization each 100 0 100 200 300 400 500 600 700 800. individual lysozyme was then further purified on a BioRex 70 EFFLUENT VOLUME ml 1. column 57 x 1 5 cm equilibrated with 0 2 M sodium phosphate FIG 2 Carboxymethyl Sephadex chromatography of duck. at pH 7 60 The column volume was 100 ml After several lysozyme The jht and second unlabeled arrow s indicate respec. column volumes of buffer had been used a switch was made to tively the points at which the sample was put onto the column. 0 8 M sodium phosphate at pH 7 55 The peak of lysozyme A and at which the gradient was started. emerged after 1 82 column volumes of 0 2 M buffer had passed. over it the peak of lysozyme B emerged after 4 61 column amino acid composition studies the partially purified lysozyme. volumes of 0 2 M buffer had passed over it Although 5 column from two egg whites shown by starch gel to have only the C type. volumes of 0 2 M buffer failed to elute lysozyme C 0 91 column lysozyme was further purified It was passed over a Sephadex. volume of the 0 8 M phosphate succeeded Each lysozyme was G 50 column 1 5 x 60 cm equilibrated with the ammonium. then placed in cellulose casing dialyzed extensively against dis acetate buffer described above and the material in the large. tilled water at 4 and lyophilized The yield of lysozyme was peak was then pooled and lyophilized Duck C prepared in this. approximately 70 mg each of A and B and 3 mg of C The low manner satisfied all but two of the purity criteria described below. yield could be anticipated since duck egg white has a low lyso it was not tested on the Amberlite column nor was it injected. zyme content in contrast to that of gallinaceous birds 21 into rabbits. Individual Egg Whites Thirty milliliters of each individual RESULTS. egg white were diluted 1 5 in the ammonium acetate buffer. homogenized filtered and stirred overnight at 4 with 2 g of Criteria of Purity of Lysozymes Each of the duck lysozymes. carboxymethyl cellulose The supernatant was decanted the tested emerged as a single symmetrical protein peak from a. resin washed and the lysozyme eluted and lyophilized as above column of Amberlite BioRex 70 The specific activity was. The residue was then dissolved in 1 ml of distilled water cen constant across the peak In addition each lysozyme moved. trifuged at 26 000 x g for 15 min and stored frozen The as a single protein zone upon starch gel electrophoresis at pH. lysozyme was at this point sufficiently pure 60 to 70 and 5 3 7 0 and 11 9 The previously described difficulties of. sufficiently concentrated 10 mg per ml to be run on starch gels trailing and concentration dependence in the pH 7 0 system 5. and detected easily by the Amido black stain On each gel a were also encountered here A single symmetrical protein peak. pooled sample purified to this same point was run as a marker was exhibited by each lysozyme in sedimentation velocity. enabling one to classify the lysosyme phenotype of each in studies Immunoelectrophoresis with an antiserum against. dividual egg white The major nonlysozyme contaminant had a whole bobwhite quail egg white produced only one precipitin. mobility at pH 5 3 which was markedly different from the ly arc with each duck lysozyme 2. sozymes as shown below The low molecular weight contami An additional immunological criterion of purity could be ap. nating material see Fig 1 Sephadex G 50 column did travel in plied to lysozymes A and B Rabbits immunized with A or B. the lysozyme region at this pH However it was soon washed produced antisera which when tested by immunoelectrophoresis. out when the Amido black stained gel was rinsed with the metha against duck egg white revealed only one precipitin arc. nol water acetic acid solvent and thus it could not interfere Speci c Activity In their capacity to lyse M luteus under the. with the determinations of the type s and amount of lysozyme conditions used the three duck lysozymes did not differ signifi. present in each egg cantly from one another or from the chicken enzyme. Duck C Lysozyme from Two Individual EggsIn order to ob 2 Lysozyme C however partially precipitated in the agar before. tain additional Duck C lysozyme for ultracentrifugation and the antiserum was added. 526 Duck Lysoxymes Vol 246 No 2,Amino acid composition of duck lysozymes.
Duck A Duck B Duck C, Amino acid Time of hydrolysis Time of hydrolysis Time of hydrolysis. 2 hrs Integralvalue 72 hrs Integralvalue,24 hrs 42 hrs 42 hrs 72 hrs 24 hrs 42 hrs. Lysine 6 57 6 08 5 96 6 6 00 6 10 6 6 68 6 50 5 60 6. Histidine 0 0 0 0 0 0 0 0 0 0 0, Arginine 13 38 12 67 12 70 13 13 60 13 74 14 15 9 15 0 14 1 15. Aspartic acid 18 75 19 00 18 92 19 18 60 18 59 19 18 95 18 50 18 70 19. Threonine 6 76 6 68 6 15 7 6 45 5 66 7a b 6 82 6 55 6 54 7a. Serine 9 65 9 46 8 26 11 8 41 6 37 115 6 8 21 8 37 7 16 9a. Glutamic acid 5 03 5 01 5 02 5 5 08 5 04 5 5 08 5 16 4 97 5. Glycine 12 00 12 00 12 00 12 12 00 12 00 12 12 00 12 00 12 00 12. Alanine 11 00 10 81 11 08 11 11 00 10 98 11 11 28 10 62 11 5 11. Valine 6 81 6 70 6 93 7c 6 72 7 05 7c 6 55 6 59 7 00 7c. Methionine 2 20 0 94 1 70 2 1 62 1 77 2 2 34 1 50 1 66 2. Isoleucine 5 47 5 65 5 60 6 5 63 5 54 6 5 26 5 32 5 65 6. Downloaded from http www jbc org by guest on November 28 2019. Leucine 7 90 8 04 8 01 8 8 16 7 98 8 7 95 7 73 7 86 8. Tyrosine 5 05 3 91 4 61 5 4 60 4 65 5 5 05 4 97 5 00 5. Phenylalanine 1 06 1 12 1 05 1 1 06 1 00 1 1 16 1 06 1 14 1. Q Extrapolated to zero time of hydrolysis, b The unusually steep decline in values between 42 and 72 hours of hydrolysis leads us to question the extrapolated values Threo. nine would still be 7 even with a much shallower slope whereas serine would give an integral value of 10. c Determined from the 72 hour hydrolysis, Amino acid composition of histidine free duck lysozymes and of chicken lysozyme.
Duck lysozymes, Amino acid Prager and Wilson this work JolEs et al Imanishi el aZ a Chicken lysozymeb. Duck A Duck B Duck C Duck IIc Duck IIId DL le DL Ze. Lysine 6 6 6 6 6 5 5 6,Histidine 0 0 0 0 0 0 0 1,Arginine 13 14 15 13 13 14r 14 14 11. Aspartic acid 19 19 19 19 18 19 19 19 21,Threonine 7 7 7 7 7 7 7 7. Serine 11 10 11 9 11 lo 11 11 10 10,Glutamic acid 5 5 5 5 5 5 5 5. Proline N D Q N D N D 2 2 2 2 2,Glycine 12 12 12 12 12 12 12 12.
Alanine 11 11 11 11 11 11 11 12,Half cystine N D N D N D 8 8 8 8 8. Valine 7 7 7 7 7 7 7 6,Methionine 2 2 2 2 2 2 2 2,Isoleucine 6 6 6 6 6 6 6 6. Leucine 8 8 8 8 8 8 8 8,Tyrosine 5 5 5 5 5 5 5 3,Phenylalanine 1 1 1 1 1 1 1 3. Tryptophan N D N D N D 6 6 6 6 6,a Reference 3,b References 15 and 16. c From the sequence 18,d Reference 22,e DL duck lysozyme.
f Duck III is specifically said 22 to have more arginine than Duck II at a time when the arginine value for Duck II was given. as 12 to 13,Q N D not determined, Issue of January 25 1971 E M Prager and A C Wilson. Cross reactions of cluck chicken and human lysozymes with antisera. to duck and chicken lysozymes,Index of dissimilarity. Antiduck Aa Antiduck B Antichick,Duck A 1 00 1 30 6 67. Duck B 1 08 1 00 7 00,Duck C 1 50 1 51 6 83,Chicken 7 gd 7 0 1 00. Human 1OOd 100 100,Q Antiserum 432 second bleeding.
b Antiserum 436 second bleeding, c Antisera A24 A54 A64 and A74 pooled in reciprocal propor. tions to their micro complement fixation titers 24 26. dTested with antiserum434 secondbleeding sincethe anti. complementarity of 432 second bleeding did not allow it to be. FIG 3 Photographs of Ouchterlony double diffusion patterns used at the concentrations needed to test for cross reactivitywith. obtained by reacting antichicken and antiduck lysozyme sera with chicken and human lysozyme. Downloaded from http www jbc org by guest on November 28 2019. chicken and duck lysozymes 1 antichicken A24 2 antiduck A 6 Antisera A24 A54 and A74 tested individually. No 432 second bleeding S antiduck B No 436 second bleeding. Ch chicken lysozyme and A R and C Duck A Duck B and not distinguishone duck enzyme from the other by this tech. Duck C lysosyme respectively nique Fig 3b Fuji0 et al 23 showed that the one duck. lysozyme which they studied with the use of an antiserum to. Molecular Size When the purification of lysozyme from pooled chicken lysozyme gave a reaction of partial identity with the. egg white was carried out a singleactive peak wasobtained on chicken enzyme. SephadexG 50 at the samepoint at which Pentex chickenlyso In micro complementfixation tests our four antichicken sera. zyme emergedwhen passedover that column Sedimentation did showdifferencesamongthe duck enzymesrelative to chicken. velocity studiesgave sZO w values of 1 93 for Duck A 1 89 for lysozyme A pool of the four seragave indicesof dissimilarity. Duck B and 1 91for Duck C in agreementwith a value of 1 92 4 24 of 6 67for Duck A 7 00for Duck B and 6 83for Duck C. obtained for the chicken enzyme under the same conditions Indices of this magnitude indicate structural relatednessyet. Thus duck lysozymesA B and C probably do not differ from considerablestructural differencesbetween chicken lysozyme. eachother or from chickenlysozyme in molecularsize and the duck enzymes in agreement with the Ouchterlony. Amino Acid Composition Table I shows the amino acid double diffusion results Similarly Maron et al 25 have. compositionof the three duck lysozymes The three enzymes observed in experimentswith antichicken lysozyme that Duck. appearto be identical in compositionexcept that Duck B hasone II and Duck III are related to chicken lysozyme although not. more arginine than Duck A and that Duck C has onemore argi very closely and that thesetwo duck lysozymesare distinguish. nine than Duck B and two fewer serinesthan Ducks A and B able from eachother by meansof an immunologicalcompetition. although the serinevalue for Duck B is open to questionand is technique. possiblyequal to 10 rather than 11 Table II summarizesthe Antisera produced in response to Duck A and Duck B gave. amino acid composition integral valuesof residuesper molecule lines of identity amongthe three duck lysozymesin the Ouch. of our three duck lysozymes of the histidine freeduck lysoaymes terlony test Fig 3 c and d indicating close structural simi. investigated by other workers and of chicken lysozyme Our larities amongthem Micro complement fixation with antisera. Duck A agreesin compositionwith Duck II and our Duck B elicited by duck lysozymesA and B gave small indices of dis. seemsto fit the compositionalvalues given for Duck III Our similarity among the duck enzymes in comparison to the indices. values and alsothosefor Duck II and Duck III differ from those given by antichicken sera tested against duck lysozyme and. given for DL 1 and DL 2 3 in the caseof lysine and of arginine antiduck seratested against chicken lysozyme Nevertheless. in the latter instance Imanishi et al 3 found no variation in real differencesare readily detected amongthe three duck lyso. arginine value betweentheir enzymes in marked contrast to our zymes as shown in Table III which summarizesthe micro. findings and to the findingsfor Duck II comparedwith Duck III complementfixation results and in Fig 4 The leukemichuman. 22 The chicken enzyme differs considerablyfrom the duck lysozyme that we tested which differs greatly in amino acid. enzymesin its content of hi dine arginine asparticacid serine sequence from bird lysozymes 27 gave no detectable com. alanine valine tyrosine and phenylalanine its sequencediffers plement fixation with either antichicken or antiduck lysozyme. from that of Duck II in 22 positions 18 sera Others have alsoobservedthat antichicken lysozyme sera. Immunological Cross Reactivity Chicken lysozyme produced react weakly 25 or not at all awith human lysozymes. spurs over all three duck lysozymeswhen tested by the Ouchter Eleetrophoretic Mobility At pH 11 9 Duck C moved most. lony double diffusion technique with antisera directed toward rapidly toward the cathodeand Duck A most slowly with Duck. chicken lysozyme Fig 3a for example indicating partial B having an intermediate mobility Chicken lysozyme moved. identity but significant structural differencesbetweenthe duck 3 N Arnheim J Sobel and R E Canfield personal com. lysozymes and that of chicken However theseantisera could munication. 528 Duck Lysoxyrnes Vol 246 No 2, Individual variation in lysozymes of duck egg white. Type of lysozyme No of eg,A and B 19,0 One egg per individual duck. Theoretical and observed lysozyme phenotype frequenctes irs. individual duck egg whites, For calculation of the theoretical values we assumeda ran. domly mating population three alleles at one locus and no selec. Downloaded from http www jbc org by guest on November 28 2019. tive pressure In this situation the Hardy Weinberglaw predicts. an a B y CX 72 2 2017 2 3r distribution, with a being the allele frequency of a p of b and y of c in the.
population 012 for example represents the frequency of the aa. genotype A phenotype whereas 2 for instance represents the. IO 20 40 80 frequency of the cib genotype AB phenotype. mpg Lysozyme Added,I No of eggs, FIG 4 Micro complement fixation with antiserum prepared Phenotype Presumed. against Duck B lysozyme The reaction mixtures contained 1 ml Theoretica 1 Observed Theoretica Observed. of l lO OOO dilution of antiserum 436 second bleeding. A aa 5 82 5 0 132 0 114,B bb 11 0 10 0 250 0 227,C cc 0 815 2 0 019 0 046. AB ab 16 0 19 0 364 0 432,AC ac 4 35 3 0 099 0 068. BC bc 5 98 5 0 136 0 114, as well as at pH 11 9 in the 3 molecules Duck B by virtue of. its 14 arginines can be expectedto have 1 unit of chargein excess. of Duck A which has 13 arginines and Duck C by virtue of. its 15 arginines can be expectedto have 1 unit of chargeabove. 0 0 that of Duck B and 2 units above that of Duck A This situa. tion would prevail at both pH 11 9and pH 5 3, FIG 5 Starch gel electrophoresis at pH 5 3 of the six possible.
duck egg white lysozyme phenotypes and of an artificial mixture Survey of Lysozymes Present in Individual Duck Eggs The. of the three duck lysozymes centermost slot The prominent partially purified lysozymesfrom 44 individual duck eggswere. less basic band is the major nonlysozyme contaminant subjectedto starch gel electrophoresisat pH 5 3 An individua1. egg white containedeither a singletype of lysozyme or a com. with the samemobility asDuck B At pH 5 3 the samepattern bination of any two of the three No singleegg contained all. held with Duck C mo ving mostrapidly and Duck A most slowly three lysozymes Fig 5 showsthe six possiblephenotypesfound. toward the cathode the caiculation of relative mobility of the as well as an artificially made mixture of the three lysozymes. three enzymesat this pH was100 for Duck A 108for Duck B Table IV showsthe frequency of each phenotype. and 116 for Duck C In this system chicken had a mobility Densitometry performedon photographsof the Amido black. intermediate between that of Duck A and Duck B This be stained starch gels showedthat when two lysozymes occurred. havior on starch is consistentwith the behavior of the three duck together in one egg they were present in very nearly equal. enzymeson the carboxymethyl Sephadexand BioRex 70 columns amounts When A and B were present we found 55oj A and. with Duck C being most basic and binding most strongly and 45 B whenA and C were present we found 58 A and 42yo. Duck A being least basicand binding most weakly C when B and C were present we found 58 B and 42 C. The electrophoretic mobility results are also consistentwith The slight deviation from equality is not surprisingbecauseany. the amino acid compositionresults Assumingthat the number streakingor trailing on our starch gelswould raisethe measured. of asparagineand glutamineresiduesis equal and that an equal amount of the more anodal slowermoving component. number of carboxyl groupsare exposed and ionized at pH 5 3 In view of the marked similarity amongthe three duck lyso. Issue of January 25 1971 E M Prager and A C Wilson 529. zymes the absence of an egg containing all three enzymes and posttranslational modifications as being responsible for the. the nearly equal amounts of each lysozyme found in those egg multiple lysozymes present in duck egg white. whites containing two of them we find it reasonable to suggest In the absence of amino acid sequence studies on our three. a model of one locus with three alleles with both alleles being ex duck lysozymes we cannot say how many amino acid substitu. pressed should two occur in one individual On that basis the tions occur among the three lysozymes The immunological. allele frequency would be as follows a 0 364 5 0 500 and c evidence indicates that they are few in number cf Reference 5. 0 136 Here a represents the allele coding for lysozyme A b for and Table III However the sequencing work 30 on Duck II. B and c for C This computation was made with 88 genes versus Duck III which may be the same as our Duck A and Duck. from 44 diploid individuals and assuming that the A phenotype B respectively on the basis of amino acid composition relative. for example is indicative of the au genotype We can then ask basicities and amounts found in a sample of pooled egg white. given the allele frequencies computed how many eggs of each indicates that more than one amino acid difference most likely. phenotype would be expected if the duck population was in exists between any two of the three duck lysozymes Although. Hardy Weinberg equilibrium at this locus Table V compares a number of reported allelic differences such as those for human. the theoretical and observed results in terms of numbers of eggs hemoglobin 31 appear to result in single amino acid differences. and frequencies cases of multiple amino acid differences have been reported for. Agreement between the theoretical and observed values is the hemoglobin fl chains of sheep 32 33 cattle 34 35 and. excellent Applying the x2 test we find we can accept the goats 36 for rabbit hemoglobin OLchains 37 and for bovine. hypothesis of three alleles at one locus at the significance level carboxypeptidase A 38 39 The 3 chains of sheep hemo. of 0 50 P 0 75 n 5 globins A and B for example differ by at least 7 residues 33. Thus in view of our results in surveying individual Peking. Downloaded from http www jbc org by guest on November 28 2019. DISCUSSION duck egg whites we feel it is reasonable to accept a multiple. allele one locus model despite the possibility of a number of amino. The principal alternatives available for explaining the presence. acid differences among the allele products Given this model it. of multiple lysozymes in Peking duck egg white are the existence. is possible that the histidine containing lysozyme Duck I re. of multiple lysozyme loci or the existence of multiple alleles at. ported by Jolles et al 1 2 is the product of an allele found in. one lysozyme locus Both these alternatives have been reported. some populations of ducks but not in the Peking ducks studied by. for other species of birds 28 29 In the case of the black swan. us Immunological techniques namely immunodiffusion and. Cygnus stratus two nonallelic genes for egg white lysozyme. micro complement fixation would enable one to determine. appear to exist since this species produces two lysozymes which. whether Duck I is closely related to Duck II and Duck III or. are antigenically entirely unrelated one rather like the chicken. whether it is indeed much more like chicken lysozyme as its. enzyme and the other rather like the goose enzyme 23 In. amino acid composition would imply Comparative electropho. contrast Baker and Manwell 29 have implicated multiple two. resis and micro complement fixation should enable one to deter. alleles at one lysozyme locus in a population of migratory quail. mine which of our three histidine free lysozymes do or do not. Coturniz species,match those reported by other workers 1 3. On the basis of a survey of individual duck eggs in which we. Another possibility is that one or the other allele may have. found no more than two lysozymes in any one egg representa. entered the domestic duck population through rather recent. tion of each of the six possible phenotypes any one lysozyme. hybridization of different breeds To shed some light on this. alone or any two together a 1 l ratio of the two lysozymes in. aspect of the problem it would be desirable to study the lyso. every heterozygote and a frequency distribution in accord with. zyme s present in the wild species the common northern mal. the Hardy Weinberg law for three randomly assorting alleles at. lard 6 9 An investigation of the lysozymes found in domestic. one locus we propose that the lysozyme polymorphism in the. and wild A platyrhynchos populations in different areas of the. duck is due to multiple alleles at one locus rather than to the. world would perhaps reveal duck lysozymes we have not found. presence of multiple nonallelic genes for lysozyme If such. in our Peking duck population and also geographic variations in. multiple loci existed in the duck one would expect some eggs if. allele frequencies Genetic tests would also be valuable in ob. not all to have all three lysozymes and one would not expect. taining further supporting evidence for the multiple allele one. agreement with the Hardy Weinberg law,locus model that we are proposing. A further possibility to account for the presence of electro. phoretically and immunologically different lysozymes in duck. egg white is posttranslational protein modification Examples Acknowledgments We thank D Wachter and J Caufield for. carrying out the amino acid analyses R Wayner for performing. of such modifications could include adding a phosphate or a. the sedimentation velocity experiments and D Goodman for. charged carbohydrate moiety to the protein neither of which. providing instructions on densitometry One of us E M P. has been reported for any lysozyme to date or perhaps of con. is especially grateful to G T Cocks for instruction in several. verting asparagine or glutamine to their respective acids an. techniques We also thank N Arnheim G T Cocks D Good. amino acid substitution would not be possible at the posttrans. man T Y Lin F C McCollum and V M Sarich for helpful. lational stage Our amino acid composition studies which show. discussions, Duck B to have one more arginine than Duck A Duck C to have. two more arginines than Duck A and thus one more than Duck REFERENCES. 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