Multiple Lysozymes of Duck Egg White

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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.
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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