THE PREPARATION AND AMINO ACID SEQUENCE OF CRYSTALLINE TURKEY EGG WHITE LYSOZY ME Thesis for the Degree of Ph. D. MICHIGAN; STATE UNIVERSITY JOHN NELSON LaRUE’ 1969 ‘J'HESIS 0-169 This is to certify that the thesis entitled THE PREPARATION AND AMINO ACID SEQUENCE OF CRYSTALLINE TURKEY EGG WHITE LYSOZYME presented by John Nelson LaRue has been accepted towards fulfillment of the requirements for Ph_-D . degree inJipshgmistry ZQEMCJIM Major professon/ Date % 4 l. Umvcrwy‘ , X W IIIIIIIII' I“ ABSTRACT THE PREPARATION AND AMINO ACID SEQUENCE OF CRYSTALLINE TURKEY EGG WHITE LYSOZYME BY John Nelson LaRue The primary and three-dimensional structures of chicken egg white lysozyme have been determined (1,2,5). X—ray crystallographic studies coupled with three—dimensional model building led to predictions which specified the amino acids involved in substrate binding and in the catalytic mechanism (5). It was desirable to examine the primary structure of a closely related avian lysozyme to determine whether or not any differences existed which might have con- formational or mechanistic significance in relation to the findings and predictions made for chicken egg white lysozyme. Crystalline and apparently homogeneous turkey egg white lysozyme was prepared by a simple procedure. The reduced and g-carboxymethylated (RCM) derivative of crystalline turkey egg white lysozyme was prepared. Amino acid analysis of this derivative showed there were a minimum of 6 amino acid differences between the chicken and turkey egg white lysozymes. 5w... . John Nelson LaRue RCM—turkey lysozyme was digested by trypsin. Amino acid analysis of the purified tryptic peptides revealed there were actually 7 amino acid differences between the two lyso— zymes distributed over 6 tryptic RCM-peptides. A peptide analogue for each RCM-chicken egg white lysozyme tryptic peptide was isolated from'the trypsin digest of RCM—turkey egg white lysozyme. This information coupled with sequence analysis of certain peptides disclosed the following differ- ences in primary structure between the turkey and chicken lysozymes respectively: Tyrs for Phes; Leuls for Hisls; Hi341 for Gln41; Lys73 for Arg73; Ala99 for Va199; Glylol for ASP1017 H1812; for Glnlgl. The presence of Glylol in turkey egg white lysozyme eliminates the possibility that this residue is involved in hydrogen bonding to the substrate molecule as was predicted for Asp101 in chicken egg white lysozyme. REFERENCES p. Canfield, R. E., J. Biol. Chem., 258, 2698 (1963). N Canfield, R. E., and Liu, A. K., J. Biol. Chem., 240, 1997 (1965). 5. Blake, C. C. F., Mair, G. A., North, A. C. T., Phillips, D. C., and Sarma, V. R., Proc. Roy. Soc., B 167, 365 (1967). THE PREPARATION AND AMINO ACID SEQUENCE OF CRYSTALLINE TURKEY EGG WHITE LYSOZYME BY John Nelson LaRue A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1969 ACKNOWLEDGMENTS The author wishes to extend his appreciation to Dr. J. C. Speck, Jr., for his assistance and guidance throughout the course of this research. The suppOrt of a United States National Institutes of Health predoctoral fellowship is gratefully acknowledged. ii TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . . EXPERIMENTAL . . . . . . . . . . . . . . . . . . . . Reagents. . . . . . . . . . . . . . . . . . . . Preparation of Turkey Egg White Lysozyme. . Preparation of the crude enzyme. . . . . . Crystallization of turkey egg white lysozyme. . . . . . . . . . . . . . End point assay of lysozyme activity . . . Initial rate assay of lysozyme activity. . Protein determination by the Lowry method. CM-cellulose column chromatography of crystalline turkey egg white lysozyme Disc electrophoresis on polyacrylamide gel Preparation of Reduced and §7Carboxymethylated Turkey Egg White Lysozyme. . . . . . . . . Preparation of Trypsin Free from Chymotrypsin Activity . . . . . . . . . . . . . . . . . Trypsin Digestion of RCM-Turkey Egg White Lysozyme . . . . . . . . . . . . . . . . . Amino Acid Analysis of Proteins and Peptides. . Preparation of samples for amino acid analysis. . . . . . . . . . . . . . . Amino acid analysis. . . . . . . . . . . . TryptoPhan determinations on intact protein . . . . . . . . . . . . . . Peptide Column Chromatography . . . . . . . . . Preparation of resins. . . . . . . . . . . Packing of ion exchange resin columns. . . Description of buffers used in the elution of peptides from ion exchange columns Description of systems used in column chromatography on ion exchange resins Detection of peptides eluted from column chromatography on ion exchange resins iii Page 10 10 14 14 15 15 16 16 16 19 19 21 22 25 25 24 24 24 24 27 28 29 29 TABLE OF CONTENTS - Continued Reaction of hydrolyzed peptides with ninhydrin . . ._. . . . . . . .H. . . Treatment of peptide peak fractions eluted from ion exchange chromatography. . Characterization of Individual Peptide Peak Fractions Eluted from Ion Exchange Column .Chromatography . . . . . . . . . . . . . . Purification of Peptides. . . . . . . . . . . . Peptide Sequencing Techniques . . . . . . . . . Subtractive Edman degradation. . . . . . . Carboxypeptidase digestion . . . . . . . . Cyanogen bromide cleavage of T-15. . . . . Chymotrypsin digestion of peptide T-7. . . Enzymatic hydrolysis of peptides . . . Determination of the net charge of peptides. . . . . . . . . . . . . . . Initial Separation of RCM-Turkey Egg White Lysozyme Tryptic Peptides. . . . . . . . . RESULTS. . . . . . . . . . . . . . . . . . . . . . Purification of Turkey Egg White Lysozyme . . . Amino Acid Content of Turkey Egg White Lysozyme Purification of the Tryptic Peptides from RCM-Turkey Egg White Lysozyme. . . . . . . Enzymatic Hydrolysis of Trypt0phan Containing Tryptic Peptides . . . . . . . . . . . . . Amino Acid Composition of the Tryptic Peptides from ROM-Turkey Egg White Lysozyme . . . . Sequence Determinations on Selected RCM-Turkey Egg White Lysozyme Tryptic Peptides. . . . Comparison of the Lytic Activities of Turkey and Chicken Egg White Lysozymes. . . . . . DISCUSSION . . . . . . . . . . . . . . . . . . . . . BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . iv Page 31 32 33 4O 4O 42 44 44 45 46 47 48 48 55 55 59 61 68 77 80 88 ! LIST OF TABLES TABLE 1. Parameters of Ion Exchange Chromatography. . . . 2. Purification and Characteristics of RCM-Turkey 8 9. 10. 11. 12. Egg White Lysozyme Tryptic Peptides. . . . . . . Enzyme Purification Summary Sheet. . . . . . . . Amino Acid Composition of RCM-Turkey Egg White Lysozyme . . . . . . . . . . . . . . . . . . . . Enzymatic Hydrolysis of the Tryptic Peptides from RCM-Turkey Lysozyme Which Contained Trypto— phan . . . . . . . . . . . . . . . . . . . . . . Amino Acid Composition of the RCM-Turkey Lyso— zyme Tryptic Peptides. . . . . . . . . . . . . . Observed Differences in Amino Acid Composition Between the Analogous Tryptic Peptides from RCM- Chicken and RCM—Turkey Lysozymes . . . . . . . Edman Degradation of Peptides T-1+2 and T-5. . . Edman Degradation of Peptides T-7 and T-7-Cht-3. Edman Degradation of Peptide T-13. . . . . . . . Edman Degradation of Cyanogen Bromide Cleavage Fragments from Peptide T-15. . . . . . . . . . . Proposed Amino Acid Sequence of RCM-Turkey Egg White Lysozyme Tryptic Peptides. . . . . . . . Page 30 36 49 56 60 62 67 7O 73 75 76 82 gr» I | g Ill ll ’1'! LIST OF FIGURES FIGURE Page 1. A fragment of the peptidoglycan from g, lysodeikticus showing points of cleavage by various enzymes. . . . . . . . . . . . . . . 3 2. Preparation of a standard curve for the Lowry protein determination . . . . . . . . . . . . . 18 3. Standard curve for tryptophan determinations according to the method of Spies and Chambers . 26 4. Elution of Whatman 3mm paper strips . . . . . . 39 5. CM-cellulose chromatography of turkey egg white 9. 10. 11. lysozyme. . . . . . . . . . . . . . . . . . . . 51 Polyacrylamide disc gel electrophoresis of turkey egg white lysozyme at different stages of the purification . . . . . . . . . . . . . . 52 Crystals of turkey egg white lysozyme . . . . . 54 Initial separation of RCM-turkey egg white lysozyme tryptic peptides . . . . . . . . . . . 58 Sequence diagram of peptide T-7 . . . . . . . . 72 Comparison of the lytic activities of turkey and chicken egg white lysozymes . . . . . . . . 79 Three-dimensional diagram of crystalline chicken egg white lysozyme. . . . . . . . . . . 86 vi INTRODUCTION The term lysozyme has generally been applied to any enzyme capable of hydrolyzing intact bacterial cells regard- less of the mechanism by which this lysis is effected. It has been suggested (1) that lysozymes should more correctly refer to that group of basic heat stable proteins which are capable of cleaving the glycosidic linkage between N-acetyl- muramic acid and. N-acetylglucosamine (Figure 1) as is found in bacterial cell walls. This definition would separate lysozymes from some of the other endoacetylmuramidases and all other bacteriolytic carbohydrases, as well as the various bacteriolytic peptidases and acetylmuramyl—L—alanine amidases. Relatively few investigators have chosen to character- ize the products produced upon lysis of bacterial cell walls by suspected lysozymes. Rather, they have simply assayed lytic activity. In this way lysozymes have been reported in phage infected E, lei_cells; various bacterial sources; in the invertebrate annelid, Nephthys hombergi; in different members of the blattid and acridid families of insects; in different plants; in fish as represented by the pike and sturgeon, and in many other invertebrate and vertebrate organs and secretions. Lysozymes are also found in human a, 31'.“er 42.2"" "' 5-“ «2-0:!- "‘ wqmzmx 5’5“... 3" V‘ 3 1.7—}. ‘fa " Iii...“ .mwmmpflumwmopcm .Q pom “mommcflam mchmHMIAIH>EMHDE lawnmom .U ammeUHCHEmmOUDHmawuwumoocw .m "AmmE>Nom>H mCHUDHUCHV mommoflamusEHmumom locum .¢ "mum mmm>mmHU mo mmuflm ucwuwMMHU gnu um pm>ao>CH mmfimncm one .pflum UHEMHSEkuoUMIz .2dz “madammousamawumumnz .Odz "mum poms mcofluma>munnm one .mmfiwucm msoflum> mm wmm>mmau mo mucflom mafi3onm wDUflumeCOmw% .2 Eouw cmo>amopflummm on» no ucmEmmuw < .a musmwm a musmflm 2.1. «124.24... 108-20-50-20- To 100w 2%...0 12 n w um>n_un_ _ :ooo-s_o-v-=_w-7o O O 042 s :38 :ofo meEmaEzua . _ ,I. a. ..,.. .4 .t .y . N I t N 1.. 2421...... 042. ..__ _., I. 5.1.0 .136, 10.: a . 1 SK .tL _ ,. av .- .. .. 1...; f r . .l . .. . .. . .1 . ‘5. .. u.. , -_ . , . . , - a . ,. . L 0 e L . . .. , h. . r v . .. .. .- . t. l P. A .u. ... o; .1. .-. .4 I-.. If. ,._.C x... . 1 n at It”. .1 .1 1C .vx. 4C 1 SI; I .x\. ..| O .J u a V. . 1 . \. .. . . L. i C. ._ \._ 3 , . LI” .. 71 i -.T. ,. . . ,.. ,. . A. ; ... .. +1 A ... l .. I. x. L _. _ . . d I r... a . . . “I I I C I. r .1 VJ S t . .I ( I y (\ S n e O r l l e S e I w E T. e b e I .l a t t g S e R a m t 0.. d t C n 5 Z r r. v S b e n l e .K Y O n. S l l a. a 0 e U t a n n C .l l S C a n... t a n r U 0 g d S V t S e 2 O a I a S .1 D. e .l m a a S d .1 V1 0 a U .I. m. . .l . U m m. h a V h U r h e n 0 m I. C n m C 8 T t N d 3 C D. e C h t C r e EDS I §DS l ODS _ weabwbDflsmn. EDS 1' ODS _ moqnmLDaSmn a .10on .10on o 0 IO I O a hu\ 7‘0 OI oh: oh: _ owxomxo :ooo-eo§-c_w-fo @lzs Islet-.. _ _ ED-.. 2qu IOOQ-§0:8-c__.o- To 1000 ”1344.1... /© 0:.70 8. organs, tissues, and secretions (spleen, kidney, milk, tears, and saliva). Human leucocytes are particularly abundant in lysozyme; however, the richest source of lysozyme to be dis- covered is the avian egg. In summary, it appears that lysozymes are ubiquitous in nature. However, judgment concerning each particular lysozyme must be reserved until it is known precisely which bond is cleaved during lysis of the bacterial cell wall. It has been shown, for instance, that the enzyme re- sponsible for the lysis of E. 99;; cells infected with the T-2 bacteriophage is a true lysozyme (2) 1.9., by demonstra— tion that carbon atom 1 of N—acetylmuramic acid (and not N-acetylglucosamine) becomes available as a reducing function during lysis of the bacterial cell wall. This enzyme is also small (M.W. approximately 14,000), alkali unstable, and possesses a maximum activity between pH 6 and 7 as does the chicken egg white lysozyme (3). Recent work (4) with the chicken egg white, human, and papaya lysozymes has shown that lysozymes may have to be even more stringently defined. Treatment of chitobiose and chitotriose oligomers with chicken egg white lysozyme or human lysozyme produced not only hydrolysis but also extensive transglycosylation to give higher molecular weight polysac— charides. Glycosidic bond cleavage by these two lysozymes results in complete retention of configuration. The papaya endoacetylmuramidase is considered a lysozyme even with its ‘higher molecular weight (M.W. approximately 28.000). However, it does not appear to catalyze transglycosylation reactions and its cleavage of glycosidic bonds proceeds with inversion of configuration. Thus, lysozymes which exclu- sively catalyze the hydrolysis of 8-1,4-N-acetylmuramic acid bonds in g, lysodeikticus cell walls may do so by entirely different mechanisms. The differences observed in the catalytic mechanisms between the different lysozymes of similar specificity prob- ably reflects the independent nature of their evolution. This is reinforced by the relatively few structural studies which have been carried out on lysozymes of diverse origins. The primary structure of T-4 bacteriophage lysozyme has been determined (5). This lysozyme was shown to be a mur- amidase as was the T-2 phage lysozyme. Its Specific activity, however, was much higher when assayed with E, ggli_cells, its natural host, than with g, lysodeikticus cells (6). It is a basic protein as is the chicken egg white lysozyme and contains 164 amino acids (M.W. equals 18,134) in contrast to 129 for the chicken enzyme (M.W. equals 14,388). The T-4 phage lysozyme is more heat labile than the chicken lysozyme. This has been attributed to the absence of cystine bridges in the phage lysozyme (two cysteine residues are present) whereas the chicken molecule has four disulfide bonds. The results of the sequence determination showed there was no common primary structure between these two lysozymes. The terminal sequences of the papaya lysozyme have recently been elucidated (7). This proved that the enzyme was actually a single polypeptide chain whose molecular weight was approximately 28,000. Certainly the primary structure of the papaya lysozyme will prove to be very dif— ferent from the chicken lysozyme whose molecular weight is 14,388. It is the chicken lysozyme which is most familiar and about which the most is known concerning its structure. The primary structure has been determined independently by Jolles (8,9) and Canfield (10,11). The three-dimensional structure was established by x-ray crystallographic analysis to 2~angstrom resolution (12) and suPports Canfield's primary structure determination rather than Jolles'. The 2vangstrom level of resolution does not show individual atoms as separ rate maxima. However, the polypeptide backbone appears as a "continuous ribbon of electron density" and many amino acid side chains were easily identified; 93g3, the four di- sulfide bridges and six tryptophan residues. When this information was coupled with the primary structure knowledge, Phillips was able to construct a three-dimensional model of the molecule. The molecule appears ellipsoidal in nature with approxi- mate dimensions of 45 x 30 x 30 angstroms. Forty-six amino acid residues of 129 total contribute to continuous helical regions of four or more residues. The alpha helix content is thus 50 per cent lower than found in myoglobin. For the most part, the acidic and basic side chains are distributed over the surface of the molecule while most of the hydro- phobic side chains are on the interior of the molecule. A closer examination reveals that residues 1-40 contain two alpha helical regions (residues 5-15 and 24-34) which fold back on each other burying several hydrOphobic residues and in so doing forming one wing of the molecule. Residues 41-45 and 50-54 form an antiparallel pleated sheet structure, enabling the hydrophobic residues 55 and 56 to be buried in the existing hydrOphobic pocket. The other wing of the lysozyme molecule is formed by residues 56-86 folding irregu— larly around the pleated sheet structure. The two wings lie at an angle to each other and the gap between then is par- tially filled by an irregular helix (residues 88-100) which acts as a hydrophobic backbone. Because the helix only partially fills the gap, the lysozyme molecule is left with a deep cleft on its surface which Phillips has implicated in substrate binding and as the catalytic site. Phillips was able to form a stable N-acetylchitotriose- lysozyme crystal complex. Because of the stability of the complex, Phillips postulated that it represented a partial enzyme-substrate complex in which only a binding function of the enzyme was revealed. X-ray examination of the complex disclosed the possibility of favorable nonpolar interactions and hydrogen bond formation between lysozyme and its substrate (residues A, B, and C in Figure 11). Through model building, Phillips also demonstrated that the cleft could accommodate a hexasaccharide substrate molecule. The spatial requirements of the model showed that only residues B, D, and F of the substrate could be N-acetylmuramic acid. The proximity of Gluss and Aspsa to the glycosidic bond ruptured between residue D and E of the substrate prompted Phillips to propose a cleavage mechanism in which Glu35 protonates the glycosidic oxygen and a negatively charged Aspsa stabilizes an intermediate carbonium ion at C—1 of substrate residue D. Support for the involvement of Glu35 and Aspsg in the catalytic mechanism of lysozyme action has recently come from two sources. By the use of a carboxyl group modifica— tion procedure (13) which converts free carboxyl groups to substituted amides, Lin and Koshland (14) were able to show that only these two acidic residues could be essential for catalytic activity. Furthermore, the blocking of Asp52 re- sulted in a concomitant loss of enzyme activity. Similar results were obtained by Parsons et al. (15) who were able to isolate a mono-esterified lysozyme which was able to bind trisaccharide but was catalytically inactive against g. lyso- deikticus cells. Unfortunately Parsons and his co-workers have not yet identified the esterified residue. Although a simplification of Phillips mechanism has been presented, it is apparent from above that some specific amino acid residues are essential for its operation. It is with this in mind, that we chose to examine the primary structure of a closely related avian enzyme. Using sequence analysis as a tool, we hoped to determine whether or not any differences between the two enzymes existed which might have conformational or mechanistic significance in light of Phillips findings and predictions. EXPERIMENTAL Reagents All concentrated acids, common inorganic salts and organic 1. chemicals were reagent grade. beta-Alanine General Biochemicals. DL-ASpartic acid, A grade California Corporation for Biochemical Research. Benzene, thiophene free Mallinckrodt Chemical Works. Bio—Gel P-2, 200 to 400 mesh (wet) BioRad Laboratories. Bromine, technical grade Dow Chemical Company. n-Butanol Mallinckrodt Chemical Works. Carboxymethyl cellulose (Cellex—CM) BioRad Laboratories. Carboxypeptidase A, DFP treated .Worthington Biochemical Corporation. Carboxypeptidase B, DFP treated Worthington Biochemical Corporation. 10 11 10. alpha-Chymotrypsin, salt free crystalline, A grade California Corporation for Biochemical Research. 11. Citric acid, monohydrate Mallinckrodt Chemical Works. 12. Cyanogen bromide Aldrich Chemical Company. 13. Dialysis tubing, Visking sausage casing (18/32 inch) Union Carbide Corporation. 14. p-Dimethylaminobenzaldehyde Matheson, Coleman and Bell. 15. Dowex (Aminex) AG 50W—X2, 200 to 325 mesh (wet) Dowex AG 50W-X2, 50 to 100 mesh (wet) Dowex AG 1-X8, 200 to 400 mesh (wet) Dowex 1-x2, 200 to 400 mesh (wet) BioRad Laboratories. 16. Ethylenediaminetetraacetic acid,,diSodium salt’ Sigma Chemical Company. 17. Ethylene dichloride (1,2-dichloroethane) Aldrich Chemical Company. 18. N-ethylmorpholine Aldrich Chemical Company reagent grade was redis- tilled through a Vigreaux column;.the fraction boiling at 136-1370 was collected. 19. L-Glutamic acid, A grade California Corporation for Biochemical Research. 20. 21. 22. 23. 24. 25. 26 27. 28. 29. 30. 31. 12 Iodoacetic acid Eastman Organic Chemicals. iso-Amyl alcohol Mallinckrodt Chemical WOrks. Leucine aminopeptidase Worthington Biochemical Corporation. Lysozyme, chicken egg white, twice crystallized Worthington Biochemical Corporation. 2-Mercaptoethanol, redistilled California Corporation for Biochemical Research. Methanol, anhydrous Matheson, Coleman and Bell. Methyl Cellosolve (ethylene glycol monomethyl ether) Fisher Scientific Company. alpha-Naphthol California Corporation for Biochemical Research. Ninhydrin Pierce Chemical Company. Papain, twice crystallized susPension Worthington Biochemical Corporation. Phenyl isothiocyanate Matheson, Coleman and Bell vacumn redistilled through a Vigreaux column; the fractiOn boiling at 990 at 14 mm pressure was collected. 2-Picoline Aldrich Chemical Company reagent grade redistilled 32. 33. 34. 35. 36. 37. 38. 39. 40. 13 through a Vigreaux column, the fraction boil- ing at 1270 was collected. Polyacrylamide disc gel electrophoresis reagents Canal Industrial Corporation. Pyridine J. T. Baker Company. Sephadex G-10 Pharmacia Fine Chemicals, Incorporated. DL-Serine, A grade California Corporation for Biochemical Research. Sulfanilic acid Matheson, Coleman and Bell. Trifluoroacetic acid Aldrich reagent grade; exhaustively oxidized at refluxing temperature with solid Croa, distilled off, dried over Na2804, decanted, and redistilled through a Vigreaux column from fresh Na2804. The fraction boiling at 650 was collected. The re- agent was stored in a desiccator over sodium hydroxide pellets (16). Trimethylamine (anhydrous) Eastman Organic Chemicals. Tris(hydroxy methyl)aminomethane Sigma Chemical Company. L-1-Tosylamido-2-phenylethyl chloromethyl ketone (TPCK) California Corporation for Biochemical Research. 14 41. Trypsin, salt free and twice crystallized Worthington Biochemical Corporation. 42. DL—Tryptophan, A grade California Corporation for Biochemical Research. 43. Urea Mallinckrodt Chemical Works reagent grade; urea solutions were deionized with Amberlite MB-3 mixed bed resin, filtered, concentrated in a rotary evaporator, washed with ethanol and then ether and finally dried in a vacumn desiccator attached to a water aspirator. Preparation of Turkey Egg White Lysozyme Eggpgration of the crude enzyme A modification of a procedure used for the purification of goose egg white lysozyme was employed (17). A convenient preparation started with 10 turkey egg whites which were separated from the yolk by decantation. The egg whites were suspended in two volumes of 0.05 M NaHgPO4 to which CM- cellulose was added (6 to 12 g per ml) with constant stirring. The mixture was stirred at 50 for 18 to 24 hours after which the CM—cellulose was centrifuged. The pellet was washed four times with 150-ml portions of 0.05 M NH4HC03 by alter- nately mixing and centrifuging. Determination of the residual lysozyme activity of the supernatant established that the adsorption of lysozyme to CM-cellulose had been 15 quantitative. In addition, no evidence of lysozyme activity was found in the 0.05 M NH4HC03 washings. Therefore both the supernatant and washings were discarded. The lysozyme activity was eluted from the CM-cellulose by alternately stirring and centrifuging with three 50—ml aliquots of 0.4 M (NH4)2C03. The eluates were combined, filtered, and lyOphilized. To insure that all of the (NH4)2C03 was removed, the lyOphilized product was dissolved in 100 ml of redistilled water, adjusted to pH 5.5 with 1 N HCl and re-lyOphilized. Crystallization of turkey egg white lysozyme The re-lyOphilized powder was dissolved in water (50 mg per ml) and adjusted to pH 5.5 with 1 N HCl. The resultant solution was made 5 per cent in NaCl and the pH was read- justed to 8.5 with 1 N NaOH. After standing overnight at 5°, the crystals began to separate. Stirring the solution caused it to become cloudy and crystallization was complete within an hour. Recrystallization was accomplished by centrifuging the crystals, removing the supernatant and repeating the above procedure. End point assay of lysozyme activity Lysozyme activity was determined by measuring the de- crease in turbidity of a suspension of M. lysodeikticus cells at 550 mu. Dried M. lysodeikticus cells (13 to 15 mg) were suspended in a solution containing 90 ml of 0.067 M sodium 16 phosphate buffer, pH 6.8 and 10 ml of 1 per cent NaCl. This gave an initial absorbance of 0.5 to 0.6 at 550 mu. The assay mixture contained 2.9 ml of the bacterial suspension and 0.1 ml of lysozyme solution. One unit of lysozyme activity was defined as that amount of enzyme which causes a decrease in turbidity of 1 absorbance unit in the time inter- val between t equals 30 and t equals 180 seconds. This assay was linear in the range of 0.01 to 0.05 absorbance units per time interval. Initial rate assay of lysozyme activity This assay was used to compare the lytic activities of chicken and turkey egg white lysozymes. In order to measure precisely the small changes in absorbance observed in the linear range, a Gilford Model 2000 automatic recording spectrophotometer was used. The initial rates of change in absorbance at 550 mu were determined at 25°. The assay mix— ture contained 290 ul of the previously described bacterial suspension and 1 to 10 ul of enzyme solution. Protein determination by the Lowry method The procedure of Lowry gt_§l. (18) was followed exactly as described. A standard protein curve was prepared from chicken egg lysozyme and is shown in Figure 2. CM-cellulose column chromatography of crystalline turkey egg white lysozyme A solution of 200 mg of twice-crystallized turkey egg \white lysozyme in 10 ml of 0.05 M (NH4)2C03 was applied to .coflusHOm cflwuoum gnu mo mumHm ca umum3 pmaaflumflcon cocamucou nufln3 Roman m uncammm mcouoflEHHHflE 0mm um pawn mum? wwamfimw on» mouscflfi on sound .waxowsv pmxflE can onsu some ou mapflmmu woven wMB m ucmmmwu mo HE o.H .musumquEmu Econ um wouDCAa OH How Unaum Op cw3oHHm muo3 mmHmEmm on» .mCHxHE umuw< .0 ucmmwou mo muoswfiam Helm coped wumB Houm3 pmaafiumflcmu mo HE H ca Umcwmucoo “mamumj oom .ooa .om .mmv wawnomwa muH£3 mmw cmxuflno wo muCDOEm c3ocx OB HmuMB suHB dufi peasant ucommmu Hocmnm smuamoofloncflaom .Q ucwmmmm .w .m ucmmmmm mo as a Spa? ¢ ucmmmmm mo HE om tz .coausaom uwmmoo mcfimeHm .U ucmmmwm .m oumuuumu EDHUOm ucou god a CH me m.+0mso ucoo uwm m.o .m ucmmmmm .N momz z oa.o ca moommz ucwo Mom N .4 ucwmmmm .a "Umummwum muw3 mucmmmmu mGABOHHOM one .Amav COHuMCAEumuoc camuoum >u3oa gnu How m>usu pumccmum m mo coaumummmnm .N musmwm ’Y‘ (‘ I“, 60 90“ uGRAM$,OF 30 I20 - I A T L: ‘l .1 7M N PROTEI Figure 2 BI- :scb A; “L m; r cw n,sv5 mvcfiscquus .s 3 . pvt 1.63 m.H .J 0L5 mcu:.. [II J \H! 1... at E J J «21.2 . #.. mm 0 . .. l 41 L . I V O_m Om_ Om_ Om_ 0m 0w Om LZ/S_w._.omb .10 mZDmmz w mucosa owb mvb vWO w_b VggO 19 a 2 x 22 cm column of CM-cellulose previously equilibrated with 0.05 M (NH4)2C03. The column was eluted with a linear gradient delivered from a two-chambered gradient system by means of a positive-displacement piston pump (Milton Roy Co., St. Petersburg, Florida). The mixing chamber contained 500 ml of 0.05 M (NH4)2C03 and the reservoir 500 ml of 0.4 M (NH4)2C03. Fractions of 5 ml were collected at a constant flow rate of 30 ml per hr. The absorbance of the column eluate was monitered at 280 mu by means of a Beckman DB SpectrOphotometer attached to a Sargent Model SRL recorder. Disc electrophoresis on pglyacrylamide gel The procedure of Reisfeld et al. (19) was followed. This is a modification of the original procedure which em- ploys a pH 4.5 B-alanine-acetate buffer and makes possible the separation of basic proteins and peptides. The sample gels contained 40 to 500 ug of protein and electrophoresis was carried out by applying a current of 8 mamp per tube for 45 minutes. Preparation of Reduced and g—Carboxymethylated Turkey Egg White Lysozyme The reduced and ghcarboxymethylated lysozyme deriva- tive was prepared by a modification of the method described by Crestfield, Moore, and Stein (20). The reaction was carried out in Bantam—ware apparatus (Kontes Glass Co., Vineland, New Jersey). A 25 ml round—bottomed flask was 20 equipped with a Claisen adapter having two 14/20 outer joints. An ebullition tube was inserted through a Teflon adapter in the center joint to the bottom of the flask and was connected to a pre-purified nitrogen source. An outlet tube was placed in the second joint through a Teflon adapter. Twice crystallized turkey egg white lysozyme (100 mg) was dissolved in a solution containing 3.61 g of deionized urea, 0.3 ml of EDTA solution (50 mg of disodium EDTA per ml), and 3.0 ml of Tris buffer, pH 8.6 (5.23 g of Tris and 9 ml of 1 N HCl diluted to 30 ml with water). The solution volume was made up to 7.5 ml with water and a solution of 8 M urea containing 0.2 per cent EDTA was added to make the final volume 12 ml. The system was flushed gently with nitrogen for 15 minutes before and after the addition of 0.1 ml of redistilled mercaptoethanol; after flushing, the system was closed. The reduction was carried out for 4 hours at room temperature. At this time a freshly prepared solution of 0.268 g of iodoacetic acid in 1.0 ml of 1 N NaOH was added to the reaction mixture. (After 15 minutes at room temperature, in the absence of light, the alkylation mixture was rapidly transferred to 18/32 inch cellulose dialysis tubing and dialyzed against deionized water-—also in the dark. Urea and buffer salts were thus removed from the modified pro- 1 tein. The precipitation of RCM-lysozyme began after 15 lThe abbreviation used is: RCM, reduced and g-carboxy— rnethylated. 21 minutes and was usually complete within one hour. After dialysis, the white, precipitated protein was centrifuged in a clinical centrifuge and the pellet was washed three times with 8-ml portions of redistilled water by suspension and centrifugation. The washed protein was then lyOphilized. Subsequent amino acid analysis of the RCM—lysozyme indicated that the reduction and g-carboxymethylation had been com- plete as no cystine could be detected. Eggparation of Trypsin Free from Chymotrypsin Activity It is possible to inactivate Chymotrypsin which is present in trypsin preparations by specifically labeling its active center with L-(1-tosylamido-2—phenyl) ethyl chloro— methyl ketone (TPCK). This treatment has no effect on tryp— sin. The procedure of Kostka and Carpenter was followed (21). Trypsin (100 mg, 4 umole) was dissolved in 33 ml of 0.001 M CaClg. To this solution was added 28.4 mg of TPCK dissolved in 0.75 ml of anhydrous methanol. The reaction mixture was titrated to pH 7.0 with 0.5 N NaOH and maintained at that pH for five hours by the automatic addition of 0.5 N NaOH from a pH-stat (Titrator TTT1, Radiometer, Copenhagen). The reaction mixture was adjusted to pH 3.0 with 1 N HCl and excess inhibitor which had precipitated was removed by centrifugation. The filtrate was transferred to 18/32 inch cellulose tubing and dialyzed at 50 against redistilled water. The dialyzed solution was lyophilized and the TPCK—treated trypsin was stored at 50 until use. 22 Egypgin Diggstion of Rpm-Turkey Egg White Lysozyme A finely divided suspension of RCM-turkey lysozyme was produced by briefly sonicating a 1 per cent mixture of the enzyme in redistilled water. The pH of the suspension was adjusted to 8.0 with 0.197 N NaOH. TPCK—treated trypsin was added in an amount equal to 2 per cent of the lysozyme by weight. The pH of the digestion mixture was maintained at 8.0 by the automatic addition of 0.197 N NaOH diSpensed from a pH-stat. After digestion at room temperature for 3 hours, the uptake of sodium hydroxide essentially stopped. The addition of more trypsin did not cause consumption of sodium hydroxide, indicating the digestion was finished. A small amount of undigested insoluble material (approximately 1 per cent) remained at the end of digestion and was removed by centrifugation. A similar observation was reported by Canfield in his work with chicken egg white lysozyme (22). The uptake of sodium hydroxide at the end of the digestion was 50 per cent of the theoretical amount if cal— culated on the basis of one hydroxide ion per peptide bond cleaved. It is apparent that the pKa values of the unmasked amino and carboxyl functions of the liberated peptides must be considered in calculating the true theoretical uptake of hydroxide ion. 23 Apino Acid Applysis of Proteins and Peptides Preparation of samples for amino acid analysis Acid hydrolysates of peptides or proteins were prepared by the addition of 2 ml of constant boiling HCl to the sample contained in a small ampule prepared from a drawn-out 18 x 150 mm Pyrex test tube. The solution was cooled in an acetone—dry ice bath and then subjected to repeated evacua- tions (water pump pressure) and flushings with pre—purified nitrogen. In this manner the dissolved oxygen was removed. After flushing with nitrogen, the tube was sealed under vacumn. Hydrolysis was carried out at 1050 in a constant temperature oil bath. Proteins were hydrolyzed for 24, 48, or 72 hours and peptides, in general, were hydrolyzed from 20 to 24 hours. In some instances, in an attempt to minimize tryptophan degradation, peptides were hydrolyzed only 12 hours. Imme- diately after hydrolysis the sample was taken to dryness on a rotary evaporator and then dissolved in pH 2.2 sample diluting buffer. When enzyme digests were analyzed, the mixtures were lyophilized and then dissolved in pH 2.2 sample diluting buffer. Due to the presence of non-volatile buffer salts, it was necessary to adjust the pH of the samples to pH 2.2 before applying them to the amino acid analyzer column. 24 Amino acid analysis All amino acid analyses were carried out according to the method of Spackman, Stein, and Moore (23) using a Spinco Model 120 amino acid analyzer. Tryptophan determinations on intact protein The method of Spies and Chambers (24) was followed for this determination. An aliquot from a stock solution of turkey egg lysozyme, estimated to contain approximately 100 ug of tryptophan was lyOphilized. The concentration of lysozyme present in the stock solution was determined by amino acid analysis of a suitable aliquot. The dried pro— tein was dissolved in 10 ml of 19 N H2504 which contained 30 mg of dissolved p-dimethylaminobenzaldehyde. The result- ing solution was allowed to stand at room temperature for 12 hours at which time 0.1 ml of 0.045 per cent NaNOa was added. After the color had deve10ped for 30 minutes, the absorbance at 590 mu was determined in a Beckman Model B Spectrophotometer. The blank solution contained 10 ml of 19 N H2804 and 0.1 ml of 0.045 per cent NaNOa. A standard curve was prepared from free tryptophan and is shown in Figure 3. Peptide Column Chromatography Preparation of resins Dowex (Aminex) AG 50W-X2 (200 to 325 mesh) resin was prepared for column chromatography by thoroughly washing 25 .mOZmz ucmu umm mso.o no as a.o cam «comm 2 as no as 0a pocnmucou coausaom xcman one .ponHEuouop mm3 15.0mm um oocmnuomnm onu pcm mouanE on How poououm Cu po3oHHo mmB unoemoHo>op HOHOU .oamEMm nuoo Op poppo mmB Nozoz ucou mom meo.o mo HE «.0 .ouson we now ousuouomEou Eoou um mcwpanw uouwd .openopaonconocflEmHmnuoElem po>aommfip mo 08 on pocwmucoo noHnB «Ommm Z we mo HE OH an po>aommflp ouoB Amemumi OOH .Ne .omv nonmoumenu mo monEmm .Awmv muoneonu cam moamm mo ponuoe onu Op mcflpuouuo mcofluocaEuouop nonm0pm>uu Mom o>uso pumpcmum .m ousmflm . n unamwh I VC II. ..I. . . . , 1 ... .. Mb I . .... .. II . .... .,.. IL . s. l I] ..1 A f A .\J m . . I, .L ...._ .3 1.. $1 I 1 .. ., I . a . e , .I f“ m s I. .s . a . ,. . I I ... u. .I.. . .3 .I“ I . I». .I I. I. I . i Z. . .1 .... L . ... q l .. .m. J. ) . _ A. . .. j mO_, 00.. . me. . Om; me. On . m_ . is .... . It» L I I. ... .L I , I . ... ..., . c._ . . I . . .L I .I. ... ... I II. . Id I. .. .I. 7 ,II. . t. , . .. . .1 t. v.1. III .I. 1 ,II I . . TI _ . I .... I\ .1 \II _I. II . _ . 4 .II. . .1 ix. m. I. . I . I . I .z 1 .. r. . . . . I vi . I .I, w). n I ll. ,. t it. It . r. _ A .... .I . I .. I .L 1 I\“ I 4... . H4 1r. r II.I ,1 I . I, . . ..t a. . . . ... , I. , C O .. II) I: I. . ) I E, I; ..t i, .... I .1 PM .... I , “i. .. .... .H I .I I; I I w; n . I s _. T., ‘ 1 _ a. IA NI - .I . I , E II c... . I 6 ,A 4 IN. ... I .. 2 I... 1c .. . . l ..c I. : V n. ..U I., I. . I I; . 0.. .1 I. x} I. t. 1 I“ . t . . . I I . .... .1. ..v ‘ . ... (II I I _ I II n... .v. w (1 r. u I ”J . I .I .1. ... _ L I.1 I I I. II n ... . I o .. I... I t .. .n I. If E J. m 0Q :. I .N . ._ 1. I1 I. In I I 1 N .r L ..,.. . 1 4U. E ... r I. ... . I. ..I1 ,II. .... C .0 3 1 , .. ...) 3 L” J 9,. L Z... 1 CI .II ..., . L I. I, . 7 z; .I. n... ... .r _. I W . I ..I. n h H; ...” .... I i .I .1 I. .. H ..C 3 pt. .u. ... C I... . I. w. i ‘L w A L. f . C. a . . .. J l I. .f. .r... ..I I. .I. «I , .. ,.I :. .L . to .L. 1.. (, z , _ I. r. I... I I. _ I U. . in .I. I I. I. .1 ...; 1 U I I. r1 .. w; .l I"... ... .. S . . . ... II. 4.. I.. .I . I 7 1. I I, If I. .I ..H r . r I.. l I, . ... . . ... .I 1.. ... . .J : 1 X .. . ..m 1.. ...... n.“ . .I “v. . . n. J , m .0 BS / mo_ 00 m4 Om mt Om SDIflOHLJEH no mgbmoz m wucmwd m _ m0 v.0 _w.. m . O O .O VgBO 27 on a sintered glass funnel with 1 M NaOH, followed by 5 M HCl, and then 2 M pyridine to establish the pyridinium ion as the cation. The resin was then equilibrated for use with 0.2 M pyridine-acetic acid buffer, pH 5.1 (25). Dowex AG SOW-XZ (50 to 100 mesh) was prepared for purification of residual peptides in the Edman degradation by washing with 1 N NaOH, followed by 1 M HCl and finally redistilled water. The resin was used in the hydrogen form. Dowex 1-X2 (200 to 400 mesh) or AG 1-X8 (200 to 400 mesh) resins were prepared for column chromatography by washing on a sintered glass funnel with water at 60°, followed by 0.5 N NaOH, water, 1 N HCl, and finally water. The resin was stored wet in the chloride form until needed. Before packing a column, the resin was washed in this order’ with water, 0.5 N NaOH, water, 1 N acetic acid, and water. The resin was then equilibrated with pH 9.4 buffer (26). gacking of ion exchange resin columns The jacketed ion exchange columns were poured in sec- tions at the operating column temperature. A suitable quantity of resin slurry was poured in the column which was then carefully filled with the initial buffer. The section was packed by pumping starting buffer through the column at a reasonable flow rate. After removal of the excess buffer, another section could be packed in a similar fashion. It was especially important to pack the Dowex 1 columns as quickly as possible to prevent formation of air bubbles. 28 Descri tion of buffers used in the elution of peptides from ion exchange columns Gradient elution from Aminex AG SOW-XZ employed two pyridine-acetic acid volatile buffers (25). First buffer: pH 5.1, 0.2 M in pyridine (64.5 ml pyri— dine and 1114 ml of glacial acetic acid diluted to a volume of 4 liters). Second buffer: pH 5.0, 2 M in pyridine}(645 ml pyridine and 575 ml of glacial acetic acid diluted to a volume of 4 liters). Dowex 1-X2 or AG 1-X8 peptide columns were eluted with an N-ethylmorpholine, d-picoline, pyridine, acetic acid sys- tem of buffers (26). First buffer: pH 9.4 (60 ml N-ethylmorpholine, 80 ml a-picoline, 40 ml pyridine, and approximately 0.5 ml glacial acetic acid to give a pH of 9.4 when diluted to 4 liters with water). Carbon dioxide was removed from the redistilled water used in preparing both the pH 9.4 and 8.4 buffers by bubbling with nitrogen. Both of these buffers were pro— tected from the air by means of an Ascarite tube attached to the dispensing bottle. Second buffer: pH 8.4 (60 ml N—ethylmorpholine, 80 ml a-picoline, 40 m1 pyridine, and about 5 ml of acetic acid diluted to 4 liters). Third buffer: pH 6.5 (60 ml N-ethylmorpholine, 80 ml a-picoline, 40 ml pyridine and approximately 57 ml of acetic acid diluted to 4 liters). 29 Fourth solution: acetic acid, 0.5 N. Fifth solution: acetic acid, 2.0 N. Qgscription of systems used in column chromatography on ion exchange resins In general, eluting buffer, from a two—chambered gradient system was delivered by means of a positive- displacement piston pump (Milton Roy Co., St. Petersburg, Florida) to a column equipped with a water jacket. The column was maintained at a constant temperature (usually 40°) by attaching the jacket to a circulating water bath. Suitable fractions were collected in a fraction collector. A description of the gradients and flow rates employed for the various resins and columns is shown in Table 1. Detection of peptides eluted from column chromatography on ion exchange resin; Alkaline hydrolysis: A suitable aliquot (usually 0.05 to 0.2 ml) was removed from each or every other fraction and carefully pipetted into the bottom of polypropylene test tubes (16 x 100 mm). The samples were evaporated to dryness in a ventilated oven at 1100 in the hood. The poly- prOpylene tubes melt if heated at higher temperatures. Each tube received 0.15 ml of 15.5 N NaOH (71.5 ml of 50 per cent NaOH solution diluted to 100 ml with water) and was then covered with a Kap—ut (Bellco) culture tube cap. Alkaline hydrolysis of the samples was carried out in the autoclave with steam at 15 psi for 20 minutes. After hydrolysis, 0.25 mEsHo> 00¢ OflN Owd ONH Ofi mMH ucmumcoo ON OOH X m.O mEDHo> OOH Om 0% On OH ow uCMHmEOU OH Oh x m.O z o.N z m.o m.m mm ¢.m mm ¢.m mm .AHEV ucwflpmuo AH£\HEV AEUV owod pau< Hmwmsm uwmmsm umwmzm umxflz mo mama mama mcoflmcwaaa owumu< Ufluwod mo mEsHo> 30Hm GEDHOU AHEV mEDHo> Homon>0Q szmumoumeouno GEDHOU mxlfi,m¢ Ho lefi Xm3oa O 5 omfi om mm Q #.d n xw>coo OH on x m.o com com OOH Q Q ummcfiq om OOH x m.o 00mm comm com a Q Hmwcflq om omfi x o.N ufio>ummwm nexus ucwflpmuw Hwo>ummmm Hwnamno ucmflpmuw AH£\HEV AEUV. cw memsm CH uwmmsm mnemmn now no mcflxflz no no make mama mcoflmcwEHn o.m mm H.m mm Imsm H.m mm umumEmflQ HmumEmHD 30am :EDHOU AHEV mEDHo> uwm0H0>mQ NEmfluamom wasmm pom m>fluwmom anusmmxmm .m>Hummmc cwupmncfiz m.m mm um wflmwuonmouuomam Momma mIB . mum Aomu HmGIOAm co coflumuuaflm Hum wn owBOHHom EU OOH Iwuumzuv m>HUHmom wanna x m.o .NIXIH XmBOQ .>£mmumouMEouco CESHOU no 6cm m>Huflmom qusmmxmm Amuauw .OmmuoHuflmom nowaunm Umuflsvmu coflumoflmauzm oz mIB Ammcmuov m>fluflmom wasmm oalo xmpmnmwm co coflumuuafim Hmm >9 UmBOHH0m Cam m>HuHmom Hnuzmmxmm Eu oh x m.o .mXIH Gd .mnmmumoumfionno GEDHOU mIB m>Huflmom accommxmm Amuauw .Ommuudmumosmucv >£mmumOumEOH£U Hmmmm ale m.m mm um mammuozmouuomam Hmmmm mla EU 00d Ammcmuov m>fluamom manna x m.o .NNIH xmBOQ .anmwumoumeonnu cEDHoo Ho Ucm m>wuamom Anusmmxmm Amudufi .Ommuudmumosmlcv unamnmoumaounu Hmmmm NIB Ammcmuov m>fluflmom wanna 0cm m>wuflmom accommxmm Umuflsvmu coflumoflmwusm oz N+HIB moflumwumuomumzo mcflnmflsmcflumfla coHumuwwausm mo ponumz mpaummm mMQHBmmm UHBmMmB MSMNOmWA HEHEZ 00m memDBIZUm m0 mUHBmHmmBUfluflmom fisosmmxmm w>fiuamom Hausmmxmm w>HuHmom nofiaunm paw Avon IMHuwSUV w>flufimom %H5mm Ucm w>flufimom agosmmxmm w>fluflwom Hausmmxmm m>fluflmom Hausmmxmm Ucm w>HuHmom SUMHHSM 0>wuwmom asusmmxmm paw w>Hufimom nofiaucm Amndnfl .Ommuodmnmosmlcv mnmmumOuMEouso memm Amuau¢ .ommnummumosmlcv mammumoumfiousu Hmmmm Uwuflsvmu coHuMUAMHqu oz omufisku coflmeMMHusm 02 EU oh x m.o .mNIH Gd .hnmmumoumEouzu GEDHOU Umuflsku coflumofiwflnsm 02 EU om x m.o .wxld w< .wsmmumoumeouno GEDHOU Umufisvon coHuMUHMHHDQ oz Amuduw .ONmuodmumosmlcv mSQMHmOuMEOHSU memm confisvwh coflumoflmausm oz md+>HIB hale mHIB male aale ma-e ma.aua Haue OHIB “MG . 58 .mmEHu whoa o3u Umummmmu mmmuoum mcu Ucm Um>OEmH mmB mumsHm one .Hmmmm mnu musam.ou Uw>umm mmsmwuucwo HMUACHHU mnu cw cofiummsmauucmu wmflufl d .xuoo Uwum>OUIHHom Escflfisam cm zufl3 mumHm Ca pawn paw onsu meMHuuch m5» m0 QHH ms» Hm>o Umoaom mm? mfluum mzu mo flaw mumsvm mLB .AQV umuwm pom Umummmullmnsu mmsmauucmo may oucH Umunwmcw 0cm ADV mafiosuuoum Ucm UwquMuIIHHom EscwEsHm mo mUHow o3u CH Ummmmu3 mm? aw .mcaoanwm ucmu mom on £ua3 mfluum mnu maficmumfloa Hmuwd .A4v 6cm one so ucflom UmquMu m nuHB wasp mmDMHHucmu HMUHCOU HEINfi m mUflmcfl paw Op uso mm3 Umusam m9 ou mfluuw mQB .mmfluum momma EEm unaumnz mo coausam .¢ musmflm 'v; , {'4 . 1 ‘ n L, t ".b IA ‘ ; in? T. .. , 7 r‘ : ,. Tie \ A i , ,; _ . h .1 ,‘ a l i. . .\l ._ 4’ 1 Ex 59 1n i-‘i ., , . _. : :‘ {W't-i 1 a; .himillrl‘ 5 C. if] : -_.________-__..__- 471. ;. 1 1.1 1'.” . V .1. .. .__-___,_____________ r,:. Y 7""W'XZH" . L, _. , _1 11:1; »2* ,4 film 1w. ‘fti‘c .’ f ;;: a ;t11.i’t;2 w, r;: , in»... 1‘ -41i11 V 7‘ ““"‘ ‘ L I q rili'i.‘€ was {- ;‘ J. CA;‘ 1 ‘ . Lttt...« .wl it: TYI—v. :T‘ , rs. ' , ‘ 1,: l - . — .— SL- 1:, r 2: uten- e: , 1t ituit ri. 11H, I, a» leflw riy, {1‘1 "‘ r, ‘v.’ Inserted Strip Wrapped Strip Figure 4 Aluminum Foil 8v 3383 91m Q R: // m. of.“ a... . u _ . u u / n m V m ” $232 35365 91m :om b mucmwm swoon. 91m 40 Peptides were eluted from Whatman 5mm paper by the following time-saving procedure. The strip to be eluted was cut to fit inside a 12-ml conical centrifuge tube with a tapered point on one end. After moistening the strip with 50 per cent pyridine, it was wrapped in two folds of aluminum foil (tapered end protruding) and inserted into the centrifuge tube (tapered end first). The square end of the strip was folded over the lip of the centrifuge tube and held in place with an aluminum foil—covered cork. A brief centrifugation in the clinical centrifuge served to elute the paper. The eluate was removed and the process repeated twice more, after which no ninhydrin positive material remained on the paper. A representation of the method is shown in Figure 4. Peptide Sequencing Techniqges Subtractive Edman degradation The procedure of Taniuchi and Anfinsen (55) was followed for all but one of the peptides degraded by the Edman method. The coupling mixture contained 2 to 5 umole of peptide in 0.5 ml of distilled water, 0.5 m1 of 2 per cent phenyl iso- thiocyanate in pyridine, and 50 ul of 25 per cent trimethyl- amine. \The mixture was placed in a 12-ml conical centrifuge tube fitted with a cork stOpper which had two openings; one for a stainless steel ebullition tube and the other for an outlet tube. The reaction mixture was flushed for two 41 minutes with pre-purified nitrogen after which the tube was sealed and incubated at 400 for two hours. The mixture was extracted five times with 4 to 5 ml of thiophene-free benzene by mixing in a Vortex mixer, followed by centrifuga- tion in a clinical centrifuge. The upper layer was removed with a Pasteur pipette and discarded, taking care not to disturb any emulsion which might have formed. After the final extraction, the mixture was lyophilized. Cyclization and subsequent cleavage of the phenylthio- carbamyl derivative was accomplished by dissolving the lyophilized product in 1.0 ml of trifluoroacetic acid. The solution was flushed with nitrogen for 1 minute; the tube was sealed and incubated at 570 for 50 minutes. The reaction mixture was taken to dryness in a vacumn desiccator over NaOH and CaClg pellets; the dried residue was extracted four times with 4 to 5 ml of ethylene chloride. The residual peptide residue remained as‘a film, layered on the inside surface of the conical centrifuge tube, as the extracts con- taining the thiazoline and phenylthiohydantoin amino acid derivatives were removed by Pasteur pipette. The residual peptide residue was lyOphilized and dissolved in a known quantity of 50 per cent pyridine. A suitable aliquot was withdrawn for amino acid analysis and the remainder was lyophilized prior to the next subtractive step. A modification of this procedure (56,57), in which the residual peptide product was purified after each cyclization 42 step, was applied to the Edman degradation of the N—terminal cyanogen bromide fragment from peptide T—15. After removal of the trifluoroacetic acid from the cyclization mixture, the residue was dissolved in 4 ml of 0.2 M acetic acid and extracted twice with an equal volume of benzene. The aqueous layer containing the residual peptide was adsorbed on a 0.5 x 6 cm column of Dowex AG 50W-X2 (50 to 100 mesh) and the resin was washed with three 4—ml portions of 0.2 M acetic acid. The peptide was then eluted with 4 ml of 1.07 M pyridine- acetic acid buffer, pH 5.4 (86 m1 of pyridine and 50 ml of acetic acid diluted to a volume of 1 liter with redistilled water). The eluate containing the peptide was lyophilized and treated as above. Carboxypeptidase digestion The procedure of Dolpheide, Moore, and Stein was fol- lowed (58). Carboxypeptidase A solution was prepared by washing 50 pl of the SUSpension of crystalline enzyme twice with cold redistilled water by suspending it in the water and then centrifuging it in the clinical centrifuge. The precipitate was dissolved in 1 M potassium bicarbonate to a concentration of 1 mg per ml. Carboxypeptidase B was used as a solution in 0.1 M sodium phosphate, pH 7.8. All diges— tions were carried out in 0.1 M sodium phOSphate, pH 7.8 at either 250 or 570. Peptide T-5 (1.5 umole) was dissolved in 1.0 ml of phosphate buffer and incubated with 2.0 ul of carboxypeptidase 45 B (6 mg per ml) for 5.75 hours at room temperature. At this time 50 pl of carboxypeptidase A (1.0 mg per ml) was added so that the molar carboxypeptidase A to substrate ratio equaled 1:1000. At the end of one hour's incubation, an ad- ditional 450 pl of carboxypeptidase A solution was added to the digest, increasing the molar carboxypeptidase A to sub— strate ratio to 1:100. Digestion at the increased carboxy— peptidase A concentration was carried out for 20.5 hours at 57°. Aliquots for amino acid analysis were removed at 6.75, 8.25, 12.25, and 27.75 hours after initiation of the diges— tion with carboxypeptidase B. The N-terminal pentapeptide derived from Chymotrypsin hydrolysis of T—7 (T—7—Cht-1) was digested with carboxy— peptidase A. The reaction mixture contained 0.45 pmole of peptide, 400 pl of phosphate buffer and 100 pl of carboxy— peptidase A (10 mg per m1). Amino acid analysis was per- formed on the entire digest after 2.5 hours at 57°. The molar enzyme to substrate ratio was 1:15. Carboxypeptidase digestion of the N-terminal octa- peptide from T-7 (T—7-Cht—2) was also carried out. The reaction mixture contained 0.56 pmole of peptide, 2.0 ml of phosphate buffer, and 150 pl of carboxypeptidase A (4 mg per ml). The digestion was carried out for 11 hours at 570 with aliquots removed for amino acid analysis at 0.67 and 11 hours. The molar enzyme to substrate ratio was 1:50. 44 Cyanogen bromide cleavage of T-15 Cyanogen bromide digestion of T-15 was carried out according to Taniuchi and Anfinsen's procedure (55) which is a modification (59) of the original procedure (40). Approximately 4 pmole of peptide T—15 was dissolved in 0.75 ml of 70 per cent formic acid to give a 1 per cent solution of the peptide. A 50-fold molar excess of cyanogen bromide (120 pmole, 12.7 mg) per methionine residue was added. The reaction mixture was kept at room temperature for 24 hours at which time 2 volumes of redistilled water were added. The solution was lyophilized to remove methyl thiocyanate and excess cyanogen bromide. The cleavage products were separated by column chromatography on a 0.5 x 50 cm column of Aminex AG 50W-X2. Chymotrypsin digestion of peptide T-7 The initial digestion mixture contained 5.8 pmole of peptide T-7, 1.25 ml of 0.1 M NH4HC03 buffer, and 200 pl of a 1 per cent Chymotrypsin solution. The molar enzyme to substrate ratio was 1:50. Digestion was carried out at 570 for 10 hours after which the digest was lyophilized and then dissolved in pH 9.4 buffer. The peptides were separated by column chromatography on a 0.9 x 100 cm column of Dowex 1-X2. Because of the low yield of the N-terminal pentapeptide (T— 7-Cht-1) and difficulty in eluting this peptide from Dowex 1-X2, the reaction conditions were changed; also changed was the method of separation. ’3 45 The digestion mixture contained 5 pmole of peptide T-7, 0.2 pmole of Chymotrypsin, and 2.25 ml of 0.1 M NH4HC03 buffer. The molar ratio of enzyme to substrate was 1:25. Digestion was carried out at 400 for 12.75 hours. The pH was adjusted to 2.7 with formic acid and the digest was chromatographed on a 0.5 x 50 cm column of Aminex AG 50W-X2. Enzymatic hydrolysis ofypeptides Peptides which gave a positive Ehrlich test were sub- jected to enzymatic digestion in an attempt to quantitate their tryptOphan content. Peptides T-6, T-15, and T-16 were digested by the combined action of papain and leucine amino— peptidase. Peptide T-9 was treated with leucine amino- peptidase only. Papain digestion of peptides was performed according to the procedure of Smyth, Stein, and Moore (41). The papain hydrolyzing mixture contained 0.5 to 0.4 pmole of peptide, 2400 pl of 0.02 M sodium phosphate buffer, pH 7.0, 500 pl of 0.01 M NagEDTA in phosphate buffer, 200 pl of freshly prepared 0.1 M NaCN in phosphate buffer, and 100 pl of papain solution (1.1 mg per ml) activated prior to use by incuba- tion for 2 hours at room temperature in the above EDTA-NaCN- phosphate buffer system. The molar enzyme to substrate ratio was 1:50. The papain digest was maintained at 400 for 18 hours at which time the pH of the solution was adjusted to 2.0 with 1 N HCl; the solution was lyophilized. 46 Peptides (0.2 to 0.4 pmole) to be digested by leucine amin0peptidase were dissolved in 0.5 ml of 0.005 M MgClg in 0.005 M Tris buffer, pH 8.5. To the dissolved peptide was added a solution containing 24 pl of a leucine amino- peptidase suspension (5 mg per ml) previously activated by incubation for 50 minutes at 400 with 0.5 ml of the Tris- MgClg buffer system (42). Hydrolysis with leucine aminopepti— dase was carried out for 18 to 24 hours at 400. If present, denatured papain precipitated during this time and was re— moved by centrifugation at the end of the digestion. The digest was diluted to 5 ml with pH 2.2 sample diluting buffer before amino acid analysis. A better procedure for the enzymatic hydrolysis of pep- tides and proteins has been described by Hill and Schmidt (45). Determination of the net charge ongpgptides The assignment of amide or free carboxylic acid func— tion to an acidic amino acid within a peptide can often be made on the basis of the net charge on the peptide. If this assignment is to be based solely on the net charge, the pep- tide must contain only a single unknown amide or acidic func- tion. The relative net charge of peptides was determined from the mobility of the peptides compared to aspartic acid, glutamic acid, and serine after high voltage electrophoresis at pH 6.5. The peptides were located by means of the ninhydrin dip. 47 Ipltial Separation ofngM-Turkey Egg White Lysozyme Tryptic Peptides The digest from 600 mg of the RCM—turkey lysozyme was adjusted to pH 2.8 with formic acid and applied with air pressure to a 2 x 150 cm jacketed column of Aminex AG 50W-X2, previously equilibrated with 0. 2 M, pH 5.1 starting buffer. The column was eluted first at 400 with 500 ml of starting buffer and the elution then was continued at 400 with a linear gradient provided by a two-chambered gradient appara- tus containing 5500 ml of starting buffer in the mixing chamber and 5500 ml of 2.0 M, pH 5.0 pyridine-acetic acid buffer in the reservoir. After completing the gradient elution, 500 ml of 2 M NH4OH was passed through the column at 50°. The flow rate during all stages of the elution was 60 ml per hour and the volume of fractions collected was 10 ml. RESULTS Purification of Turkey Egg White Lysozyme The purification of egg white lysozymes, in general, takes advantage of the basic nature of these proteins and of their abundance in egg whites compared to other basic proteins. Therefore, a 90 fold purification of turkey egg white lysozyme was achieved by batch adsorption of the basic egg white proteins to CM-cellulose, washing with ammonium bicarbonate, and finally elution of the lysozyme activity with an ammonium carbonate solution. AS seen in Table 5, this was the principal step in the purification procedure. The crystallization and recrystallization of turkey lysozyme served to remove minor basic protein contaminants without a Significant increase in Specific activity. This was demon- strated by both CM-cellulose column chromatography and poly- acrylamide disc electrOphoresis of the enzyme before and after crystallization (Figures 5 and 6 respectively). The twice crystallized turkey lysozyme gave a single peak on CM-cellulose column chromatography and a single band on disc electrOphoresiS. By these two criteria the enzyme was homo- geneous. The appearance of the twice crystallized enzyme is shown in Figure 7. 48 49 mfihucw UmNHH oaa oo.mm was m.mm ma ooa.oa mama mm.m -HMummuo amaze mumsam om om.ma cma a.m cm oom.ma oma oma mmoHsHHmoizu in 54.00 oom.ma o.mm ooa oom.am mm cam acausafia cowumoflw >uw>flpu¢ 02 Hz >Hm>oomm mums: H2 H2 sauna paom uawaummm Hmuoe .mm ucmo umm Hmuoa mums: coaumuwmwusm cwmuoum >ua>wu0¢ mEDHo> amum Emmmm Mm¢zzbm ZOHBummmn mnu pcm mOUNAvmzv fl mo.o mo HE oom pmcfimucoo.quEmnu mcflxHE maa .pmumflum> Hmnamno i03u m Eoum pmum>wamp unmapmum Hmmcfla m Lua3 pmusam mm3 :EDHOU one .moomavmzv S mo.o nuw3 Uwumunwaflsvm >Hw50fi>mum mmoHDHHwoizo mo cEsHoo EU mm x N m 0» Goddamn mm3 noomaamzv Z no.0 mo HE OH ca 05>N0m>H mmw mmxusu mo 05 Com mo COHHDHOm ¢ .wE>NOm>H muw£3 mmm wmxusu mo wsmmumODMEoufio wmoasaawoizo .m musmwm 51 .LNBICIVHE) 20:234va :50 W *0. o mh_ O' '0. 0 V. o (x! m whfimfih mmmsSz ZO_._.omo mmonmm ms;sz _ _ a . ,ms>sz ,omN_j<$>mo _ ll moi; m._ nYm 3.. nYm m.h 082v Ill \ (ill l').\l\llll\ll \Ilii (I‘ll): I. mb _ _ r M mmouwm m=sM_LL>4m9 poumooH oHoB wxmom one .pouooHHou oHoB mcofluomum HEIOd paw uson Mom HE om mm? coflusao on» mo wommum Ham mcwusp oumu 30am one .oom um CEDHOU onu nmsounu pommmm oHoB m0¢mz 2 N mo HE oom .cofiusao unofipmum onu mcfluoamaoo Houm¢ .HH0>HomoH onp CH o.m mm .uomwsfl Ufiom owuoUMIonflpflnwm z o.N mo HE 00mm paw Honfimno mnwxfie onu ca Momwzn mcwuHMuw mo HE 00mm pocfimunou nUMnB msumummmm ucoeomnm poHoQEanIo3u m Scum ucoflpmum Hmocfla m an poBoHHom mp3 mane .oow ume.m mm .Hommsn mcfluumum peom oeuoom locapwu>m z N.o mo HE oom nun? pouzao mm3 CEDHOU one .NNIzOm w¢ XocHE¢ mo chHou EU oma x N m Op poflammm mm3 oE>N0m>H eoxusuizom no me oom Eonm umomflp aflwmmuu one .mopwumom Uflpmeuu ofiwuomea oufln3 mmo eonHDuIZUm mo noflumummom HmfluwcH ..m ousmflm 58 00h mum—2:2 ZO_._.onx Abiv G-ro an sea} no 11811“ SL1; :. «a: Edam 2; UHDQmSt my if,“ ..M ,<. 11:..n 1, «_nm _ . . ,._; . . . .. .- c in; .. . .nw.n Hm. 51v :Punfiu j oN>HCnntuio I on; on oucmnn,» «no uzcn moanu and ;; ...... . «7:; 4,. L... .L r . ”.../.4. .__l,v wqfiJu.i 0... 9m 0.. 004 00m 00m mmmZCS $030.93”. m oucmhd OO. VBQOBBWCS V1 £5910 Tum 59 Enzymatic Hydrolysis of TryptOphan Containing Tryptic Peptides In an attempt to resolve whether there were six or seven tryptophan residues in the turkey lysozyme molecule, aliquots of those purified tryptic peptides which gave a positive Ehrlich test (T—6, T-9, T-15, T-16) were hydrolyzed enzymatic- ally. The results are shown in Table 5. No peptide was com- pletely degraded by this treatment-—perhaps because of a relatively inactive preparation of leucine amin0peptidase. In this context the results are not wholly satisfactory. Peptide T-9, which was digested only with leucine amino- peptidase, released two moles of tryptophan for each mole of §fcarboxymethylcysteine and asparagine. This is precisely what was eXpected if the sequence of the turkey lysozyme peptide was the same as its chicken lysozyme analogue: Trp- Trp-CMCs-Asn-ASp-Gly-Arg. This confirmed the results from acid hydrolysis of the peptide in which 1.6 moles of trypto- phan were recovered per mole of peptide. Peptide T-15 also contained two trypt0phan residues. Approximately two moles of tryptOphan were released for each mole of isoleucine, valine, and arginine--amino acids which were recovered as single residues from the acid hydrolysate of T-15. Similarly, the analogous chicken lysozyme peptide contains two tryptOphan residues. 3The abbreviation used is: CMC, §fcarboxymethylcysteine. 'EABLE FROM 5. 60 ENZYMATIC HYDROLYSIS OF THE TRYPTIC PEPTIDES RCM-TURKEY LYSOZYME WHICH CONTAINED TRYPTOPHAN Peptide Amin° ACid T-9 T-15 T-16 T-6 Trp 1.90 2.00 1.16 1.24 Lys 0.51 (1) His -- (1) Arg 0.11 (1) 1.07 (1) 11.02 (1) S-Carboxymethyl- 1.00 (1) 0.82 (1) cysteine Asp 0.26 (2) 0.22 (2) 0.10 (1) -- (1) Thr 0.22 (1) Ser (Asn) 0.90 (0) 1.47 (1) 1.21 (1) Gly 0.12 (1) 0.95 (5) 0.50 (1) 1.45 (2) Ala 2.61 (5) 1.00 (1) 1.14 (2) Val 1.00 (1) 0.06 (1) 0.75 (1) Met 0.56 (1) Ileu 0.87 (1) 0.97 (1) Leu 1.00 (1) Tyr 0.65 (1) Note: The amino acids released by enzymatic hydrolysis are exPressed as molar ratios based on the moles of §fcarboxy— methylcysteine, valine, alanine, and leucine for peptides T-9, T-15, T-16, and T-6 respectively. Following in paren- thesis, is the integral value as determined from acid hydrolysis. 61 Peptide T—16 quantitatively released a single trypto- phan residue for each alanine, isoleucine, and arginine residue. Also, approximately one mole of tryptophan was recovered for each mole of leucine in peptide T-6. Therefore it seemed likely that each of these two peptides contained a single tryptOphan residue as do their chicken lysozyme counterparts. These results indicate that the four Ehrlich positive peptides isolated from trypsin digestion of RCM—turkey lyso- zyme most likely contain six tryptophan residues which com— prise all of the tryptophan present in turkey lysozyme. Amino Acid Copposition of EB? Tryptic Peptides from RCM—Turkey Egnghite Lysozyme Amino acid analysis of the purified tryptic peptides from RCM-turkey lysozyme was a powerful tool in this investi- gation. The results as shown in Table 6 served as (1) a test of homogeneity for purified peptides, (2) an unequivocal means of confirming the amino acid composition of the intact protein, and (5) a method by which amino acid sequence dif- ferences between the turkey and chicken lysozymes could be detected. An inspection of Table 6 shows that the molar ratios of amino acids in the purified peptides are nearly intergers, confirming the homogeneity of these peptides. This was the most stringent criterion of purity available because neither single peaks from column chromatography nor single bands TABLE 6. rAMINO ACID COMPOSITION*0F THE RCM-TURKEY LYSOZYME TRYPTIC PEPTIDES Amino Acid Peptides T-1 T-1+2 T-5 T-4 Lys His NH3 Arg CMC Asp Thr Ser Glu Pro Gly Ala Val Met Ileu Leu Tyr Phe +(1) 0.95 (1) 1.01 (1) 1.17 (1) 1.01 (1) 0.87 (1) 0.99 0.96 (1) +(1) (1) (1) (5) (1) (1) 2.07 1.94 0.99 (1) (1) (2) (1) (2) ‘ (1) Trp continued TABLE 6 — continued 65 Amino Peptides Acid T-6 T-7 T-8 T-9 Lys 0.97 (1) NH3 1.54 (1) 5.76 (5) 5.96 (5) 1.51 (1) Arg 1.05 (1) 1.04 (1) 1.04 (1) CMC 1.00 (1) 1.05 (1) Asp 0.74 (1) 5.00 (5) 4.10 (4) 1.91 (2) Thr 1.79 (2) 2.01 (2) Ser 0.98 (1) 0.94 (1) 1.92 (2) Glu 1.08 (l) 1.00 (1) Pro Gly 2.05 (2) 2.01 (2) 1.01 (1) Ala 2.06 (2) 1.08 (1) Val 0.94 (1) Met Ileu 1.98 (2) Leu 1.05 (1) 1.01 (1) Tyr 0.99 (1) 0.96 (1) Phe 1.80 (2) Trp (1) 1.6 (2) continued TABLE 6 - continued 64 Amino Peptides Acid T—10 T-11 T-12 T—15 T-14 Lys 1.01 (1) 1.00 (1) +(1) His NH3 5.48 (5) 0.82 (1) Arg 1.00 (1) 1.00 (1) CMC 5.09 (5) Asp 5.92 (4) 2.04 (2) 1.00 (1) Thr 0.87 (1) 0.99 (1) Ser 1.00 (1) 5.90 (4) 0.90 (1) Glu Pro 0.88 (1) 1.05 (1) Gly 1.09 (1) 2.97 (5) Ala 5.14 (5) 2.96 (5) Val 1.05 (1) 1.02 (1) Met 1.02 (1) Ileu 2.18 (2) 1.05 (1) Leu 5.08 (5) Tyr Phe Trp 1.4 (2) continued 65 TABLE 6 - continued Amino Peptides Acid T—15 T-16 T-17 T-17+18 Lys 0.95 (1) His 0.85 (1) NHa 1.86 (1) Arg 0.89 (1) 1.05 (1) 1.00 (1) CMC 1.05 (1) 0.90 (1) 0.99 (1) Asp 1.16 (1) Thr 1.06 (1) Ser Glu Pro Gly 1.22 (1) 1.07 (1) 1.00 (1) Ala 1.19 (1) Val 0.81 (1) Met Ileu 0.81 (1) Leu 1.00 (1) Tyr Phe Trp (1) *The amino acid compositions for the purified tryptic pep- tides are expressed as molar ratios based upon the average number of pmoles per residue as determined from amino acid analysis of their acid hydrolysates. The integral number of amino acid residues per peptide is given in parenthesis. Serine and threonine are uncorrected for destruction during acid hydrolysis. The integral tryptophan values were de- termined from enzymatic hydrolysis (Table 5). 66 from paper electrophoresis or paper chromatography are re- liable or sufficient proof of homogeneity. The ambiguities arising from amino acid analysis of the reduced and §fcarboxymethylated turkey lysozyme were resolved. Summation of the individual amino acid residues over all tryptic peptides (Table 6) confirmed the presence of 8 g-carboxymethylcysteine residues and 20 aspartic acid residues per mole of protein. All other amino acid values previously determined for the intact protein (Table 4) were verified by this procedure. Coupled with the results from the enzymatic hydrolysis of the tryptOphan containing pep— tides, these results establish that turkey lysozyme contains 129 amino acid residues--the same number found in the chicken lysozyme. The amino acid compositions of the tryptic peptides from RCM-turkey egg white lysozyme and RCM—chicken egg white lysozyme were compared. This revealed that an analogue for each of the reduced and S—carboxymethylated chicken egg white lysozyme tryptic peptides had been isolated. In addi- tion, two tryptic dipeptide analogues, T—1+2, and T-17+18 were also separated from the trypsin digest of RCM-turkey lysozyme. Furthermore, as shown in Table 7, this comparison demonstrated there were a minimum of seven amino acid se- quence differences between the primary structures of these two lysozymes and that these differences were distributed over six tryptic peptides. ll".\ ‘i. 4 ‘31 66 from.paper electrophoresis or paper chromatography are re- liable or sufficient proof of homogeneity. The ambiguities arising from amino acid analysis of the reduced and Sycarboxymethylated turkey lysozyme were resolved. Summation of the individual amino acid residues over all tryptic peptides (Table 6) confirmed the presence of 8 §fcarboxymethylcysteine residues and 20 aspartic acid residues per mole of protein. All other amino acid values previously determined for the intact protein (Table 4) were verified by this procedure. Coupled with the results from the enzymatic hydrolysis of the tryptOphan containing pep- tides, these results establish that turkey lysozyme contains 129 amino acid residues--the same number found in the chicken lysozyme. The amino acid compositions of the tryptic peptides from RCM-turkey egg white lysozyme and RCM-chicken egg white lysozyme were compared. This revealed that an analogue for each of the reduced and §fcarboxymethylated chicken egg white lysozyme tryptic peptides had been isolated. In addi— tion, two tryptic dipeptide analogues, T-1+2, and T—17+18 were also separated from the trypsin digest of RCM-turkey lysozyme. Furthermore, as shown in Table 7, this comparison demonstrated there were a minimum of seven amino acid se- quence differences between the primary structures of these two lysozymes and that these differences were distributed over six tryptic peptides. 67 TABLE 7. OBSERVED DIFFERENCES IN AMINO ACID COMPOSITION BETWEEN THE ANALOGOUS TRYPTIC PEPTIDES FROM RCM-CHICKEN AND RCM-TURKEY LYSOZYMES Tryptic Amino Acid Present Amino Acid Present Peptide in Turkey Lysozyme in Chicken Lysozyme T-1+2 Tyrosine Phenylalanine T-5 Leucine Histidine T-7 Histidine Glutamine T-10 Lysine Arginine T—15 Glycine and Valine and Alanine ASpartic Acid T—16 Histidine Glutamine 68 It is to be emphasized that certain sequence differences between analogous peptides cannot be detected by comparison of their amino acid contents as determined from acid hydroly- sates. Acid hydrolysis destroys the ability to recognize differences in amide content between peptide analogues whose composition is otherwise the same. For example, the substi- tution of aspartic acid for aSparagine would not be detected. Also, this comparative technique does not distinguish between peptide analogues of identical amino acid composition and varying sequence. The C-terminal tetrapeptide sequence of cytochromes p_is known to vary in this way (44). Sequence Determinations on Selected ROM-Turkey Egg White Lysozyme Tryptic Peptides To Show homology between two proteins, it is first necessary to establish their primary structures independent- ly. In the case of turkey lysozyme this would involve: (1) determining the sequence of each tryptic RCM-peptide-- including those whose amino acid compositions were the same as their tryptic RCM-chicken lysozyme analogues and (2) cor- rect alignment of the tryptic peptides through a set(s) of overlapping peptides. This exhaustive study was not under- taken and only certain of the tryptic RCM-turkey lysozyme peptides, known to differ in amino acid composition from their chicken lysozyme counterparts, were sequenced. gpptide T-1+2 (corresponds to residues 1 through 5 from chicken egg white lysozyme): The sequence of this 69 pentapeptide was established by three steps of the Edman degradation (Table 8). The specificity of trypsin placed arginine as the C—terminal residue after lysine had been shown to be N—terminal. The sequence is: Lys—Val-Tyr-Gly- Arg. Thus it is shown that tyrosine, rather than phenyl- alanine, is the third residue in tryptic peptide T-1+2 from RCM-turkey lysozyme. Peptide T-5 (corresponds to residues 15 through 21 from chicken egg white lysozyme): The sequence of the four N—terminal residues of this peptide was shown to be Leu- Gly—Leu-Asx-peptide by successive Edman degradations (Table 8). Treatment of peptide T—5 with carboxypeptidase B and A released 0.94 moles of arginine and 0.96 moles of tyrosine per mole of peptide (aliquot removed for analysis after 5.75 hours digestion with carboxypeptidase B and 1.00 hours digestion with carboxypeptidase A). A trace of aSparagine was also present at this time and small but progressively larger quantities were released as the digestion continued to 27.75 hours. The ammonia content of the peptide as de— termined from acid hydrolysis (Table 6), indicated that only one of the two aspartic acid residues was present as aspara- gine. No aspartic acid was released by carboxypeptidase treatment. Thus the sequence of peptide T-5 is: Leu-Gly- Leu-Asp-Asn-Tyr-Arg. This places leucine, instead of histidine, at residue 15 of tryptic peptide T—5 from RCM- turkey lysozyme. 70 neon ocfiEm omone .D.z an Uoumoflpcfl oum oocHEHouop no: mpflom OCHE< .0ocfla luopcs oum coflumpmnmop caspm onu mo mommum unouommap um Uo>OEoH ouo3 nUHnB mospflmou .mpnosuflumcou oHQmum onu Eoum nonmasuamu mm ospflmon Mom moaofii wo H0985: ommuo>m onu coms pommn weapon HMHOE mm commonmxo oum mosam> Had "ouoz 00.a am.0 06.0 00.H 00.0 a 00000 00.0 «mid ma.0 00.0 >0.0 0 00000 >0.0 H0.H qum «0.0 00.0 0 00000 00.0 mmqm 00.0 00.0 .a.z a 00000 mle m0 coaumpmumoa cmEpm 00.0 «0.a «0.5 No.0 00.5 >¢.a 0-9 00 coapnmoaeoo mmqm HH.0 00.0 00.5 00.0 0 00000 00.0 .wmqm 00.5 00.H 00.0 0 00000 00.0 H0.0 0a.a >0.a maid a 00000 N+Hle mo coflumpmumon cmfipm a0.0 ao.a AH.H ao.a 00.0 0+Hua no coaunmoaeoo Mme son Hm> eaw and mud nmz m>A Ufl0< ocflfié UoEHomnom mfimeamc< mle QZfi N+HIB meHBmmm ho ZOHB0EoH oHoB nonnB mospflmou 0000 0:050 omone .mpnoDUHumcoo oanmum on» Some poumasuaoo mm ospwmou mom moaoaj m0 quEsc ommuo>m onu com: @0009 mOHuMH MMHOE mm pommoumxo ohm mosam> Had "ouoz 00.0 00.0 00.0 00.0 0 00000 0040 00.0 00.0 .0.2 a 00000 0- 000. 70. m0 Sofiumpmnmon swapm 00.0 00.0 00.0 00.0 0-000.0ia 00 00000000500 00.0 00.0 00.0 00.0 00.0 00.0 0-0zuieua 00 00000000200 mm 00.0 00.0 00.0 00.0 ai0nou0ie 00 00000000200 00.0 00.0 00.0 mmqm 00.0 00.0 00.0 00.0 0 00000 00.0 00.0 .wmqm 00.0 00.0 00.0 00.0 00.0 0 00000 qum 00.0 00.0 00.0 00.0 00.0 00.0 00.0 a 00000 hie mo newumpmnmon cmfipm 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 00.0 0-0 00 00000000500 0:0 0H0. :00 000 use 000 00¢ 002 000 002000000 00000000 0004 00000 Mlunvlwle 92¢ NIB mmflHemmm m0 ZOHB¢Q4MOHQ ZfiZQm .m mam¢9 74 minutes digestion at 570. This extended the sequence of T-7 through the first eight N—terminal residues to: Phe-Glu— Ser-Asn-Phe—Asn-Thr—His-peptide. rAlso isolated from the chymotryptic digestion of peptide T-7 was the C-terminal tetrapeptide fragment, T-7-Cht-3 (Table 9). ElectrOphoresis of this peptide at pH 6.5 showed that it had a net positive charge with an Rf of 4.5 compared to serine. If aspartic acid had been present, the net charge should have been zero. Therefore, T-7-Cht-5 contains asparagine and not aspartic acid. Two stages of the Edman degradation showed the sequence of this peptide to be: .Ala-Thr—Asn-Arg (Table 9). Combined with the previous results, the sequence of peptide T-7 is: Phe-Glu-Ser-Asn-Phe-Asn-Thr-His-Ala-Thr-Asn—Arg. Thus it is established that residue 41 is histidine, rather than glutamine, in peptide T-7 from RCM-turkey lysozyme. Peptide T-15 (corresponds to residues 98 through 112 from chicken egg white lysozyme): The Edman degradation of peptide T-15 suggested the N-terminal tetrapeptide sequence was: Ileu-Ala-Ser-Gly-peptide (Table 10). However, dif— ficulty in removing serine at the third stage caused the results from the fourth stage of the Edman degradation to be less than satisfactory. Therefore, the peptide was cleaved with cyanogen bromide to isolate smaller fragments which might be more amenable to the Edman degradation. 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O“: O J ass: WTE Wl;| I335 5653 been OthlHEd by a imp ‘~_:r". .'t.-"':‘i'I'}O a ‘d 3 .ar gs. Ya- T 'T "7 {E 'y'rL-i -. {Bf-1 er I I '6; (3. Y" I 79 gem-3:. as P ‘3 Lab ‘revi usly DDS . man (4L1; w1_ ; '1L J «ice vzlva i T * ‘ YifO t0 ]YSLJ Le, H weter eke the r u 0 a *U ('0 J0 I: 3' P . 0596‘“ procedg ;B reduce reported 1801 and D n'l-Z U T— 0242? :00 ,... LOO i 159» d LU . ZYME ‘U‘: 20:73 U u f 0 751- ‘1 :R" 7; r tic L“$3’ 0‘ i-J ft‘TulLfi th0 1y 1”L f f H. vrdxrfl w iSLC;HL I 000 Fheyf- e, I: TT* 0:; :51 Fyi? y 3 5f :0 i e :“0r;t1039s 0 W r ”Sr 0? e 6 1trffpd S 11431.” mu- .Lt ‘ L ‘ ":1 .’ sine ‘ f‘eaéfif u e v» u :‘r‘. Irrir 1 V * um peé—Lj tiyat :33} 3.1: (3. “z. "r- on 8 Wm: oqu whi 8p 6 regent asima~c lysrxryme .(Tmb 1.. .5 - NIW StCDCtU“ ,0? . -6 1:1:- °Cl'O V assumpf1 F I I. aswnmémt for tL;~'r.-;¢ b-Se%g%n :39 l r. L .uGRAMS ?OF H. Figure 16: I my ““f--+m l||\|\|! 1‘1 Fl _ 0 _ _ _ mmo m:<_0 0 0 J 00 n0, ‘0 . J .. ; / ... [3.5. Q 01mg” M .D A _ . H a z . ; N O .. u 1 O_ O V / w 4‘ ...-0 .000. 09 00.0 9.0 8.0 mto 00.0 2.0 m§Huumm¢ucm¢uozonmuaumuav mus om om ma AmuauuwmucmumuancmHo5u>s|Hm> mus a mmq ale mGDUmem mo wucmsvwm mowpmmm HGQEDZ mMQHBmmm UHBQNMB HSVNOmNA MBHMZ 00w WMMZDBIEUm m0 WUZWDOMW QHU¢ OZHEd QmmOmOMQ .NH mgmdfi .owcfianmpcs mum mfiwuomma ouwns mom >mxusu msu ca ucmeMMflp mum nuflSB wowom OGflEM one .mmoconmm IMHU Umamcmflmmp may omamw>mu m5mo~mcm moaummm oflummuu man no mammamcm UHUM OGHEM ouwnB HNfi 6cm mp mmDUHmwM um mum mcoflummuxm ecu “mahnomma muH£3 mmm cmxoflno How Add.0fiv vamwmcmo an omcHEprmo mmonu mum mammnucmumm casuMB mmucmsvmm one .mflmmsucmumm cflnuHB ommoHocw uoc wmsofimwu whom ocHEm mmocp mo mucmsvmm map ownmflanmumm mamhamcm mucmsvmm .mEMNowMH hmxusulzom mo umwmflo aflwm>uu mcu Eonm mowumom Uflumhuu mfiwuomMH cwxofizo Izom comm Mom osmoncm cm mo coHumHomH map so comma ma wocmswmm oomomoum one "muoz % a Asqu many m 3.2192933 SIB ONH m AmumumaHumnaumHmlmnmuam>nmmdlunanmawv male m Ammqnozov male N AmhcrcmlmuavwadlxmuummumHmn oHDUoHOE thnomea oufln3 mmo monks“ onu cw uconomwfio on on :3onm moflum ocwEm one .m can a moDUHmou oumuumnsm coo3uon mHDUUo oE>N0m>H en omm>moao .m can .m .D .U .m .< ooaoan oum ouohumnsm oUHHMnUUMmmxon onu mnflmflumeoo mosoflmou opflumnuummocofi xflm one .BOHHom :H n3onm on omone .oHDUoHOE oamuumnam onu Ou mcwocon comonoen ca Ono Emficmnoofi oeueamumo on» a“ oo>HO>CH on On ooumHDumom mono cwmuuoo Uco mocon oowmasmfio “sow onu ousooum uonu omonu How umooxo oouuHEo coon o>on mcwono oowm one .osan cw n30nm ma oHDUoHOE oE>N0m>H on» mo aflono ocwumomeaom one .Uouoflmoo we oumuumnsm m can ofieuomwa oan3 mmo noxoenu Mo noduoo IHounfi on» nown3 CH neov mmflaafinm en mcwzono o Eonm ooumooo we ousmem mane .oEMNomea ouflnz mmo noxUHnU onwaaoumeuo mo Eoumoflp Hmnowmcofiflolooune .fid ousmem 87 The most noteworthy of the apparent amino acid sequence differences occurs at residue 101. X-ray crystallographic examination of chicken lysozyme complexes with certain competitive inhibitors has implicated the Asplol carboxyl group in substrate binding through hydrogen bonds to either substrate residue A or B (Figure 11). Substrate binding by turkey lysozyme has not been examined. Nevertheless, this enzyme is a better catalyst for hydrolyzing fl. lysodeikticus cell walls than is the chicken enzyme and therefore must bind some substrates efficiently. The effect of replacing Asplol by glycine on substrate binding can be viewed in two ways: (1) the carboxyl group of Asplol is involved in binding substrate to the chicken lysozyme, and the turkey lysozyme binds substrates somewhat differently; or (2) Asplol is not important for binding substrate to the chicken lysozyme. Certainly glycine cannot participate in this type of binding regardless of whether the conformations of the two enzymes are the same or not. We cannot know which of these possi- bilities is correct without further knowledge of the turkey lysozyme structure. It is evident that structural investigations of this nature on lysozymes from closely related species may have considerable significance, not only for lysozyme evolution, but also for deciding between various possible catalytic mechanisms. BIBLIOGRAPHY 1. 10. 11. 12. 15. 14. . Maass, D., and Weidel, W. Biochim. Biophys. Acta, . Weidel, W., and Katz, W. Z. Natruforsch, 16b, 156 . Dahlquist, F. W., Borders, Jr., C. L., Jacobson, G., . Inouye, M., and Tsugita, A., J. Mol. Biol., 22, . Tsugita, A., Inouye, M., Terzaghi, E., and Streisinger, . Howard, J. B., and Glazer, A. N., J. Biol. Chem., 244, . Jolles, J., Jauregui-Adell, J., and Jolles, P., . Jolles, P., Jauregui-Adell, J., and Jolles, J., .Lin, T., and Koshland, Jr., D. E., J. Biol. Chem., BIBLIOGRAPHY Strominger, J. L. and Ghuysen, J., Science, 156, 215 (1967). 78, 569 (1965). (1961). and Raftery, M. 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I., and Hindley, J., J. Mol. Biol., 15, 894 (1965). 90 Fieser, L. F., in A. H. Blatt (Editor), Organic Synthesis, Collect. Vol. II, John Wiley and Sons, Inc., New York, 1945, p. 55. Bailey, J. L., Techniques in Protein Chemistry, American Elsevier Publishing Co., New York, 2nd ed., 1967, p. 25. Bailey, J. L., Techniques in Protein Chemistry, American Elsevier Publishing Co., New York, 2nd ed., 1967, p. 21. Taniuchi, H. and Anfinsen, C. B., J. Biol. Chem., 241, 4566 (1966). Konigsberg, W., and Hill, R. J., J. Biol. Chem., 257, 2547 (1962). Konigsberg, W., in C. H. W. Hirs (Editor), Methods in Enzymology, Vol. XI, Academic Press, New York, 1967, p. 461. Dolpheide, T. A. A., Moore, S., and Stein, W. H., J. Biol. Chem., 242. 1855 (1967). Steers, E., Jr., Craven, G. R., Anfinsen, C. B., and Bethune, J. L., J. Biol. Chem., 240, 2478 (1965) Gross, E., and Witkop, B., J. Biol. Chem., 257, 1856 (1962). Smyth, D. G., Stein, W. H., and Moore, S., J. Biol. Chem., 258, 227 (1965). Hill, R. 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