Lactoperoxidase

ARCHIVES

OF

BIOCHEMISTRY

h,\iD

BIOPHYSICS

113, 540-547 (1966)

Lactoperoxidase VI. lmmunochemical

Studies

PETER Department

Rochester,

on Lactoperoxidase

2. AI,I&:K

from

AA-D MAILTIn-

the Milk

of Several

Species’

JIORRISON

of Microbiology, I’nir?ersity of flochester School of Medicine and Dentistry, Sew York, and the Department of Biochemistry, City of Hope Medical Center, Duarte, California Received

October

1, 1965

The production of specific allti-enzyme iu response to immunization with ptwified lactoperoxidase could be demonstrated by assay-s of peroxidase activity on the supernatant fluids and precipitates obtained from quantit,ativc precipit,in determinations. Spect,rophotometric analysis of specific precipit.ates formed in antibody excess for :I distinctive ant,igen marker showed all the hemoprotein added as antigen to be qrlantitatively precipitated by antibody. Laet.operoxidase isolated from the milk of rrlminaltts such as the goat. and sheep were examined for their cross reactivity with anti-bovine lactoperoxidase and found to be immlmochemically indistinguishable from cow’s milk lactoperosidase in their immunodiffllsion and qllantitative precipitin behavior. Bovine lact,operoxidase is immunologically distinct from bovine transferrin, ceruloplasmin, red protein, and lactollin as well as dog myeloperoxidase. Exposlwe of lactoperoxidase to proteolytic enzymes or 8 N urea does not result in any change detectable by imrnlltlodiffusiot~. The diffrlsion coefficient of bovine milk lactoperoxidase was estimated by irnmllllodiffusion to be 5.2 X 1OF cm SY.-~

Preparations of crude and purified factoperoxidase, a minor protein component isolated from VOW’S milk (l-6), have been employed in several studies as ant,igens (7-10). Antisera t)o the crude enzyme preparation have provided valuable reagents used t.o monit,or the fate of antigeni? cont~amnant,s during the course of lactoperoxidase purification (7, 8). Amisera to purified enzyme have facilit,ated the detection and isolation of lactoperoxidase from tissues other than the mammary gland (9, 10). Such monospecific sera permitted t,he immunological det8ect8ion and specific identificat,ion of lact’o-peroxidase in the salivary glands of steer as well as cows. Anti-lactoperoxidase has also been employed t’o demonstrate that a protein antigenically related but not, ident,ical to bovine lactoperoxidase is present in pig salivary glands (10).

In earlier studies, evidence for t.he speeificity of antisera to lactoperoxidase was provided by immunodiffusion and immunoelertrophoretic analyses as well as by t.hc use of chemical and physicochcmic~al met’hods t,o establish the purit’y of antigen (6-10). It, was the purpose of t.hc present study to establish t,he specifi&y of t,he inmumochennc~al rpactions of lact’operoxidase by use of quantitat.ivc m&hods. The immunochcmi(~al rea(~tions of the enzyme isolated from bovine, goat’ and sheep milk were compared and found t,o bc identical. In contrast,, the iron binding “red proteins” obtained from the same sources were also examined and were shown to be c~loscly related t’o one another but irrlnlurlologic:tlly distinct from lwtjoperoxidase.

1 This work was srlpported GM 089G4 from the I1.s.P.H.S.

-4ntisera. Crude and purified bovine Iactoperoxidase as well as crtlde bovine red protein isol:tt,ed

by research

grant

IMMUNOCHEMICAL

STUDIES

from fresh, raw cow’s milk were lrsed as antigens. Antisera were prepared in rabbits by intramuscular injectioll. One ml of complete Freund’s adjttvant (I>ifco Laboratories) containing 1 mg protein per ml was injected at weekly intervals for 3 weeks. Animals were bled 3 weeks after the last injection to provide antisera Ri3 and R76 lactoperoxidsse). Animals (nntiprirified bovine imm~~nieed against crrlde bovine red protein provided antisera Ki8, It70, Rll, and R12. Postimmallization bleedings obtained from these anmals after booster stimldation were pooled to provide anti-CBRP It7879 alld It1112. The preparat ioli of antiserrim Rl, an anti-crllde lartoperoxidase, has already been described (8). III order to obtaitl an antibody preparat,ion free of peroxidase activity dr~e to the presence of hemoglobin colltamination, t,he globldin fraction \vas obtained from antiserlun I(76 by ammollirml srdfatc precipii ation. Sertun diltlted 1 to 2 with saline was brollght to 50’;; salt saturation b> addition of an eqllal voltme of fully saturated ammonium srdfate solution (adjllsted to pH 7.0). Fractionation was carried out at 4°C. Precipitated globldins were washed twice ill cold, half-satItrated nmmolliurn sulfate. After dissolving in were reprecipitated. The saline, the glohrdins procedure \vas repeated lllltil a globlllill fraction was obtained free of significant absorption at 412 rnp as well as free of any ability to catalpse the oxidation of grlaiacol. ilntigrns. Prlrified lact,operoxidase was prepared from fresh raw cow, goat and sheep’s milk hy a simplified procedure employing iolr exchange chromatography and gel filtration (6,s). The milks rlsed were pooled samples obtained from several animals. The pllrified enzymes showed a 412/280 rnp absorption ratio of 0.93, 0.92, and 0.90, respectively, for the cow, goat, and sheep lact,operoxidases. A vallle of 114 for the millimolar extinctioll roelficient, at, 412 mp for the bovine lact,operoxidase (5, 6) was also asslimed for the gnat and sheep enzymes. This vnlne was employed to estimate the concentration of spectrophotometrically lactoperoxidase in antigen solrltions. Crklde red proteitl \vas obtained at the same time the lactoperoxidase was isolated and comprised those basic milk proteins which were removed by 0.5 dl anmonilml hydroxide after t,he elutioll of lactoperoxidase by 0.5 fiI sodirtm acetate (5, 8). Lactollin isolat,ed from cow’s milk (15) was generously provided by Dr. Morton I,. Groves. Bovine transferrin and ceruloplasmin were cornmercial samples pllrchased from Pentex Inc. I)og myeloperoxidase was kindly provided by Dr. S. Klebanoff. Samples of human milk and c,)lostr\Im and were obtained through the cooperation assistalIce of La 1,eche Leagrle of San Gabriel \‘nl-

541

ON LACTOPEROXIDASE

ley. Rabbit milk was obtained hy thawing frozen lactating rabbit mammary glands purchased from Pel-Freez Biologicals Inc. (Rogers, Srkansas). Extracts of rabbit mammary and rat salivary glands examilred by immurrodiffusion were prepared by the bldfer-cholate extraction procedure already described in detail (10). Quantitative precipitin determination.s. A ynarltitative precipitin determination was carried oat as described by Kabat and Meyer (lo), employing 1.0 ml of a solution of antibody globulin prepared from antiserllm 1~76. The total nitrogen of washed specific precipitates was determined by the Markham micro-Kjeldahl method (16). Peroxidase activity of washed specific precipitates was determined by stlspending insoluble complexes in 2.5 ml of 0.02 dl phosphate bluffer (pH 5.9) and assaying 0.1.ml aliqlmts of t.he sllspensiolr for enzymatic activity. Precipitin clirves for cow, goat and sheep lactoperoxidases were carried out with anti-bovine lactoperoxidase employing 0.5 ml of antiserrlrn 1:7(i. Washed specific precipitates were dissolved ill 0.05 ml of 0.5 ~1; sodillm hydroxide which was then diluted to 1.5 ml with 5 111sodium carbonate. The opt,ical density of dissolved precipitates was determined at 280 and 412 mp. Recovery of hemoprotein from specific precipitates was estimat,ed from the absorption at, 412 mp. A millimolar extinction coefficient of 101 was employed for the lactoperoxidase in 5 &1 sodirml carbonate (pH 11.3). Assay of lactoperoxitlaae actiaity. Peroxidase activity was determined essent,ially as described by Maehly (5, 17). The rate of oxidation of guaiaco1 was followed spectrophotometrically. One lmit of peroxidase activity was defined as that amolmt of enzyme giving a AOD at 470 mp = 0.001 per minute lmder the assay conditions described (17). Immunodiffusion

and

immlLnoelectropho~e.~i~.

Immunoelectrophoresis and immunodiffusion were carried orit, by procedures already described in detail (8). The lact,operoxidase-antilactoperoxidase band could be readily identified in diffrlsion blue benzidine positive plat,es b> its intense staining. Plates t horollghly washed iI1 saline were stained for peroxidase activity by immersion in a solution of 25 ml ethanol satlirated with benzidine hydrochloride, li ml water, 2 ml glacial acetic acid, 0.5 ml of 309; hydrogen peroxide, and 0.5 ml pyridine (18). The diffusion coeflicient of plu-ified bovine lactoperoxidase was estimated immunochemicall~ by the method of Allison and Humphrey (19). When equivalent amormts of lactoperoxidase and specific antibody were diffused from rectangular trollghs at right angles to one another, a band was

542

ALLEN

AND

MORRISON

FIG. 1. (A, B, C): Immunodiffusion analysis. Antiserum against purified bovine lactoperoxidase from cow’s milk (R73, anti-BLP) and anti-crude bovine red protein (anti-BRP R1112) placed in center wells. Peripheral wells contain crude bovine lactoperoxidase (CR BLP), purified lactoperoxidase (LP1114), bovine red protein (BRP), bovine serum (Bov. Ser.), bovine eeruloplasmin (B. cer.), bovine transferrin (B. t.r.), laetoperoxidase and red protein from goat’s milk (GLP) and (GRP), whole raw sheep’s milk (SM). FIG. ID. Immunoelectrophoresis of partially purified lactoperoxidase obtained from ion exchange chromatography of goat’s milk (Prep. G8). Ant,igen placed in center well. Puri fied lactoperoxidase from cow’s milk placed in upper trough labelled Bovine LP; antiserum to crude bovine lactoperoxidase (anti-CLP Rl) in lower trough. obtained with a slope of 1.17. The diffusion coefficient (D,) of lactoperoxidase isolated from cow’s milk was calculated from the expression 1.17 = Xb/X, = (DO/Da)““, where Xa and X, are the distances of a point of precipitation from the antibody and antigen troughs, respectively, and Db is the diffusion coefficient of rabbit anti-lact,operoxidase. The value of Db was taken to be equal to 3.8 X 1@7 cm2 set-i, the value of the diffusion coefficient of rabbit gamma globulin. Treatment with proteolytic enzymes or urea. Purified bovine lactoperoxidase was incubated with the proteolytic enzymes trypsin, chymotryp-

sin or Nagarse.z The amount of proteolytic enzyme was adjusted so that it was l’i: of the weight of lactoperoxidase. Treatment was carried out, in 0.1 M phosphate buffer (pH 8.0) for 24 hours at 25”27”C. Trypsin soy bean inhibitor was then added to the tryptic digest in an amount equal to the weight. of trypsin employed. Chymotryptic and Nagarse digests were quantitatively dilut.ed about a-fold with 0.1 M phosphate buffer (pH 7.4) and examined by immunodiffusion. One mg -. .._ 2 Nagarse, a lyophilized, crystalline bacterial protease obtained from Nagarse and Co., Ltd.. Osaka, Japan.

IMMUNOCHEMICAL

STUDIES

0.1%

0.2%

52%

58%

4

ON LACTOPEROXIDASE

543

1% Added Activity Remaining In Supernate % Added Activity Found In Precipitate

+

I

pg, BOVINE LACTOPEROXIDASE

ADDED

FIG. 2. Quantitative precipitin curve obtained with purified bovine lactoperoxidase employing 1.0 ml of a solut,ion of the globulin fraction prepared from antiserum R73. Assays of lactoperoxidase activity recovered in precipitates and from supernatants of reaction mixtures are shown in upper portion of figure. per ml sollltions of lactoperoxidase were also made 4 and 8 ll4 with respect to urea and allowed to stand 48 hours at room temperature. Aliqllots were removed at various times during treatment, dialysed against saline and examined by immunodiffusion and immunoelectrophoresis. RESULTS

Antisera R’76 and R73 obtained from rabbits immunized with purified cow’s milk lactoperoxidase give only a single band of precipitation when diffused against crude or purified enzyme (Figs. lA, lB, and Fig. 5). That the antibody present in serum R73 is directed against lactoperoxidase is shown by the ability of the globulin fraction obtained from this serum to precipitate essentially all of the enzymic activity added as antigen. As shown in Fig. 2, in t’he region of antibody excess only 0.1 and 0.2% of the peroxidase

activity added remained behind in supernatant#s while 52 and 58 % of the added acti\,ity could be found in insoluble antigenantibody complexes precipitated. The failure of excess antibody to effect a complete inhibition of catalytic activitjy permits specific identification of the lactoperoxidase-ant’ilactoperoxidase system in immunodiffusion and immunoelect’rophoresis. Thus when crude enzyme was electrophoresed and arcs of precipit’ation developed with antiserum Rl, only a single arc took on an intense blue staining upon exposure to the benzidine-peroxide reagent, used to detect peroxidase activity (Fig. 3). Antisera R73 and R76 gave only a single band in immunoelectrophoresis when diffused against raw whole milk and crude or purified lactoperoxidase. This arc gave a positive peroxidase stain.

544

ALLEN

AN11 MORRISON

Fro. 3. Immut~oelec~rophoresis of 30, 59, and 118 pg of lactoperoxidase obtained from cow’s milk, in wells labelled A. B, aud C, respectively. .4ntiserLun It1 in troughs. Arcs of lactoperoxidase-antilactoperoxidase precipitation stained bltle 1)~ benzidinc-peroxide stain.

A specificity for lactoperoxidase is fmther demon&rat,ed by hemoprot’ein estimations on specific precipitates formed in the region of antibody excess with 0.5 ml antiserum R76. Hemoprot’ein determinations were carried out on specific precipitates formed with less than 200 pg cow, goat, and sheep lac$operoxidase. Antigen recoveries based on hcmoprotein estimat8ions on specific: preripitates are listred in Table 1. As es& mated ;;pectrophotjometjricaIly, X8-1 10 %I of t,he hemoprotein added as antigen u-as precipitated by ant8iserum R76 prepared against, the bovine milk enzyme. Lact,operoxidase obtained from sheep and goat’s milk was examined by immunodiffusion for cross reactivity with antiserum R76. As shown in Fig. 1B and Fig. 5, the cow, goat, and sheep antigens each give single bands of precipit,at)ion which show caomplete fusion wit(h one another. The immunological identit,y of t)he cow and goat,

milk enzymes could also be demon&rated by of modified immunoelecthe tjechnique trophoresis described by Oaserman (20). One arc of precipitation given by t,he interaction of partially purified goat, lactoperoxidase w&h antiserum Rl (anti-crude enzyme) is seen in Fig. ID, to fuse complet’ely with the band given by purified cow’s milk enzyme diffused from the upper trough. Dat#a obtained from quantitat)ivc precipit’in detwminations are in agreement with the immunodiffusion findings showing the goat, sheep and POW enzymes to be immunologically indistinguishable. As shown in Fig. 4, the lartoperoxidases isolated from the milk of cows, goats, and sheep are comparable on a weight, basis in their abilit,y to remove a&-lactoperoxidase from serum R76. All 3 antigens show a comparable quantitative precipit,in behavior and are immunochemically indistinguishable from one another.

IMMCNOCHEMICAL TBBLE HEMOPKOTEIN

STUDIES

I

I~ECOVERIES

ox

SPECIFIC

PKECIPITATES~ Source of lactoperoxidase

Cow’s

milk

Goat’s

milk

Antigen

addedh (PLP)

59 99 50 83 125

Sheep’s milk

Antigen

recoveredC (Pd

Z Antigen

Recovered

52 99 55 76 123

88 100 110 92 99

a Precipitates formed in region of antibody excess with 0.5 ml antisertlm R76 prepared against bovine milk enzyme. h Amorlnt of antigen calculated from millimolar extinct,ion coefficient of 114; 1 pg lactoperoxidase/ml is equivalent to 0.0014 011 rlnits at 412 mr. c Antigen recovered calculated from 01) at 412 mp corrected for change in absorbancy shown bJ lactoperosidase in 5 111carbonate.

2 400 Q ‘F 2\ 300 2 s q

200

OCOW'S MILK LACTOPEROXIDASE ciGOAT'SMlLK LACTOPEROXIOASE

Q

.SHEEP'SMlLK

LACTOPEROXIOASE

L too

200

300

I 400

kg. LACTOPEROXIDASE ADDED FIG. 4. Quantitative precipitin cllrves obtained with ptu-ified lactoperoxidase of cow’s, sheep’s and goat’s milk employing 0.5 ml ant,iserum R76. Optical density of specific precipitat,es determined at 280 mu in 5 X carbonate solution.

That’ lactoperoxidase is immunologically distinct from other m&alloprot,cins found in bovine milk or serum is demonstrated by immunodiffusion analysis. The bovine an& gens, red protein, ceruloplasmin and transferrin, failed to show any reaction mit#h anti-lactoperoxidase serum R73 (Figs. IA and 1B). Whole bovine serum also showed no

ON LACTOPEROXIDASE

545

reaction. The failure of goat, sheep and con lactoperoxidase to give a band of precipitation when diffused against anti-bovine red protein R79 (Fig. 5) further demonst,rates that red prot)ein and lactoperoxidase are antigenic*ally distinct. Additional preparat,ions which were cxamined by bot,h the Ourhterlony and l’reel methods showed no reaction with ant’i lactoperoxidasc. These included extract)s of rabbit mammary and rat, salivary glands, rabbit milk, 30 samples of human milk 01 cbolostrum as well as wystalline dog mycllopcroxidase and crystalline bovine lactollin. As determined by immunodiffusion and immunoclec%rophoresis no change in im. munoc~hemic~al behavior of bovine la&peroxidase could be det&ed as a result ot tJreat.ment wit,11 trypsin, chymotrypsin 01 Sagarse. Simil:\rly, exposure to 4 and 8 31 urea at ‘27°C for 4S hours did not, result in my change detec%ablc by immunodiffusion. The diffusion coefficient of lactoperoxidasc isolat’ed from cow’s milk as eetimat8ed by immunodiffusion was found t,o be 5.2 X IO-’ cm SC(*-~. Preparations of red prot,ein obtained from goat and sheep’s milk were diffused against, antiserum R1112 prepared against bovine red protein. Both sheep and goat’ antigens showed the format8ion of a strong band of precipitat’ion (Fig. 1C). While some preparat)ions of bovine red protein showed a caomplete fusion of bands, others showed a slight, very faint spurring over the band given by the sheep and goat antigens. DISC1JSSION

Immunizat,ion of rabbits with preparations of crude or purified lactoperoxidase obtained from raw cow’s milk gives rise to the production of specific ant,ibodies. The presenw of anti-lactopcroxidase in immune sera could be demonstrat,ed in several ways. Assays of cat#alyt,ic ac%ivitSy on supcrnatant8s and specific precipitates obt#ained during t#he course of quant,itatjive precipitation (Fig. 2) showed that little or no peroxidase remained in the supcrnat,ant,s while more than 50% could be recovered in suspension of washed insoluble specific precipitates. Quantitative rec*overy of peroxidase ac%ivity from enzymo-

546

ALLEN

AND

MORRISON

FIG. 5. Immunodiffusion of lactoperoxidase. Purified lactoperoxidase prepared from cow’s milk (B), sheep’s milk (S), and goat’s milk (G) placed in peripheral wells; antiserum to bovine lactoperoxidase (K76) and antiserum to crude bovine red protein (R79) placed in center wells. Plate st,ained with Buffalo Black dye. antienzyme precipitates would noD necessarily be expected since partial inhibit’ion of enzymahic activity by antibody has been found for other enzyme antigens arting on low molecular weight substrates (cf. 21 for review). The findings wit’h lactoperoxidase are comparable to data obtained by Deutsch and Seabra (22), with t’he crystalline beef liver catalase-anticatalase system. Assays of catalase activity on suspensions of catalase-anticatalase specific pre cipitates showed inhibition of 3560% of the original act’ivity. A specific&y directed against lactoperoxidase could be further established by hemoprot,ein estimations on specific precipit’ates. Since lactoperoxidase contains heme as an integral part, of it’s structure, this distinct#ive constituent could be employed to estimate quantitatively amounts of antigen precipitated. Spectrophotomet’ric analysis of specific precipitates for hemoprotein has been previously employed by Orlando, Levine and Kamen (23) to establish t,he specificity of antibody produced in response to purified Rhodospirillum rubrum, heme protein. As shown in Table I, 88-100 % of the hemoproOein added as ant,igen was preripi-

t’ated by antibody and could be recovered from antibody complexes formed with lactoperoxidase from cow’s milk. Within experimental error, t,he heterologous goat and sheep ant,igens were also quantit#atively recovered. Immunodiffusion and quantitative precipitin data (Figs. 4 and 5 and Table I) showing cow, goat and sheep lact,operoxidases to be immunochemically indistinguishable from one another, stand in marked contrast to findings obtained wit,h the pig enzyme (10). Peroxidase isolated from porcine salivary glands was found to have a lower isoelectric point and was shown by immunodiffusion to be antigenically related but not. identical to lact’operoxidase isolated from bovine milk or bovine salivary glands (9, 10). Antigenic relationships are thus consistent with phylogenet,ic relationships among these species (24). Immunological relationships found among the lactoperoxidases of these various species is comparable to that reported for other milk proteins. The cY-la&albumins as well as the P-lactoglobulins of ruminants such as cows, sheep and goats were shown by ,Johke et al. (25) to be very closely relat’ed t)o one anot,her but,

IMMUNOCHEMICAL

STUDIES

immunologically distinct from those of nonruminant’s such as the pig. The diffusion coefficient of cow’s milk lact’operoxidase estimat,ed by immunodiffusion to be 5.2 X 1O-7 cm WC-’ is in good agreement with the value of 5.95 X 1CF7 cm set-’ obtained by Theorell and Pederson, who used independent methods (3). It has been suggested that the ironcont’aining “red protein” which is always isolated from milk with lactoperoxidase might be a derivative or degradation product of the enzyme (4). As determined by immunodiffusion, this protein from t,he cow, goat, and sheep milk is clearly immunologically distinct from t’he enzyme. Moreover, treat#ment#of the enzyme wit.h urea or proteolytic enzymes resulted in no change in the immunological properties of the enzyme. Thus, t#woproteins are clearly distinguishable. The results of t,reatment of lactoperoxidase with 8 M urea contrasts markedly with the results obtained with oc-amylase (26). oc-Amylase showed a marked change in immunodiffusion properties while lactoperoxidase showed no change after t’his treatment. As det,ermined by immunodiffusion, preparations of red prot’ein obtained from cow, goat and sheep milk are very closely related. Whether the faint spurring shown by some bovine preparat’ions over the band given by t#he goat and sheep antigens is attributable to a t.race antigen& contaminant, or has some other basis remains t)o be established. REFERENCES 1. THEORELL, H., AXD AKESON, A., Brkiv. Kemi Minera/. Geol. li’B, No. 7 (1943). 2. THEORELL, H., AND PAUL, Ii. G., Arkiv. Kemi Mineral. Geol. 18A, No. 12 (1944). 3. THEORELL, H. AXD PEDERSON, K., in “The (A. Tiselius, ed.). Almqvist Svedberg” and Wiksells, Uppsala (1944). 4. POLIS, D., AND SHMUKLER, H. W., J. Biol. Chem. 201, 475 (1957).

ON LACTOPEROXIDASE

547

5. MORRISON, M., HAMILTON, H. B., AND STOTZ, E., J. Bid. Chem. 228, 767 (1957). 6. MORRISON, M., AWD HULTQUIST, D., J. Biol. Chem. 238, 2847 (1963). 7. ALLEN, P. Z., AXD MORRISON, M., Federation Proc. Abstr. 22, 264 (1963). 8. ALLEN, P. Z., AND MORRISOS, M., 4rch. Biothem. Biophys. 102, 106 (1963). 9. MORRISON, M., AND ALLEN, P. Z., Biochem. Bioph,ys. Res. Commun. 13, 490 (1963). 10. MORRIS~X, M., ALLEN, I?. Z., BRIGHT, J., BPI’DJAYASIXGHE, W., Arch. Biochem. Biophy.s. 111, 126 (1965). 11. S~RENSEX, M., AXD SORENSEN, S. P. L., Compt. Rend. I’rav. Lab. Carlsherg 23, 55 (1939). 12. GROVES, b. I,., J. Am. Chetn. See. 82, 3345 (1960). 13. GORDON, W. G., GROVES, M. L., AND BASCH, J. J., J. Am. Chem. Sot. 2, 817 (1963). 14. DERECHIN, S. S., AND JOHNSON, P., Sature 194, 473 (1962). 15. GROVES, M. L., B.~scH, J. J., AND GORDON, W. G., Biochemistry 2, 814 (19G3). 1G. KABAT, E. A., ANU MAYER, M., “Experimental Immunochemistry” Thomas, Springfield, Illinois (1961). 17. MAEJILY, A. C., in “Methods Analysis”

(D.

Glick,

ed.),

of Biochemical Vol.

1, p. 386.

(1954). Johu Wiley & Sons, New York. 18. CONNELLY, J. L., MORRISON, M., AND STOTZ, E., J. Biol. Chem. 233, 743 (1958). 19. ALLISON, A. C., AND HUMPHREY, J. H., Immunology 3, 95 (1960). 20. OSSERMAN, E. F., J. Immunol. 84, 93 (1960). 21. CINADER, B., Ann. A(‘. Y. Bead. Sci. 103, 495 (1963). 22. DE~~I~cH, H. F., AXD SEABRA, A., Chew

J.

Biol.

214, 455 (1955).

23. ORLAXDO, J. A., LEVIXE, L., BND KAMEN, M. D., Biochim. Biophys. Acta 46, 126 (1961). 24. SLOAN, R. E., JENNESS, R., KENI-ON, A. L., AXD REGEHAR, E. A., Corn. Biochem. Physiol. 4, 47 (1901). 25. JOHKE, T., HBGEMAN, E., AND LARSON, B. L., J. Dairy Sci. 47, 28 (1964). 26. MORRISON, M., ALLEN, P. Z., BRIGHT, J., AND JAYASINGHE, W., 111, 126 (1965).

Arch.

Biochem.

Biophys.