The Separation and Properties of a Mucoprotein from a Lymphatic Organ' BY E. L. Hsss, LY. XYALAAND AILENEHGRR.\KEN RECEIVED APRIL 14, 195.3
A method for t h e separation of a high niolecular \\right muroprotcin from bovinc p&tinc tonsils is givcii. Chemical and physical data, including sedimentation and diffusion coefficients, intrinsic viscosity, partial specific volume, isoclcctric point, electrophoretic behavior, ultraviolet absorption charxteristics, anti nitrogeli, phosphorus, hexose, hexose:mirie, hexuronic acid and lipid contents are reported
Introduction Little is known of the composition or properties of the proteins of lymphoid tissue. Xucleoproteins have been separated from the thymus2-* and characterized5t6as nucleohis tones. Xore recently Abrams and Cohen' reported electrophoretic and chemical analyses of the human tonsil and calf thymus extracts. Since these workers did not report carbohydrate analyses, no information is available regarding carbohydrate content of their extracts. Several workers have isolated carbohydrate containing protein fractions from animal Mucoprotein I from human serum is the better characterized one due to the careful and complete work of Winzler and his associates.l1S1? Mucoproteins have also been prepared from gastric saliva'; arid urine.l6 Urinary mucoprotein and ovoinucoid have been better characterized than the others due to the I-ccent work of Tarnm and IIorsfal116and of Fredericq and Deutsch.I7 T h e conditions of separation, the yield, and some chemical and physical properties of a mucoprotein isolated from bovine palatine tonsils will be dis-
Experimental palatiric tonsils were securctl from freshly sl;iuglit c . r d arii~nals.'~After excision the gland\ \vet-e iiiinicrwi irnrnediatcly in cracked ice. T h e reinov:ll of n i u r l e a l i i 1 riteriial connective tissue was accomplishcrl withiti :t fciv hours after slaughter. Each bovine tonsil consists of tiro lobes of lymphoid tissue folded over a mucosal surfacc colihviiic:
tinuous with the pharyiigeal mucosa. The tonsilar mucosa was exposed and scraped in order t o rciiiove mucus and any infected areas. The cleaned tonsil w i ~ sthen washed with cold 0.13 iV NaC1, frozen and stored a t -56' until used. Electrophoresis determinations were run in a conventional Tiselius assembly equipped with a Philpot-Svensson schlieren optical s y ~ t e m . ' Buffer ~ ~ ~ ~ compositions, ionic strength, pH and the potential gradient employed varied throughout this study and will be stated in each individual case. The experiments were performed in an all-glass cell assembly at a t y i p e r a t u r e of 0.4". Couductivities were measured at 0.0 ; the mobility values therefore obtain a t 0.0°.2i The conductivity of the buffer was used.for mobility calculations. The values reported were calculated front tracings of enlargements of photographs of the descending patterns in the usual Intrinsic viscosity measurements were ttiade in 0.1 .V sodium acetate (pH 7.3) a t 37.0 i- 0 . 0 3 O in il Fisher-Irany viscometer, accordiiig t o methods discusscd previously,*3 except that the mean velocity gradient 6 varied from 21 to 70 set.-' in the present work. Sedimeiitation constants were measured in the standard 4 - t u r b i n e centrifuge in the laboratory of Prof. J . 51' Williams a t the University of Wisconsin. A speed of 50,400 r . p .m . was employed and the average temperature was 24 Values of the sedimentation constant were determined from the slope of log xi '0's. tp, where xi is the distance of the axis of rotation from the boundary and t; is the time of centrifugation corrected for the viscosity variation of the solvent medium with temperature. The sedimentation coefficients have been reduced t o the value in water a t 20' (s201\)21,23 and are recorded in Svedberg units. Diffnsion nieasurcnients were made in the electrophorcsis assembly described above. Duplicate determinations rcsult from using both limbs of the cell. The boundaries iverc sharpened according to the method of Kahn and PolThe height aiid arca, the second moment, the inflectioii point and the successive analysis methods were used for calculatiotis. 27 The zero time correction suggested 'ny K d i n and Polsotit6 and by Longsworth28 was applied to thcbc nie;isureiiicnt,. Thc diffusion experinicnts were pcrformed ;it 0,4', corrected for tcmper-ature and viscosity values according to Ihc q u a effects, aiiti rrportvcl as Dt,,,\ tioii,29
11) Presented before t h e Division of Biological Chemistry o f (tic American Chemical Society a t t h e 121st Nalional Meeting, April 3 , 1952, in Xilwaukee. (2) L. Lilienfeld. Z. p h y s i o l . C h e r n . , 18, 1 7 3 (1893). 3 ) I. Bang, Beilr. C j e m . Physiol. Path., 5, 317 (100.4), 1 R. 0. Carter and J. Hall, THIS J O U R N A L , 62, 119-1 f L'IkO] Gosting aiid L1orriszg atid Lotigs\vorth3° have s1ioir.n that J R. 0. Carter, ibid., 63, 1060 (1941). these corrections arc rcasoiiahly valid. ) J . L. Hall, i b i d , 63, 794 (1041). Partial specific voluiiies were calculatcd according to the I A. Abrams and P. P. Cohen, J . Bioi. Cl;c,ii , 177, 43!1 i I$i.&!JJ. method describcd by Kraenicr2*from a plot of 5 ~ i lz's. I'nhere J L. 1'. Hewitt. Biochem. J . , 31, 1834 (10373. WI is the weight fraction of protein and 'L the specific volume I C . Rimington, i b i d . , 34, 931 (1940). of the solution. Thc specific volumes were dctcrmined a t ' H I S J O V R N A I'73, . 381fi < 1 0 ~ 0 , . r , J. TV Mchl a n d R J . m'inzler .I I { ! , >i / k c i n . , (19) J . S I.r ('olloid Cliem., 61, 816 of slaughter. T h e resulting cotitaminatioii w i t h blood serum proteins (1917). ititrurluces problems which are largely a\.oided by the use of tomils ( 2 7 ) H S c u r a r h C ' h u m . Ke;ss., 3 0 , :%37 \ l $ l i 2 ) , from kosher-slaughtered h r e f which a r e s i r t u a l l g Iree o f blood. We ) I, CY.~.~>tlL!s\V
