Further investigations on the carbohydrate moiety of egg albumin

.PRELIMINARY NOTES

311

chondromucoprotein used to prepare Sample 9 showed the incorporation of only 7 t~moles of bound hydrazine per gram. If this is corrected for protein content, it would represent at the most IO/~moles of hydrazine per gram of chondroitin sulfate. These results are incompatible with an ester bond. This work was supported in part by a research grant from the U.S. Public Health Service. Department of Medicine, BONNm ANDERSON* College of Physicians and Surgeons, Columbia University, PHILIP HOFFMAN New York, N.Y. (U.S.A.) KARL MEYER x p. HOPFMAN AND K. MEYER, Federation Proc., 21 (I962) lO64. I H. MUIR, Biochem. J., 69 (1958) I95. a S. M. PARTRIDGE, H. F. DAVIS AND G. S. ADAIR, Biochem. J., 79 (I96I) 15. 4 S. M. PARTRIDGE AND D. F. ELSDEN, Biochem. J., 79 (196I) 26. 5 I. MALAWISTAAND M. SCHUBERT, J. Biol. Chem., 230 (I958) 535. 6 j, VoN K6HLER AND M. SCHWABE, Z. Naturforsch., I6b (I96I) 492, v M. B. MATHEWS, Biochim. Biophys. Act.a, 58 (I962) 92. s j. D. GREGORY AND L. RODIN, Biochem. Biol~hys. Res. Commun., 5 (I961) 430. 9 D. H. SPACKMAN, W. H. STEIN AND S. MOORE, Anal. Chem., 30 (I958) II9o. m M. M, RAPPORT, K. MEYER AND A. LINKER, J. Ant. Chem. Sue., 73 {I95I) 2 4 1 6 . It S. HIRANO, P. HOFFMAN AND K. MEYER, J. Org. Chem., 26 (1961) 5 0 6 4 . se C. H. W. HIRS, W. H. STEIN AND S. MOORE, J. Biol. Chem., 2 i i (1954) 941. IS E. JORPES, Biochem. Z., 204 (1928) 354. t4 B. H. NICOLET, L. A. SHINN AND L. J. SAIDEL, J. Biol. Chem., 142 (I942) 6o9. l~ O. O. BLUMENFELD AND P. M. GALLOP, Biochem., i (1962) 947.

Received April 12th, 1963 * Fellow of the Arthritis and R h e u m a t i s m Foundation.

Biochim. Biophys, Acta, 74 {I963) 3 o 9 - 3 I I REPRINTS OF PRELIMINARY NOTES

that have appeared in non-specialized issues since the last mucoproteins and mucopolysaccharides issue of Biochimica et Biophysica Acta PN

I243

Further investigations on the carbohydrate moiety of egg albumin* It has been suggested that the bond linking carbohydrate to protein in egg albumin is that of an N-(~-aspartyl)glycosylaminO, ~ of N-acetylglucosamine a-5. Partial acid hydrol~is of a purified glycopeptide from the protein gave rise to small amounts of a substance, containing glucosamine and asparticacid but no mannose, which behaved electrophoretically and chromatographically like 2-acetamido-I-~-(L-/~-aspartamido)z,2-dideoxy-D-glucose6.Further high-tension paper-clectrophoreticstudies (Whatman 3 MM, 4o V/cm, 6o rain,p H x.87) of partial acid hydrolysates (2 N HCI, I2 rain, lOG °) of the latter compound and of the glycopeptide from egg albumin have revealed a great similarityin the "fingerprint" obtained by staining the paper strip with the ninhydrin reagent described previously' (Fig. x). In this Figure, Spot 2 in each case (brown with ninhydrin) was given by a substance which had the same mobility as 2-acetamido-I-~-(L-/~-aspartamido)-z,2-dideoxy-D-glucose; Spot 3 (blue-purple) the same mobility as aspartic acid and Spot 8 (purple) the same mobility as glucosamine. Spot 8 was also Elson-Morgan positive in each case. The identity of the remainder * First publication: Biovhim. Biophys..4eta, 7I (I963) 505 .

Biochim. Biophys. Aaa, 74 {x963) 3II-314

312

PRELIMINARY NOTES

of the spots is at present unknown but it is possible that Spot 7 was produced by 2-amino-x-fi-(L-fl-aspartamido)-x,z-dideoxy-D-glucose. Spot 9, which occurs solely in that electropherograrn from the egg-albumin glycopeptide, might be produced by a similar deacetylated derivative but containing a further residue of glucosamine with a free amino group. Paper chromatography in phenol, of similar partial acid hydrolysates of both substances, has revealed in each case the presence of a substance staining brown with ninhydrin with an Rv value of 0.37, the same as that of the unhydrolysed model compound. Similar electrophoretic experiments with partial aci.d bydrolysates of 2-(L-flaspartamido)-2-deo~,-D-glucose 8 have indicated the presence of glucosamine, aspartic acid, a compound staining purple with ninhydrin with minimal mobility, and some of the original substance. Paper chromatography, in phenol, of a partial acid hydrolvsate of 2-(L-fl-aspartamido)-2-deoxy-D-glucose gave rise to a substance (Re----- o.o4) staining brown with ninhydrin, similar to the original substance. +

w

S

~, W W W

PBC= origin

I

1 2

S

S

S

7

8

9

o o o 3

4

5 6

8~

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~

c~

wS

S

S

W

W WW

B

Fig. 1. " F i n g e r p r i n t s " o b t a i n e d b y p a p e r electrophoresis ( W h a t m a n 3 MM, 4 ° V/cm, 60 min, p H 1.87) of partial acid h y d r o l y s a t e s (z N HC1, 12 min, Ioo °) of (A) egg-albumin glyeopeptide and (B) 2-acetamido-I-fl-(L-fl-aspartamido)-I,z-dideoxy-D-glucose. The strips were stained with ninhydrinL A purple colour is represented b y an open area, and brown b y a black area: W ----weak, S = strong.

By comparing ultraviolet-absorption measurements in water at 205 m/, with those of N-acetylglucosamine, we have found that the glycopeptide with aspartic acid as the only amino acid constituent contains 3.85 peptide bonds. Tiffs value was based on weight measurements of the lyophilised glycopeptide after correction for moisture content and absorption due to carboxylate ion 9. This value would be expected if there were one amide bond in the carbohydrate-protein linkage, together with the three N-acetylglucosamine bondsL We do not agree with the recent suggestion 1° of the presence of four glucosamine residues in the protein. The infrared spectrum of the glycopeptide (solid phase, NaCl) revealed the presence of absorption bands due to amide bonds and the anticipated absence of bands due to esters. Molecular-weight estimations of the pure egg-albumin glycopeptide have been carried out by ultraviolet-absorption measurements in ethanol-water (3° :7 o, v/v) after substitution of the free **-amino group (of the asparagine moiety) ,.~dth the benzyloxycarbonyl groupL The spectrum of this substance is represented in Fig. 2, showing three peaks, a, b and c. The mnlar extinction coefficients for the comparable three peaks exhibited by i-fl-(N-benzyloxycarbonyl-L-fl-aspartamido)-I-deoxyD-glucose 8 were 167 for Peak a, 205 for Peak b and IX9 for Peak c; for N-benzyloxycarbonylglucosamine I66 (a), 213 (b) and I I5 (c); for N-benzyloxycarbonyl-a-methylglucosaminide I68 (a), 215 (b) and 114 (c); for N-benzylox-ycarbonyl-a-amino-~hydroxyadipic acid n x73 (a), 206 (b) and I I 5 (c); for N-benzylo:,ycarbonylaspartic-fi semialdehyde 1~ x68 (a), 209 (b) and I t 9 (c). [l'he last compound was kindly given Biochim. Biophys. Acta, 74 (1963) 3 t t - 3 t 4

PRELIMINARY NOTES

313

by Dr. G. H. TAIT.) The molecular weight of the N-benzyloxycarbonyl glycopeptide was calculated from these data and from measurements of the absorbancy of a weighed quantity in a known volume of solution. Corrections were applied for the moisture content of the material and tot the small contribution made to the absorption by the glycopeptide itself. Values of 1710, 175o and 166o were obtained for Peaks a, b and c, respectively. The mean value for the glycopeptide unsubstituted by the benzyloxycarbonyl group is thus 157o, in good agreement with the value ot

04I u.

| 0.3 r

b

< 0.2

0.1 ~

I

I

I

I

I

3g 38 37 Wavenumber ( kcycles/cm)

41

40

Fig. 2. Spectrum of the N - b e n z y l o x y c a r b o n y l derivative of egg-albumin glycopeptide (2.31 m g / m l in I cm cell) in ethauol--water (3o: 7 o, v]v). T h e results were obtained using ~. U n i c a m SP-7oo recording spectrophotometer.

158o found by KAVERZNEVAAND BOGDANOVla by titration methods. The molecular weight required for a glycopeptide consisting of 5 mannose, 3 N-acetylglucosamine and I asparagine residues is I55I. A radioisotope-dilution method indicated the presence of 5 moles of mannose in the whole protein 14 in agreement .adth most estimates obtained by colorimetric assay. However, other workers, using colorimetric procedures, have stlggested the presence of 6 residues l°, 15 which might be explained in one of two ways: either the protein contains one single mannose residue not bound to the main heterosaccharide, which is very unlikelyl, or egg albumin from different strains of hen contains different amounts of mannose. Moreover, colorimetric procedures, which may be non-specific, have to be interpreted with caution. We acknowledge the willing technical assistance of Mr. V. BHOVROO and the financial assistance of the U.S. Public Health Service.

Department of Chemical Pathology, St. Mary's Hospital Medical School, London (Great Britain)

A. P. FLETCHER* R. D. MARSHALL A. NEUBERGER

1 p. G. JOttANSEN, R. D. MARSHALL AND A. NEUBERGER, Biochem. J., 78 (I961) 518. a R. H. NUENKE AND L. W. CUNmNGHAM, J. Biol. Chem., 236 (1961) 2452. s G. S. MARKS, R. D. MARSHALL AND A. NEUBERGER, Biochem. J., 85 (I962), 15 P. 4 V. P, BOGDANOV,E. D. KAVERZNEVAAND A. ANDREJEVA, Biochim. Biophys. Acla, 65 (1962) 168. 6 I, YAblASltINA AND M. MAKINO, j. Biochem. Tokyo, 5I (1962) 359. * Junior Research Fellow (Medical Research Council).

Biochim. Biophys. Acta, 74 (1963) 3 I I - 3 1 4

314

PRELIMINARY NOTES

6 G. S. MARKS, R. D. MARSHALLAND A. NEUBERGER, Biochem. J., 87 (1963) 274. 1 A. P. FLETCHER, G. S. MARKS, R. n . MARSHALLAND A. NEUBERGER, Biochem. J., 87 (x963) 265. s G. S. MARKS AND A. NEUBERGER, J. Chem. Soc., (1961) 4872. 9 H. ROSENZECK A~D P. DOTV, Proc. Natl. Acad. Sci. U.S., 47 (1961) 1775. to H. H6RMANN AND R. GOLLWlTZER, Ann. Chem., 655 (1962) 178. u W. G. LAVER, A. NEUBERGER AND J. J. SCOTT, J. Chem. Soc., (1959) 1474. 12 h. NEUBERGER AND G. It. TAIT, J. Cheryl Sot., (I962) 3963. to E. D. KAVERZNEVAAND V. P. BOGDANOV,Biokhimiya, 27 (I962) 273. 14 C FRANCOIS, R. D. L'~/I.ARSHALLAND A. NEUBERGER, Biochem. J., 83 (I962) 335. lS y . C. LEE AND R. MONTGOMERY,Arch. Biochem. Biophys., 95 (I96I) 263.

Received February I5th, 1963 Biochim. Biophys. Acta, 74 (2963) 311-314

PN 1234

An intermolecular defect of collagen in experimental lathyrism* The experimental disease, osteolathyrism, induced in a variety of animals by agents such as fl-anfinopropionitrile is characterized by mesenchymal deformities l, loss of tensile strength, and dramatic increases in collagen solubility-°. It has been proposed that the large pool of extractable collagen is derived from old insoluble fibrils transformed to an extractable state S. A contrary view holds that newly synthesized molecules are prevented from_ pol3anerizing to fibrils3. While the molecular dimensions, conformation and fibril-forming ability of lathyritic collagen seems not to be grossly altered ~ there is evidence for a failure of intramolecular cross-linking characteristic of the maturation process 4,5

500 f

I 400

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I I III rig ||!

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t

2

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,

3

,

4

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7

1

2

, 7 74,

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6

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Fig. I. Thermal gelation of NaCI purified normal ( . . . . (7 days at 37°).

)

and lathyritic

(. . . . . . )

collagen

* First publication: Biochim. Biophys. Acta, 71 (t963) 25 o.

Biochim. Biophys. Acta, 74 (t963) 3x4-3t6