Isolation and characterization of two structural isomers of N-acetylneuraminyllactose from bovine colostrum

BIOCHIMICA ET BIOPHYSICAACTA

I

BBA 25674 ISOLATION AND CHARACTERIZATION OF TWO STRUCTURAL ISOMERS OF N-ACETYLNEURAMINYLLACTOSE FROM BOVINE COLOSTRUM MICHAEL L, SCHNEIR* A~D MAX E. RAFELSON, JR.** Department of Biochemistry, Presbyterian St. Luke's Hospital, Chicago, Ill. (U.S.A.) and Department of Biological Chemistry, University of Illinois, College of Medicine, Chicago, Ill. ( U.S.A .) (Received June 28th, 1966)

SUMMARY A method has been described for the isolation of two structural isomers of N-acetylneuraminyUactose from bovine colostrum. The two isomers were shown to be N-acetylneuraminyl-(2 -~ 3)-fl-D-galactopyranosyl-(i -, 4)-D-glucopyranose and N-acetylneuraminyl-(2 -~ 6)-fl-D-galactopyranosyl-(i -~ 4)-D-glucopyranose.The latter isomer had not previously been reported to occur in bovine milk or colostrum. Although both isomers were cleaved completely b y the purified neuraminidase (EC 3.2.1.18) from influenza virus, the isomers differed in their pH optimum, Km and V. The mode of linkage of N-acetylneuraminic acid to lactose appears to influence both the rate of formation and breakdown of the enzyme-substrate complex.

INTRODUCTION NAN-LAC is a suitable substrate for the enzyme neuraminidasO (N-acetylneuraminate glycohydrolase, EC 3.2.1.19) as it is one of the simpler compounds available containing NANA in glycosidic linkage. NAN-LAC was first isolated from rat m a m m a r y gland by CAPUTTOAND TRUCCOz, and later in more substantial yields from bovine colostrum b y KUHN AND BROSSMER~,~8. Less complicated isolation procedures were subsequently developed ~-6, including a 7-step procedure from this laboratory ~. Although our NAN-LAC preparations appeared to be 3-NAN-LAC, this study showed them to be heterogeneous and to consist of 3-NAN-LAC, 6-NAN-LAC and an unidentified NANA-containing oligosaccharide. NAN-LAC isolated by the methods of KUHN AND BROSSMERa,~ and of HEIMER AND MEYER4 has also been reported to be heterogeneousS, 9. The present study describes the isolation and characterization of the two isomers of NAN-LAC and their behavior as substrates for the neuraminidase of influenza virus. Abbreviations : NAN-LAC, N-acetylneuraminyllactose of unknown or undesignated structural configuration; 3-NAN-LAC and 6-NAN-LAC, the 2--~ 3 and z-+ 6 isomers of NAN-LAC, respectively; NAN, N-acetylneuraminic acid, * Present address : Department of Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pa. (U.S.A.). John W. and Helen H. Watzek Memorial Chairman of Biochemistry, Presbyterian St. Luke's Hospital, Chicago, Ill. (U.S.A.). * *

Biochim. Biophys. Acta, 13o (1966) i-xi

2

M. L. S C H N E I R , M. E. R A F E L S O N , JR.

MATERIALS A N D M E T H O D S

N euraminidase preparations Purified preparations of these enzymes were isolated from influenza virus according to the method of WILSON AND RAFELSON1°.

Paper chromatography Two solvent systems were used for descending chromatography on W h a t m a n 3MM paper; (A) ethyl acetate-pyridine-acetic acid-water (5:5 :I :3, v/v) ; (B) ethyl acetate-pyridine-water (2:I:I, v/v). Zones were detected with dips of periodatebenzidine n (for compounds with vicinal hydroxyl groups) and silver nitratO 1 (for reducing compounds), and with a p-dimethylaminobenzaldehyde spray (for NANA and NANA-containing compounds). The spray was prepared by diluting the Ehrlich reagent (see below) with two volumes of water. After spraying, the chromatogram was sandwiched between glass plates and heated 3 min in an autoclave at 19 lb/inch ~, I22 °.

Analysis for NANA Total NANA, both free and bound, was determined b y adding I ml of Ehrlich reagent (2 g p-dimethylaminobenzaldehyde in 20 ml conc. HC1 plus 80 ml of ethylene glycol) to 2 ml of sample containing 5-20o #g of NANA and heating in an autoclave for 15 rain at 122 ° and 19 lb/inch 2. This assay is more sensitive and reproducible than the assay 1~ performed at Ioo °. Material positive to this test is referred to as Ehrlich-positive material. The thiobarbituric acid procedure of WAt~REN1~ was employed to measure NANA released from glycosidic linkage b y neuraminidase or b y acid hydrolysis (o.oi M He1, 15 min, 80°). The extent of cleavage of NAN-LAC was also determined indirectly ~4by measuring with the Ehrlich assay the amount of bound NANA remaining after borohydride reduction of an enzyme reaction mixture. If complete cleavage were achieved, all of the NANA was reduced b y the borohydride treatment and no Ehrlich reaction was obtained. NaBH~ (4 ° mg in 0.2 ml of water) was added to the sample to be analyzed and the mixture was kept at room temperature for I h. o.2 ml of conc. HC1 and i ml of Ehrlich reagent were added and the reaction carried out as described above.

Analysis for lactose, glucose and galactose Lactose was determined b y the orcinol procedure of WINZLER15 employing one-half the usual volumes of reagent and sample. Glucose and galactose were determined respectively with the Glucostat and Galactostat kits (Worthington Biochemical@ The hydrolysis of NAN-LAC to glucose and galactose was carried out with Dowex 50 (H +) resin as follows: 2 ml of water was added to a test tube containing 2oo mg of washed, dried (IOO°) Dowex 50 (H +) resin and Io mg of NANLAC. The tube was sealed and heated at Ioo ° for 4 h. After removal of the resin by filtration, 5o mg of sodium benzoate was added to the combined filtrate and the washings, the p H adjusted to 7.o with N a O H and the volmne made to 50 ml. The solution was stored for 24 h at 4 ° before analysis for glucose and galactose. Glucose and galactose standards were prepared from refrigerated stock solutions containing o.I % sodium benzoate. Biochim. Biophys. Acta, I 3 o (1966) 1 - i i

ISOMERS OF N-ACETYLNEURAMINYLLACTOSE

3

Periodate oxidations Periodate oxidations were performed at o ° in the absence of light in unbuffered solutions. The p H range during the oxidations was 3.0-4.5. The amount of each sample to be oxidized was adjusted so the quantity of sodium metaperiodate (o.o5 M) was in 2-fold excess over the expected consumption. Periodate consumption was determined according to DYERis and formaldehyde production b y the chromotropic acid method 17. Formic acid was determined as follows : a I-ml aliquot of the oxidation mixture (IO ml) was transferred to a beaker to which were added o.I ml of ethylene glycol (o.17 raM), I ml o.oi M HC1 and I ml of water. The solution (pH 1.3) was titrated to p H 9.0 with o.oi M NaOH. A comparison of the titration curves for sample and blank showed the amount and the pKa of the acid liberated. RESULTS

Isolation of NAN-LAC Skimmed bovine colostrum (first and second milkings post-partum) contains 14-2o % total solids and 1.1-1. 3 % Ehrlich-positive material. Lyophilized colostrum was reconstituted (280 g of colostrum solids per 1 of water) and dialyzed for 24 h against 5 1 of distilled water. The dialysis and all subsequent procedures were carried out at 4 °. The diffusate (pH 6.1) was passed through a column (8 cm x 43 cm) containing 135o g (wet weight) of Dowex I - X 2 (20o-400 mesh) resin in the acetate form. The 2% cross-linked resin has twice the capacity of 4% cross-linked resin and 6 times the capacity of 8 % cross-linked resin for retaining Ehrlich-positive material. An additional 5 1 of diffusate m a y usually be passed through such a column before Ehrlich-positive material is detected in the emuent fluid. The column was washed with 6 1 of water and eluted with a concave pyridyl acetate gradient system 7. The elution system consisted of a mixing vessel containing I 1.2 1 of o.o85 M pyridyl acetate and a reservoir containing 4 1 of o.15 M pyridyl acetate. The column flow-rate was IO ml/min and 25-ml fractions were collected. Fractions containing Ehrlich-positive material were pooled and lyophilized to yield a white, fluffy solid. The dried material was mixed with absolute methanol until no further material appeared to dissolve, and filtered through a sintered glass funnel of medium porosity. Anhydrous diethyl ether (3-4 volumes) was added to the clear filtrate to yield a white, flocculent precipitate. This suspension was mixed continuously while successive portions were filtered through a sintered glass funnel, the precipitate washed with ether, and the funnel with precipitate placed immediately in a desiccator attached to a water aspirator. After removal of residual ether the funnel was placed in an appropriate flask and connected to a lyophilizing apparatus for several minutes. The dried NAN-LAC (1.5-2 g from 560 g of colostrum solids) was placed in a closed vial and stored in a desiccator at --15 ° . I t is important that exposure to the atmosphere be minimized in order to obtain a dry, easily transferable product. Trace amounts of pyridine m a y be removed b y additional methanol-ether fractionations; this is preferred to the use of Dowex 50 (H +) resin as originally recommended ~.

Separation of NAN-LAC isomers The NAN-LAC was rechromatographed on a column of Dowex I - X 2 (2oo-4oo mesh) resin in the acetate form. In a typical separation, 300-500 mg of the crude

Biochim. Biophys. Acta, 13o (1966) I - i i

4

M . L . SCHNEIR, M. E. RAFELSON, JR.

material was dissolved in 3oo ml of distilled water (4 °) and passed through the column (2.5 cm × IOO cm). The column was washed with 2 1 of distilled water and eluted with the gradient system consisting of 900 ml of o.15 M pyridyl acetate (reservoir) and 25oo ml 0.085 M pyridyl acetate (mixer). The flow-rate was i ml/min and 5-ml fractions were collected. The elution pattern is shown in Fig. I. Peaks I and I I were identified respectively as 3-NAN-LAC and 6-NAN-LAC. Peak I I I appears to be a NANA-containing oligosaccharide. The appropriate fractions were pooled, lyophilized and fractionated three times with methanol-ether. About 80% of the material applied to the column was recovered as 3-NAN-LAC, 6-NAN-LAC and the unidentified compound in ratios of approx. 5 : i ' I . It is also possible to obtain separation of the isomers b y preparative paper chromatography with Solvent A. However, the recovery is less than 5 ° % and the products are contaminated with a basic substance derived apparently from the chromatography paper.

2©

k3 0.2

,

I O~

20

40

60 80 Tube number

,% 100

1 2,, 3., 4 }1 ~ ~imethy~Periodatebenzidine rnlno

I

L

P"

/

z°ldehyde

5,. 6 Silver nitrate

I

120

Fig. I. Fractionation on D o w e x - I of N-acetylneuraminyllactose: I, z --> 3 isomer; II, 2 --> 6 isomer; I I I , N A N A - c o n t a i n i n g oligosaccharide. Fig. 2. C h r o m a t o g r a m of 2 mg each of 3- and 6-NAN-LAC in Solvent A: 6-NAN-LAC (I, 4 and 5) ; 3-NAN-LAC (2, 3 and 6).

Characterization of isomers Both isomers were obtained as white, amorphous powders and were homogeneous b y the criteria of high-voltage electrophoresis (pH 6.8-8.6) and paper chromatography in Solvent A of 2 mg or more of either isomer. The R3-NAN-LAC of 6-NAN-LAC in Solvent A is 0.75 (Fig. 2). Pertinent analytical data for the two isomers are collected in Table I. Both isomers as isolated contained 5-1o% moisture and were dried in vacuo for 16 h at room temperature over P2Os prior to analysis. Samples once dried appeared to regain their original moisture even when stored in a desiccator. The preparations have persistently contained o.6-1.5 % ash which has resisted removal b y ion-exchange Biochirn. Biophys. Acta, I3o (1966) 1 - I I

ISOMERS

OF

N-ACETYLNEURAMINYLLACTOSE

TABLE I ANALYTICAL DATA FOR ISOMERS OF N - A C E T Y L N E U R A M I N Y L L A C T O S E

Calculated

Analysis

Found z -> 3 i s o m e r

2 --> 6 i s o m e r

Lactose (orcinol) N-acetylneuraminic acid : by Ehrlich assay released by o.oi M HCI released by neuraminidase

54.2 %

46.6 ~: 2 %

47 ~: 2%

48.8 % 48.8 % 48.8 %

46 ± 2 % 44 ± 2 % 48.8 %

47 4- 2 % 47 ± 2 % 48.8 %

Glucose

28.4 %

24 %

25 %

Galactose

28. 4 %

22 °/o

Neutralization equivalent

21%

633.6

6Ol.O

6o9-7

-[-16.8 °

+27.9 °

[~]~ (c = I.O, in water)

--

pK a

--

2.45-2.50

2.50-2.55

C* H* N* Ash Moisture

43.6% 6.20 °~ 2.21% ---

43.7% 6.31% 2.65 % o.6-1.5 % 5-1o %

43.6% 6.24% 2.36 % 0.6-0.5 % 5-1o %

* Elemental analyses were performed by Midwest Micro Laboratory, Indianapolis, Ind., "U.S.A.

resins: preliminary spectrographic analyses indicate that the preparations contain O.l-O.3% total Ca + Mg + Mn. The amount of NANA released by the hydrolysis of either isomer at 80 ° in o.oi M HC1 was about 90% of theoretical. This is presumed to be a quantitative recovery, of the NANA, since there was a 7.5% destruction of NANA subjected to the same conditions. Moreover, a quantitative recovery of NANA from both isomers was achieved by the milder conditions of neuraminidase hydrolysis. Complete enzymic hydrolysis was confirmed by the "indirect" Ehrlich assay and by paper chromatography of enzyme reaction mixtures.

#

II

It

0

e

e

Periodate-benzidinedip Fig. 3. Chromatogram of hydrolysate of 3-NAN-LAC in Solvent B: i, authentic NANA; 2 and 3, 3-NAN-LAC hydrolysate; 4, authentic lactose. B i o c h i m . B i o p h y s . Mcta, 13o (1966) i - i i

6

M.L.

NANA

SCHNEIR,

M. E. RAFELSON,

and lactose were isolated from i.o g of 3-NAN-LAC

after hydrolysis

o . o i M HC1. T h e h y d r o l y s a t e

was mixed with N,N-dioctylmethylamine

with

the

chloroform

to remove

HC1, t h e r e s i d u a l

and the aqueous layer passed through

chloroform

a column of Dowex

JR.

in

and extracted

extracted

with

ether

I-X 8 (200-400 mesh) resin

in the acetate form. The effluent fluid which contains the lactose was lyophilized, the dried material dissolved in 0.75 ml of water and crystallization effected by the addition of 2.25 m l of a b s o l u t e

ethanol.

The

NANA

pyridyl acetate system, the appropriate

was eluted

lized from glacial acetic acid. The lactose and NANA were identified by paper chromatography The

infrared

those published

spectra

from

the column

with

the

fractions lyophilized and the NANA recrystal(463 a n d 360 m g r e s p e c t i v e l y )

( F i g . 3) a n d b y m e l t i n g p o i n t d e t e r m i n a t i o n s .

for 3-NAN-LAC

and

6-NAN-LAC

were

identical

with

b y t{UHN AND BROSSMER ls.

TABLE II P E R I O D A T E CONSUMPTION AND FORMIC ACID AND F O R M A L D E H Y D E PRODUCTION OF N - A C E T Y ' L NEURAMINYLLACTOSE

ISOMERS

AND

RELATED

Compound

COMPOUNDS

I rain I h

2 h

4h

24 h

48 h

Theory

N-Acetylneuraminyl(2 -+ 3)-lactose

Periodate F o r m i c acid Formaldehyde

2.7 I.o i.o

2.9 I.I 0.9

3.o 1.2 0.9

3.2 1. 3 0.9

4.1 1.6 0.9

4.I 2.0 i .o

4.o 2.0 i.o

N-Acetylneuraminyl(2 -+ 6)-lactose

Periodate F o r m i c acid Formaldehyde

3-3 1.2 I .o

3 .8 1. 5 I .o

4. I 1.8 ---

4-4 2.o I .o

5.4 2. 7 I .o

5.9 2.9 I .o

6.o 3.o i .o

N-Acetylneuraminic acid

Periodate F o r m i c acid Formaldehyde

2.9 o.9 i.o

2.9 I.o 0.9

2.9 I.I i.i

2.9 I.O I.O

2.9 I.O i.o

2.9 i .o I.I

2.0 I.O I.O

2-Methoxy-N-acetylneuraminic acid

Periodate Formic acid Formaldehyde

1.9 I.O 0.9

1.9 I.O I.O

2.0 I.o --

1.9 o.9 I.O

2.o I.O I.O

2.0 I.o I.o

2.0 I.O i .o

2-Methoxy-N-acetyln e u r a m i n i c acid m e t h y l ester

Periodate F o r m i c acid Formaldehyde

1.8 o.9 o.9

1.9 I.O I.O

2.0 o.9 I.O

2.o I.O o.9

1.9 o.9 I.O

2.0 I.o I.O

2.0 I.O I.O

N, 7 -+ o - D i a c e t y l n e u r a m i n i c acid

Periodate F o r m i c acid Formaldehyde

I.O o.o I .o

0.9 o.o I .o

i .o o.o I .o

i.o o.o 0.9

i .o o.o I .o

i.o o.o I .o

i.o o.o I .o

Colomin ic acid*

Periodate F o r m i c acid

1.5 0.7

1.6 o.8

1. 7 0.9

1.9 0.9

2.o I.O

2.o i.o

2.0 I.O

Lactose

Periodate F o r m i c acid

2.o 0. 7

2.8 0.8

3.3 0.9

3 .6 o.9

3.9 I.O

4.0 I.O

4-0 I.O

E t h y l e n e g lycol

Periodate F o r m i c acid

I. I o.o

--

--

--

--

I. I o.o

I. I o.o

---

I.O o.o

Glycerol

Periodate Formic acid

1.8 0.9

---

---

1.8 0.9

1.8 I.O

2.o I.o

2.o i.o

Erythritol

Periodate Formic acid

2.9 2. I

---

---

2.9 2. I

2.9 2. I

---

3.0 2.0

Galactitol

Periodate Formic acid

4.7 3.9

4.7 3-8

---

4.7 3-9

4 .8 3.9

5 .0 4 .0

5 .0 4.0

* M o l e c u l a r w e i g h t d e t e r m i n e d as 3200 a n d c o r r e s p o n d s t o i i N A N A residues.

Biochim. Biophys. Acta, 13o (1966) i - n

ISOMERS OF N-ACETYLNEURAMINYLLACTOSE

7

Periodate oxidations were used to elucidate the linkage of NANA to galactose in both NAN-LAC isomers. For the evaluation of the periodate oxidation data, a series of model compounds were examined. These included straight-chain polyols, NANA, 2-methoxy-N-acetylneuraminic acid and its methyl ester, N, 7 ~ O-diacetylneuraminic acid, colominic acid (2 ~ 8 polymer of NANA) and lactose (Table II). The usefulness of the straight-chain polyols resided in their correspondence to the polyollike chain of 3- and 6-NAN-LAC, i.e., carbon atoms 7, 8 and 9 (Fig. 4). Under the CHzOH

H i OH

.V_oi, [4

H

OH

H-c -OH

CHzOH

(o)

.o I.

CH~C-N-C -H

H I OH OH l 6 I I I I H-C -..C -.-,, .C .-I-^CH~OH r I

J

IO I

H

I~

H i

H00C--C

0

I

CH'C-N-C-HoH

° HI

n i OH,

o-~z

....

H -C-H

OH

i:

i

H

"T ° H 0H

CHz0H

H - C - C --,-" C --P,ell.OH I

H

I

l

H

(b)

Fig. 4. Proposed structures of N-acetylneuraminyl-(2 --~ 3)-lactose (a) a n d N - a c e t y l n e u r a m i n y l (2 -+ 6)-lactose (b) ; dotted lines show sites of periodate oxidation.

conditions of the periodate oxidations, the consumption of periodate and release of formic acid b y the straight-chain polyols was immediate, the same values being obtained in I min as in later time periods. NANA, 2-methoxy-N-acetylneuraminic acid and its methyl ester, and N, 7 -~ O-diacetylneuraminic acid also showed an immediate periodate consumption. Colominic acid, although showing a somewhat slower rate of oxidation, gave similar results. With the exception of NANA, all results corresponded to those expected. NANA consumed an additional mole of periodate which could not be accounted for either as formic acid or formaldehyde. A recent report 19 suggested that the third mole of periodate oxidized NANA between carbon atoms 6 and 7; however, formic acid measurements were not performed to establish whether or not an additional mole of formic acid was produced. We consider it unlikely that NANA exists to any extent in the open-chain form since polarographic studies s° indicate that NANA exists in the pyranose structure and we did not observe in the present study the formation of a second mole of formic acid. The additional periodate uptake was not observed in the glycosides of NANA, the aglycone groupings apparently conferring stability to the NANA moiety. Lactose gave the results expected for its cyclic pyranose structure as did other disaccharides and trisaccharides studied. The periodate oxidation was gradual as observed for isoinositol and other cyclic compounds. Biochim. Biophys. Acta, 13o (1966) i - I I

8

M. L. SCHNEIR, M. E. RAFELSON, JR.

The consumption of periodate and the release of formic acid and formaldehyde by 3-NAN-LAC were commensurate with the values expected. A consumption of 4 moles of periodate per mole of 3-NAN-LAC and the release of 2 moles of formic acid and I mole of formaldehyde supported the conclusion that the structure of 3-NAN-LAC was N-acetylneuraminyl-(2 -~ 3)-fl-D-galactopyranosyl-(i ~ 4)-D-glucopyranose. The acidic conditions of the oxidation solution did not result in hydrolysis of 3-NAN-LAC or 6-NAN-LAC to NANA and lactose. 6-NAN-LAC gave expected values for periodate consumption and the release of formic acid and formaldehyde. The consumption of 6 moles of periodate and the release of 3 moles of formic acid and I mole of formaldehyde supported the proposed structure. No other linkage of NANA to the galaetose moiety of lactose will give such results. The galactose moiety of 3-NAN-LAC should remain unoxidized by periodate. A complete recovery of galactose subsequent to periodate oxidation would further support the proposed linkage. These experiments are shown in Table III. A number of control experiments were necessary for the interpretation of the results. These included the hydrolysis of lactose in the presence and absence of NANA and/or iodate TABLE III RECOVERIES OF GLUCOSE AND GALACTOSE FROM PERIODATE-OXIDIZED AND NON-OXIDIZED LACTOSE AND N-ACETYLNEURAMINYLLACTOSEISOMERS

Compound

Lactose* Lactose + N A N A (equimolar)* 3-NAN-LAC * 6-NAN-LAC* Lactose + iodate** Lactose + iodate + NANA** 3-NAN-LAC + iodate* * 6-NAN-LAC + iodate** Lactose + NaIO4*** 3-NAN-LAC + NaIO4*** 6-NAN-LAC + NaIO4***

% of calculated Glucose

Galactose

lOl. 3 lO4. 5 85 . 2 86. 4 91. 4 9o. 7 76.o 75.8 o o o

lO2. 4 87.0 76 . 2 76.4 85. 9 72.2 67. I 67.2 o 59.6 o

* Lactose (24 mg), lactose (24 rag) + N A N A (44 rag), 3-NAN-LAC (4 ° rag) and 6-NAN-LAC (4 ° mg) were hydrolyzed directly as described in the experimental section. 5 ml of precooled o.I M N a l O 4 was mixed with o.I ml of ethylene glycol (o.17 mmoles) for 2 rain and t h e n added to the sugar solutions to provide a source of iodate comparable to t h a t obtained in the periodation experiments. Following hydrolysis with Dowex-5o (H+), the solutions were passed t h r o u g h a column (3 cm × IO cm) of D o w e x - I resin in the acetate form to remove iodate. The column was washed with w a t e r and the combined effluent and washings lyophilized and analyzed for glucose and galactose as described in the experimental section. *** Oxidized 24 h at o ° and processed as the iodate-containing samples described above.

and the hydrolysis of 3- and 6-NAN-LAC in the presence of iodate to determine what effects these substances might have on the recoveries of glucose and galactose. The recoveries of glucose and galactose from the hydrolysis of lactose by Dowex-5o (H+) were essentially quantitative, whereas the recovery of galactose from the hydrolysis of lactose in the presence of an equimolar quantity of NANA was significantly de.Biochim. Biophys. Acta, 13o (1966) I - i i

9

ISOMERS OF N-ACETYLNEURAMINYLLACTOSE

pressed (87 % of calculated). Iodate decreased the recovery of galactose from lactose, and iodate plus NANA further depressed the recovery to 72% of the calculated amount. The recoveries of galactose from 3- and 6-NAN-LAC were 76 % of theoretical and were reduced to 67 % by the addition of iodate. Thus, the recovery of galactose from the hydrolysis of 3-NAN-LAC in the presence of iodate is 67 % of calculated and should be compared with the value of 72 % obtained for lactose hydrolyzed in the presence of NANA and iodate. In the final experiments lactosel 3-NAN-LAC and 6-NAN-LAC were oxidized with periodate for 24 h, the excess periodate reduced to iodate with ethylene glycol and the oxidized materials hydrolyzed for the determination of the hexoses. The results were as expected in that the glucose moiety of all three compounds was totally destroyed as was the galactose of lactose and 6-NANLAC. About 60 % of the galactose was recovered after the periodation of the 3-NANLAC. This is taken to represent a quantitative recovery in view of the results of the control experiments and supports further the postulated structure of 3-NAN-LAC.

Stability The N-acetylneuraminic acid-lactose bond (either 2 ~ 3 or 2 -~ 6) was stable at pH's 5.0, 7.o and 9.0 whether buffered solutions (o.I M buffers) of the isomers were frozen, refrigerated or maintained at 37 °. In unbuffered solutions containing 200 #g/o.2 ml of either isomer (pH 3.2), extensive autohydrolysis was observed, 2 %, 20% and 60% hydrolysis respectively after 2, 24, and 96 h. Drying at 78°, prolonged storage in a desiccator at room temperature or passage of an aqueous solution through a column of Dowex 50 (H +) resin resulted in degradation to lactose, NANA and several faster and slower moving components observable in Solvent A. Well-dried 3- and 6-NAN-LAC appear stable indefinitely when stored in a desiccator at --IO to --15 °. N eur aminidase experiments Table IV presents a comparison of the properties of the two NAN-LAC isomers as substrates for the neuraminidase isolated from the JAP 3o5/1957 strain of influenza virus. Michaelis constants (Kin) and maximal velocities (V) were estimated by the method of LINEWEAVER AND B U R K il. TABLE IV COMPARISON OF ISOMERS OF N-ACH;TYLNEURAMINYLLACTOSEAS SUBSTRATES FOR N E U R A M I N I D A S E *

pH Optimum Kra (moles/l) V (mmoles/h per mg protein)

3-NAN-LAC

6-NAN-LAC

6.5 2" lO -4 7.2

4.5 I. io -~ o.45

* Purified enzyme from influenza virus ( J A P 3o5/1957) (ref. IO). DISCUSSION

The isolation procedure described here is simple and 2 g or more of the NANLAC isomer mixture can be isolated from bovine colostrum in a single chromatographic run on a column of Dowex-i resin. This yield is IO times that obtained by Biochim. Biophys. Acta, 13o (1966) i - i i

I0

M. L. SCHNEIR, M. E. RAFELSON, JR.

our earlier method v. The two isomers, 3-NAN-LAC and 6-NAN-LAC, can be separated by rechromatography of the isomer mixture on a second column of Dowex-i resin. Hitherto, the 6-isomer of N-acetylneuraminyllactose had not been isolated from bovine colostrum and had been found only in human milk 22. The isomers as finally isolated satisfy a number of criteria of purity including two that we believe to be the most critical, namely, that both isomers are completely cleaved by neuraminidase and that paper chromatography of 2 mg or more of each isomer shows a single spot. Total cleavage of both isomers by neuraminidase was shown by direct analysis of the liberated NANA, by the "indirect" Ehrlich assay and by paper chromatography of the enzyme reaction mixture. With regard to chromatographic purity, it is not usual to employ milligram amounts of material to check homogeneity; examination of microgram amounts of NAN-LAC by paper chromatography gives specious evidence for homogeneity. The periodate oxidation studies and the other analytic data presented support the structures of the NAN-LAC isomers as being respectively, N-acetylneuraminyl(2-+3)-fl-D-galactopyranosyl-(I->4)-D-glucopyranose and N-acetylneuraminyl-(2-->6)fl-D-galactopyranosyl-(I-->4)-D-glucopyranose.

K U H N AND BROSSMER 23 a n d K U H N AND

GAUHE~4 have reported that permethylation of 3-NAN-LAC isolated from bovine colostrum gave 2,4,6-trimethylgalactose in a yield of 5 6 % and that 6-NAN-LAC isolated from human milk gave 2,3,4-trimethylgalactose (44 % yield). Recently, RYAN et al. 25 have presented qualitative evidence that NANA is linked to the 3-position of the galactose moiety of neuraminyllactose sulfate. It is clear that the mode of attachment of NANA to lactose influences the velocity of the neuraminidase reaction (Table IV). If the Km (assumed here to be equal to Ks) is the equilibrium constant of the reaction forming the enzyme-substrate complex and V is a measure of the velocity constant of the breakdown of this complex, it then appears that the nature of the bond (2 -->3 vs. 2 -+ 6) influences the velocity of the reaction in two distinct ways, namely by an effect on both the formation and breakdown of the enzyme-substrate complex. That such an effect is possible is further supported by the finding that the K m and V of the reaction for colominic acid (2 --> 8 NANA polymer) differ from those obtained with either NAN-LAC isomer 26. It would appear necessary to compare maximum velocities if a meaningful comparison of the rate of splitting of two substrates is desired, since it is quite possible that the same maximum velocities may be obtained despite a difference of several orders of magnitude in the Km values. It may also be noted that the optimal pH for the enzymic cleavage of the two isomers is quite different, 3-NAN-LAC having a pH optimum of 6.5, whereas pH 4.5 is optimal for 6-NAN-LAC. This may explain the earlier failure to observe the enzymic splitting of the 6-isomer 2v. ACKN OWLEDGEMENTS

The authors wish to thank Drs. H. L. KEIL and R. D. JONES for their able assistance and the Kenyon Brothers, Elgin, Ii1., for their generosity in supplying bovine colostrum. This investigation was supported in part by grants from the U.S. Public Health Service, National Institutes of Health (AI-o4435) and the Commission on Influenza, Armed Forces Epidemiological Board, U.S. Army Research and Biochim. Biophys. Ac/a, I3o (1966) I II

ISOMERS OF N-ACETYLNEURAMINYLLACTOSE

II

Development Command (MD 2293 ). These studies were based in part on a dissertation by M. L. SCHNEIRin partial fulfillment of the requirements for the Ph.D. degree in the Graduate College, University of Illinois at the Medical Center, Chicago. REFERENCES I M. E. RAFELSON, M. SCHNEIR AND V. W. WILSON, Argh. Biochem. Biophys., lO 3 (1963) 424 • 2 R. CAPUTTO AND R. E. TRUCCO, Nature, 169 (1952) lO61. 3 R. KUHN AND R. BROSSMER, Angew. Chem., 68 (1956) 211. 4 R. HEIMER AND K. MEYER, Biochim. Biophys. Aeta, 27 (I958) 490. 5 L. W. MAYRON AND C. TOKES, Biochim. Biophys. Acta, 45 (196°) 6Ol. 6 H. NOLL, T. AOYAGI AND J. ORLANDO, Virology, I (196o) 141. 7 M. SCHNEIR, R. WINZLER AND M. E. RAFELSON, Biochem. Prepn., 9 (1962) I. 8 P. MATHIEU, L. COLOBERT, O. CREACH AND R. FONTANGES, Ann. Inst. Pasteur, i o i (1961) 539 R. A. GIBBONS, Bioehem. J., 89 (1963) 38o. IO V. W. WILSON AND M. E. RAFELSON, Bioehem. Prepn., io (1963) 113. i i I. SMITH, Chromatographic and Electrophoretic Techniques, Vol. I, Interscience, N e w York, 196o. 12 I. WERNER AND L. ODIN, Acta Soc. Med. Upsalien., 57 (1952) 230. 13 L. WARREN, J. Biol. Chem., 234 (1959) 1971. 14 J. N. WALOP, TH. A. C. ]3OSCHMAN AND J. JACOBS, Biochim. Biophys. Acta, 44 (196°) 185" 15 R. WINZLER, Methods Biochem. Anal., 2 (1955) 269. 16 R. DYER, Methods Biochem. Anal., 3 (1956) I I I . 17 M. LAMBERT AND A. C. NEISH, Can. J. Res., 28]3 (195 o) 83. 18 R. KUHN AND R. BROSSMER, Angew. Chem., 7 ° (1958) 25. 19 G. ]3. PAERELS AND J. SCHUT, Biochem. J., 96 (1965) 787 • 20 ]3. ROBERT, M. E. RAFELSON AND L. ROBERT, Nature, 191 (1961) 596. 21 H. LINEWEAVER AND D. ]DURK, J. Am. Chem. Soe., 56 (1934) 658. 22 R. KUHN, Proc. 4th Intern. Congr. Biochem. Vienna, x958, Vol. I, Pergamon, London, 1959, p. 67. 23 R. KUHN AND R. ]DROSSMER, Chem. Ber., 92 (1959) 166724 R. KUHNAND A. GAUHE, Chem. Ber., 98 (1965) 395. 25 L. C. RYAN, R. CARUBELLI, R. CAPUTTO AND R. E. TRUCCO, Bioehim. Biophys. Acta, i o i (1965) 252 • 26 V. W. WILSON, JR., Doctoral Thesis, University of Illinois College of Medicine~ Chicago, IlL (U.S.A.), 1965 . 27 R. KUHN, Bull. Soc. Chim. Biol., 5 ° (1958) 297. 28 R. KUHN AND R. BROSSMER, Chem. Ber., 89 (1956) 2o13.

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