THE JOURNAL OF BIOLOGICAL CHE.MIGTRY Vol. 239, i’h. 2, February 1964

Printed in U.S.A.

Periodate Oxidation of the Glycoprotein Fetuin*

ROBERT G. &mot

From the Department of Biological Chemistry, Harvard Medical School, and the Baker Clinic Research Laboratory, New England Deaconess Hospital, Boston 15, kfassachusetts

(Received for publication, August 21, 1963)

Previous reports have described the isolation of the glycopro- tein fetuin from fetal calf serum in a high degree of purity and have dealt with several aspects of its structure (Z-5). These in- vestigations have indicated that the carbohydrate portion of fetuin consists of three branched heteropolysaccharide units at- tached at internal positions along a single polypeptide chain (3, 5). It has been shown that each of these carbohydrate units has a molecular weight of approximately 3500 and a similar mono- saccharide composition (3). The sequential arrangement of some of the sugar residues in t.hese polysaccharide units has been determined from their release by graded acid hydrolysis and by the action of glycosidases, as well as from a study of several oligo- saccharides isolated from partial acid hydrolysates of the protein

(1, 3). It was the purpose of the present investigation to obtain fur-

ther information in regard to the sequence and linkages of these sugar residues by submitting both the native and sialic acid-free protein to periodate oxidation. The monosaccharides resistant to destruction by periodate were estimated, and the products of those sugars oxidized by periodate were identified after sodium borohydride reduction.

In addition, a sequential degradation of the monosaccharides of fetuin was achieved by repeated application of a procedure consisting of periodate oxidation followed by sodium borohydride reduction and mild acid hydrolysis. In this manner, the peptide portion of fetuin was obtained with only hexosamine and a small amount of mannose attached.

The structure of the carbohydrate units of fetuin and the identit,y of the sugar involved in t.he carbohydrate-peptide linkage are discussed in the light of these and previously reported results.

EXPERIMENTAL PROCEDURE

Preparation of Z’etuin-Fet.uin was isolated from pooled fetal calf serum by fractionation with ethanol at low temperature, as previously described (2). Sialic acid-free fetuin was prepared by mild acid hydrolysis (2).

Periodate Oxidation-Oxidation was carried out with sodium metaperiodate in 0.05 M sodium acetate buffer at pH 4.5 and 4” in the dark (6). The protein concentration was approximately 5 mg per ml (0.1 pmole of protein per ml), and molar ratios of periodate to protein varying between 100: 1 and 300: 1 were em- ployed. Aliquots were taken at several times for the determina-

* This work was supported by a grant from the American Heart, Association and bv Grant A-5363 from the National Insti- tute of Arthritis and Metabolic Diseases, National Institutes of Health, United States Public Health Service. A preliminary report has been published (1).

t This work was done during the tenure of an Established Investigatorship of the American Heart Association.

t.ion of periodate consumption and formaldehyde liberation, as well as for sugar analyses. In the samples taken for analyses of the sugar components, t,he oxidation was terminated by addition of an excess of ethylene glycol, and this was followed by extensive dialysis, first against 0.1 M NaCl and then distilled water at 2-4”. Controls treated similarly except for the addition of the protein to the periodate after the ethylene glycol showed no destruction of the sugars upon subsequent analysis. Concentration by lyo- philization was generally avoided because of the decreased solu- bility of the oxidized protein.

Determination of Period& Consumption-The arsenite method of Fleury and Lange (7) was used to follow periodate consump- tion. Aliquots (1 ml) of the reaction mixture were analyzed at various times. In order to exclude the possibility of a direct interaction of either arsenite or iodine with the protein, blanks containing the protein and all the reagents except periodate were analyzed. These did not differ significantly from blanks in which the protein was not present.

Formaldehyde Determination-The chromotropic acid reaction (8) was used for the estimation of formaldehyde at several times on aliquots (0.2 to 0.3 ml) of the periodate reaction mixture. To these samples were added in succession 0.2 ml of 2.5 N H2S04, 0.2 ml of 0.15 M sodium arsenite, and water to make a total vol- ume of 1.0 ml. After a period of 10 minutes, 10 ml of the chromotropic acid reagent were added and the color reaction was performed as described (8). In this manner, formaldehyde in the range of 0.04 to 0.20 pmole was determined. Erythritol oxidized with periodate for 1 hour was used as a formaldehyde st.andard. Blank determinations were performed in which the protein was added to the periodate after the addition of the H2S04 and arsenite in order to correct for the small amount of color produced in the chromotropic acid reaction by the un- oxidized protein.

Sodium Borohydride Reduction of Oxidized Protein-In order to identify the products of periodate oxidation, the oxidized, dialyzed protein was treated with sodium borohydride before acid hydrolysis. The molar ratio of borohydride to protein was approximately 2000: 1, and the sodium borohydride was added in an equimolar sodium borate buffer at pH 8.0 to give a final concentration of both borohydride and buffer of about 0.15 M.

The reaction was carried out at 0” for 12 hours and terminated by lowering the pH to 5 by the addition of acetic acid. Subse- quently, the reaction mixture was dialyzed at 2-4” against 0.1 M

NaCI, followed by distilled water. In control studies, fetuin not subjected to periodate oxidation was treated with sodium borohydride in a similar manner.

Paper Chromatography-Descending chromatograms were run on Whatman No. 1 paper in the following solvent systems: py-

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,568 Periodate Oxidation of Fetuin Vol. 239, No. 2

0 3 6 9 12 3 6 9

HOURS

FIG. 1. Periodate consumption of native (0) and sialic-acid free fetuin (X). Left, sodium periodate concentration, 0.01 RI;

right, 0.03 M. The protein concentration was 10m4 M in all oxidations. Other conditions were as described in text.

ridine-ethyl acetate-water-acetic acid (5: 5 : 3 : 1)) as described by Fischer and Nebel(9); I-butanol-ethanol-water (10:1:2);n-butyl acetate-acetic acid-water (3 : 2: 1) ; and 1-butanol-acetic acid- water (4: 1:5). Sugars and sugar alcohols were located on the chromatograms with silver nitrate (lo), the silver oxide back- ground being removed by liquid s-ray fixer (11). For the detec- tion of sialic acid and its reduced oxidation products, the re- sorcinol (12), Ehrlich (la), and thiobarbituric acid (13) reagents were also employed. The chlorine, starch-iodine method (14) was used for the location of N-acetylserinol (2-acetamido-1,3- propanediol). Chromatographically pure N-acetylserinol was prepared by the N-acetylation of serinol (Nutritional Biochemi- cals Corporation) by a modification (15) of the method of Rose- man and Daffner (16).

Sugar Analyses-Many of the carbohydrate analyses were per- formed as previously described (2, 4). For the determination of neutral sugars, hydrolysis was carried in 1 N sulfuric acid for 8 hours at 100”. Galactose and mannose were determined by quantitative paper chromatography of the neutral sugar fraction (2). Hexosamines were determined by the Elson-Morgan reac- tion after hydrolysis in 4 N HCl for 6 hours at 100” (4) and sep- aration on Dowes 50 ion exchange columns (17). Sialic acid was determined in the intact protein by the resorcinol (18) or direct Ehrlich (19) reactions, or after mild acid hydrolysis by the thiobarbituric acid assay (20). For chromatographic study, sialic acid or its periodate degradation products were released by weak acid hydrolysis and separated on Dowex 1 columns as previously described (2).

Glycerol Determination-For the estimation of glycerol, aliquots (containing 0.1 to 0.6 pmole of glycerol) from the neutral sugar fractions from hydrolysates were chromatographed on washed Whatman No. 1 paper in butanol-ethanol-water (10: 1:2) for 24 to 30 hours. Glycerol, which separated well from hexoses and other sugar alcohols in that period (Rgiactose = 3.93, Rerythritol = 1.42), was located by staining adjacent guide strips on which standards had been spotted. The areas containing the glycerol were cut out, placed in test tubes, and eluted with 5.0 ml of mater. ilppropriate aliquots, containing 0.02 to 0.2 pmole, were then taken for the determination of glycerol by a micromodifica- tion of the method of Lambert and Neish (21). Glycerol stand- ards and appropriate paper blanks were analyzed in the same manner for each chromatographic sheet.

Protein Determination-Protein was determined either by op-

tical density measurements at 278 rnp or by the method of Lowry et al. (22), with fetuin as a standard. After prolonged or repeated periodate oxidation, these methods were no longer re- liable, owing to the progressive destruction of tyrosine. In order to estimate the pept’ide content of such preparations, the measure- ment of valine, an amino acid unaffected by periodate, was per- formed. For t,his determination, samples were hydrolyzed for 48 hours at 105” in constant boiling HCl (23) and chromato- graphed in the butanol-acetic acid-water system. In this sys- tem, valine was separated from all other amino acids, since fetuin does not contain methionine (23). The chromatograms were stained with ninhydrin, and the color of the valine spots was eluted and measured in a manner previously described (3). The peptide content was calculated from the valine content of native fetuin, determined in the same manner.

RESULTS

Periodate Consumption-The consumption of periodate at vari- ous times during the oxidation of native and sialic acid-free fetuin is shown in Fig. 1. Although the initial periodate con- sumption was more rapid at the higher periodate to protein ratio, the final level achieved was approximately the same at both periodate concentrations. In both native and sialic acid-free fetuin, there was a rapid initial reaction, followed by a prolonged, more gradual phase. The amount of periodate consumed per mole of protein by the native preparation was greater than that consumed by the sialic acid-free protein at all times at both periodate concentrations. After the initial rapid phase, the slopes of the periodate consumption by the native and sialic acid- free protein became the same, and the two curves were separated by approximately 7 to 8 moles of periodate per mole of prot.ein.

Formation of Formaldehyde-Periodate oxidation of the native protein under the same conditions as in the left-hand portion of Fig. 1 resulted in the rapid liberation of 13 to 14 moles of formal- dehyde. The following values were determined for the native fetuin: 5 minutes, 13.5 moles per mole of protein; 30 minutes, 13.9; 120 minutes, 13.7; 6 hours, 13.7; and 21 hours, 13.8 moles of formaldehyde per mole of protein. Essentially no formalde- hyde was released from the sialic acid-free fetuin at any of these times, and only 0.40 mole per mole of protein was determined even after 21 hours.

Monosaccharide Analyses of Oxidized Proteins-The percent- ages of the monosaccharides recovered after periodate oxidation

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February 1964 R. G. Spiro 569

for various periods of time are given in Ta,bles I and II for both native and sialic acid-free fetuin at two different periodate con- centrations. The sialic acid, as judged by paper chromato- graphic analysis, was completely destroyed at both periodate concentrations. No significant destruction of hexosamines took place in either native or sialic acid-free fetuin during the entire oxidation period studied, as determined by the Elson-Morgan reaction and confirmed by chromatography. In the native pro- tein, even over a prolonged period, very little destruction of galactose or mannose was observed, and the ratio of galactose to mannose remained essentially constant. In the sialic acid-free protein, however, there was a rapid and essentially complete de- struction of galactose. During the same period of time there was only a small destruction of mannose. The preferential de- struction of galactose was reflected in the pronounced decrease in the ratio of galactose to mannose (Tables I and II).

TABLE II E$ect of periodate oxidation on monosaccharides of jetuin

(3 X 10m2 M sodium metaperiodate, 1GF M protein)

Monosaccharides recovered

Fetuin preparation

Length of oxidation

- Sialic acids

.- HCGX- amines

Galac- tose iannose

Mactose to mannose

ratio

_-

Vative hrs

0 0.25 1.0 3.0 6.0

10.0 24.0 48.0

% 100

0 0 0 0 0 0 0

% 100

98 85 99 88 92 91

102

%

100

113 103 110

99 96 91 88

1.59 1.53 1.51 1.42 1.46 1.57 1.59 1.59

Identification of Products of Periodate Oxidation-Although there was a rapid and complete destruction of sialic acid by peri- odate, as judged by the disappearance of sialic acid on chro- matography, the continued reactivity of the oxidized protein in the calorimetric determinations of this sugar (Table III) sug- gested the persistence of chromogenic parts of the sialic molecule. That the color given by the oxidized native fetuin was due to re- maining fragments of the sialic acid molecule and not due to some other unrelated products of periodate oxidation was indi- cated by the observations that the sialic acid-free protein failed to give any color with these reactions, either before or after periodate oxidation.

Sialic acid- free

0 100 1.0 93 3.0 93 6.0 110

% 100 109 98 98 91 95 91 88

100 17 11 0

100 84 84 85

1.45 0.29 0.19

-

TABLE 111

tuin as Effect of periodate oxidation on sialic acid content of fe, determined by several methods

Per cent of original sialic acid

Treatment Length of oxidation

After periodate oxidation, there was an approximately Z&fold increase in the color given by the resorcinol reaction at 580 rnp both in the periodate-oxidized and in the periodate-oxidized, sodium borohydride-reduced protein (Table III). There was also a change in shape of the absorption spectrum, with a shift of the maximum from 580 to 630 rnp.

The color given in the thiobarbituric acid assay at 549 rnp was decreased to approximately 50% in both the periodate-oxidized and oxidized-reduced preparations. The direct Ehrlich reaction also showed a decrease of approximately 50% in the color in- tensity at 565 rnp after periodate oxidation. However, after sodium borohydride reduction of the periodate-oxidized protein,

Thiobarbi- Direct turic acid Ehrlich

assay reaction

Periodate oxidation

bus % % 0 100 100 0.25 0 231 3.0 0 233 6.0 0 217

24.0 0 213

% % 100 100 52 51 47 45 48 47

Periodate oxidation followed by sodium borohydride reduc- tion

48

0 100 100 100 100

0.1 0 283 57 441 1.0 0 274 53 412

TABLE I Effect of periodate oxidation on monosaccharides of fetuin there was a 4-fold increase in the color product 31 1 in this reaction

(10e2 M sodium metaperiodate, 1OP x protein) over that given before oxidation (Table III). This increase in color was not accompanied by a change in the shape of the ab- sorption curve.

The chromogenic products of sialic acid oxidation could be re- leased with 0.025 N sulfuric acid at 80” for 1 hour from both the oxidized and the oxidized-reduced protein. To characterize fur- ther the oxidation products of the sialic acid, the acidic products of this sugar obtained after this mild hydrolysis were adsorbed on a Dowex 1 (formate) column and were eluted with 0.3 N formic acid. When the periodate-oxidized protein was treated in this manner, the Dowex 1 eluates gave less than 5 y. of the color given by the three sialic acid methods before passage through these columns. However, the Dowes 1 eluates from the periodat.e- oxidized, sodium borohydride-reduced protein showed a high recovery (80 to 90%) of the chromogenic sialic acid products, presumably because of the greater stability of the reduced forms during the column separation.

When the Dowex 1 eluates from the oxidized-reduced protein

Monosaccharides recovered T 2&.ctose to

mannose ratio

Fetuin Length of preparation oxidation

- Sialic acids

HeXOS- Galac- amines tose

hrs

0 0.5 1.5 4.0 8.0

24.0

_- %

100 0 0 0 0 0

% % %

100 100 100

98 83 98

94 97 115

99 98 11B 94 88 98 97 84 83

0 100 100 100 0.5 100 16 89 1.5 103 11 85 8.0 105 8 80

24.0 104 0 71

I’

Native

Sialic acid- free

1.51 1.28

1.27

1.27

1.36

1.53

1.44

0.26 0.19 0.14

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570 Periodate Oxidation of Fetuin Vol. 239, No. 2

TABLE IV Glycerol present after periodate oxidation and sodium borohydride

reduction of fetuin

Glycerol present Length of oxidation

Native fetuin Sialic acid-free fetuin

hrs pmole/?ng native protein

0.1 0.16 (7.7)* 0.042 (2.0) 1.0 0.13 (6.3) 0.22 (10.6) 3.0 0.14 (6.8) 0.24 (11.6) 6.5 0.17 (8.2) 0.28 (13.5)

* Figures in parentheses indicate residues per mole of protein.

were examined by paper chromatography in the butyl acetate- acetic acid-water system, they showed no component at the level of sialic acid, but contained several faster moving substances which could be demonstrated by the silver stain. These had migrations in relation to N-acetymeuraminic acid, RNAN,l of 1.35, 1.75, and 2.48. The two slower components also reacted on paper with the resorcinol, Ehrlich, and thiobarbituric acid re- agents. The substance with an RNAN of 1.35 reacted most in- tensely with all of these stains. The Dowex 1 eluates obtained from mild acid hydrolysis of native fetuin showed on chroma- tography only N-acetylneuraminic acid, whereas in the eluates from the periodate-oxidized, reduced, sialic acid-free fetuin, no components were detected.

It is of interest that the humin formation evident upon acid hydrolysis of native fetuin in 1 N H&JO* was still observed after periodate oxidation with or without subsequent sodium boro- hydride reduction. After a mild acid hydrolysis, the humin- forming material was found in the Dowex 1 eluates.

Further information regarding the products of periodate oxida- tion was obtained by examination of the neutral fractions of the hydrolyzed periodate-oxidized, sodium borohydride-reduced na- tive and sialic acid-free fetuin by chromatography in the butanol- ethanol-water system. No erythritol or threitol was detected in either preparation at several time intervals up to 24 hours. On the other hand, substantial quantities of glycerol were present in both the native and the sialic acid-free fetuin (Table IV). In the sialic acid-free protein, the amount of glycerol corresponded closely to the amount of galactose destroyed and would be the product expected from this sugar when located in nonreducing terminal positions.

The rapid appearance of glycerol in the native protein could only be attributed to the sialic acid, since no other sugars were destroyed in sufficient amounts to account for its formation. The glycerol from the native fetuin, moreover, was produced more rapidly than glycerol from the sialic acid-free protein (Table IV). This is consistent with the faster destruction which sialic acid undergoes in the native preparation, compared to that of galactose in the sialic acid-free protein (Fig. 1; Tables I and II). The glycerol obtained from the oxidized-reduced native protein could be released by weak acid hydrolysis in 0.025 N sulfuric acid at 80” for 1 hour. There was no evidence for the simultaneous release of any oligosaccharides, which would be expected if the glycerol originated from the oxidation of a sugar located in a position internal to an unoxidized sugar residue.

Hydrolysis, without prior periodate oxidation, of native fetuin

1 The abbreviation used is: NAN, N-acetylneuraminic acid.

and of native fetuin reduced with sodium borohydride revealed no glycerol, indicating that its appearance must be related to the periodate oxidation. It is of interest to point out that examina- tion of this sodium borohydride-reduced fetuin showed no chro- matographic evidence of galactitol, mannitol, or hexosaminitols, indicating that none of the sugar components of fetuin have free reducing groups.

Serial Periodate Oxidation of Carbohydrate Portion of Fetuin- The techniques of periodate oxidation followed by sodium boro- hydride reduction can be used in the degradation of carbohydrate polymers because of the increased acid lability of the acetal bonds present in the resulting polyalcohols (24). These bonds can be cleaved without breaking the glycosidic bonds between the un- oxidized portions of the molecule. Since essentially all but the nonreducing terminal sugar components of fetuin were shown to be resistant to periodate oxidation, an attempt was made to se- quentially degrade and release the sugar residues of fetuin by the technique of periodate oxidation followed by sodium boro- hydride reduction and mild acid hydrolysis. Each step in the degradation process consisted of an initial periodate oxidation at a molar ratio of periodate to protein of 300: 1 to 600: 1, followed by dialysis. The dialyzed material was then reduced with sodium borohydride and again dialyzed. Subsequently the ace- tal bonds were cleaved by hydrolysis in 0.05 N H&04 at 80” for 1 hour. The hydrolysate was then neutralized with sodium hy- droxide and the process was repeated, beginning with another periodate oxidation.

At the conclusion of each step, the protein was analyzed for the sugars remaining. In addition, after each mild acid hydroly- sis, a neutralized sample was taken to determine the components released. This sample was placed on a small charcoal-Celite col- umn to remove the protein. The columns were washed with water to recover alcohols and monosaccharides, and eluted with 30% ethanol to detect any oligosaccharides which might have been released. The water fraction was subsequently desalted through a mixed bed ion exchange resin.

The analysis of the protein after each of four steps is given in Table V. The first step resulted in the complete destruction of sialic acid and the release of only glycerol and the previously described acidic chromogenic products of the sialic acid oxidation. The second step destroyed the galactose entirely and resulted in the release of glycerol. The protein was recovered at the con- clusion of this step with all of the hexosamines and most of the mannose present.

TABLE V Effect of serial periodate oxidation on carbohydrate portion of fetuin

This procedure involved periodate oxidation followed by so- dium borohydride reduction and mild acid hydrolysis in each step, as described in text.

Length of oxidation

Per cent of original monosaccharides remaining step employed

in step Sialic acids Galactose M~IXIOS~ Hexosamines

hrs % % % %

1 0.5 0 96 98 101 2 9.5 0 0 81 102 3 56 0 0 75 53 4 55 0 0 11 (0.3)* 40 (2.3)

* Figures in parentheses indicate residues of sugar remaining per carbohydrate unit of fetuin.

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The third step destroyed approximately half of the hexosa- mines and yet left 75% of the mannose intact. As indicated in Table V, a prolonged oxidation period was required to obtain maximal destruction of the hexosamines in this step. This slow oxidation is consistent with the rate of oxidation of methyl K- acet,ylglucosaminide under similar oxidation conditions (6). After the mild acid hydrolysis, both glycerol and N-acetylserinol (2-acetamido-1,3-propanediol) were identified. The N-acetyl- serinol was obtained in the following manner. After the mild acid hydrolysis of the third step, an aliquot was taken and re- duced further with sodium borohydride. The sample was then passed through a Dowex 50-X4 (H+ form) column (200 to 400 mesh), and a Dowex l-X8 (formate form) column (200 to 400 mesh) in succession. Protein was then removed on a small char- coal-Celite column, and the effluent and water wash were taken to dryness in a vacuum rotator. The boric acid from the sodium borohydride was volatilized as methyl borate. Paper chroma- tography of the sample in the Fischer-Nebel system revealed a spot with the chlorine, starch-iodine stain which migrated to the level of the standard N-acetylserinol, with an Rserinoi of 1.74 and

and RN aoetylgluoosamine of 1.30. The N-acetylserinol is believed to represent the upper portion of the oxidized N-acetylglucosamine, having been formed by reduction of N-acetylserinal (2.aceta- mido-2-deosyglyceraldehyde) after its release from the protein by mild acid hydrolysis. The glycerol released at this step pre- sumably represents the lower 3 carbon atoms of the oxidized A--acetylhexosamines.

The fourth step resulted in a small further decrease in the hesosamine content and a marked destruction of mannose. The protein obtained after this fourth step contained 6.8 residues of herosamine and 0.9 residue of mannose per mole. Each of the 3 carbohydrate units of fetuin would therefore contain at the end of this step 2.3 residues of hexosamine and 0.3 residue of man- nose. Chromatographic examination of the remaining hexosa- mines indicated the presence of glucosamine and galactosamine in approximately the same ratio, 8 : 1, in which they occur in the native protein (2), further pointing to the random distribution of the two hesosamines in t,he structure of fetuin (4).

No erythritol or threitol was noted after any of the four steps of this procedure. When the sialic acid-free fetuin was sub- mitted to this serial periodate oxidation technique for three steps, the results obtained were similar to those in Steps 2 through 4 of the procedure as applied to the native protein.

DISCUSSION

These periodate oxidation studies have provided further in- formation regarding the structure of the carbohydrate units of fetuin and have confirmed the monosaccharide sequence previ- ously proposed on the basis of enzymatic release and acid hy- drolysis studies.

The rapid oxidation of all of the N-acetylneuraminic acid resi- dues is consistent with their terminal location in the oligosac- charide chains. The destruction of the 13 to 14 residues of sialic acid of fetuin mas accompanied by a consumption of approxi- mately 35 moles of periodate and the liberation of 13.5 moles of formaldehyde. Since no formaldehyde was released from the sialic acid-free protein, if may be presumed to originate from the sialic acid, as has been reported during periodate oxidation of the free sugar (25). It would appear that the initial rapid consump- tion of periodate in the native protein (Fig. 1) should be at- tributed entirely to the oxidation of the sialic acid residues, as

no other sugars are destroyed in that time. This would indicate the consumption of approximately 2.7 moles of periodate per mole of sialic acid oxidized.

It is difficult to correlate precisely the amount of periodate consumed with the quantity of a particular sugar destroyed in a complex polymer such as a glycoprotein. Not only may over- oxidation of the sugars be taking place, but oxidation of several amino acids has been reported to occur in peptide-containing ma- terial (26, 27). The slower phase observed in the periodate consumption curves (Fig. 1) of both the native and the sialic acid- free protein was probably the result of these reactions.

Little information is yet available in regard to the periodate consumption of glycosides of N-acetylneuraminic acid or to the exact nature of the oxidation products of either free sialic acid or its glycosides. Varying amounts of periodate consumption by N-acetylneuraminic acid have been reported in previous studies, ranging from 2 to more than 3 moles of periodate per mole of this sugar (25, 28, 29). The identification in the present study of several acidic products from the oxidation of the sialic acid would indicate that the periodate cleavage of this sugar is a complicated process. Of interest was the rapid appearance of glycerol in the oxidized-reduced fetuin, which appeared to be a product of the sialic acid oxidation. Although it is not known from which car- bons of the original sialic acid molecule the glycerol is derived, the most likely origin would be from carbons 5, 6, and 7. A basis for this derivation of the glycerol is the belief that P-formyl- pyruvic acid is the chromogen formed as the product of periodate oxidation in the thiobarbituric acid assay for sialic acid (20, 30), indicating that oxidative cleavage between C-4 and C-5 may take place. If the sialic acid is assumed t,o exist in the pyranose form (31), oxidative cleavage between C-7 and C-8 should also take place, in addition to that between C-8 and C-9.

The continued reactivity of the oxidized sialic acid with the color reactions used in the determination of this sugar gives some information in regard to the chemistry of these as yet incom- pletely understood reactions. Since a large part of the sialic acid molecule could be destroyed without eliminating color forma- tion in t,hese reactions, they cannot be used as indices of the integrity of the sialic acid molecule. The chromogenic part of the molecule would appear to reside in its upper portion and to include C-l, since the chromogens could be adsorbed on Dowex 1. However, the lower portion of the molecule may have some in- fluence on color formation, since there was a decrease in color formation in some of the reactions and a marked enhancement of color in others after periodate oxidation (Table 111).

The observation that the galactose of the native fetuin was not appreciably destroyed by periodate oxidation despite its rapid and complete destruction in the sialic acid-free protein strongly suggests that the terminal sialic acid residues are linked to the galactose and that this linkage must involve carbon 3 of the galactose. The periodate studies, moreover, would rule out the existence in substantial amounts of sialic acid linked to sugars other than galactose, since the galactose is protected from oxida- tion by sialic acid and there are approximately equimolar amounts of sialic acid and galactose present in fetuin (2).

The rapid phase of periodate oxidation of the sialic acid-free fetuin (Fig. 1) involved the consumption of about 26 moles of periodate per mole of protein, and was accompanied by the de- struction of the 12 to 13 residues of galactose present in fetuin. This would indicate the consumption of 2 moles of periodate per mole of galactose destroyed, since there was essentially no de-

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572 Periodate Oxidation of Fetuin Vol. 239, No. 2

AcH(Qo H(L>o<y$o

H H H OH H HNAc FIG. 2. Structure of an oligosaccharide chain of fetuin: X-acetylneuraminyl-(2 --f 3).6.n-galactopgranosyl-(1 ---) 4)-N-aeetyl-

n-glucosamine.

NAN NAN NAN NAN

Ghl GL GL GL.1

GLAC GA GIL GAAC I I I I

(Man, Man, Man, GNAc)-GNAc - peptide

FIG. 3. Proposed structure of a heteropolysaccharide unit of fetuin. Gal, galactose; GXL4c, N-acetylglucosamine; Man, mannose. The sequence of the sugars enclosed in parentheses is not yet known.

struction of hexosamine or mannose in this time interval. These results are consistent with the location of galactose in nonreduc- ing terminal positions in the sialic acid-free protein.

No hexosamine oxidation was noted in either the native or the sialic acid-free protein. Since all the hexosamine residues in fetuin have been shown to be A;-acetylated (4)) this is consistent with previous work indicating the location of hexosamines in- ternal to the galactose and linked at C-4 in the oligosaccharide chains of the carbohydrate units (4). The lack of hexosamine oxidation would, moreover, suggest that the 2 amino sugar resi- dues present in the internal portion of each of the carbohydrate unit,s cannot be linked at position 6 unless also involved as branching points.

The method of sodium borohydride reduction of a periodate- oxidized carbohydrate polymer to form a polyalcohol (32, 33) has been used to advantage in the present structural study of a glycoprotein, both for obtaining information in regard to sugar linkages through an identification of the reaction products, and also as a means of degrading the carbohydrate unit’s by selective acid hydrolysis of the acetal bonds present (24). The resistance to periodate oxidation of essentially all of the sugars in fetuin except those in terminal position has made the serial periodate oxidation technique (periodate oxidation, sodium borohydride reduction, mild acid hydrolysis) an alternate tool for the se- quential removal of the monosaccharide residues.

h previous st,udy demonstrated that the sequence of sugars in the oligosaccharide chains of the carbohydrate units of fetuin is sialic acid + galactose + N-acetylhexosamine (4). This con- clusion was based on the studies utilizing graded acid hydrolysis and selective cleavage with glycosidases, as well as the isolation of N-acetyllactosamine from partial acid hydrolysates. The re- sults of the serial periodate oxidation procedure used in the present study are consistent with such a sugar sequence. All of the sialic acid was destroyed in the first step, all of the galactose in the second step, and a large proportion of the hexosamine in the third step (Table V).

On the basis of information obtained in the present study and those previously reported, the structure of the oligosaccharide chains of fetuin may be drawn as in Fig. 2. The linkage of the sialic acid to the galactose may be considered to be an a-ketosidic

bond on the basis of its susceptibility to neuraminidase. This enzyme has been described as an oc-ketosidase (34, 35)) although this assignment of cr-specificity has been questioned (36). The linkage of the galactose to AT-acetylglucosamine is depicted in the ,8 configuration on the basis of its susceptibility to ,L-galac- tosidase and the isolation of N-acetyllactosamine (4). In each of the 3 polysaccharide units of fetuin there would be four such chains attached to an internal portion consisting of 3 residues of mannose and 2 of N-acetylhesosamine (Pig. 3) (4).

It is of interest that the linkage of sialic acid to galact,ose has also been shown to be 2 + 3 in sialyllactose (34) isolated from another bovine source, COW colostrum (37). Moreover, an en- zyme capable of synthesizing sialyllactose with this 2 + 3-linkage has recently been reported to be present in rat mammary gland (38). The linkage of sialic acid to galact,ose has also been in- vestigated in the cyl acid glycoprotein of human plasma with differing results (29, 39) ; it is possible that in this particular glycoprotein more than one type of sialic acid + galactose linkage may exist (36).

The present periodate study has permit,ted a further under- standing of the internal portion of the carbohydrate units of fetuin to which the oligosaccharides are attached. Previous studies have indicated that the 3 mannose residues and the 2 additional N-acetylhesosamine residues of each carbohydrate unit make up this internal portion (I, 4). The results with the serial periodate osidation technique used in the present study were consistent with this concept of an internal portion consisting of mannose and hexosamine, since after the third step cf this procedure the protein was obtained with only mannose and hesos- amine present (Table V).

From the periodate oxidation studies, some understanding of the linkages of the mannose residues can be obtained. Since only a small amount of mannose destruction took place in either native or sialic acid-free fetuin (Tables I and II), it is likely that most of the mannose residues are linked at position 3 or serve as branching points, or both. In the serial periodate procedure, a large portion of the mannose became susceptible to oxidation after destruction of the hexosamines in the oligosaccharide chains, suggesting that these mannose residues were protected by linkages from the hesosamines. The small amount of de- struction which took place before the release of the hexosamines in the serial oxidation technique, and which was also observed in the periodate oxidation time studies of the native and sialic acid- free proteins, could be interpreted as the slow oxidation of a man- nose residue located in an unbranched position at the nonreducing end of the internal portion of the carbohydrate unit. Since no erythritol was observed, even after most of the mannose was destroyed in the fourth step, it is unlikely that linkages to C-4 of mannose occur in this internal portion.

This study has provided some informat,ion in regard to the

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February 1964 R. G. Spiro 573

sugar involved in the linkage of the polysaccharide units to the protein. After four steps of serial periodate oxidation, the pro- tein was obtained with approximately 2 residues of hexosamine and substantially less than 1 residue of mannose for each of the polysaccharide units, suggesting that an N-acetylhexosamine residue is involved in the carbohydrate-peptide linkage. This is consistent with the isolat,ion from partial acid hydrolysates of fetuin of several oligosaccharides containing all 3 mannose resi- dues of the carbohydrate unit, and yet having N-acetylhexosa- mine on the reducing end (1). Since it, has been shown in the present study that there are no sugars with free reducing groups present in the intact protein, it is likely that C-l of the N-acetyl- hesosamine is involved in this linkage.

It is of interest that the participation of glucosamine in the glycopeptide bond of human y-globulins has recently been sug- gested on the basis of periodate oxidation studies (40) and that this sugar has also been implicated in the carbohydrate-peptide linkage of ovalbumin (41).

SUMMARY

Periodate oxidation studies have been performed on native and sialic acid-free fetuin. The periodate consumption, formal- dehyde liberation, and the amount of destruction of the various monosaccharides have been determined at several time intervals. In addition, some of the products of periodate oxidation have been identified after sodium borohydride reduction of the oxi- dized protein. In native fetuin there was a rapid and complete destruction of the sialic acid with no significant destruction of the galactose, whereas in the sialic acid-free protein, galactose was completely destroyed. This would indicate that all of the galac- tose is located internal to the peripheral sialic acid residues and that the linkage of the sialic acid is to carbon atom 3 of galactose. In both native and sialic acid-free fetuin preparations, there was no destruction of hexosamines and only a small amount of man- nose destruction.

A serial periodate oxidation technique involving the repeated treatment of the protein with periodate, followed by sodium borohydride reduction and then mild acid hydrolysis, was em- ployed to degrade the carbohydrate units. This process per- mitted a sequential destruction and release of the monosaccharide residues of fetuin and made it possible to obtain the peptide portion of this protein with only 2 residues of hexosamine and substantially less than 1 residue of mannose attached per car- bohydrate unit.

The products released during the various steps of this serial treatment included glycerol and several acidic oxidation prod- ucts of sial;c acid. These acidic products were shown to react with several of the calorimetric reagents used in the determina- tion of sialic acids. N-Acetylserinol was identified subsequent to hesosamine oxidation by an additional reduction of the re- leased oxidation products. No threitol or erythritol was found at any step of this procedure.

These periodate oxidation studies, in conjunction with previous investigations, suggest that the sequence of the oligosaccharide chains of the 3 carbohydrate units of fetuin is N-acetyl- neuraminyl(2 + 3)fl-n-galactopyranosyl-(I 3 4)-N-acetyl-n- glucosamine, and that these chains are attached to an internal portion consisting of mannose and additional N-acetylhexosamine residues. The periodate studies have indicated that it is likely that mannose serves as branching points of these carbohydrate units and that carbon 1 of N-acetylglucosamine is involved in the glycopeptide bond.

Acknowledgments-The author would like to thank Miss Margaret Hines and Mrs. Margaret Warner for valuable tech- nical assistance.

1. 2. 3. 4. 5. G.

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Robert G. SpiroPeriodate Oxidation of the Glycoprotein Fetuin

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