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A modification of determination for glucosamine and galactosamine in glycoprotein with the amino acid analyzer
ANALYTICAL BIOCHEMISTRY 70, 537-541 (1976)
A Modification of Determination for Glucosamine and Galactosamine in Glycoprotein with the Amino Acid Analyzer Application to Total Acid Hydrolyzate of Rat Renal Glomerular Basement Membrane K I M I E M U R A Y A M A , NORIKO S H I N D O , AND HIKARU K O I D E
Central Laboratory of Medical Science and Department of Medicine, Juntendo University, School of Medicine, Bunkyo, Tokyo, Japan Received May 8, 1975; accepted September I 1, 1975
A procedure was described for the rapid determination of glucosamine and galactosamine in total acid hydrolyzate of rat renal glomerular basement membrane (GBM) by the use of amino acid analyzer. Glucosamine and galactosamine, as well as other amino acids in glycoprotein hydrolyzate, were well identified and estimated simultaneously by using a short column of HITACHI I-PF-B spherical resin, eluted with a pH 6.09 buffer containing 8% methanol at 38°C. Total time consumed for elution of galactosamine was 60 rain. This method is ideal for the separation of small amount of galactosamine from hydroxylysine-rich materials.
The technique of gas-liquid chromatography has been used for the analysis of the neutral sugars, hexosamines, and neuraminic acid in biological materials (1-3). An excellent separation of all these compounds was obtained. However, by this procedure it was difficult to determine quantitatively hexosamines when they were present in small quantities in glycoprotein. On the other hand, amino acid analyzer has been used for determination of hexosamines (4,5). Chromatography was carried out on the long column (0.9 x 69 cm) for the separation of acidic and neutral amino acids. In such cases hexosamines were resolved behind aromatic amino acids. Glucosamine was eluted ahead of galactosamine. We have encountered, however, a difficulty in the determination of hexosamines with amino acid analyzer because hexosamines were eluted over a broad area. Thus, we developed a modification of the amino acid analyzer method that permitted the separation and quantitation of glucosamine and galactosamine along with all of the normal protein amino acids. 537 Copyright© 1976by Academic Press, Inc. All rightsof reproductionin any form reserved.
538
MURAYAMA, SHINDO AND KO1DE
MATERIALS AND METHODS
Hexosamine and amino acid standards. Glucosamine hydrochloride and galactosamine hydrochloride were products of Wako Chemical Co., Ltd. Amino acid calibration mixture was a product of Takara Kosan Co., Ltd. DL-allo-5-hydroxylysine hydrochloride was purchased from Fluka AG, Chemische Fabrik. The standard amino acid calibration mixture was made by diluting the above solution to 0.1 /xmol/ml in 0.01N HC1. Glucosamine and galactosamine were also made to 0.1 /zmol/ml in 0.01 N HC1. Analysis of glucosamine and galactosamine. A Hitachi amino acid analyzer model KLA-5 (Hitachi Ltd.) was used for the experiments. The short column (0.9 × 24 cm) for the resolution of hexosamines and basic amino acids was packed with Hitachi I-PF-B (No. 2615) spherical resin. 1 The eluant buffers were prepared according to modified Benson's buffer (6), i.e., sodium concentration, 0.50M; citrate concentration, 0.166 M; concentrated HCI, 10 ml; caprylic acid, 0.4 ml; 25% Brij solution, 16 ml; final volume, 4000 ml. The pH was adjusted to 6.09. Other eluting buffers, pH 4.05, 5.05, and 5.76 were prepared by adding HCI. In order to obtain the best resolution, the 3, 5, 8, 10, or 15% methanol were added to pH 6.09 buffer. The buffer was pumped at 60 ml/hr and ninhydrin solution was pumped at 30 ml/hr. Column temperature was 38°C. After every analysis the column was regenerated with 0.5 N N a O H and equilibrated with eluting buffer. Hydrolysis of GBM for the release of hexosamines. Rat glomerular 1 This resin is 10% cross-linked, the spherical particle s having a diameter of 13 -+ 2/zm. The exchange capacity is 4.3 mEq/g of resin.
I pH 405 I J ~ 30
60 rain I
pH S.CS ~
30
60 rnin I
pH 576 1 3 ~ 6 0 I
pH 609
~ 30
I GIcNH= F](;. I. E l u t i o n
rnin
60rain lI: GaI'NH=
profile ofglucosamine and galactosamine at various pH of the eluting buffer.
D E T E R M I N A T I O N FOR G L U C O S A M I N E AND GALACTOSAMINE
539
rain 7O
2-_-2-_2-_-2-_ :---~ .
6O 5O
-.
4O
-a . . . . .
.<~.: -_
"-4
I--.a
3O
0
i 40
i 5.0
i 60
pH
FIc. 2. Effect of pH on the retention time of amino acids and hexosamines. ©, Glc-NH2; O, GaI-NHz; [], Tyr; I Phe; A, OH Lys; A, allo-OH Lys.
basement membrane (GBM) was prepared by the modification of Spiro's method (7) with the use of 150 and 200 mesh sieves. Lyophilized GBM (5.55 mg) were hydrolyzed in 2 ml of 4 N HC1 for 6 hr at 100°C. The hydrolyzate was evaporated to dryness over NaOH pellets in vacuum, dissolved in 0.02 N HC1 at concentration to 1.36 mg/ml of GBM.
RESULTS Figure I presents the elution profile of the hexosamines at the various pH of the eluting buffers. At pH 4.05 glucosamine and galactosamine were poorly resolved and both hexosamines occurred as unsymmetrical peaks. It was observed that hexosamines were resolved completely and eluted as symmetrical peaks with an increase in the pH of the eluting buffer. As can be seen in Fig. 2, which shows the retention time of amino acids and hexosamines in the various pH, tyrosine and phenylalanine overlapped the peaks of glucosamine and galactosamine, respectively, at pH 4.05 buffer. When the pH 6.09 buffer was used, the elution time of tyrosine and phenylalanine was decreased remarkably, but that of hexosamines was not changed. Therefore, glucosamine was separated from phenylalanine completely. However, galactosamine was overlapped on the hydroxylysine peak. TABLE 1 ANALYTIC
CONDITIONS
Column Size Resin Column Temperature Eluant Buffer Flow Rate
OF HEXOSAMINES
WITH
AMINO
ACID
ANALYZER
0.9 x 24 cm Hitachi I-PF-B Spherical Resin 38°C 0.5 M Sodium citrate, pH 6.09 containing 8% methanol Buffer, 60 ml/hr Ninhydrin, 30 ml/hr
540
MURAYAMA, SHINDO AND KOIDE
-
H
~i
±
t
l-t
!l
i
~ ,ii,
:+',
~
i~
ill.i ~ :,! ':i
i5
l ii~ :!i ::i i~li
!!!!!11~!~:t ~
..
~ ~;.:
,.
FIG. 3. Resolution of amino acids and hexosamines from standard solution containing 0.05-/xmol quantities.
For successful resolution of these compounds, methanol was added to pH 6.09 buffer. When the methanol concentration was more than 8%, galactosamine was resolved successfully from hydroxylysine. However, at higher methanol concentration, the peaks of glucosamine and galactosamine were not so well separated. The useful conditions for analysis of hexosamines are summarized in Table 1. Figure 3 shows a typical chromatogram of standard solution, which contains hexosamines and amino acids. Total time required for analysis of " hexosamines was within 60 min. Under the same conditions, acid hydrolyzate of GBM was analyzed as presented in Fig. 4. This sample was not treated with enzyme to remove protein and therefore contained a full spectrum of commonly occurring amino acids and hexosamines. GBM contains 1.06% of glucosamine and 0.15% of galactosamine. DISCUSSION
Previously, we have investigated the carbohydrate composition of GBM by using gas-liquid chromatography. Galactosamine could not be identified because it was present in small quantities in GBM. We have found that amino acid analyzer is a most satisfactory instrument for determining small quantities of glucosamine and galactosamine in
DETERMINATION
F O R G L U C O S A M I N E AND G A L A C T O S A M I N E
541
.t/7
, ! . . . . . :. , :
• .~,'~ ..,.
i ; I
"•
-i. '. ;. . . '.
' 1. 1 '
~ : Ii. . . . .
I
0
!i
"
.z.
30
.
.
.
.
60
.
.
.
.
,
min
-o.ol
, , ,
FIG. 4. GBM.
Determination
~.~..,
I..~_
of hexosamines
~ L. ~,~ . ~
of GBM
~,.4-.r-,~
hydrolyzate.
Sample
load was
0.68 mg
of
glycoprotein hydrolyzate such as GBM, which contains a great amount of hydroxylysine. The addition of 8% methanol to pH 6.09 buffer achieved successful resolution of galactosamine and hydroxylysine. Furthermore, the use of a short column and of high pH buffer made this method time-saving. Elution profile of amino acids appeared to be sensitive to small difference in the pH of the eluting buffer. However, in the hexosamine analysis, the difference of pH affected the elution pattern rather than the elution time. REFERENCES 1. 2. 3. 4. 5. 6. 7.
Bhatti, T,, Chamber, R., and Clamp, J. R. (1970) Biochim. Biophys. Acta. 222, 339. Richey, J. M., Richer, H. G., Jr., a n d S c h r a e r , R. (1964)Anal, Biochem. 9, 272. Sweely, C. C., a n d W a l k e r , B. (1%4) Anal. Chem. 36, 1461. Peterson, D. E. a n d B e r n l o h r , R. W., (1970)Anal. Biochem. 33, 238. Rosan, B. (1972)Anal. Biochem. 48, 624. Benson, J. V., Jr., (1972) Anal. Biochem. 50, 477. Spiro, R. G. (1967) J. Biol. Chem. 242, 1915.
A Modification of Determination for Glucosamine and Galactosamine in Glycoprotein with the Amino Acid Analyzer Application to Total Acid Hydrolyzate of Rat Renal Glomerular Basement Membrane K I M I E M U R A Y A M A , NORIKO S H I N D O , AND HIKARU K O I D E
Central Laboratory of Medical Science and Department of Medicine, Juntendo University, School of Medicine, Bunkyo, Tokyo, Japan Received May 8, 1975; accepted September I 1, 1975
A procedure was described for the rapid determination of glucosamine and galactosamine in total acid hydrolyzate of rat renal glomerular basement membrane (GBM) by the use of amino acid analyzer. Glucosamine and galactosamine, as well as other amino acids in glycoprotein hydrolyzate, were well identified and estimated simultaneously by using a short column of HITACHI I-PF-B spherical resin, eluted with a pH 6.09 buffer containing 8% methanol at 38°C. Total time consumed for elution of galactosamine was 60 rain. This method is ideal for the separation of small amount of galactosamine from hydroxylysine-rich materials.
The technique of gas-liquid chromatography has been used for the analysis of the neutral sugars, hexosamines, and neuraminic acid in biological materials (1-3). An excellent separation of all these compounds was obtained. However, by this procedure it was difficult to determine quantitatively hexosamines when they were present in small quantities in glycoprotein. On the other hand, amino acid analyzer has been used for determination of hexosamines (4,5). Chromatography was carried out on the long column (0.9 x 69 cm) for the separation of acidic and neutral amino acids. In such cases hexosamines were resolved behind aromatic amino acids. Glucosamine was eluted ahead of galactosamine. We have encountered, however, a difficulty in the determination of hexosamines with amino acid analyzer because hexosamines were eluted over a broad area. Thus, we developed a modification of the amino acid analyzer method that permitted the separation and quantitation of glucosamine and galactosamine along with all of the normal protein amino acids. 537 Copyright© 1976by Academic Press, Inc. All rightsof reproductionin any form reserved.
538
MURAYAMA, SHINDO AND KO1DE
MATERIALS AND METHODS
Hexosamine and amino acid standards. Glucosamine hydrochloride and galactosamine hydrochloride were products of Wako Chemical Co., Ltd. Amino acid calibration mixture was a product of Takara Kosan Co., Ltd. DL-allo-5-hydroxylysine hydrochloride was purchased from Fluka AG, Chemische Fabrik. The standard amino acid calibration mixture was made by diluting the above solution to 0.1 /xmol/ml in 0.01N HC1. Glucosamine and galactosamine were also made to 0.1 /zmol/ml in 0.01 N HC1. Analysis of glucosamine and galactosamine. A Hitachi amino acid analyzer model KLA-5 (Hitachi Ltd.) was used for the experiments. The short column (0.9 × 24 cm) for the resolution of hexosamines and basic amino acids was packed with Hitachi I-PF-B (No. 2615) spherical resin. 1 The eluant buffers were prepared according to modified Benson's buffer (6), i.e., sodium concentration, 0.50M; citrate concentration, 0.166 M; concentrated HCI, 10 ml; caprylic acid, 0.4 ml; 25% Brij solution, 16 ml; final volume, 4000 ml. The pH was adjusted to 6.09. Other eluting buffers, pH 4.05, 5.05, and 5.76 were prepared by adding HCI. In order to obtain the best resolution, the 3, 5, 8, 10, or 15% methanol were added to pH 6.09 buffer. The buffer was pumped at 60 ml/hr and ninhydrin solution was pumped at 30 ml/hr. Column temperature was 38°C. After every analysis the column was regenerated with 0.5 N N a O H and equilibrated with eluting buffer. Hydrolysis of GBM for the release of hexosamines. Rat glomerular 1 This resin is 10% cross-linked, the spherical particle s having a diameter of 13 -+ 2/zm. The exchange capacity is 4.3 mEq/g of resin.
I pH 405 I J ~ 30
60 rain I
pH S.CS ~
30
60 rnin I
pH 576 1 3 ~ 6 0 I
pH 609
~ 30
I GIcNH= F](;. I. E l u t i o n
rnin
60rain lI: GaI'NH=
profile ofglucosamine and galactosamine at various pH of the eluting buffer.
D E T E R M I N A T I O N FOR G L U C O S A M I N E AND GALACTOSAMINE
539
rain 7O
2-_-2-_2-_-2-_ :---~ .
6O 5O
-.
4O
-a . . . . .
.<~.: -_
"-4
I--.a
3O
0
i 40
i 5.0
i 60
pH
FIc. 2. Effect of pH on the retention time of amino acids and hexosamines. ©, Glc-NH2; O, GaI-NHz; [], Tyr; I Phe; A, OH Lys; A, allo-OH Lys.
basement membrane (GBM) was prepared by the modification of Spiro's method (7) with the use of 150 and 200 mesh sieves. Lyophilized GBM (5.55 mg) were hydrolyzed in 2 ml of 4 N HC1 for 6 hr at 100°C. The hydrolyzate was evaporated to dryness over NaOH pellets in vacuum, dissolved in 0.02 N HC1 at concentration to 1.36 mg/ml of GBM.
RESULTS Figure I presents the elution profile of the hexosamines at the various pH of the eluting buffers. At pH 4.05 glucosamine and galactosamine were poorly resolved and both hexosamines occurred as unsymmetrical peaks. It was observed that hexosamines were resolved completely and eluted as symmetrical peaks with an increase in the pH of the eluting buffer. As can be seen in Fig. 2, which shows the retention time of amino acids and hexosamines in the various pH, tyrosine and phenylalanine overlapped the peaks of glucosamine and galactosamine, respectively, at pH 4.05 buffer. When the pH 6.09 buffer was used, the elution time of tyrosine and phenylalanine was decreased remarkably, but that of hexosamines was not changed. Therefore, glucosamine was separated from phenylalanine completely. However, galactosamine was overlapped on the hydroxylysine peak. TABLE 1 ANALYTIC
CONDITIONS
Column Size Resin Column Temperature Eluant Buffer Flow Rate
OF HEXOSAMINES
WITH
AMINO
ACID
ANALYZER
0.9 x 24 cm Hitachi I-PF-B Spherical Resin 38°C 0.5 M Sodium citrate, pH 6.09 containing 8% methanol Buffer, 60 ml/hr Ninhydrin, 30 ml/hr
540
MURAYAMA, SHINDO AND KOIDE
-
H
~i
±
t
l-t
!l
i
~ ,ii,
:+',
~
i~
ill.i ~ :,! ':i
i5
l ii~ :!i ::i i~li
!!!!!11~!~:t ~
..
~ ~;.:
,.
FIG. 3. Resolution of amino acids and hexosamines from standard solution containing 0.05-/xmol quantities.
For successful resolution of these compounds, methanol was added to pH 6.09 buffer. When the methanol concentration was more than 8%, galactosamine was resolved successfully from hydroxylysine. However, at higher methanol concentration, the peaks of glucosamine and galactosamine were not so well separated. The useful conditions for analysis of hexosamines are summarized in Table 1. Figure 3 shows a typical chromatogram of standard solution, which contains hexosamines and amino acids. Total time required for analysis of " hexosamines was within 60 min. Under the same conditions, acid hydrolyzate of GBM was analyzed as presented in Fig. 4. This sample was not treated with enzyme to remove protein and therefore contained a full spectrum of commonly occurring amino acids and hexosamines. GBM contains 1.06% of glucosamine and 0.15% of galactosamine. DISCUSSION
Previously, we have investigated the carbohydrate composition of GBM by using gas-liquid chromatography. Galactosamine could not be identified because it was present in small quantities in GBM. We have found that amino acid analyzer is a most satisfactory instrument for determining small quantities of glucosamine and galactosamine in
DETERMINATION
F O R G L U C O S A M I N E AND G A L A C T O S A M I N E
541
.t/7
, ! . . . . . :. , :
• .~,'~ ..,.
i ; I
"•
-i. '. ;. . . '.
' 1. 1 '
~ : Ii. . . . .
I
0
!i
"
.z.
30
.
.
.
.
60
.
.
.
.
,
min
-o.ol
, , ,
FIG. 4. GBM.
Determination
~.~..,
I..~_
of hexosamines
~ L. ~,~ . ~
of GBM
~,.4-.r-,~
hydrolyzate.
Sample
load was
0.68 mg
of
glycoprotein hydrolyzate such as GBM, which contains a great amount of hydroxylysine. The addition of 8% methanol to pH 6.09 buffer achieved successful resolution of galactosamine and hydroxylysine. Furthermore, the use of a short column and of high pH buffer made this method time-saving. Elution profile of amino acids appeared to be sensitive to small difference in the pH of the eluting buffer. However, in the hexosamine analysis, the difference of pH affected the elution pattern rather than the elution time. REFERENCES 1. 2. 3. 4. 5. 6. 7.
Bhatti, T,, Chamber, R., and Clamp, J. R. (1970) Biochim. Biophys. Acta. 222, 339. Richey, J. M., Richer, H. G., Jr., a n d S c h r a e r , R. (1964)Anal, Biochem. 9, 272. Sweely, C. C., a n d W a l k e r , B. (1%4) Anal. Chem. 36, 1461. Peterson, D. E. a n d B e r n l o h r , R. W., (1970)Anal. Biochem. 33, 238. Rosan, B. (1972)Anal. Biochem. 48, 624. Benson, J. V., Jr., (1972) Anal. Biochem. 50, 477. Spiro, R. G. (1967) J. Biol. Chem. 242, 1915.