11334
Langmuir 2008, 24, 11334-11337
Quantitation of Surface-Reducing-End Covalently Bound Polysaccharides via Hydrazinolysis and Deamination Antonio Peramo* and Garrett Matthews Department of Physics, UniVersity of South Florida, Tampa, Florida 33620 ReceiVed July 20, 2008. ReVised Manuscript ReceiVed September 11, 2008 Depending on the method of deposition, reactive sites of polysaccharides on substrates may not be available when their reducing ends have been used to covalently bind them to the substrates. Here we present a method that allows surface density measurements of reducing-end covalently bound polysaccharides in a procedure that cleaves the polysaccharide chain from the surface via hydrazinolysis and deamination, leaving on the surface a disaccharide that is later radiolabeled with an aldehyde in a reaction with enamine formation. The method described has the advantage that it may be used with any polysaccharide patterned to any surface exposing an amino-terminated monolayer by reductive amination of their galactosamine or glucosamine repeating units. We illustrate the technique with the quantitation of glycosaminoglycans (GAGs) on silanized glass surfaces.
* To whom correspondence should be addressed. E-mail: aperamo@ umich.edu. Current address: Department of Materials Science and Engineering, University of Michigan.
in equimolar quantities. Other known methods include HPLC5 and different electrophoretic methods,6 including fluorophoreassisted carbohydrate electrophoresis (FACE). These techniques were unavailable for use with the GAGs in our glass surfaces because the reducing ends of the polysaccharides were no longer available for further reaction or because they could not be implemented directly on the surfaces. Enzymatic elimination of the GAGs from the surface is also possible. The products, typically small oligos (tetra and disaccharides), can then be analyzed by mass spectrometry (MS) or FACE. However, this methodology would require the use of very well known standards for each type of possible saccharide produced by the enzymes whereas MS analysis of sulfated oligomers is considered to be a complex task.7 As an alternative method to quantitate the GAGs directly on the surfaces, we considered the possibility of using fluorescence quantitation techniques. However, these techniques require the titration of the primary antibody to establish the GAG/antibody ratio. Additional titration is required if a fluorescently labeled secondary antibody is used, and the use of standards is necessary to relate the number of molecules to the observed intensity. This process is generally expensive and time-consuming and may require specialized equipment such as confocal microscopy. For the above reasons, we decided to develop a different technique in which the surface coverage of the GAGs could be inexpensively measured on the surface with high sensitivity and without the use of their reducing ends. To illustrate our methodology, it is convenient to look at Figure 1A, where a simple schematic of the deposition of the GAGs is represented. As shown, the final GAG is linked using a secondary amine to the substrate. A simple procedure for the quantitation of the surface density of these GAGs is to use a radiolabeled aldehyde that reacts with the secondary amine present in the covalent link between the surface and the GAG to form an enamine. The idea is that a procedure via enamine formation could produce a feasible technique that works for all types of glycosaminoglycans, regardless of disaccharide composition. However, because,
(1) Fan, V. H.; Tamama, K.; Au, A.; Littrell, R.; Richardson, L. B.; Wright, J. W.; Wells, A.; Griffith, L. G. Stem Cells 2007, 25, 1241–1251. (2) Peramo, A.; Albritton, A.; Matthews, G. Langmuir 2006, 22, 3228–3234. (3) Peramo, A.; Meads, M. B.; Dalton, W. S.; Matthews, G. Colloids Surf., B, published online Aug 12, http://dx.doi.org/10.1016/j.colsurfb.2008.07.019. (4) Peramo, A.; Meads, M. B.; Wright, G.; Dalton, W. S.; Matthews, G.Proceedings of the NSTI Nanotechnology Conference; 2006; Vol. 2, pp 754-757.
(5) Toida, T.; Shima, M.; Azumaya, S.; Maruyama, T.; Toyoda, H.; Imanari, T.; Linhardt, R. J. J. Chromatogr., A 1997, 787, 266–270. (6) Volpi, N.; Maccari, F.; Linhardt, R. J. Electrophoresis. 2008, 29, 3095– 3106. (7) Kuberan, B.; Lech, M.; Zhang, L.; Wu, Z. L.; Beeler, D. L.; Rosenberg, R. D. J. Am. Chem. Soc. 2002, 124, 8707–8718.
1. Introduction There is a growing consensus that studies of cell responses in vitro to biomolecules should be performed with the molecules replicating their in vivo orientation and not only in free diffusion in solution.1 Although the molecules can be deposited by adsorption, a more convenient method that allows better control of the orientation is achieved through covalent bonding to the substrates. In addition to controlling the orientation of the molecules, an important parameter to measure is the surface density, which is of interest because biological activity may change with the density of the deposited molecules. The origin of the work presented here was related to the need to find a rapid and suitable method for calculating the surface density of the glycosaminoglycans deposited in substrates, as we reported in previous work.2 There, we described a controlled method for the deposition of glycosaminoglycans on glass silanized surfaces. Once prepared, the GAG-coated substrates were used to study in vitro cancer cell adhesion.3,4 The GAGs were bonded to an amino-terminated monolayer of 3-aminopropyltriethoxysilane (APTES) through their reducing ends, producing an orientation that mimics that of the GAGs bound to the backbone of the proteins, the proteoglycans. After the deposition, we wanted to calculate the GAG surface density, but the main difficulty was the lack of reducing ends. As is well known, the technique of choice to quantitate polysaccharides is by the use of their reducing ends, which provide an easy target for chemical reaction. Methods for the quantitation of polysaccharides exist, but they are not useful or not easily implemented on surfaces. For instance, chromophores linked to the reducing ends are used in the detection and quantitation of polysaccharides in suspension. Saccharides are also quantitated by the reaction of radiolabeled sodium borohydride (NaB3H4) with the reducing end of the saccharide
10.1021/la802315s CCC: $40.75 2008 American Chemical Society Published on Web 09/27/2008
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Langmuir, Vol. 24, No. 20, 2008 11335 Scheme 1. Detailed Deacetylation and Deaminationa
a
Figure 1. Simplified scheme of the procedures for [14C]-acetaldehyde radiolabeling of polysaccharide surfaces.(A) APTES is first fixed to glass in ethanol, forming an amino-terminated layer that is further modified with different GAGs for 24 h in the presence of cyanoborohydride (B). (C) In 1), the deacetylation of GAG chains is performed by hydrazinolysis. In 2), the deamination reaction is shown. Deamination at pH 1.5 is performed only for HS GAGs. The nitrosamine formed then reverts to the secondary amine by denitrosation in a reaction overnight (D). (E) Radiolabeling can be performed by enamine formation using [14C]-acetaldehyde.
chemically, glycosaminoglycans8 contain acetamido groups, it would be necessary to eliminate all of them before using the radiolabeled aldehyde. For this purpose, we devised a method in which the GAG chains were first deacetylated and then deaminated. Tritiated sodium borohydride could be used after hydrazinolysis and deamination of the polysaccharide chains, but this procedure is inconvenient in that it does not work for certain type of sugars containing acids as part of their disaccharide structures, for instance, heparan sulfate or heparin. Our methodology is applicable to all of them. The main advantage and significance of the method described here is that it requires only inexpensive chemicals and a scintillation counter and provides an easy method by which to quantitate surface densities, as described in a recent report on heparinized surfaces.9 We emphasize that our technique is substrate-independent and can be used with any polysaccharide with galactosamine or glucosamine repeating units that has been linked to the surface using their reducing ends.
2. Materials and Methods 2.1. Materials. Heparan sulfate (Seikagaku America, M.M. 11 kDa), keratan sulfate (Seikagaku America, M.M. 13 kDa), chondroitin sulfate C (Seikagaku America, M.M. 60 kDa), and chondroitin sulfate A (Sigma Aldrich, M.M. 25 kDa) were the mucopolysaccharides used. [14C]-Formaldehyde with a specific activity of 52 mCi/mmol was purchased from Perkin-Elmer. [14C]-Acetaldehyde with a specific activity of 52 mCi/mmol was purchased from American Radiolabeled (8) Lindahl, U.; Hook, M. Annu. ReV. Biochem. 1978, 47, 385. (9) Kett, W. C.; Osmond, R. I. W.; Stevenson, S. M.; Moe, L.; Coomb, D. R. Anal. Biochem. 2005, 339, 206–215.
The example corresponds to a chondroitin sulfate C sample.
Company. Radioactivity was measured in 5 mL of scintillation fluid on a Beckman-Coulter scintillation counter. 2.2. GAG Surface Deposition. Glass coverslips were modified in a two-step procedure, as we previously described2 using the silane agent APTES (3-aminopropyltriethoxy-silane) and are conceptually reproduced in Figure 1A,B. This modification initially produces an NH2-terminated submonolayer or monolayer on the glass surface that is later incubated for 24 h at room temperature in solutions of 0.1 µg/mL of HS, KS, CSC (all from Seikagaku America), and CSA (Sigma-Aldrich) in PBS 1X with NaBH3CN (Acros Organics) at a concentration of 3 µg/mL. 2.3. Deaminative Cleavage of N-Acetylated Glycosaminoglycans. After the GAGs were attached to the surface as described in paragraph 2.2, they were N-deacetylated by treatment with hydrazine and then cleaved with HNO2 at pH 4.0 and 1.510,11 (Figure 1C.1 and Scheme 1). This reaction sequence cleaved the glycosaminoglycans at their N-acetyl-D-glucosamine or N-acetyl-D-galactosamine residues. For N-deacetylation, GAG samples were treated with a solution of excess anhydrous hydrazine and hydrazine sulfate depositing 200 µL of the mixture on the surfaces. The solution was prepared by dissolving 1 mg of NH2-NH2 · H2SO4 in 1 mL of anhydrous NH2-NH2. Glass coverslips were placed inside small straight-sided glass jars (Fisher Scientific) and heated to 90 °C in a sand bath for 10 h. Afterward, samples were cooled, rinsed with water to eliminate residual hydrazine, and quickly dried in a stream of nitrogen. Deamination was performed with the addition of HNO2 that was freshly prepared and kept at 0 °C. Nitrous acid at pH 1.5 was prepared by extracting the supernatant of the centrifuged mixture of equal amounts of separately kept and ice cooled solutions of H2SO4 0.5 M and Ba(NO2)2 0.5M. Nitrous acid at pH 4.0 was prepared by mixing 5 mL of 5.5 M NaNO2 and 2 mL of 0.5 M H2SO4. Samples for all GAGs were deaminated with 200 µL of nitrous acid at pH 4.0. In the particular case of HS samples, an additional deamination step with nitrous acid at pH 1.5 was necessary. Samples were deaminated for 1 h.12 Samples were abundantly rinsed with water to wash off of the surface the saccharide chains that were cleaved and dried in a stream of nitrogen. 2.4. Denitrosation of Polysaccharide Surfaces. This step is represented in Figure 1D and Scheme 2. Given that nitrous acid also attacks the secondary amine linking the silane and the GAG, forming an N-nitrosamine, it was necessary to perform a denitrosation step. This was done by depositing on the surface, at room temperature and overnight, a solution containing an excess mixture of NaN3, SC(NH2)2, and H2SO4 prepared by dissolving 7.15 mg of NaN3, 85.3 mg of SC(NH2)2, and 100 µL of H2SO4 in 10 mL of acetic acid.13 (10) Shaklee, P. N.; Conrad, H. E. Biochemistry 1984, 217, 187–197. (11) Shaklee, P. N.; Conrad, H. E. Biochem. J. 1986, 235, 225–236. (12) Shively, J. E.; Conrad, H. E. Biochemistry 1970, 9, 33–43.
11336 Langmuir, Vol. 24, No. 20, 2008 Scheme 2. Detailed Reaction of Denitrosation
Scheme 3. Detailed Reaction of Radiolabeling via Enamine
2.5. [14C]-Acetaldehyde Radiolabeling of GAG Surfaces. This step is represented in Figure 1E and Scheme 3. The remaining monosaccharide or disaccharide substrates were radiolabeled as follows. Previously denitrosated coverslips were covered with 200 µL of a solution containing 1 mM NaBH3CN and 0.2 mM 14CHO14CH in CH CN overnight at room temperature to keep 3 3 acetaldehyde and GAG in a 100:1 molar ratio. After incubation, samples were thoroughly rinsed with CH3CN first and water afterward and dried in a stream of nitrogen. During the reaction, samples were kept in a desiccator containing P2O5 to avoid moisture and hydrolysis of the formed enamine. 2.6. [14C]-Acetaldehyde Radiolabeling of Control APTES Surfaces. Experiments were repeated using control APTES surfaces only, with no glycosaminoglycans, as seen in Figure 2. One set of samples were subjected to hydrazinolysis, deamination, denitrosation, and radiolabeling, but the other set was subjected only to radiolabeling. As an additional background control, clean glass surfaces were also reacted with [14C]-acetaldehyde.
3. Results and Discussion The general schematic of the method developed to evaluate the surface density of GAGs is shown below in Figure 1 containing a total of seven steps that are summarized as follows: (1) After cleaning, the surface is silanized using APTES at a concentration
Figure 2. APTES control surfaces subjected to hydrazinolysis, deamination, denitrosation, and radiolabeling (A) did not produce any labeled material because of the nonreactivity of the silanol with the acetaldehyde. In contrast, direct reaction between the primary amine and the acetaldehyde could be carried out via reductive amination (B).
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of 0.86 mM for 15′. (2) Immediately after, GAGs are deposited by reductive amination with cyanoborohydride for 24 h. (3) GAG chains contain acetamido groups that are eliminated by hydrazinolysis at 90 °C for 10 h. (4) Deaminative cleavage of the GAG chains is performed with nitrous acid for 1 h. For HS, an additional step is necessary at lower pH for full chain cleavage. (5) At this point, there is only one remaining disaccharide whose bond with the silane has to be denitrosated overnight. (6) Radiolabeling is performed overnight using an aldehyde in a reaction with enamine formation. As can be observed in Figure 1B, in the first part of the surface modification GAGs are deposited by the well-known method of reductive amination,14 using cyanoborohydride, of the Schiff base produced in the reaction between the primary amine and the aldehyde. Solutions of APTES (0.86 mM) were used during the work with GAGs to prepare the surfaces. The secondary amines present in the GAGs may also react to form an enamine, thereby masking the true surface density. The process of deamination15 of N-deacetylated GAGs is necessary given that several acetamido groups are present in the GAG chains. As mentioned, N-deaminative cleavage of deacetylated polysaccharides cleaves the glycosaminoglycans at their N-acetylD-glucosamine or N-acetyl-D-galactosamine residues, leaving only a disaccharide or a monosaccharide attached to the surface.16 Exploiting the fact that the secondary amine can react with a ketone or aldehyde, a final reaction produces a 14C-labeled enamine. The reaction conditions used for the deacetylation and deamination are the standard ones in the literature, with the only change made here to extend the reaction times for the deamination to 1 h. The deamination process is also beneficial to enamine formation for two reasons related to the use of small aldehydes in the reaction. First, given the structural limitations of the GAG-APTES chain, the formation of the enamine tautomeric group will be favored (versus the formation of the imine group) by using small aldehydes or ketones and minimizing steric hindrance. Second, it has been shown that, in general, reaction yields are higher by using aldehydes and that in some reactions enamine does not form by using ketones.17 The rate of enamine formation essentially depends on two factors: the basicity of the amine and steric hindrance. All previous considerations favored the use of the aldehydes. Secondary reactions such as aminal formation were beyond the scope of this study and were not analyzed; however, aminals do not form in reactions involving noncyclic amines. A secondary reaction involves the formation of an Nnitrosamine in the secondary amine in Figure 1C and Scheme 2 that quantitatively reverts to the secondary amine by denitrosation of the N-nitrosamine with the removal with a trap (sodium azide) of the nitrous acid produced in the denitrozation in the presence of a good nucleophile (thiourea) under highly acidic conditions.18 The reactant concentrations were in molar excess in several cases. Conrad and co-workers used the following values for their reactions: 300 µg of GAG/20 µL of NH2-NH2/0.2 mg of NH2-NH2 · H2SO4, which represents a 5-30 molar excess of GAG over hydrazine sulfate when performing the reactions in (13) Dix, L. R.; Oh, S. M. N. Y. F.; Williams, L. H. J. Chem. Soc., Perkin Trans. 2 1991, 8, 1099–1104. (14) Jentoft, N.; Dearborn, D. G. J. Biol. Chem. 1979, 254, 4359–4365. (15) Shively, J. E.; Conrad, H. E. Biochemistry 1970, 9, 33–43. (16) Shively, J. E.; Conrad, H. E. Biochemistry 1976, 15, 3932–3942. (17) Dyke, S. F. The Chemistry of Enamines; Cambridge University Press: Cambridge, U.K., 1973. (18) Williams, D. L. H. In Nitrosamines and Related N-Nitroso Compounds; Quantitative Aspects of Nitrosamine Denitrosation; Loeppky, R. N., Michejda, C. J., Eds.; American Chemical Society: Washington, DC, 1994.
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Langmuir, Vol. 24, No. 20, 2008 11337
Table 1. GAG Surface Coverage by [14C]-Acetaldehyde Radiolabelinga
a
GAG
surface density (sites/µm2)
HS KS CSA CSC
478.5 ( 59.5 766.4 ( 142.5 690.4 ( 136.8 1060.2 ( 290.2
15′ deposited APTES surfaces were used in all cases.
closed reaction vials in suspension. However, 20 µL of pure anhydrous hydrazine does not yield the volume necessary to cover a glass coverslip. Thus, the reactions were designed to deposit 200 µL total volume on the surfaces, and the solution used contained the same ratios of NH2-NH2 and NH2NH2 · H2SO4 employed by Conrad. The benefit of using small molecules like acetaldehyde is to have easier access to reactive sites, an important factor given that the reaction is performed on a surface. In this case, the azeotropic elimination of water is not possible, but given the small volumes and molar quantities of the species over the surface, it suffices to eliminate water using a desiccator containing P2O5 or any other strong desiccant. After reaction, samples are rinsed with acetonitrile and blown dry in a stream of nitrogen. To avoid moisture and possible reversibility of the reaction, samples were immediately immersed in a scintillation liquid, and their radioactivity was measured. Surface coverage obtained for polysaccharide surfaces is shown in Table 1. Essentially the results indicate that the ratio of deposited GAG per amino terminal present on the surface is low and that there are small differences between GAGs, with larger molecules such as CSC having slightly higher surface densities. Given that denitrosation is a fully quantitative process, improvements in the values can be made in the reaction yields of enamine formation by the strong elimination of water during enamine formation. A secondary aspect of the reaction, shown in Figure 2A, is the possibility of a competing process occurring by the reaction between excess primary amines on the APTES surface, previously unreacted with GAGs, and the acetaldehyde. This possibility is eliminated because the deamination also cleaves primary amines on the APTES molecules. The reason is that the primary step in the deamination reaction is the nitrosation of the enamine,19 RNH2 + HNO2 f RNH2NO+ + OH-, and continues in subsequent steps with the final elimination of N2 and the conversion of the alkane to an aldehyde eliminated in subsequent washes. The conditions for the production of other species are complex and include the formation of NO and NO2. As a result, the deamination process cleaves all NH2 groups that may remain on the surface and on the GAG chains or converts them to nitrosamines that are later eliminated with the denitrosation procedure. To test this assessment (Figure 2), APTES control surfaces were subjected to direct hydrazinolysis, deamination, denitrosation, and radiolabeling, and the levels of radioactivity were similar to that of control glass coverslips that did not have amines on their surfaces, in contrast to control APTES samples (19) Horton, D.; Philips, K. D. Carbohydr. Res. 1973, 30, 367–374.
Table 2. Surface Coverage by [14C]-Acetaldehyde Radiolabeling of Control APTES and Glass Surfaces (without Glycosaminoglycans)a surface
surface density (sites/µm2)
APTES (treatment) APTES (no treatment) glass (no treatment)
127.2 ( 58.4 12 477 ( 549.7 36.9 ( 7.3
a 15′ deposited APTES surfaces of 0.86 mM concentration were used in all cases. Treated surfaces were subjected to hydrazinolysis, deamination, and denitrosation. Nontreated surfaces were directly subjected to [14C]acetaldehyde.
not subjected to hydrazinolysis and deamination that showed lowered levels of radioactivity after the formation of the Schiff’s base, as shown in Figure 2B. The levels of radioactivity shown in Table 2 indicate two things: The first is that hydrazinolysis treatment of an APTES surface effectively eliminates the amino groups from the surface (with levels on the order of clean glass samples) and thus no contribution from amino groups to the surface density of GAG can be expected. The second is that when APTES surfaces are not treated with hydrazine the reaction produces higher levels of radioactivity than in the case of no hydrazinolysis treatment.
4. Conclusions The determination of surface densities of biomolecules is necessary given that their orientation and density affect the biological activity of the molecules and the cellular response to the functionalized substrates. With this aim, we have presented a technique for the determination of glycosaminoglycan surface densities on glass silanized substrates. This quantitation method can be used with any N-acetylated sugar bound to surfaces exposing an amino-terminated monolayer. The method uses known chemical procedures for glycosaminoglycan chain cleavage, and it requires only inexpensive chemicals and a scintillation counter. In particular, our method could be useful in the precise quantitation of glycans deposited on glass slide microarrays.20 Finally, we emphasize that our method has certain advantages over other techniques that cannot measure surface densities when the reducing ends are not available (radiolabeling with tritiated sodium borohydride), are not suitable for direct use on surfaces (electrophoresis), or have limitations depending on the size, type, or orientation of the deposited molecules (XPS and AFM) or that are expensive and time-consuming, such as fluorescence intensity. Acknowledgment. We thank Dr. William S. Dalton for access to the facilities of the Moffitt Cancer Center. The Moffitt Cancer Center is a nationally recognized comprehensive cancer research center. We gratefully acknowledge funding provided for this work by American Cancer Society Institutional Research Grant No. 032. LA802315S (20) Nimrichter, L.; Gargir, A.; Gortler, M.; Altstock, R. T.; Shtevi, A.; Weisshaus, O.; Fire, E.; Dotan, N.; Schnaar, R. L. Glycobiology 2004, 14, 197– 203.
