Gel properties as a hydrogel of crosslinked poly (l -ornithine) using organic crosslinking agents

Polymer Gels and Nerworks 3 (1995) 71-84 0 1994 Elsevier Science Limitrd Printed in Northern Ireland. All rxhts reserved 0966-78~2/YS/$OY~50 ELSEVIER

096fb7822(94)00023-9

Gel Properties as a Hydrogel of Crosslinked Poly (L-ornithine) using Organic Crosslinking Agents Hiroyuki Yamamoto Institute

& Yuuki Hirata

of High Polymer Research, Faculty of Textile Science and Technology, Shinshu University, Ueda 386, Japan

(Received 3 March 1994; revised version received 3 June 1994; accepted 4 June 1994)

ABSTRACT Gel formations of water-soluble cationic ornithine polypeptides were examined using organic aliphatic crosslinking agents such as dialdehydes and diketones in water systems. When l/20-.5 equivalent molar amounts of organic crosslinking agents were added to the ornithine polypeptide systems, the corresponding gels were formed. Among the organic crosslinking agents used, glutaraldehyde was the most effective for the gel formation. As a whole, the molecular weight of the samples, the amino acid compositions, the crosslinking agents used, the molar ratios between crosslinking agents and functional residues, and system pH levels were ,found to play roles in the gel formation. The gels formed were characterized by swelling properties and by the selective adsorption ability of some amino acids. The polyornithine gels exhibited reversible, but hysteretic swelling in a water-acetone mixed solvent. Due to the cationic S-amino moieties which remain unreacted, the acidic amino ucid, aspartic acid, was adsorbed into the gels’ matrix, exhibiting the predominant adsorption. Biodegradable characteristics of the copoly (ornithine tyrosirze) [copoly (Orn Tyr)] gels by chymotrypsin were also investigated.

INTRODUCTION It has been widely observed in aqueous systems that water-soluble proteins remain insoluble, or exhibit only surface adhesion, in biological systems.’ With respect to the insolubilization and adhesion phenomena of the natural biopolymeric systems, a variety of explanations 71

72

H. Yamamoto.

Y. Hirata

including the well-known mechanism called auto-crosslinking have been proposed to explain the insolubilization of proteins secreted by marine invertebrates. Auto-crosslinking occurs mainly between the tyrosyl residues of protein and the free lysyl amino groups of other protein molecules, with an oxidation enzyme forming an intermolecular crosslink. Apart from the biological interests, crosslinked polymers in watery systems have long been an important class of materials, and are used in a diverse assortment of applications as hydrogels including medical wound dressings; progress has been made in developing approaches to the description of the molecular structure of crosslinked polymers.“,4 The interaction of glutaraldehyde with poly(L-lysine) (PLL) as a model for the tanning process of hides has also been reported.5,6 The number of methylene groups (n) on the side chains of a poly-a-amino acid is essential in the establishment of particular characteristics such as chain conformation and reactivity. For instance, at high values of pH in aqueous solutions PLL (n = 4) is completely a-helical at room temperature, while poly(L-ornithine) (PLO, n = 3) is only partially so, revealing a decreasing stability of a-helix accompanying a shortening of the side chain.’ We have investigated the polymer chemistry of adhesive proteins in watery systems. During the investigation we conducted insolubilizing experiments of some barnacle adhesive protein models’ using an oxidase, and also reported the insolubilizing experiments including gel formation of simplified water-soluble lysine (Lys) polypeptides”-” using organic crosslinking agents. In our ongoing study we reported the insolubilizing results of ornithine (Orn) polypeptides in a previous paperI and describe here the gel formation and its properties as a biohydrogel of water-soluble Orn polypeptides using organic crosslinking agents, dialdehydes and diketones.

EXPERIMENTAL Materials Dialdehydes (glyoxal and glutaraldehyde), diketones (2,4-pentanedione and 2,5hexanedione), and enzymes chymotrypsin (lot no. APJ 1868) and trypsin (lot no. PTM 122.5) were purchased from Wako Pure Chemical Industries, Ltd. Most experiments were carried out in distilled water and waterorganic solvents mixed media. The pH of the polypeptide solutions in

Gel properties as a hydrogel of crosslinked poly(L-ornithine)

Molecular

Weights

TABLE 1 for Poly(L-ornithine)

Sample

and Copoly(Orn’

Molecular weight”

73

Tyr’)

Degree of polymerization

Poly(L-ornithine)

Copoly(Orn’

a An average

14000 57 000 140 000

70 290 720

25 000

140

Tyr’)

value from the viscosity

equations

(see Ref. 12).

distilled water was brought to pH 3-12 by the addition of NaOH or HCl (O*Ol-0.1 M), and was measured with a Beckman digital pH meter. The phosphate buffer (0.02 M) was used when necessary. PLO and copoly (Om Tyr)

PLO and copoly (Orn’ Tyr’) were synthesized previous article.‘* The water-soluble polypeptides ment are listed in Table 1. Preparation

as described in a used in this experi-

of solid and swelling gels

Solid gel

As an example, to an alkaline solution containing 5 mg of PLO (25.6 pmol residue) in 2 ml of water (over pH 1l), 50.9 mg (53.3 ~1) of 25% glutaraldehyde (128 pmol) in 2 ml of distilled water was added and the mixture was allowed to stand without stirring giving a solid gel after 40 h. Swelling gel

As an example, to a solution containing 20 mg of PLO (102 pmol residue) in 0.1 ml distilled water (pH 5), (5.1 pmol of glutaraldehyde was added. The gels formed were washed with distilled water, centrifuged (15 000 rpm for 30 min at 2O”C), washed with ethanol, centrifuged (15 000 rpm for 30 min at 20°C) again, and dried.

H. Yamamoto,

74

Gel Preparation

of Ornithine

DP

Sample

290

PLO

TABLE 2 Containing Polypeptides at 20°C Crosslinking agents

Glutaraldehyde

Glyoxal

2,SHexanedione 2.4.Pentanedlone 720

2,SHexanedione 2,4-Pentanedlone

Copoly(Om

Tyr)

a + + red-brown;

140 + orange;

Glutaraldehyde f

Y. Hirata

pale yellow; -

by Organic

Reaction time (h)

Crosslinking

Agents

Gel formation

Colorization”

pH

Equiv. mol

2-3 12 2-3 12 2-3 12

1 1 l/20

20

solid

20

solid

0.5

swelling’

l/20

0.25

swellmg’

1

20

fragile

1

20

fragile

2-3 12 2-3 12 2-3 12 2-3 12

l/2 l/2 l/2 l/2 112 l/2 l/2 l/2

20

swelling’

20

swelling‘

20

fragile

2-3

112

20

fragile

20

swellmg”

20

swelling’

20

fragile

20

fragile swelling‘

++ ++ * f

+ ++ ++ ++ ++ +

colorless.

h high swelling. ’ low swelling.

Table 2 summarizes the experimental (solid and swelling) of PLO and copoly

conditions to prepare (Orn Tyr) gels.

two kinds

Method The turbidity, judged from the decrease of transmittance at 65Onm and 25°C was measured using a Jasco UVIDEC-1 spectrophotometer with stirring. The degree of swelling was determined from the ratios of (W,-W,)/W,, where W, is the weight of the swelled gel and W, is the net weight of the gel material after it was completely dried. The adsorption of amino acids was determined by calorimetry as follows. The PLO-gel used was 5-10mg when dried, and the aqueous solutions containing the equivalent molar amino acids [aspartic acid (Asp), alanine (Ala), Lys, and tryptophan (Trp)] were added to the pendant amino groups in the gel matrix. After stirring the mixture for lo-24 h, the remaining amino acids in the bulk were determined quantitatively from the ninhydrin development at 570 nm and from the absorption at 280 nm in the case of Trp.

Gel properties as a hydrogel of crosslinked poly(L-ornithine)

75

The biodegradable experiments were carried out as follows. First, the swelling copoly (Orn’ Tyr’)-glutaraldehyde (l/20) gel was prepared as described above and was immersed throughout in distilled water in order to remove unreacted crosslinking agents, and then chymotrypsin solution (400units) was added to the gel system at pH 8.

RESULTS

AND DISCUSSION

Crosslinking

Since the crosslinking reaction of PLO by the organic crosslinking agents was reported in detail in our previous article,12 we will avoid a repetitive description here, including only additional information about gel formation properties. When the organic crosslinking agents were used to insolubilize polypeptide samples, in most cases a rapid precipitation or gel formation was observed by spectroscopy.‘” When the crosslinking reaction was carried out in the solvent (system in the pH region above approximately 10*5), the reaction was almost complete after 0.5 h at pH 12. When the equivalent molar ratios of glutaraldehyde to amino groups were low, l/10-1/5, the crosslinking reaction took over 2 h; on increasing the ratio to l/2 the reaction was almost completed after about 0.5 h, forming a solid gel in both cases. Likewise, the crosslinking reaction using 2,5-hexanedione was almost completed after about 4 h. However, when comparing the dialdehydes and the diketones, the crosslinking reactions of the former were much faster (as an example, see Fig. 1). Gel formation

When the aqueous PLO and copoly(Orn’ Tyr’) systems containing dialdehydes were gently mixed (viscosity) or mechanically stirred (turbidity), the systems became turbid, exhibiting a phase separation. On the other hand, when allowed to stand without mixing or stirring the Orn-containing polypeptides and dialdehyde systems formed soft and solid gels after 20-40 h. When the ratios of the added glutaraldehyde to PLO residues were less than l/5, soft gels formed, and when the ratios were more than 1 (excess), solid gels formed. In cases where we used excess aliphatic dialdehydes (glutaraldehyde or glyoxal) as a crosslinking agent for copoly(Orn’ Tyr’) in distilled water, solid gels were also obtained. The copolypeptide gels formed with

76

H. Yamamoto,

Y. Hirata

Time(h)

Fig. 1. Change of turbidity dehyde and 2,Shexanedione 650 nm: A, glutaraldehyde;

of PLO (DP 290 sample) with l/2 equivalent glutaralat pH 11 in water by measuring the transmittance at 0, 2,Shexanedione. Polymer concentration: 0.25 g/dl.

glutaraldehyde were brownish red, while the copolypeptide gels formed with glyoxal were virtually colorless. System pH levels play a role in the gel formation. The gel formation in the alkaline pH region was much faster, but even in the acidic pH region the gel formed, albeit slowly. The gel formation in the neutral pH region was also easily realized. Accordingly, pH values are not so influential on gel formation. The PLO (DP 290 and 720 samples) formed soft gels with diketones (2,4+entanedione and 2,5hexanedione), while the copoly(Orn’ Tyr’) did not. When we used a lesser amount of the crosslinking agents, no gels were formed, as summarized in Table 2-wherein the molar ratios between the Orn residues and the crosslinkers, the reaction conditions, and color of the gel are listed. Swelling properties

Figure 2 shows the photographs of the PLO (DP 290 sample) gel crosslinked by l/20 equivalent amount of glutaraldehyde. The dry crosslinked PLO-glutaraldehyde (l/20) with cu. 5 mm diameter swells to a 16-fold apparent surface area (cn. 20mm diameter) in distilled water. Figure 3 shows the time course of the change in the degree of

77

Gel properties as a hydrogel of crosslinked poly(L-ornithine)

Fig. 2.

Swelling

of PLO

(DP 290 sample)-l/20 Drying; (b) in distilled

equivalent water.

glutaraldehyde

gel.

(a)

swelling of the PLO (DP 290 sample)-glutaraldehyde (l/20) gel by changing the sunken medium from water to organic solvent (acetone, ethanol, or 1,4-dioxane) and inversely from organic solvent to water. The PLO (DP 290 sample)-glutaraldehyde (l/20) gel exhibited mostly reversible expansion-contraction when immersed alternatively in water

0

10

20

30

40

50

60

70

Time (min)

equivalent glutaraldehyde Fig. 3. Swelling time course of PLO (DP 290 sample)-l/20 gel at 25°C in organic solvents and in water: A, in water and A, acetone; 0, in water and 0, ethanol; 0, in water and n , dioxane.

H. Yamamoto,

78

Y. Hirata

80 -

I

0’

0

1

10

I

20 Swelling

Fig. 4. dehyde

30

40

degree

Change of swelling degree of PLO (DP 290 sample)-l/20 equivalent glutaralgel by changing acetone ratio in acetone-water mixed media: A, starting in water; A, starting in acetone.

and in organic solvent media. Figure 4 shows the change in the degree of swelling of the PLO (DP 290 sample)-glutaraldehyde (l/20) gel after 30 min (swelling equilibrium) by changing the solvent composition in the water-acetone mixed media. The gel with a swelling degree of about 33 in pure water started to contract at an acetone concentration of about 60 ~01% and terminated at over 90 ~01% acetone, and, conversely, the shrunken gel with a swelling degree of about three started to expand at over 80 ~01% acetone and terminated at about 40 ~01% acetone, reverting mostly back to the initial swelling degree. The expansion-contraction was reversible but exhibited a hysteresis in the water-acetone mixed solvent system in both weight measurement (after 30 min, Fig. 4) and tube diameter measurement (after 24 h, not shown of the most typical swelling hydrogels. here). This is one Copoly(Orn’ Tyr’)-glutaraldehyde (l/20) exhibited similar swelling properties in distilled water (2*6-fold surface area).

Gel properties as a hydrogel of crosslinked poly(L-ornithine)

79

The PLO (DP 290 sample)-2,Shexanedione (l/l) gel formed by adding one equivalent molar hexanedione was fragile but exhibited a swelling gel property. On the other hand, the PLO (DP 720 sample)2,Shexanedione (l/l) formed in the same way was much stronger and, it can be seen from the swelling time course that the gel expanded to the swelling degree of cu. PO after l-3 min in distilled water and contracted to a degree of 3-5 when immersed for 1 min in acetone. This behavior of the PLO-2,Shexanedione gel in water and in acetone was also reversible. Selective adsorption

of amino acids in PLO gels

In our previous article we have reported the selective adsorption of anionic (Asp and Glu), neutral (aliphatic Ala and aromatic Trp), and cationic (Lys) amino acids in the matrix of the PLL-glutaraldehyde gel in order to understand the fundamental character of the cationic crosslinked PLL gel. In order to understand the side chain length effects of the crosslinked PLL homologue series, we examined the adsorption characteristics of the crosslinked PLO gel. Figure 5 shows the adsorption time course of Asp (pKa values of 1.9, 3.7 and 9.6) at pH 7 and 25°C in the matrix of the PLO (DP 290 sample)-glutaraldehyde (l/10) gel. When equimolar amounts of Asp were added to the &amino groups of Orn residues in the gel, the adsorption of the Asp reached a final maximum value after 10 min when 47% of the Asp had been adsorbed (Fig. 5(a)). This was predominantly due to free P-COO- (COO;) with a higher pZ& value of 3.7 (see Fig. 6(b). When half amounts each of Asp and Trp were added to the &amino groups of Orn residues of the above gel, the adsorption of the Asp and Trp reached final values after 10 and 2 min, respectively, when 63% of the Asp and 5% of the Trp had been adsorbed (Fig. 5(b)). Figure 7 shows the pH dependence of the Asp adsorption of the PLO (DP 290 sample)-glutaraldehyde (l/10 and l/20) gels, together with the results of the PLL (DP 240 sample)-glutaraldehyde (l/10) gels. The PLO-glutaraldehyde gels adsorbed the acidic amino acid Asp in a wide neutral pH region (pH 5-9), exhibiting an optimal pH at 7, but adsorbed a few equivalent molar Asp even at pH 3 and 11 due to a weak interaction. This is shown schematically in Fig. 6(a). The origin of another weak interaction between the cr-amino group (plYa3 9.6) of the _4sp and the crosslinked PLL and PLO gel matrices at pH 11 is unclear. However, from the independent adsorption results using benzoic acid

80

H. Yamamoto,

Y. Hirata

70

a 60

2

50

0’ ;a 40 b 9 z

30

E 2 20 5 10

0

0

I

I

10

20

1

30

’

/I-

40 Time

10

0

20

30

40

( min)

Fig. 5. Adsorption time course of Asp and Trp in PLO (DP 290)-l/10 equivalent glutaraldehyde gel matrix at pH 7 and at 25°C: 0, Asp; & Trp. (a), Free S-amine, 23.7 pmol residues and added Asp, 24 pmol; (b) free b-amine, 23.7 pmol residues and added Asp and Trp, 12 pmol, each. The amounts of adsorption of amino acids in (b) were half that in (a).

and the PLO-GA gel, the Donnan equilibrium between the ionic solute and the cationic gel is estimated to participate in the adsorption to a small extent (to be reported later). The results of the adsorption of amino acids in the PLO-gel were summarized in Table 3, together with our earlier results with the -

NHYHCO-

-

NHYHCO-NHYHCO-

(yH2)3

(yH2)3

(yH2)3

vH3’

YH3’

rH3’ I

~

I

7002H

;~O$CH2

fyO>CH;cooi

’ .C, I ( NH,+ H

1 c ’ $.+<\ NH3+ H

Fig. 6. Two explanatory representations of the adsorption of Asp in PLOglutaraldehyde gel matrix (for COO, and COO; see text). Bold dashed lines stand for strong electrostatic interaction and dotted lines stand for weak supplemental interaction.

Gel properties as a hydrogel of crosslinked poly(L-ornithine)

81

aoti 70 t

2 60 t

3

I

I

I

I

I

I

I

4

5

6

7

8

9

10

11

PH Fig. 7. pH dependence of adsorption of Asp in PLO (DP 290 sample) and PLL (DP 240 sample)-l/l0 and l/20 equivalent glutaraldehyde gels; free w-amine, 23.7 pmol residues and added Asp 20pmol. 0, PLO (DP 290 sample)-l/l0 equivalent glutaraldehyde gel; A, PLO (DP 290 sample)-l/20 equivalent glutaraldehyde gel; X, PLL (DP 240 sample)-l/l0 equivalent glutaraldehyde gel.

The Adsorption

Sample

t_-Aspartic

Quantities

pKa (COOOH)

acid

1.94 3.70

L-Tryptophan r_-Lysine r_-Alanine n Free &amine

of Amino

23.7 pmol

TABLE 3 Acids Adsorption Gels PH

2.38 2.18 2.34

3 5 7 9 11 7 7 7

residues

and added

in PLO-glutaraldehyde

(GA)

Amount of adsorption (%) PLO-GA (l/IO) PLO-GA (l/20) 5 40 48 34 3 5 5 6 amino

acids 24 pmol

8 40 51 40 4 5 7 each.

82

H. Yamamoto,

Y. Hirata

PLL-gel. As can be seen from the results in Table 3, 5-6 molar % of cationic Lys and neutral Ala and Trp were adsorpted in the gel matrix. This 5-6% adsorption was due to COO, with a lower pKu, value [Fig. 6(a)]. Figure 6 illustrates the adsorption of amino acids in the crosslinked PLO-gel matrix due to the predominant electrostatic interaction. These findings suggest that: (1) P-carboxylate of Asp contributes, predominantly and selectively, to the ionic attraction (highest 51%); (2) in amino acids with zwitter ion, the lesser interaction contributed by a-carboxylate could be anticipated to adsorb acidic (Asp), neutral (Ala and Try) and cationic (Lys) amino acids (5-6%) in the cationic gel matrix; (3) when the adsorption results of the PLO (DP 290 sample)glutaraldehyde (l/20) gel is compared with the PLL (DP 240 sample)glutaraldehyde (l/20) gel, the amounts of Asp adsorpted are almost the same, exhibiting no side chain length effect, and (4) the crosslinking matrix itself formed by an organic crosslinking agent such as glutaraldehyde determines the adsorption characteristics. On understanding the results in Table 3, the most nebulous point is the chemical structure of the crosslinked bridge part. Although some chemical structures have been proposed,‘3 this has not yet been exactly determined because of the self-condensation reactivity of proteins and glutaraldehyde.‘4*‘s We are now trying to determine the chemical structure of the bridge part using simpler crosslinked PLL- and PLO-glutaraldehyde systems by the NMR techniques, and we will report on this elsewhere. Enzymatic

cleavage

of copoly(Om’

Tyr’) gels

In order to consider application as biodegradable hydrogels, copoly(Orn’ Tyr’)-glutaraldehyde gel was treated with an enzyme chymotrypsin (EC 3.4.21.2), which is an endopeptidase and digests the peptide bond at the tyrosine position. The chymotrypsin digested the copoly(Orn’ Tyr’)-glutaraldehyde gel matrix after 60 h and the gel matrix was completely reduced to a liquid, while the *enzymes trypsin and papain did not digest the copolypeptide’ gel. Biodegradation of cationic polypeptide hydrogels will be reported in detail in the near future. This gel formation, due to the crosslinking reaction, might not be directly related to the natural observation of the biological adhesion process described in the Introduction. However, the results themselves are interesting, and might offer some clues to understand biological

Gel properties as a hydrogel of crosslinked poly(L-ornithine)

adhesion, process.

which

clearly

includes

the

crosslinking

and

83

gel formation

CONCLUSIONS We have reported some data on gel formation as biohydrogel formulations using ornithine, containing polypeptides as a second case of cationic polylysine homologues. Based on the experimental results, we propose the following conclusions for a simple crosslinked polypeptide gel system: (1) dialdehydes react with S-primary amines in the side chains forming an intermolecular crosslinking; (2) excess dialdehydes without stirring form solid gels; (3) less dialdehydes or diketones form swelling soft gels; (4) swelling gels cause a reversible expansioncontraction accompanying hysteresis in water-acetone mixtures; (5) gels containing pendant ornithine residues in their matrix adsorb predominatingly acidic amino acid Asp; and (6) the copoly(Orn’ Tyr’)glutaraldehyde gel matrix is destroyed by chymotrypsin digestion exhibiting biodegradation. Finally, these findings have the prospect of being used to develop new material formulations as biohydrogels or biodegradable gels.

ACKNOWLEDGEMENT This work Foundation

was supported in part for the Electrotechnology

by a grant of Chubu.

from

the

Research

REFERENCES 1. Manly, R. S. (ed.), Adhesion in Biological Systems. Academic Press, NY, 1970. 2. Lindner, E., In Marine Biodeterioration: An Interdisciplinary Study, ed. J. D. Castlow & R. C. Tipper. Naval Institute Press, Annapolis, MD, 1984. 3. Dickie, R. A., Labana, S. S. & Bauer, R. S. (eds), Cross-Linked Polymers. American Chem. Sot., Washington DC, 1988. 4. Labana, S. S. & Dickie, R. A. (eds), Characterization of Highly Crosslinked Polymers. American Chem. Sot., Washington DC, 1984. 5. Blauer, G., Harmatz, D., Meir, E., Swenson, M. K. & Zvilichovsky, B., The interaction of glutaraldehyde with poly(cr, L-lysine), n-butylamine, and collagen. I. The primary proton release in aqueous medium. Biopofymers,

14 (1975) 258.5-98.

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H. Yamamoto. Y. Hirala

6. Swenson, M. K., Meir, E., Yanai, P. & Zvilichovsky, B., The interaction of glutaraldehyde with poly((~, L-lysine), n-butylamine, and collagen. II. Hydrodynamic, electron microscopic, and optical investigations on the reaction products. Biopolymers, 14 (1975) 2599-612. 7. Grourke, M. J. & Gibbs, J. H., Comparison of helix stabilities of poly-L-lysine, poly-L-ornithine, poly(t_-diaminobutyric acid). and Biopofymers, 10 (1971) 795-808. 8. Yamamoto, H. & Nagai, A., Polypeptide models of the arthropodin protein of the barnacle Balanus balanoides. Marine Chem., 37 (1992) 131-43. 9. Yamamoto, H., Kuno, S., Nagai, A., Nishida, A., Yamauchi, S. & Ikeda, K., Insolubilizing and adhesive studies of water-soluble synthetic model polypeptides. Int. J. Biol. Macromol., 12 (1990) 305-10. 10. Yamamoto, H., Tanisho, H., Ohara, S. & Nishida, A., Cross-linking and gel formation of water-soluble lysine polypeptides. An insolubilization model reaction for adhesive proteins. Int. J. Biol. Macromol., 14 (1992) 66-72. 11. Yamamoto, H. & Tanisho, H., Gel formation and its properties as hydrogel of cross-linked lysine polypeptides using organic cross-linking agents. Materials Sci. and Engng, 1 (1993) 45-51. 12. Yamamoto, H., Hirata, Y. & Tanisho, H., Cross-linking and insolubilization studies of water-soluble poly(L-ornithine). Int. J. Biol. Macromol., 16 (1994) 81-5. 13. Monson, P., Puzo, G. & Mazarguil, H., etude du mecanisme d’etablissement des liaisons glutaraldehyde-proteines. Biochimie, 57 (1975) 1281-92. 14. Kawahara, J., Ohmori, T., Ohkubo, T., Hattori, T. & Kawamura, M., The structure of glutaraldehyde in aqueous solution determined by ultraviolet absorption and light scattering. Anal. Biochem., 201 (1992) 94-8. 15. Kawahara, J., Shimizu, M., Orita, H., Nagawa, Y. & Hattori, S., The structure of glutaraldehyde in aqueous solution and its cross-linking mechanism with proteins (II). Polym. Preprints Jpn., 41 (1992) 3065-7.