Reaction of proteins with glutaraldehyde

ARCHIVES

OF

BIOCHEMISTRY

AND

Reaction

BIOPHYSICS

126,

of Proteins

A. F. S. A. HABEEB Department

of Biochemistry,

16-26

(1968)

with

Glutaraldehyde

AND

R. HIRAMOTO’

St. Jude Children’s Research Hospital and Units, Memphis, Tennessee Zl8101 Received

November

20, 1967;

accepted

January

University

OJ Tennessee

Medical

2, 1968

Glutaraldehyde was found to react with the a-amino groups of amino acids, the N-terminal amino groups of some peptides and the sulfhydryl group of cysteine. The phenolic and the imidazole rings of tyrosine and histidine derivatives were partially reactive. With proteins such as bovine serum albumin, ovalbumin and human gamma globulin, glutaraldehyde reacted predominantly with the c-amino groups of lysine to form mainly intermolecular cross-linkages. Some reaction, however, did occur with tyrosine, histidine, and sulfhydryl residues. The soluble aggregated proteins were capable of reacting with antibodies against their respective native proteins. Glutaraldehyde was also capable of conjugating ovalbumin to bovine serum albumin and the product was found to contain only minor contaminants of aggregat,ed ovalbumin and bovine serum albumin.

Glutaraldehyde has been introduced as a fixative for electron microscopy and cytochemistry (1). It was found to be an excellent cross-linking agent which resulted in insolubilization of proteins and gave a relatively undistorted fixation of cellular structures. At the same time cellular ensymic activities were maintained. Moreover, glutaraldehyde has been used: (a) to cross-link crystals of carboxypeptidase-A to give an insoluble network with reduced, but considerable enzymic activity (2, 3), and (b) to prepare water insoluble derivatives of trypsin with substantial enzymic activity (4). Since the above investigations were mainly concerned with preparing insoluble products, characterization of the reaction products of glutaraldehyde with proteins was lacking. The work presented in this paper was undertaken to study the reactivity of functional groups of amino acid residues in proteins with glutaraldehyde. As a preliminary, (1) the reaction with glutaraldehyde of model compounds such as amino acids and 1 Present address: ology, The University Birmingham, Alabama

Department of Alabama 35233.

of Medical

peptides was performed. (2) The now modified proteins were characterized by t’heir elution behavior on a Sephadex G-200 column and by their sedimentation coefficients. (3) Both intra- and inter-molecular crosslinks in the modified proteins were determined. (4) In order to assess whether the modified proteins were drastically changed from the native proteins, quantitative precipitin tests were performed with antibodies against the respective native proteins. (5) The ability of glutaraldehyde to effect intermolecular conjugation of ovalbumin with bovine serum albumin was examined. MATERIALS Bovine serum albumin (BSA) was obtained from Pentex, Inc.; human gamma globulin (H-rG), ovalbumin (OA), tyrosine, lysine, histidine, and leucylglycylphenylalanine from Nutritional Biochemicals Corporation; N-acetyl tyrosine ethyl ester monohydrate, 5,5’-dithiobis(nitrobenzoic acid) (DTNB), glycylglycylvaline, and 5-aminotetrazole monohydrate from Aldrich Chemical Company; N-acetyl histidine from Calbiochem; glycyltyrosine from Sigma Chemical Company; trinitrobenaenesulfonic acid (TNBS) from Eastman Kodak Company; 56% glutaraldehyde from Fisher Scientific Company; O-nitrobenzenesulfenyl chloride from Cycle Chemical Corporat,ion;

MicrobiCenter, 16

REACTION and Sephadex G-206 Pharmacia Chemical

and Blue Company.

OF Dextran

PROTEINS 2000 from

METHODS

A. Analytical Methods for Determining the Extent of Reaction of Functional Groups with Glutaraldehyde in Model Compoundsand Proteins 1. Pree amino groups. The free amino groups were determined by the trinitrobenzenesulfonicacid (TNBS) method using borate buffer, pH 9.0 (4, 5). Absorbance of the solution was read at 335 rnp and the percentage of modified free amino groups was calculated. Solutions of the respective unmodified model compounds or proteins served as standards containing 100% free amino groups, as determined by TNBS. Protein concentration was derived either from the nitrogen content determined by Kjeldahl digestion and followed by Nessler’s reagent, or from the absorption at 280 rnp (l.O-cm light path) of 1 mg/ml protein solutions, based on nitrogen determination. 2. Sulfhydryl content. The sulfhydryl content was determined using the reagent DTNB of Ellman (6) as described by Habeeb (7). 3. Phenolic and imidazole groups. The method of Sokolovsky and Vallee (8) was employed. This depends on the reaction of 5-diazo-lH-tetrazole with tyrosine and histidine to form bisazo derivatives, which maximally absorb at 480 and 550 rnp, respectively. 4. Tryptophan. Proteins and glutaraldehydetreated proteins were dried in a vacuum desiccator, dissolved in 1 ml 99% formic acid and then reacted with 0-nitrobeneenesulfenyl chloride in formic acid for 3 hours (9). The absorbance was read at 365 mp and used to calculate the tryptophan content.

B. Reaction of Glutaraldehyde with: 1. Model compounds. Five milligrams of the model compound (amino acid, amino acid derivative or peptide) were dissolved in 10 ml of 0.1 M phosphate buffer, pH 6. One ml of this solution was diluted with 9 ml water, and 1 ml of the diluted solution used to measure the concentration of functional groups at zero time. To the remaining 9 ml of the solution of model compound in phosphate buffer, 1 ml of a 5% glutaraldehyde solution in water was added and the solution was mixed. At. intervals, 1 ml samples were removed and diluted with 8 ml water. Duplicate aliquots of the 1 ml of the diluted solutions were used to determine the functional groups by methods described previot~sly. The percentage of reacted functional groups was calculated from the relative proportion of the

WITH

GLUTARALDEHYDE

17

absorbance of the solutions at zero time and at regular intervals. 2. Proteins. One hundred mg of protein was reacted in 10 ml 0.1 M buffer (sodium acetate, pH 4.0 and 5.0; sodium phosphate, pH 6.0 and 7.0), containing 0.5yo glutaraldehyde, at room temperature for W, 1, and 2 hours. At the end of the reaction, an amount of NaHS03, equivalent to 80% of the glutaraldehyde, was added. The preparations were first dialyzed exhaustively against water and then against 0.05 M borate buffer, pH 8.0 in 0.05 M KCl. 3. Conjugation of bovine serum albumin and ovalbumin. Seventy mg of BSA and 45 mg O-4 were dissolved in 0.1 M sodium phosphate buffer, pH 6.0 or 7.0. Then glutaraldehyde was added to give a concentration of 0.5% in a total volume of 10 ml. After reacting for 2 hours at room temperature, NaHS03, equivalent to 80% of glutaraldehyde, was added. The solution was dialyzed against water and then borate buffer.

C. Concentration of Glutaraldehyde-Treated Protein Xolutions Freeze drying was unsuitable since insoluble products resulted; therefore, solutions were concentrated by dialysis against solid sucrose. The sucrose that diffused through the dialysis bag was removed by dialysis against water and finally against borate buffer.

D. Chromatography of Glutaraldehyde-Treated Proteins on Sephadex G-ZOOColumns Sephadex G-200 was allowed to swell for 7 days in borate buffer, pH 8.0 (0.05 M in borate and 0.05 M KCl) and was packed into a chromat,ographic tube (1.5 X 120 cm). About 10 mg of the protein in 1 ml of the borate buffer, pH 8, was applied to the column and eluted with borate buffer. Fractions of 2 ml/tube were collected and analyzed by mess uring the absorbance at 280 rnp. This column served as an analytical tool to study the elution behavior of the modified proteins. For preparative purposes a column of Sephadex G-200 (2.5 X 94 cm) was used. Elution was performed with borate buffer, collecting 3 ml/fraction and the eluate was read at 280 rnp. Tubes containing protein peaks were pooled and the solution was concentrated and used for further studies.

E. Physical Methods 1. Ultraviolet absorption spectra of glutaraldehyde-treated proteins. In order to detect the changes in the chromophoric groups accompanying reaction with glutaraldehyde, the ultraviolet absorption spectra of the modified proteins were measured in borate buffer, pH 8.0, using a Cary recording spectrophotometer, model 14.

18

HABEEB

AND

2. Sedirr~~tation coeficients. Sediment,at,ion experiments were performed using 3 protein con centrations at 59,780 rpm at 20” in a Model E ultracentrifuge using an AN-D rotor. The protein was dialyzed against phosphate buffer, pH 7.5, ionic strength (CL) = 0.1, for 24 hours. Sedimentation coefficients were calculated for zero protein concentration on a salt free basis at 20” (d,.u). In addition the sedimentation coefficient was determined for aggregated BSA in 1 M propionic acid, supplemented with 0.2 M ?JaCl (to overcome any sedimentation potential due to the highly acidic solvent). This dissociating solvent was used to assess whether aggregation was due to covalent intermolecular cross-linkages or noncovalent in nature. 3. Optical rotation studies. In order to ascertain whether intramolecular cross-linkages were formed in the monomeric species (obtained from Sephadex chromatography) after reaction with glutaraldehyde, the specific rotation at various urea concentrations was compared to that of OA. Optical rotation studies were performed in a Carl Zeiss polarimeter at room temperature at 365 rnp in one decimeter polarimeter tubes. Solutions of ovalbumin, or glutaraldehyde-treated ovalbumin (2 mg/ml) in 0.15 M sodium chloride were adjusted to pH 11.5, with NaOH and contained urea at concentrations from O-7 M in a total volume of 3 ml.

F. Immunological

I1 IRAMOTO BSA conj ligate, 1 ml of supernatant (in the region of antibody excess) was added to 1 ml of anti-OA antiserum (if OA-BSA conjugates were tested initially with ant,i-BSA antiserum). The mixture was incubated at 40” for 30 minutes and then at 4” for 2-3 days. The precipitate was washed with cold saline, dissolved in 3 ml 0.1 N NaOH and the absorbance read at 280 rnp. From the absorbance of total precipitat,e (ant.ibody + antigen), the percent of contaminating OA polymer was calculated from a precipitin curve of OA polymer and anti-OA antiserum. Similar methods were adopted to estimate the per cent of BSA polymer as a contaminant inOA-BSA conjugate. 2. Double diffusion in agar. The Ouchterlony technique (10) was performed by pouring 15 ml of 1% (w/v) Noble agar, buffered with physiological saline, pH 7.2-7.4 and containing l/10,000 merthiolate, into a Petri dish. Antisera and antigens were placed in wells and the precipitin lines were allowed to develop in a moist atmosphere at room temperature for l-3 days. 3. Immunoelectrophoresis. Immunoelectrophoresis was performed on microscope slides covered with a film of 1.5% Noble agar in barbital buffer, p = 0.1 and pH 8.4 containing l/10,000 merthiolate. After electrophoresis of the antigen (10 mA per slide for 1.5 hours), the antiserum was placed in a trough and the precipitin lines allowed to develop at room temperature.

Methods

1. Quantitative precipitin determination. Antisera to BSA and OA were obtained from hyperimmunized rabbits. To 1 ml of anti-native protein serum were added increasing quantities of the native or modified protein in 1 ml borate buffer. The mixtures were incubated for 30 minutes at 40”, then at 4” for 2-3 days. After centrifugation, the precipitate was washed twice with cold physiological saline, dissolved in 3 ml 0.1 N NaOH and the absorbance read at 280 rnp. The absorbance due to the antigen was subtracted to give the absorbance of the precipitated antibody. From the amount of antibody precipitated and the amount of antigen, the molar ratio of antibody/antigen was calculated (in the region of antibody excess) per 69,000g for BSA or modified BSA. Precipitin curves were performed with conjugated OA-BSA, using anti-B&4 antibody or antiOA antibody. After removal of antigen-antibody complex, the supernatant was tested by the Ouchterlony technique. Anti-OA antiserum was used in the first case and anti-BSA antiserum in the latter, to assess any contamination by polymers of OA or BSA, respectively. To determine quantitatively the amount of contamination of polymers of OA and BSA or both, which may be present in OA-

RESULTS

Reaction of Glutaraldehyde

with:

1. Model compounds. Table I gives the percentage of reacted functional groups of model compounds with glutaraldehyde. The complete reaction of the N-terminal amino groups of the peptides, glycyltyrosine, leucylglycylphenylalanine and glycylglycylvaline, and both the a-amino group and sulfhydryl of cysteine occurred in 30 minutes. Whereas extensive reaction of the a-amino group of glycine and the CY- and e-amino groups of lysine took place with glutaraldehyde, only partial reaction of the cr-amino groups of histidine and tyrosine occurred. In addition, partial reaction occurred with the phenolic ring of N-acetyl tyrosine ethyl ester and glycyltyrosine. The imidazole ring of histidine was somewhat reactive, while that of N-acetyl histidine was affected very little, if at all. 2. Proteins. Considerable reaction occurred with the free amino group in the

REACTION

OF

PROTEINS

WITH TABLE

RKKTION

OF

Model

Glycine Lysine Cysteine Leucylglycylphenylalanine Glycylglycylvaline Tyrosine N-Acetyl tyrosine-ethyl Glycyltyrosine Histidine N-Acetyl histidine

FUNCTIONAL

GROUPS

compound

ester

98-100 37-37-37 -

BSA I

80

OA 70

60

2

H-YG 50

B 6 2

40

I: 3 Cl 8 8 z E

30

20

IO

.5

I

1.5

2.0

TIME OF REACTION (HOURS)

FIG. 1. Reaction of bovine serum albumin, ovalbumin and human y globulin with glutaraldehyde as a function of time and pH; A--A human y globulin at pH 4; ovalbumin: O--O pH 4; A---A pH 5; and l l pH 6; bovine serum albumin; X--X pH 4; O-0 pH 6, and +m pH 7. For other conditions, see text.

first hour, but slowed down thereafter (Fig. 1). Human y globulin gave only a soluble product when reacted at pH 4.0, and no NaHS03 was added at the end of the reaction. If the reaction was performed at either pH 5.0, 6.0 or 7.0, the product was insoluble. Although the reaction of glutaraldehyde with the amino groups of OA did

WITH

Imidazole

ring

-

81-8&92 73-73-78 93-100 lOIt 100-100 20-29-39 -

90

:.5

COMPOUNDS

groups

a The first number in each column represents percent the second aft,er 1 hour and the third after 2 hours.

x $ w f: 2 2

I

OF MODEL

Free amino

19

GLUTARALDEHYDE

GLUTARALDEHYDE~ Phenolic

ring

Sulfhydryl

-

100 -

10-16-16 20-19-M

30-39-30 5-4-4 reaction

with

glutaraldehyde

group

after

30 minutes,

not seem to be pH dependent, modification of the free amino groups of BSA was pHdependent between pH 4.0 and 7.0. An insoluble product resulted when BSA was reacted with glutaraldehyde at pH 5.0, whereas soluble, modified BSA formed at other pH values. Whenever glutaraldehyde reacted with OA, HrG, or BSA, the solution exhibited a straw color. The conjugation of OA to BSA by glutaraldehyde at pH 6 or 7 gave a soluble product, while at pH 4, the conjugate was insoluble. Whereas the major reaction occurred with the free amino groups, partial reaction took place with the phenolic ring of t’yrosine, imidazole ring of histidine in BSA and sulfhydryl group of OA (Table II). Although not reported in Table II, tryptophan residues were found to be unaffected by glutaraldehyde treatment of BSA or OA. Behavior of Glutaraldehyde-Treated Proteins on Analytical Sephadex G-200 Column The elution profiles of glutaraldehydetreated OA, BSA and HrG are shown in Fig. 2. Heterogeneity of the reaction products was evidenced by the presence of two species: one species was eluted at the void volume of the column (designated I) while the other (designated II) was eluted at a volume similar to the native protein. Wit’h BSA and HrG, the fast eluting component was predominant after reacting for 30 minutes, whereas with OA, a considerable amount of the slowly eluting material persisted after reaction for the sametime period.

“0

HABEEB

AND

HIRAMOTO

TABLE Rsac~rrori

WITH

OF GLUTSRALDEHYDE

FREE

SERUM

GL-BSA

GL-BSA

OA GL-OA

ALBUMIN

GROUPS, AND

HISTIDINE

AND

TYROSINE

OF BOVINE

OVALBUMIN~

Number of unreacted amino acids, residue/mole protein

Protein BSA GL-BSA

II

AMINO

Free amino group

4a 4b 4c 6a 6b 6c 7a 7b 7c 4a 4c

Histidine

61 24 15 11

17.8 16.3 13.2 -

18.2 17.5 14.9 -

18 11 7 11 10 7 21 16 11

13.0 13.0 12.3 12.0 11.3 12.5 5.6 5.3 6.8

14.0 14.3 14.5 14.7 13.8 15.0 7.0 5.8 7.2

a GL refers to glutaraldehyde-treated pH of the reaction; a, b, and c refer thesis is from literature (11).

(7.4)

Sulfhydryl

(9.8)

protein; the number after the protein to reaction times of x, 1, and 2 hrs, respectively.

The elution pattern of conjugated OABSA on a preparative Sephadex G-200 column is shown in Fig. 3. Only one peak was obtained, free of the slowly eluting component. The contents of the tubes designated by the arrow were pooled, concentrated and used for further studies. PHYSICAL

Tyrosine

METHODS

C’ltraviolet Spectra of Gluturaldehyde-Treated Proteins The ultraviolet spectra of the modified BSA slid OA in borate buffer, pH 8, showed a shift in their maxima from 280 rn@ to 265 rnp (Fig. 4). There was also an increase in the value of E :k: at 280 rnN, indicating some change in the chromophoric groups.

symbol refers to the Number in paren-

GL-BSA4aJ in 1.0 M propionic acid, a highly dissociating solvent, supplemented with 0.2 M N&l. The sedimentation coefficient of 9.6S, though somewhat lower than 14.45, was much greater than that of the monomer. It is reasonable to assume that in the 1.0 M propionic acid, some unfolding or expansion of the protein molecule occurred and contributed to a value of 9.6s for the sedimentation coefficient. The slowly eluting components (minor components of reaction of glutaraldehyde with proteins) GL-BSA4a,II and GL-OA4a,II, were monomers having sedimentation coefficients of 5.0s and 3.8S, respectively. The fact that they were not native proteins was evident from the U.T:. absorption spectrum of GL-BSA4a,II which showed a characteristic maximum at 264 rnp (Figure 4), instead of the normal maximum at 280 w.

Sedimentation Coeflicients of GlutaraldehydeTreated Proteins Figure 5 gives the sedimentation coefficients as a function of concentration for GL-OA4a,II; GLBSA4a,II; GL-BSA4b,I; GL-OA4cJ; and conjugated OA-BSA. The sedimentation patterns indicated the homogeneity of GL-OA4aJI and GLOA4c,I while the conjugated OA-BSA showed some asymmetry. Sedimentation coefficients showed that the rapidly eluting material (from the Sephadex column) was an aggregate; s!o.~ of GL-BSA4bJ was 14.45. Evidence that the aggregated material was formed by covalent intermolecular linkages was derived from the sedimentation pattern of

4.1 3.2 2 .R

Optical Rotation Results that the molecular from the between (Fig. 6) specific dicative

from specific optical rotation indicated monomer GL-OA4a,II exhibited intracross-linkages, since it was stabilized unfolding effect of urea at concentrations 1 and 5 M. On the other hand, native OA showed marked increase in the negative optical rotation in presence of urea, inof unfolding.

Immunological Results Quantitative precipitin BSA with glutaraldehyde

curves. Treatment at pH 6 or pH 7, had

of no

REACTION

OF

PROTEINS

WITH

GLUTARALDEHYDE

21

to that of the native protein. All antibodies to native proteins were precipitable with the modified aggregated proteins. The molar ratio of antibody/antigen versus the antigen concentration shown in Fig. 8 for BRA or modified BSA and anti-BSA antibody system, indicates that t,here was almost a 50y0 reduction in the ability of antibody to combine with modified antigen. This phenomenon may indicate that some determinant groups on the antigen were either damaged or masked. When several preparations of GL-BSA were compared,usinganti-BSAantibodyin an Ouchterlony plate with BSA, precipitin lines developed, fusing with the BSA line. However, complete identity of the reaction was not established because BSA gave a spur (Fig. 9). The Ouchterlony results indicated that the modified protein did not possess all the antigenic determinant,s present on native BSA. Conjugated OA-BSA gave one precipitin arc with either anti-BSA or anti-OA antiserum by immunoelectrophoresis (Fig. lo), indicating the presence of antigenic determinant,s of both BSA and OA on the same prot)ein conjugate. Precipitin curves of OA-BSA conjugate, with anti-BSA, or anti-OA antiserum, are given in Fig. 11. After precipitating OA-BSA (in the range of 0.084.63 mg/ml) with anti-BHA antiserum t,he supernatants

02

0 t

Fro. 2. The elution profile of glutaraldehydetreated BSA, OA and HyG on a (1.5 120 cm) Sephadex G-200 column (2 ml per tube). GL-BSA4a and GL-BSA4c represent glutaraldehyde-treated BSA at pH 4 for 35 and 2 hr, respectively. Similar symbols are used for ovalbumin (OA) and hllmarr -, globulin (HyG).

X

effect on the amount of antibody precipitated at equivalence with aggregated BSA; it only increased the antigen concentration at equivalence [Fig. 7). Similar results were obtained with ovalbumin treated with glutaraldehyde. The slowly elrlting component gave a precipitin curve similar

FIG.

3. The elution pattern of OA-BSA conjtlgate (3 ml per tube). The conjugation was perfarmed 6. For other condit,ions see text,. -_---.- .~ a.t, ..~ ~JH I

2”

IIABEEB

AND

HIRAMOTO

m-

0.8 -

0.7 : 5 g 0.64 2 0.5 -

0.4 -

03 -

0.2 -

O.l0.0

_-

250

300

---.

I

350

1 400

WAVE LENGTH

FIG. 4. The ultraviolet absorption spectra of glutaraldehyde-treated bovine serum albumin in borate buffer pH 8. BSA (0.95 me/ml) . . . ; GL-BSAGc,I (0.27 mg/ml) - -; GL-BSA4aJ (0.41 mg/ml) p; GL-BSA4b,I (0.43 mg/ml) -.-; GL-BSA4c,I (0.47 mg/ml) -----; GL-BSA7c,I (0.27 mg/ml) -. . ‘--; CL-BSA4aJI (0.135 mg/ml) - -.-. The numeral after GL-BSA indicates the pH at which reaction was conducted; a, b, c, time of reaction ($5, 1 and 2 hr, respectively); “I” and “II” represent the rapidly elut,ing and slowly eluting components, respectively, obtained from t.he Sephadex column. were reacted with anti-OA in an Oucht,erlony plate (Fig. 12). Weak precipitin lines showed at high concentrations of OA-BSA conjugate. The calculated amount of contaminating aggregated OA amounted to 70/ when a precipitin curve was performed on the supernatants, as described in the experimental section. On the other hand supernatants, after precipitating OA-BSA conjugate with anti-OA, gave very weak precipitin lines with anti-BSA antiserum in an Ouchterlony plate. The calculated contaminating aggreagted BSA was about 5%. FIG. 5. The sedimentation coefficients of gilltaraldehyde-treated bovine serum albumin, oval bumin and OA-BSA conjugate as a function of protein concentration in phosphate buffer, pH 7.5 and fi of 0.1. With GL-BSA4a,I v---v, sedimentation was performed in 1 M propionic acid supplemented wit,h 0.2 M NaCl. Symbols as in Fig. 4.

Since glutaraldehyde is a bifunctional aldehyde, its mechanism of reaction is likely to be analogous to formaldehyde. FraenkelConrat, et al. (12, 13, 14) have studied ex-

REACTION 300

OF

PROTEINS

WITH

GLUTARALDEHYDE

23

r

230 t 220

L

I I

I I 2 3 CONCENTRATION

I , 4 5 OF UREA

I 6

I 7

M

FIG. 6. Specific optical rotation of ovalbumin O---O and GL-OA4a, II k----8 in urea solutions of different concentration at pH 11.5.

CONCENTRATION

OF ANTIGEN

CONCENTRATION

OF ANTIGEN.

mq/ml

FIG. 8. The molar ratio of antibody/antigen of bovine serum albumin and glutaraldehyde-treated BSA on reaction with anti-BSA antibody. 0, BSA; l , GL-BSAGb,I; A, GL-BSA7cJ; 13 GLBSABaJ. Symbols as in Fig. 4.

lmqlml)

FIG. 7. Quantitative precipitin curves. Top, ovalbumin derivatives and anti-ovalbumin antibodies, ovalbumin A---A ; GL-OA4aJ X-X; GL-OA4cJ O---O; GL-OA4a, II O---O. Bottom, bovine serum albumin derivatives and anti-BSA antibodies; BSA A-A; GL-BSABa, I X-X; GL-BSABb,I O--O ; GL-BSA6c.I l ~ l ; and. GL-BSA7c ,IVm~m-mm-V.

FIG. 9. Double diffusion in agar; central contained anti-BSA antibodies, wells 1-6 tained, respectively: BSA; GL-BSA4a,I; BSA4c,I; BSA; CL-BSAGc,I, and GL-BSAGa,I.

tensively the reaction of proteins with formaldehyde. They observed that the first step in the reaction involves the free amino groups with the formation of amino methyl01 groups which then condense with other functional groups (e.g., phenol, imidazole, and

indole groups) to form methylene bridges. Reaction also occurred between formaldehyde and the sulfhydryl group of cysteine (15). In this work glutaraldehyde has been shown to be very reactive towards the N-

well conGL-

2-k

IIABEEB

AND

terminal amino groups of peptides as \vell as the a-amino groups of amino acids. The sulfhydryl group of cysteine was also very reactive. While the imidazole group of histidine was partially reactive with glutaralde-

+ FIG. 10. Immuuoeleotrophoresis conjugate. Top trough contained antibodies and bottom trough bovine serum albumin antibodies.

of OA-BSA anti-ovalbumin contained anti-

LP . . t$1.2 . LO c HII~AMOTO

A:TI-MA

.

CONCENTRAT,ON OF OA-WA

FIG. 11. Quantitative BSA conjugat,e with OA antibody.

CONJUGATE

ANTIBODY

(mg/ml)

precipit.in curves ant.i-BSA ant,ibody

of OAor anti-

FIG. 12. Double diffusion in agar of supernatant obtained from precipitin curve of: (A) OA-BSA conjugate and anti-BSA antibody (Fig. 11). Central wells contain supernatants obtained from the following concentration of OA-BSA in mg/ml: 1, 0.08; 2, 0.158; 3, 0.315; 4, 0.63, and 5, 0.44. The wells on right contain BSA at 0.118 mg/ml concentration. The wells on left contain anti-OA antibodies. (B) OA-BSA conjugate and anti-Oh antibody. Central wells contain supernatants obtained from same concentrations of OA-BSA as in (A) with the exception that well 6 contains 0.88 mg/ml. The wells on right rontain OA at 0.125 mg/ml concent,ration. The wells on left, contain anti-BSA antibodies.

REACTION

OF

PROTEINS

hyde, that in N-acetyl histidine was slightly reactive, if at all. It seems that the presence of a free amino group was necessary for glutaraldehyde to react with the imidazole group. The phenolic ring of tyrosine was partially but equally reactive in N-acetyl tyrosine ethyl ester and glycyltyrosine. As with model compounds the most reactive groups in proteins were the free amino groups. Whereas about 90% of the free amino groups in BSA were modified with glutaraldehyde at pH 6 or 7 in 2 hours, only 70% were modified with formaldehyde in 7 days at 37” (5). The limited reactivity of the free amino groups and sulfhydryls of ovalbumin may result from steric hinderante. Steven and Tristram (16) found that only three c-amino groups of lysine were reactive with 1-fluoro-2,4dinitrobenzene and that denaturation by ethanol increased the availability of the c-amino groups for reaction. Alexander (17) observed that the sulfhydryl groups of OA reacted with Nethyl maleimide only when denatured with Duponol. The reaction of glutaraldehyde with proteins produced two species (monomers and aggregates) which were successfully fractionated on Sephadex G-200 columns. The monomer constituted the minor component and it had a sedimentation coefficient similar to that of the native protein. Measurements of specific optical rotation of monomer at different urea concentrations indicated the presence of intramolecular cross-linkages. On the other hand, the soluble aggregates were the major species and were formed through intermolecular cross-linking. They exhibited about a three-fold increase in sedimentation coefficient, as compared to their native counterparts. The formation of intra- and intermolecular cross-links with proteins on reaction with bifunctional reagents was reported by various workers, depending on the conditions of their experiments. The bifunctional reagent p ,p’-diAuoro-m ,m’-dinitrodiphenyl sulfone formed intramolecular cross-links with BSA at a concentration of 1 %, whereas at higher concentrations, insoluble aggregates resulted due to intermolecular cross-reaction (18). Diethyl malonimidate dihydrochloride pro-

WITH

GLUTARALDEHYDE

25

duced intramolecular cross-links at pH 8.5, with BSA at concentrations between 1.5 and 6%. At a concentration of 20%, however, both soluble and insoluble aggregates occurred (19). Hartman and Wold (20) found that the bifunctional reagent dimethyladipimidate, when reacted with ribonuclease, formed monomers, dimers and higher aggregates. Ultraviolet absorption spectra of glularaldehyde-treated OA and BSA exhibited a new maximum at 265 rnp and an increase in absorbance at 280 mp, dependent on the individual protein and extent of reaction with glutaraldehyde, (e.g., GLBSA7c, E7: ,$ at 280 rnp = 2.62 compared to 0.66 for BSA). The blue shift as well as the hyperchromism seem to have been due to reaction with tyrosine. Ko such spectral changes were observed when salmine, devoid of tyrosine and tryptophan, was reacted with glutaraldehyde. This conclusion was also supported by the finding from chemical analysis that some tyrosine residues reacted with glutaraldehyde. The low recovery of tyrosine in ovalbumin, as determined by 5-diazo-lH-tetrazole, may indicate steric hinderance and is consistent with results found by Sokolovsky and Vallee (S). From amino acid analysis after acid hydrolysis, Quiocho, and Richards (3) found that 60% of the lysine groups in carboxypeptidase A crystals were modified by glutaraldehyde. Our results were derived by the TNBS method (5) in which the assay for free amino groups is performed under mild condibions, such that hydrolysis of covalently blocked t-amino groups is unlikely. The fact that Quiocho and Richards (3) did not observe reaction with tyrosine or histidine may have been due to the labilit,y of glutaraldehyde-modified tyrosine or histidine on acid hydrolysis. However, their results showed 6.8 residues of histidine in glutaraldehyde-treated carboxypeptidase-A as compared to S.0 residues for native enzyme. Intermolecularly cross-linked bovine serum albumin or ovalbumin retained the ability to precipitate all the respective antibodies from an antiserum, although quantitative changes were apparent. There was a

26

HABEEB

AND

reduction of the combining Dower its demon&rated by a decrease in the molar ratio of antibody/antigen. When glutaraldehyde was able to crosslink ovalbumin to bovine serum albumin, the conjugate was found to be about 88% pure. This property may be extended to other proteins and it would be of interest to study the antigenic competition which occurs in some cases when two antigens are injected simultaneously in an animal (21). Moreover, glutaraldehyde may be useful as a substitute for formaldehyde for the inactivation of bacteria, toxins, and viruses to immunize humans and animals against bacterial and viral infections. -

A

ACKNOWLEDGMENT This work was supported in part by the American Lebanese Syrian Associated Charities (ALSAC), by National Science Foundation Grant GB4162, the National Institutes of Health Grant AI-W352-01 and the John A. Hartford Foundation Inc. of New York. REFERENCES 1. SABATINI, D. S., BENSCH, K., AND BARRNETT, R. J., J. Cell Biol. 17, 19 (1963). 2. QUIOCHO, F. A., AND RICHARDS, F. M., Proc. iv&Z. Acad. Sci. U.S. 62, 833 (1964). 3. QUIOCHO, F. A., AND RICHARDS, F. M., Biochemistry 6, 4062 (1966). 4. HABEEB, A. F. S. A., Arch. Biochem. Biophys. 119, 264 (1967).

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J. Am. Chem. Sot. 70,2673 (1948). 13. FRAENKEL-CONRAT, H., AND OLCOTT, H. S., J. Biol. Chem. 174, 827 (1948). H., AND MECHAM, D. K., 14. FRAENKEL~ONRAT, J. Biol. Chem. 177, 477 (1949). 15. FRENCH, D., AND EDSALL, J. T., Adv. Protein Chem. 2, 277 (1945). 16. STEVEN, F. S., AND TRISTRAM, G. R., Biochem. J. 70, 179 (1958). 17. ALEXANDER, N. M., Anal. Chem. 30, 1292 (1958). 18. WOLD, F., J. Biol. Chem. 236, 106 (1961). 19. DUTTON, A., ADAMS, M., AND SINGER, S. J., Biochem. Biophys. Res. Commun. 23, 730 (1966). 20. HARTMAN, F. C., AND WOLD, F., J. Am. Chem. Sot. 88,389O (1966). 21. ADLER, F. L., Progr. Allergy 8, 41 (1964).