A N A L Y T I C A L BIOCHEMISTRY 68, 7 9 - 8 6

(1975)

Quantitation of Alkylamino Side Chains Coupled to Agarose Beads 1 M I N O T I SHARMA 2 AND W I L S O N ROY S L A U N W H I T E , JR.

Department of Biochemistry, Schools of Medicine and Dentistry, State University of New York at Buffalo, Buffalo, New York, 14214 Received November 27, 1974; accepted April 4, 1975 Agarose, after activation with cyanogen bromide, was derivatised with various diamines. Activation was carried out most conveniently in alkaline phosphate buffer. After conversion of free functional groups to chromophoric groups, direct spectral analyses indicated much greater incorporation (213 _+ 15 (SD, n = 6) tzmoles/ml packed gel) of amines of various sizes than previously reported (1.5-10 /xmoles/ml gel). Agarose coupled with [~4C]spermine (sp act, 0.2 /zCi/mmole) showed incorporation of 177 and 190 /zmoles/ml of gel for radiometric and spectral analysis, respectively. Elemental analyses of the activated gel before and after coupling with the amine gave an incorporation of 121 /zmoles/ml gel from the difference and 324 tzmoles/ml gel from the total nitrogen content, assuming the loss of one atom and no atoms of nitrogen, respectively, during coupling. Thus, the high incorporation of amines into the matrix is observed in three independent methods.

The importance of affinity chromatography as an effective tool in purification of protein has been widely recognised, but the chemistry involved in the introduction of spacers 3 and/or ligands4 into the matrices of the supporting medium is not yet fully understood. We wish to report some of our observations on the preparation of oJ-aminoalkyl agaroses and subsequent coupling with cortisol. The probable course of events during activation of agarose with cyanogen bromide and coupling with free amino functional groups is shown in Scheme (1). The three main products of the coupling step are postulated to be an isourea derivative, an N-substituted irnidocarbonate or a carbamate, or a 1 Supported by USPHS Grant No. G M 21045. 2 Department of Biochemistry, State University of New York at Buffalo, P. O. Box U, Station B, Buffalo, New York, 14207. 3 Refers to covalently bound aminoalkyl groups that separate the matrix from the hormone derivatives. 4 Refers to the hormone derivatives that couple with the free amino end of the spacers. 79 Copyright © 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

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SHARMA AND SLAUNWHITE

~

- O C O N H ~

Activation step

~

OH OH

J ~ ~:~o/C=NH O\

LIntermediate ~-oH ~ cyanate structure

~ Coupling step

Carbamate (inert)

~O )C~- NH O Imidocarbonate

H2N-Protein

Imidocarbonate (reactive)

O--~I---NH-Protein Isourea derivative OH NH

~ : ~ C : N - Protein

N- substituted imidocarbonate

~ : H ~-NH-pr°tein N-substituted carbamate

mixture of these. The assumption of which product is formed (2, 3) influences one's conclusion. For example, we analysed a sample of gel before and after the coupling step with 1,12-dodecanediamine for its nitrogen content. The amine content was calculated to be 121/xmoles/ml gel from the difference, assuming an isourea derivative, or 324 /zmoles/ml gel from total nitrogen content, assuming either an imidocarbonate or a carbamate form. Spectroanalyses of the gels with amines of various sizes after conversion of free functional groups to chromophoric groups by a modification of the reported procedure (4) gave, in our hands, 213 ± 15 (SD) /xmoles/ml gel. Others have reported values (in/~moles/ml of packed gel) of 1.5-3.0 (4); 10-15 (5,6). Even the lower limit of our elemental analyses, however, is far above these reported values. When [14C]spermine was coupled to cyanogen bromide-activated agarose, radiometric and spectral analysis showed incorporation of 177 and 190 tzmoles/ml gel, respectively. This confirms our spectral method. These values are also between those calculated from elemental analyses of nitrogen and indicate that there is probably a mixture of products from the coupling step. MATERIALS Agarose gel in bead form (Sepharose 4B) was obtained from Pharmacia; cyanogen bromide, 3,3'-diaminodipropylamine, and trinitrobenzene sulfonic acid (TNBS) from Eastman Organic Chemicals; 1,12-

QUANTITATION OF ALKYLAMINO SIDECHAINS

81

dodecanediamine and 1,6-hexanediamine from Aldrich Chemical Company, Inc.; sodium salt of thyroxine and spermine from Sigma Chemical Company; sodium salt of 125I-T4 from Abbott Nuclear and [14C]spermine from New England Nuclear. Methanol, A,C.S. certified, from Fisher Scientific Co. was used in case of water insoluble amines. Ultraviolet spectra were recorded in 1-cm quartz cells on a Cary 15 spectrophotometer. Phosphate buffer 5 M, pH ! 1.9, was prepared from 3.33 moles of K~PO4 and 1.67 moles of K~HPO4.

METHODS

Activation. Agarose was washed free of fines with distilled water. An aqueous suspension was centrifuged under low speed in a refrigerated International centrifuge (model PR-J) in a graduated centrifuge tube for 5 min. Then, 10 ml of packed agarose was transferred to a small beaker using 10 ml phosphate buffer. In a well-ventilated hood a solution of cyanogen bromide was prepared by dissolving 1.0 g (100 mg/ml packed gel) in 5.0 ml distilled water with stirring at room temperature for about 30 min. The clear solution was cooled to 4°C and added to the gel at 0°C with slow stirring over a 2-rain period. This temperature rose to 5°C and the pH decreased to 11.5. After 10 min, including the time of addition, the gel was promptly filtered with suction on a sintered glass funnel and washed immediately with 100 ml sodium bicarbonate solution (0.25 M, pH 8.6). Coupling (a) 3,3'-Diaminodipropylamine (2 mmole/ml packed gel), previously adjusted to pH 10 with 6 N hydrochloric acid~ was diluted to the volume of the gel in distilled water and cooled to 4°C. For example, for ! 0 ml of agarose 8-9 ml of an aqueous solution of the amine is titrated to the desired pH before dilution to 10 ml. The cold solution was added to the sodium bicarbonate-washed gel in the funnel and stirred with a glass rod. The procedure of filtration, washing with bicarbonate solution, and addition of amine solution to the activated gel must be extremely quick in order to avoid loss in activity. The coupling was performed by rotating the gel suspension end-over-end in a cylindrical vessel (25 ml glass vial with screw cap is convenient for 10 ml gel) at 4°C overnight. The gel was then filtered on a sintered glass filter with suction and washed with 10 vol of cold 0.25 M sodium bicarbonate solution at pH 8.6, 100 vol of water to neutrality, 10 vol of 0.1 N hydrochloric acid followed by 100 vol of water to neutrality. The gel was stored at 4°C as a suspension in distilled water with 0.02% sodium azide as a preservative. (b) 1,12-Dodecanediamine (1 mmole/ml packed gel) was dissolved in methanol under stirring and adjusted to pH 10 with 6 N HC1. Total vol-

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SHARMA AND SLAUNWHITE

ume was equal to the volume of gel used. Coupling was performed as in (a). While rotating end-over-end at 4°C, some amine precipitated out. At the end of the reaction, precipitated amine was dissolved using 10 vol of 0.1 N methanolic HC1. The gel was filtered on sintered glass, washed with 100 vol of methanol followed by 100 vol of distilled water to neutrality and stored as above. (c) Sodium salt of Iazs-T4 diluted with sodium salt of T4 (125/~mole/ml packed gel; SA, 960 cpm//xmole) was dissolved in 0.1 Y methanolic KOH. The pH of the solution was adjusted to 10 using sodium bicarbonate solution at pH 8.6. T h e volume of the solution was equal to the volume of the get used. After coupling as described in (a) the gel was filtered on a sintered glass filter with suction and washed with 10 vol of 0.1 N methanolic K O H and methanol until the wash showed no radioactivity. The gel was then washed with 100 vol of distilled water and stored as above. The T4 content in the gel was determined by counting the gel in a Gamma Scintillation Spectrometer (Packard model 3001). 1,6-Hexanediamine and [14C]spermine (sp act, 0.2//zCi/mmole) were coupled as in (a). Spermine content was determined by counting 0.1 ml gel with 10 ml aquasol in a liquid scintillation counter (Packard TriCarb, model 2425). An equal amount of nonsubstituted agarose was used as a background control. (Counting efficiency was 45%.) Coupling of the aminoagarose with cortisol hemisuccinate was done according to the procedure of Rosner and Bradlow (6). Spectral analyses of TNBS derivatives. T N B S derivatives were prepared according to the procedure of Failla and Santi (4). The gels were washed with water until the washings showed no absorbancy at 335 nm. The packed gel of amino agarose (0.2 ml) was diluted to 10 ml with glacial acetic acid and refluxed for 2 hr giving a transparent solution. The T N B S derivative of 1,12-dodecanediamine was prepared by reacting 2.5 mmoles of T N B S with 1 mmole of the diamine. The molar extinction coefficient was found to be 1.37 × 104 in agreement with the value of 1.4 × 104 reported for other amines (4). Preparation of samples for elemental analyses. A known volume of packed gel, both before and after coupling with a diamine, was washed free of buffer with water followed by mixtures of water and acetone of increasing acetone concentration terminating with pure acetone. Acetone-washed gels were dried under vacuum over phosphorus pentoxide to a constant weight. A sample of each was sent for elemental nitrogen analysis (Galbraith Laboratories, Inc.).

RESULTS AND DISCUSSION Table 1 presents the results of determination of amine content in the agarose matrix by three independent methods.

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Q U A N T I T A T I O N OF A L K Y L A M I N O S I D E C H A I N S

TABLE 1 INCORPORATION OF COVALENTLY BOUND AMINE INTO CYANOGEN BROMIDE-ACTIVATED SEPHAROSE 4B

Amine coupled H2N--(CH2)3--NH--(CH2)3--NH2

Concentration of amine mmole/ml gel 2

H2N--(CH~)6--NHJ . . . . . . H~N--(CH2) 12--NH 2 . . . Spermine (H2N--(CH2) 3--NH--CH~--CH)e

2 . . l .

[14C]spermine [125I]thyroxine

2 0.13

H2N--(CH2)12--NHz

2

1

Analyticalmethods Spectral analyses h max 335 nm acetic acid) "

Incorporation p.moles/ml

" (c)

213 -+ 9 (SD), (n = 3) 226 127a 196b 223 13l

"

190

Isotopic labeling "

177 65

Elemental analysis

12l a 324e

a After coupling with cortisol hemisuccinate (5 /~moles/ml gel) in presence of dicyclohexylcarbodiimide in anhydrous dioxane. b Treated with dicyclohexylcarbodiimide in anhydrous dioxane. c In 50% acetic acid. a Calculated from the difference in % N of activated sepharose (2.63%) and amino coupled sepharose (4.19%). Calculated from the % N of amine coupled sepharose. s A control which omitted the CNBr showed

Quantitation of alkylamino side chains coupled to agarose beads.

A N A L Y T I C A L BIOCHEMISTRY 68, 7 9 - 8 6 (1975) Quantitation of Alkylamino Side Chains Coupled to Agarose Beads 1 M I N O T I SHARMA 2 AND W I...
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