A Labeling, Detection, and Purification System Based on 4-Hydroxyazobenzene-2-carboxylic Acid: An Extension of the Avidin–Biotin System Heike Hofstetter, Margherita Morpurgo, 1 Oliver Hofstetter, Edward A. Bayer, and Meir Wilchek 2 Department of Biological Chemistry, Weizmann Institute of Science, 76100 Rehovot, Israel Received March 27, 2000 We introduce a new nonradioactive, chromogenic label based on 4-hydroxyazobenzene-2-carboxylic acid (HABA), which is suitable for bioanalytical applica- tion, e.g., detection, localization, isolation, and purifi- cation. The HABA label is superior to other systems where it is difficult to separate labeled from unlabeled molecules or to determine the amount of label. HABA is readily detected spectroscopically by its absorption at 350 nm or by its interaction with avidin that results in a red shift to 500 nm. The HABA reagents described can be conjugated to a variety of functional groups on biomolecules and purified thereafter by affinity chro- matography on an avidin column. The interaction of the HABAylated biomolecules with their correspond- ing targets is detected with high-affinity anti-HABA antibodies or with avidin. The nonradioactive, chro- mogenic HABA-based reagents form a homogeneous system that can complement or replace systems where facile quantification of the label is desired. © 2000 Academic Press Key Words: 4-hydroxyazobenzene-2-carboxylic acid (HABA); label; avidin; anti-HABA antibody; avidin– bi- otin system. The chemical modification of biomolecules with low- molecular-weight probes, such as fluorescein (1), rho- damine (1), digoxygenin (DIG, 3 Ref. 2), dinitrophenol (3), biotin (4, 5), or radiolabels (6) is a fundamental tool in life sciences for the detection, localization, quantifi- cation, and isolation of interacting partners. The two major methods currently in use are the avidin– biotin and the DIG system. Both suffer from the lack of chromophores, rendering it difficult to follow the modification reaction and to quantify the labels (7, 8). To preserve the biological activity of the modified target molecules, only limited amounts of labeling reagent can be used. Consequently, the de- gree of modification within a preparation is usually not homogeneous, resulting in a mixture of highly modified, mildly modified, and unmodified molecules. Furthermore, biotin, a naturally occurring vitamin, is present in many body fluids. This may interfere with the detection using the corresponding proteins (avidin or antibodies) and lead to erroneous results in some applications. The azo-dye 4-hydroxyazobenzene-2-carboxylic acid, known as HABA, absorbs light at 356 nm and binds to the biotin-binding site of avidin with an affinity constant of K d = 10 -6 M. The binding results in a red shift to 504 nm that is used for the quanti- fication of avidin (9). HABA is also commonly used for the determination of the degree of biotinylation of molecules by its displacement by the biotin moiety. The resulting back shift of the absorption maximum represents on a molar basis the amount of biotin that has interacted with avidin (9). This method proved to be inaccurate and only allows estimations (10). Bio- tinylated proteins need to be digested for better ac- cessibility of the biotin, and the displacement reac- 1 Permanent address: Dipartimento di Scienze Farmaceutiche, Universita di Padova, Via Marzolo 5, 35131 Padua, Italy. 2 To whom correspondence should be addressed: Fax: +972-8-946- 8256, +972-8-934-4112. E-mail: Meir.Wilchek@weizmann.ac.il. 3 Abbreviations used: DIG, digoxygenin; BSA, bovine serum albu- min; DCC, N, N'-dicyclohexylcarbodiimide; HABA, 4-hydroxyazo- benzene-2-carboxylic acid; HSA, human serum albumin; HRP, horse- radish peroxidase; KLH, keyhole limpet hemocyanin; PBS, phosphate-buffered saline; TEA, triethylamine; TSTU, N, N, N' , N' - tetramethyl-(succinimido) uronium tetrafluoroborate. 354 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Analytical Biochemistry 284, 354 –366 (2000) doi:10.1006/abio.2000.4617, available online at http://www.idealibrary.com on