266 7.2  Bioconjugation

to a bead for optical and magnetic tweezers experiments, chemically modifying surfaces in

order to purify a mixture of molecules.

7.2.1  BIOTIN

Biotin is a natural molecule of the B-​group of vitamins, relatively small with a molecular

weight roughly twice that of a typical amino acid residue (see Chapter 2). It binds with high

affinity to two structurally similar proteins called “avidin” (found in egg white of animals) and

“streptavidin” (found in bacteria of the genus Streptomyces; these bacteria have proved highly

beneficial to humans since they produce >100,000 different types of natural antibiotics,

several of which are used in clinical practice). Chemical binding affinity in general can be

characterized in terms of a dissociation constant (Kd). This is defined as the product of all

the two concentrations of the separate components in solution that bind together divided by

the concentration of the bound complex itself and thus has the same units as concentration

(e.g., molarity, or M). The biotin–​avidin or biotin–​streptavidin interaction has a Kd of 10⁻¹⁴

to 10⁻¹⁵ M. Thus, the concentration of “free” biotin in solution in the presence of avidin or

streptavidin is exceptionally low, equivalent to just a single molecule inside a volume of a very

large cell of ~100 μm diameter.

KEY POINT 7.1

“Affinity” describes the strength of a single interaction between two molecules. However,

if multiple interactions are involved, for example, due not only to a strong covalent

interaction but also to multiple noncovalent interactions, then this accumulated

binding strength is referred to as the “avidity.”

These strong interactions are very commonly used by biochemists in conjugation chem­

istry. Biotin and streptavidin–​avidin pairs can be chemically bound to a biomolecule using

accessible reactive groups on the biomolecules, for example, the use of carboxyl, amine, or

sulfhydryl groups in protein labeling (see in the following text). Separately, streptavidin–​

avidin can also be chemically labeled with, for example, a fluorescent tag and used to probe

for the “biotinylated” sites on the protein following incubation with the sample.

7.2.2  CARBOXYL, AMINE, AND SULFHYDRYL CONJUGATION

Carboxyl (–​COOH), amine (–​NH2), and sulfhydryl (–​SH) groups are present in many

biomolecules and can all form covalent bonds to bridge to another chemical group through

the loss of a hydrogen atom. For example, conjugation to a protein can be achieved via cer­

tain amino acids that contain reactive amine groups—​these are called “primary” (free) amine

groups that are present in the side “substituent” group of amino acids and do not partake

in peptide bond formation. For example, the amine acid lysine contains one such primary

amine (see Chapter 2), which under normal cellular pH levels is bound to a proton to form

the ammonium ion of –​NH3

+​. Primary amines can undergo several types of chemical conju­

gation reactions, for example, acylation, isocyanate formation, and reduction.

Similarly, some amino acids (e.g., aspartic acid and glutamic acid) contain one or more

reactive carboxyl groups that do not participate in peptide bond formation. These can be

coupled to primary amine groups using a cross-​linker chemical such as carbodiimide (EDC

or CDI). The stability of the cross-​link is often increased using an additional coupler called

“sulfo-​N-​hydroxysuccinimide” (sulfo-​NHS).

Chemically reactive sulfhydryl groups can also be used for conjugation to proteins. For

example, the amino acid cysteine contains a free sulfhydryl group. A common cross-​linker

chemical is maleimide, with others including alkylation reagents and pyridyl disulfide.