Abstract
N-Hydroxysulfosuccinimide-linked biotins (sulfoNHS-biotins) are water soluble biotin tags commonly used to conjugate a biotin moiety to proteins by rapid N-acylation of primary amines. Unexpected O-acylation by sulfoNHSbiotin on tyrosine of Angiotensin II (Ag-II) and an acylation on third site unable to characterize were identified by LC-MS in addition to the expected N-acylation on the N-terminal of Ag-II. The N-acylation only undergoes incomplete hydrolysis in 0.1% formic acid not at pH 7.2 and 8.0, while two unexpected acylation hydrolyze at both conditions, but their hydrolysis in 0.1% formic acid was much more rapid. Dithiothreitol treatment selectively catalyzed hydrolysis of both of the unexpected acylation but not the N-acylation of Ag-II. The maximum yield of O-acylation of the Ag- II tyrosine was 99% at pH 7.2 and 95% at pH 8.0 as compared N-acylation of lysine when reacted with excess sulfoNHS-biotin with these yields of 94% at pH 7.2 and 96% at pH 8.0. Acylation of the third uncharacterized site of Ag-II showed maximum yield of approximately 17% at pH 7.2, but higher yield (≥ 47%) at pH 8.0 within 30 min. The unexpected O-acylation of the Ag-II tyrosine occurred within 1 min at either pH 7.2 or pH 8.0, as rapidly as the N-acylation, while the other unexpected acylation required more time to complete at pH 8.0.
Highlights
Labeling proteins with a molecular tag has been a standard way to capture or detect proteins in the study of cellular events; such as, intracellular transport, synthesis, processing, secretion and degradation
Both pH 7.2 and pH 8.0 only catalyzed the hydrolysis of the O- acylation of tyrosine, and more hydrolysis was observed in pH 8.0 than in pH 7.2 at 10 days
The process was slower in both pH 7.2 and pH 8.0 when compared to the hydrolysis in 0.1% formic acid (FA), and it was observed that the alkaline hydrolysis continued as time increased until all O-acylation of tyrosine was eventually consumed, which can take more than 1 month at 4oC
Summary
Labeling proteins with a molecular tag has been a standard way to capture or detect proteins in the study of cellular events; such as, intracellular transport, synthesis, processing, secretion and degradation. N-hydroxysulfosuccinimide linked biotins (sulfoNHSbiotins) are one type of popular tagging reagent for covalently attaching a biotin molecule to a protein or other molecule [1]. They contain an amine reactive group in one end, a biotin on the other end, and a spacer arm in between to reduce steric hindrance of the tagged protein that may interfere with subsequent binding of the biotin by streptavidin. The charged sulfonate (–SO3-) group on the NHS ring has no effect on the reaction chemistry, but makes the reagents water soluble and prevents sulfoNHS esters from permeating cell membranes. The sulfoNHS moiety rapidly reacts with ε-NH2 of lysine side chains and the α-NH2 of the N-terminal amino acid of a peptide or protein, under slightly alkaline conditions (pH 7.0-8.5) to yield stable amide bonds [3]
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