Improved sonolytic hydrolysis of peptides in aqueous solution with addition of 1,4-benzenedithiol

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Abstract

Described here is the sonolytic hydrolysis of peptides achieved by treatment of aqueous solution to which the radical scavenger 1,4-benzenedithiol (1,4-BDT), which has hydrogen donating ability, has been added. Mass spectrometric analysis of the products of sonolytic hydrolysis gave information about amino acid sequence of the peptides without any byproducts. The additive 1,4-BDT improves the sonolytic hydrolysis of peptides in terms of the rate of hydrolysis reaction and the amount of additive required when compared to catechol, a previously reported additive. The sonolytic hydrolysis of peptides differs from both acid hydrolysis and hydrogen atom-induced dissociation named matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD), in characteristics. We propose a mechanistic reaction for the sonolytic hydrolysis of peptides, based on the mechanisms of both acid hydrolysis and MALDI-ISD processes. The sonolytic hydrolysis of peptides upon addition of hydrogen donating radical scavengers can be rationalized via the attachment of a hydrogen atom to the carbonyl oxygen with subsequent hydrolysis.

Introduction

Ultrasound sonolysis has been utilized in a wide variety of solution processes such as wastewater treatment, homogenization of biological tissues and hydrolytic degradation of organic pollutants [1], [2]. The sonolytic degradation of organic compounds occurs via radical processes due to the presence of active species generated through ultrasound irradiation in aqueous solution. Such active radical species are formed via acoustic cavitation processes such as bubble yielding, growth and strong collapse [3], [4]. Irradiating aqueous solutions with ultrasound results in hydroxyl radicals (HOradical dot) and hydrogen radicals (Hradical dot) forming via strong collapse of bubbles, a process called “transient cavitation” [3], [4]. These radicals are generated from water pyrolysis produced by the high temperature and high pressure conditions in the bubble [3]. Hydroxy radicals oxidize organic compounds in aqueous solution because of their strong oxidative properties, and thus ultrasound irradiation causes oxidation reactions. It should be noted however, that ultrasound irradiation results not only in oxidation but also in other reactions such as hydrolysis.

We recently reported the sonolytic hydrolysis of peptides in aqueous solution upon addition of the reductive additive catechol [5]. Although the application of ultrasound irradiation resulted in the formation of oxidation products via hydroxyl radicals, ultrasound irradiation of peptides upon addition of reductive reagents resulted in hydrolytic products reflecting the amino acid sequence without any side reactions [5]. The sonolysis of peptides demonstrated a couple of degradation characteristics as follows:

  • (1)

    Xxx–Pro residues are more susceptible than other residues.

  • (2)

    Sonolytic hydrolysis is not accompanied by dehydration as a side reaction.

These reaction characteristics mean that the method has the potential to allow it to be applied as a new technique for amino acid sequencing of peptides. To this end, matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) was used for the detection of hydrolytic products. However, a large amount of catechol in the reaction often suppressed ionization in MALDI MS. Here we report the use of a new additive, 1,4-benzenedithiol (1,4-BDT), to improve the ionization suppression and irradiation time. Compared to our previous work using catechol [5], 1,4-BDT decreased the amount of additive needed and required less ultrasound irradiation time. We also propose a mechanism for the sonolytic hydrolysis of peptides on the basis of the acid hydrolysis mechanism [6], [7], [8] and laser-induced hydrogen transfer reaction [9].

Section snippets

Sonolysis

The sonication was performed with a Branson model 450 sonifier (Danbury, CT, USA) under air or Ar atmosphere conditions. The frequency and maximum power of the sonifier were 20 kHz and 400 W, respectively. The irradiation horn was immersed into the sample solution 10 mm below the surface. The vessel used for the solution was 40 mm deep and 10 mm wide. The average ultrasound power delivered to the aqueous solution was 50 W. Analytes were prepared as an aqueous solution at 10 pmol/μl. Hundred micromolar

Advantages of 1,4-benzenedithiol as an additive for the sonolysis of an aqueous solution of peptides

The sonolytic products obtained with air and argon atmospheric conditions were compared to each other prior to the experiments. Although the rate of sonolytic reaction under argon atmosphere was lower than that under air, there was no difference in sonolytic products. To compare with previous study [5], therefore, we performed all experiments in air.

Fig. 1 shows the changes in mass spectra of an aqueous solution of ACTH18–39 to which 1,4-BDT was added upon treatment with ultrasound for 0, 3, 9,

Conclusions

In this study we have investigated sonolytic hydrolysis by treating aqueous solutions of peptides containing a radical scavenger (or hydrogen donating reagent) 1,4-BDT. The analysis of hydrolytic products with MALDI MS combined with ultrasonic degradation demonstrated that the hydrolytic products of peptides were useful for determining amino acid sequence. Whereas the previous additive catechol caused ion suppression in MALDI ionization because of the large amount of reagent necessary, the

Acknowledgement

This work was supported by Grant-in-Aids for Scientific Research (C) (20550081, 23550101) from the Japan Ministry of Education, Culture, Sports, and Technology.

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