Elsevier

Analytical Biochemistry

Volume 263, Issue 2, 15 October 1998, Pages 214-220
Analytical Biochemistry

Regular Article
Simplified Endoproteinase Assays Using Gelatin or Azogelatin,☆☆

https://doi.org/10.1006/abio.1998.2819Get rights and content

Abstract

Endoproteinase assays with gels containing incorporated gelatin have shown that gelatin is an exceptional substrate for studying enzymes from different sources. However, due to its solubility in trichloroacetic acid, gelatin is not suited to “in-solution” assays carried out in the classic manner (by reading the absorbance of supernatants of hydrolysis reactions after the substrate has been precipitated with trichloroacetic acid). In this paper we demonstrate that gelatin can be used for such analyses by using isopropanol as precipitating reagent. Alternatively, azogelatin, which is precipitated by trichloroacetic acid, can be used. Azogelatin also serves as a very good substrate. One problem with using gelatin (or any nonderivatized protein) as substrate for measuring the activity of crude enzyme preparations is that protein contaminants in the enzyme preparation are hydrolyzed. The resulting peptides are impossible to differentiate from those released from the gelatin substrate. This problem is obviated when azogelatin is used, since its peptides are detected at 440 nm, where nonderivatized peptides do not absorb. Unlike some azo-derivatized proteins, azogelatin is soluble from pH 3.0 to 9.0. This, together with the fact that it is hydrolyzed by many different endoproteinases, makes it suitable for many applications.

References (17)

  • N. Zhang et al.

    J. Cereal Sci.

    (1995)
  • B.D. Robertson et al.

    Anal. Biochem.

    (1988)
  • N. Zhang et al.

    J. Cereal Sci.

    (1995)
  • R. Arnon

    Methods Enzymol.

    (1970)
  • U. Tisljar et al.

    Anal. Biochem.

    (1986)
  • T-M. Enari et al.

    Proc. Eur. Brew. Conv. Brussels

    (1963)
  • W.C. Burger et al.

    Anal. Biochem.

    (1976)
  • B.L. Jones et al.

    J. Am. Soc. Brew. Chem.

    (1997)
There are more references available in the full text version of this article.

Cited by (52)

  • Gene expression of microbial gelatinase activity for porcine gelatine identification

    2021, Food Chemistry
    Citation Excerpt :

    Furthermore, this calorimetric method was used for the recombinant protein assay as it is more accurate, specific and reliable than the previous analyses. In the previous studies, Jones et al. (1998) performed general protein reading at 280 nm while Mazotto et al. (2011) conducted the gelatinase assay for Bacillus at 660 nm. However, the minimum amount of 50 ng enzyme with 0.1 to 100 μg gelatine is required in this gelatinolytic method to estimate the change in the substrate concentration linearly (Osathanunkul et al., 2013).

  • Impact of in situ produced exopolysaccharides on rheology and texture of fava bean protein concentrate

    2019, Food Research International
    Citation Excerpt :

    The supernatant was then collected and treated according to a previously reported method (Loponen, Sontag-Strohm, Venäläinen, & Salovaara, 2007). Azogelatin was prepared based on an established method (Jones, Fontanini, Jarvinen, & Pekkarinen, 1998). The proteolytic activity was analyzed in triplicate at three pH values (5.0, 4.5, and 4.0), and the enzyme activity was defined according to Loponen et al. (2007).

  • Localisation and development of proteolytic activities in quinoa (Chenopodium quinoa) seeds during germination and early seedling growth

    2014, Journal of Cereal Science
    Citation Excerpt :

    Samples were immediately frozen and lyophilised. Azogelatin was prepared according to the method of Jones et al. (1998). Solution A was prepared by dissolving 20 g porcine skin gelatin (300 bloom) in 275 ml H2O containing 4 g NaHCO3 and heated until dissolved.

View all citing articles on Scopus

Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may also be suitable.

☆☆

Bergmeyer, H. U.

2

To whom correspondence should be addressed at U.S. Department of Agriculture, Agricultural Research Service, Cereal Crops Research Unit, 501 N. Walnut St., Madison, WI 53705.

3

Present address, Roihuvuori College of the Helsinki (Finland) Polytechnic Institute, Prinsessantie 8, 00820, Helsinki, Finland.

View full text