Continuous assay of the hydrolytic activity of human immunodeficiency virus-1 protease

Suresh C. Tyagi; Carol A. Carter

doi:10.1016/0003-2697(92)90290-N

Continuous assay of the hydrolytic activity of human immunodeficiency virus-1 protease

Suresh C. Tyagi
, Carol A. Carter

Microbiology and Immunology

Stony Brook University

Research output: Contribution to journal › Article › peer-review

5 Scopus citations

Abstract

A rapid sensitive method for the quantitation in vitro of HIV-1 protease activity has been developed. A fluorogenic compound, Nα-benzoyl-Arg-Gly-Phe-Pro-MeO-β-naphthylamide, which contains Phe-Pro, a dipeptide bond recognized by HIV-1 protease, was used as substrate. The substrate was hydrolyzed by HIV-1 protease into a fluorescent naphthylated product (Pro-MeO-β-naphthylamide). Fluorescence due to the release of Pro-MeO-β-naphthylamide was measured continuously by spectrofluorometry. This oligopeptide was found to be a good substrate for HIV-1 protease. The K_m and k_cat for the hydrolysis of Nα-benzoyl-Arg-Gly-Phe-Pro-MeO-β-naphthylamide by HIV-1 protease were calculated to be 2.0 ± 0.2 mm and 75 ± 6 s^-1, respectively. These values are comparable with those of other natural substrates of HIV-1 protease. The method is highly sensitive, reproducible, and suited to a variety of applications, including the analysis of large numbers of samples for detailed enzymological studies.

Original language	English
Pages (from-to)	143-148
Number of pages	6
Journal	Analytical Biochemistry
Volume	200
Issue number	1
DOIs	https://doi.org/10.1016/0003-2697(92)90290-N
State	Published - Jan 1992

Access to Document

10.1016/0003-2697(92)90290-N

Cite this

@article{f634c3810e0a4adf858548ef3874a975,

title = "Continuous assay of the hydrolytic activity of human immunodeficiency virus-1 protease",

abstract = "A rapid sensitive method for the quantitation in vitro of HIV-1 protease activity has been developed. A fluorogenic compound, Nα-benzoyl-Arg-Gly-Phe-Pro-MeO-β-naphthylamide, which contains Phe-Pro, a dipeptide bond recognized by HIV-1 protease, was used as substrate. The substrate was hydrolyzed by HIV-1 protease into a fluorescent naphthylated product (Pro-MeO-β-naphthylamide). Fluorescence due to the release of Pro-MeO-β-naphthylamide was measured continuously by spectrofluorometry. This oligopeptide was found to be a good substrate for HIV-1 protease. The Km and kcat for the hydrolysis of Nα-benzoyl-Arg-Gly-Phe-Pro-MeO-β-naphthylamide by HIV-1 protease were calculated to be 2.0 ± 0.2 mm and 75 ± 6 s-1, respectively. These values are comparable with those of other natural substrates of HIV-1 protease. The method is highly sensitive, reproducible, and suited to a variety of applications, including the analysis of large numbers of samples for detailed enzymological studies.",

author = "Tyagi, \{Suresh C.\} and Carter, \{Carol A.\}",

year = "1992",

month = jan,

doi = "10.1016/0003-2697(92)90290-N",

language = "English",

volume = "200",

pages = "143--148",

journal = "Analytical Biochemistry",

issn = "0003-2697",

number = "1",

}

TY - JOUR

T1 - Continuous assay of the hydrolytic activity of human immunodeficiency virus-1 protease

AU - Tyagi, Suresh C.

AU - Carter, Carol A.

PY - 1992/1

Y1 - 1992/1

N2 - A rapid sensitive method for the quantitation in vitro of HIV-1 protease activity has been developed. A fluorogenic compound, Nα-benzoyl-Arg-Gly-Phe-Pro-MeO-β-naphthylamide, which contains Phe-Pro, a dipeptide bond recognized by HIV-1 protease, was used as substrate. The substrate was hydrolyzed by HIV-1 protease into a fluorescent naphthylated product (Pro-MeO-β-naphthylamide). Fluorescence due to the release of Pro-MeO-β-naphthylamide was measured continuously by spectrofluorometry. This oligopeptide was found to be a good substrate for HIV-1 protease. The Km and kcat for the hydrolysis of Nα-benzoyl-Arg-Gly-Phe-Pro-MeO-β-naphthylamide by HIV-1 protease were calculated to be 2.0 ± 0.2 mm and 75 ± 6 s-1, respectively. These values are comparable with those of other natural substrates of HIV-1 protease. The method is highly sensitive, reproducible, and suited to a variety of applications, including the analysis of large numbers of samples for detailed enzymological studies.

AB - A rapid sensitive method for the quantitation in vitro of HIV-1 protease activity has been developed. A fluorogenic compound, Nα-benzoyl-Arg-Gly-Phe-Pro-MeO-β-naphthylamide, which contains Phe-Pro, a dipeptide bond recognized by HIV-1 protease, was used as substrate. The substrate was hydrolyzed by HIV-1 protease into a fluorescent naphthylated product (Pro-MeO-β-naphthylamide). Fluorescence due to the release of Pro-MeO-β-naphthylamide was measured continuously by spectrofluorometry. This oligopeptide was found to be a good substrate for HIV-1 protease. The Km and kcat for the hydrolysis of Nα-benzoyl-Arg-Gly-Phe-Pro-MeO-β-naphthylamide by HIV-1 protease were calculated to be 2.0 ± 0.2 mm and 75 ± 6 s-1, respectively. These values are comparable with those of other natural substrates of HIV-1 protease. The method is highly sensitive, reproducible, and suited to a variety of applications, including the analysis of large numbers of samples for detailed enzymological studies.

UR - https://www.scopus.com/pages/publications/0026516866

U2 - 10.1016/0003-2697(92)90290-N

DO - 10.1016/0003-2697(92)90290-N

M3 - Article

C2 - 1595889

AN - SCOPUS:0026516866

SN - 0003-2697

VL - 200

SP - 143

EP - 148

JO - Analytical Biochemistry

JF - Analytical Biochemistry

IS - 1

ER -

Continuous assay of the hydrolytic activity of human immunodeficiency virus-1 protease

Abstract

Access to Document

Other files and links

Fingerprint

Cite this