Optical probe for the cytochrome P-450 cholesterol side chain cleavage enzyme

• US 5,208,332 A
• Filed: 12/20/1991
• Issued: 05/04/1993
• Est. Priority Date: 04/05/1991
• Status: Expired due to Fees

• Current Assignee: United States Of America As Represented By The Department Of Energy
• Original Assignee: United States Department of Energy
• Inventors: Whaley, Thomas W., Simpson, Daniel J., Unkefer, Clifford J., Marrone, Babetta L.
• Primary Examiner(s): Cintins, Marianne M.
• Assistant Examiner(s): PRIOR, KIMBERLY
• Application Number:
  US07/811,217
• Time in Patent Office: 501 Days
• Field of Search: 544/102, 544/35, 544/37, 544/347, 549/223, 549/16, 546/102, 546/104
• US Class Current: 544/102
• CPC Class Codes: C12Q 1/60 involving cholesterol

Abstract

An optical probe enables the study of enzyme activity by absorbance spectroscopy or by sensitive fluorescence methods. In particular, the probe provides the ability to monitor the activity of cytochrome P-450_scc enzyme, the rate limiting enzyme for steroid biosynthesis. Located on the inner mitochondrial membrane, P-450_scc catalyzes the conversion of cholesterol to pregnenolone and isocapraldehyde by sequential oxidations of the cholesterol side chain. The fluorogenic probe includes a cholesterol-like steroid linked to a chromophore through a linking group. The chromophore is selected to have little optical response when linked to the steroid substrate and an enhanced optical response when cleaved from the substrate and linking group. Thus, a fluorescent anion that can be optically detected is generated by the side-chain cleavage reaction during steroidogenesis.

Claims

1. An optical probe for use in quantifying the activity of the P-450_scc enzyme, wherein the probe comprises Y-L-Q, where Y is a cholesterol-like steroid selected from the group consisting of ##STR1## L is a linking group selected from the group consisting of ##STR2## Q is a chormophore selected from the group consisting of ##STR3##

2. An optical probe according to claim 1, consisting essentially of the fluorogenic substrate 22-Phenoxazonoxy 5-cholene-3b-ol.