Determination of Activity of the Enzymes Hypoxanthine Phosphoribosyl Transferase (HPRT) and Adenine Phosphoribosyl Transferase (APRT) in Blood Spots on Filter Paper

Kasie Auler1, Robyn Broock1, William L. Nyhan1

1 Biochemical Genetics Laboratory, University of California San Diego, San Diego, California
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 17.19
DOI:  10.1002/0471142905.hg1719s86
Online Posting Date:  July, 2015
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Abstract

Hypoxanthine‐guanine phosphoribosyl‐transferase (HPRT) deficiency is the cause of Lesch‐Nyhan disease. Adenine phosphoribosyl‐transferase (APRT) deficiency causes renal calculi. The activity of each enzyme is readily determined on spots of whole blood on filter paper. This unit describes a method for detecting deficiencies of HPRT and APRT. © 2015 by John Wiley & Sons, Inc.

Keywords: Lesch‐Nyhan syndrome; HPRT; APRT; radiolabeled enzyme assay

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Blood Spot to Determine Enzyme Activity
  • Support Protocol 1: Preparation of Quality Control Samples
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Blood Spot to Determine Enzyme Activity

  Materials
  • 1.4 mM 14C‐adenine (see recipe)
  • 0.704 mM 14C‐hypoxanthine (see recipe)
  • 10 mM 5‐phosphorylribose‐1‐pyrophosphate (PRPP; see recipe)
  • 4.2 mM perchloric acid (HClO 4; see recipe)
  • Normal HPRT control blood, dried on collection cards (see protocol 2Support Protocol)
  • HPRT‐deficient control blood, dried on collection cards (see protocol 2Support Protocol)
  • Patient blood samples, dried on collection cards
  • 50 mM HEPES buffer (see recipe)
  • 4 N KOH (see recipe)
  • 50% methanol, freshly prepared (see recipe)
  • 37°C water bath
  • Blood HPRT Bench Working Worksheet (Fig. )
  • 1.5‐ml microcentrifuge tubes
  • 3‐mm hole punch
  • 20‐μl and 200‐μl micropipet tips
  • Vortex mixer
  • Timer
  • Microcentrifuge (e.g., Fisher Scientific, model 235V)
  • Small glass rod
  • pH paper
  • Kimwipes
  • 100 μl Hamilton syringe
  • Thin layer chromatography tank
  • Ruler
  • Polygram Cel 300 PEI/UV 254 thin layer chromatography plates (Macherey‐Nagel, cat. no. 801063)
  • Whatman #1 filter paper sheets
  • Hair dryer
  • Scintillation vials
  • EcoLume scintillation fluid (MP Biomedicals, cat. no. 0188247005)
  • Scissors
  • Tweezers
  • Beckman LS3801 liquid scintillation counter

Support Protocol 1: Preparation of Quality Control Samples

  Materials
  • Blood from normal patient in 10‐ml heparin tubes
  • Blood from HPRT‐deficient (Lesch‐Nyhan disease) patient in 10‐ml heparin tubes
  • Blood‐spot collection cards (e.g., Whatman 903 protein saver card; Fisher Scientific, cat. no. 05‐715‐121)
  • Wax paper
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Figures

Videos

Literature Cited

Literature Cited
  Nyhan, W.L., Barshop, B.A., and Al‐Aqeel, A.I. 2012. Atlas of Inherited Metabolic Diseases, 3rd Edition. Hodder Arnold, London.
  Page, T.M., Broock, R.L., Nyhan, W.L., and Nieto, L.H. 1986. Use of selective media for distinguishing variant forms of hypoxanthine phosphoribosyl transferase. Clin. Chim. Acta 154:195‐202.
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