::ALT BioScience:: biomedical technologies focused on oral health, neurodegenerative diseases and nucleotide photoaffinity labeling

by J. Curt Pendergrass Ph.D.
President, ALT Inc.

This work is based in large part on the previous research of Dr. Krishnamoorthy Kuppannan Ph.D., Department of Chemistry, University of Kentucky, Lexington, 40508. In his 2000 dissertation entitled "Studies on the Regulation of G-Proteins: Enhancement of Exchange at the GTP/GDP Regulatory Site in p21ras", Dr. Kuppannan, worked out virtually all of the specifics for photolabeling of recombinant H-Ras (WT) (Product #P2138) from Panvera Corporation, Madison, WI (http://www.panvera.com/catalog/P2138.asp). A manuscript detailing this work is now in progress .

Materials:

Ras Wild-type GST (http://www.cytoskeleton.com/G21G01.htm).

H-Ras-GST protein, human recombinant, product # G21G01 from Cytoskeleton Inc. (http://www.cytoskeleton.com) Denver CO.

	The human recombinant protein is 49 kD consisting of the H-Ras protein (21 kD) and a 28 kD GST tag at the amino terminal of the protein. The human H-Ras p21 protein has been produced in a bacterial expression system. The protein is supplied as a lyophilized powder. When it is reconstituted in distilled water to 1 mg/ml, the protein is in the following buffer: 2 mM Tris pH 7.6, 0.5 mM MgCl₂, 0.5% sucrose, 0.1% dextran. Protein concentration is determined by the Advanced Protein Assay
	Purity is determined by scanning densitometry of proteins on SDS-PAGE gels.
	Samples are >90% pure, the major contaminant running at 28 kD is GST protein.

	The biological activity of H-Ras-GST was monitored by measuring the ability of the protein to bind to and hydrolyze GTP.

Guanosine 5'-[g³²P]triphosphate [g] 4-Azidoanilide ([g³²P]GTPgAA)

For Pricing See Product Order No. GP01 and GPH01 (High specific activity)


	High Specific activity: GPH01: 60-80 Ci/mmol: 2200-2960 GBq/mmol; 1-4 mCi/ml, 37-148 MBq/ml, triethylammonium salt in absolute methanol.

Buffers: All buffers and metals were from Sigma-Aldrich Chemical Company, St. Louis, MO (http://www.sigmaaldrich.com)

	Tris 2-Amino-2-(hydroxymethyl)-1,3-propanediol, THAM, Tris base, Trometamol, MDL number: MFCD00004679, 77-86-1, minimum 99% (titration), Powder T1378
	BIS-TRIS 2-Bis(2-hydroxyethyl)amino-2-(hydroxymethyl)-1,3-propanediol, Bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane, MDL number: MFCD00002853, 6976-37-0, SigmaUltra, >98% (titration) B7535
	MES monohydrate 4-Morpholineethanesulfonic acid monohydrate, 2-(N-Morpholino)ethanesulfonic acid, MDL number: MFCD00006181, 145224-94-8, minimum 99.5% (titration) M8250
	Sodium phosphate monobasic Monosodium phosphate, Sodium dihydrogen phosphate, mono-Sodium phosphate, MDL number: MFCD00003527, 7558-80-7, minimum 99.0% S0751
	Sodium phosphate dibasic Disodium hydrogen phosphate, , MDL number: MFCD00003496, 7558-79-4, SigmaUltra, minimum 99% S7907

Metals:

	Aluminum chloride hexahydrate, MDL number: MFCD00149134, CAS number: 7784-13-6, minimum 98% A3017
	Chromium(III) chloride hexahydrate, MDL number: MFCD00149660, CAS number: 10060-12-5, minimum 98% C1896
	Iron(III) chloride hexahydrate, MDL number: MFCD00149712, CAS number: 10025-77-1, minimum 97%, F1513
	Magnesium chloride hexahydrate, MDL number: MFCD00149781, CAS number: 7791-18-6, ACS Reagent, 99.0-102.0% M9272
	10 mM stock metal solutions were freshly prepared in sterile, distilled and deionized water.

Experiment #1. Effect of Buffer and pH on [g³²P]GTPgAA Photolabeling of H-Ras-GST

Procedure:

20 mM stock buffer solutions were prepared in sterile, distilled and deionized water and the pH adjusted to the desired value by addition of 1.0 N HCl with stirring by pH meter. For sodium phosphate buffers, 20 mM stock solutions of NaH₂PO₄ and Na₂HPO₄ were admixed together to the desired pH.
1 µg H-Ras-GST was incubated for 30 min on ice at 4°C in 20 mM BisTris, MES, Tris, or sodium phosphate buffer at the indicated pH in a 50 µl reaction volume in 1.7 ml microcentrifuge tubes.
Afterwards, 10 µl of stock [g³²P]GTPgAA prepared in ddH₂O was added to a final test concentration of 10 µM and samples gently vortexed to mix.
H-Ras-GST was incubated with [g³²P]GTPgAA for 30 sec. followed by 60 sec of irradiation 4,000 µW/cm² at 254 nm with a hand-held UV lamp held 4 cm from the surface of the test mixture.
Immediately after photolysis, samples were quenched with 20 µl of protein solubilizing mixture containing 3.6 M urea, 10% SDS, 2.5% (w/v) DTT, 125 mM Tris-HCl, pH 6.8 and 0.05% (w/v) pyronin Y (tracking dye).
Proteins were resolved on a 7-11% discontinuous SDS-PAGE, stained with Coomassie brilliant blue and destained overnight.
The gel was air dried and ³²P photoincorporation determined using a Packard Biosciences Cyclone Phosphoimager (2-5 min exposure to screen).

Results:

	20 mM MES buffer, pH 5.0 gave optimum photolabeling of 1 µg of H-Ras-GST with 10 µM [g³²P]GTPgAA as previously reported by Dr. Krishnamoorthy Kuppannan Ph.D for recombinant p21ras (WT) from Panvera Corporation, Madison, WI. (see Figure 1 A and 1 B below).
	According to the manufacturer, Tris pH 7.6 is the optimum buffer for H-Ras-GST GTP hydrolysis. However, in this experiment Tris pH 7.5 buffer gave one the lowest levels of P-32 photoincorporation observed. By comparison, [g³²P]GTPgAA photolabeling in 20 mM MES pH 5.0 was almost 4-fold higher than Tris-HCl, pH 7.5. This disparity between the optimum pH and buffer for [g³²P]GTPgAA photolabeling and optimum pH and buffer for GTP hydrolysis is a prime example of the need to always optimize photolabeling conditions.
	Figure 1 A. Cyclone Phosphoimager scan of SDS-PAGE analysis of 1 µg of H-Ras_GST photolabeled with 10 µM [g³²P]GTPgAA as described above.

Figure 1 B. Plot of the quantified data from Figure 1 A above.

Experiment #2. Effect of Increasing Concentrations of FeCl₃, AlCl₃, CrCl₃ and MgCl₂ on [g³²P]GTPgAA Photolabeling of H-Ras-GST in 20 mM MES, pH 5.0 Buffer

Procedure:

1 µg H-Ras-GST was incubated for 30 min on ice at 4°C in 20 mM MES, pH 5.0 buffer with 10, 20, 30 or 40 µM Fe³⁺, Al³⁺, Cr³⁺ or Mg²⁺ or no metal ion in a 50 µl reaction volume in 1.7 ml microcentrifuge tubes.

Afterwards, 10 µl of stock [g³²P]GTPgAA prepared in 20 mM MES, pH 5.0 was added to a final test concentration of 10 µM and samples gently vortexed to mix.

H-Ras-GST was incubated with [g³²P]GTPgAA for 30 sec. followed by 60 sec. of irradiation 4,000 µW/cm² at 254 nm with a hand-held UV lamp held 4 cm from the surface of the test mixture.

Immediately after photolysis, samples were quenched with 20 µl of protein solubilizing mixture containing 3.6 M urea, 10% SDS, 2.5% (w/v) DTT, 125 mM Tris-HCl, pH 6.8 and 0.05% (w/v) pyronin Y (tracking dye).

Proteins were resolved on a 7-11% discontinuous SDS-PAGE, stained with Coomassie brilliant blue and destained overnight.

The gel was air dried and ³²P photoincorporation determined using a Packard Biosciences Cyclone Phosphoimager (2-5 min exposure to screen).

Results:

	Among the trivalent and divalent metal ions tested, Al³⁺ produced the greatest increase in [g³²P]GTPgAA photolabeling of H-Ras-GST. In fact, [g³²P]GTPgAA photolabeling of H-Ras-GST was elevated at all Al³⁺ concentrations tested. At 40µM Al³⁺ photolabeling was increased by 58% compared to the buffer control with no added metal. These results were in agreement with Dr. Krishnamoorthy Kuppannan's reported increase in [g³²P]GTPgAA photolabeling of recombinant H-Ras (WT) in the presence of Al³⁺ at similar concentrations. In fact, Dr. Kuppannan found that Al³⁺ increased [a³²P]8N₃GTP photolabeling of H-Ras even more so than with [g³²P]GTPgAA.
	In contrast to Al³⁺, Cr³⁺ produced a 20% increase in [g³²P]GTPgAA photolabeling of H-Ras-GST at the lowest concentration of 10µM but this returned to near control levels at the highest concentration of 40µM.
	In the presence of increasing concentrations of Fe³⁺, [g³²P]GTPgAA photolabeling of H-Ras-GST was decreased relative to the control with no added metals.
	Mg²⁺ at 10µM and 20µM produced a 16% and 27% increase respectively, in [g³²P]GTPgAA photolabeling of H-Ras-GST.

Figure 2 A. Cyclone Phosphoimager scan of SDS-PAGE analysis of 1 µg of H-Ras_GST photolabeled with 10 µM [g³²P]GTPgAA in the presence of increasing concentrations of FeCl₃, CrCl₃, AlCl₃ and MgCl₂ as described above.

Figure 2 B. Plot of the quantified data from Figure 2 A above.

References:

Scheidig AJ, Burmester C, Goody RS.The pre-hydrolysis state of p21(ras) in complex with GTP: new insights into the role of water molecules in the GTP hydrolysis reaction of ras-like proteins. Structure Fold Des. 1999 Nov 15;7(11):1311-24.

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10574788&dopt=Abstract

http://journals.bmn.com/journals/list/browse?uid=JSTR.st7b01&rendertype=text

Technical Information or General Inquiries

Contact Dr. Anjan Bhattacharyya, Ph.D.
Radiochemicals Laboratory Director
E-mail: ajbhatta@altcorp.com


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