METALLOPROTEINS

http://www.nature.com/nature/journal/v460/n7257/pdf/460813a.pdf


www.nature.com/nature
Vol 460 Issue no. 7257 13 August 2009

Proteins can catalyse a remarkably wide range of chemical reactions. Yet the main differences among polypeptides are in the side chains of naturally occurring amino acids, which account for only a small proportion of the possible chemical functionality. The diversity of function is instead made possible partly because proteins can incorporate cofactors — such as small organic molecules, single metal atoms or clusters that contain metal and non-metal atoms — into their active sites.

Almost half of all enzymes require the presence of a metal atom to function. These ‘metalloproteins' have captivated chemists and biochemists, particularly since the 1950s, when the first X-ray crystal structure of a protein, sperm whale myoglobin, indicated the presence of an iron atom.

Much is now understood about how metal clusters are assembled, how metal ions or clusters are introduced into target proteins, and which metal ions are commonly found in metalloenzymes. In addition, we are much closer to understanding the mechanisms by which metalloenzymes catalyse such a range of complex chemical reactions. But, despite more than half a century of research by chemists, biochemists and cell biologists, many discoveries remain to be made.

The articles in this Insight highlight some of the most exciting current research on metalloproteins, including how enzymes containing complex metal clusters metabolize small gaseous molecules, how proteins containing iron–sulphur clusters are assembled, and how metalloenzymes containing a single metal ion catalyse the halogenation of small organic molecules.

We are pleased to acknowledge the financial support of AstraZeneca in producing this Insight. As always, Nature carries sole responsibility for all editorial content and peer review.

Joshua Finkelstein, Senior Editor

In Situ Cell Death (Apoptosis) Detection by TUNEL labeling

by Boehringer Mannheim (Catalog No. 1684809),
modified by Josiah N. Wilcox andJosé C. Rodriguez - Emory University - April 1996

Protocol for Frozen Sections:

Warm 150ml 4% Paraformaldehyde/1x PBS to RT.

Fix slides in it, 20 min., RT.
1x PBS rinse, 2 times.
1x PBS, 30 min., RT. Begin chilling Triton/SSC on ice.
0.1% Triton/ 0.1% Sodium Citrate, 2 min., 4°C.
All slides: 1x PBS rinse, 2 times (+ 10 min for those non-pos.control slides).
(Pos. control slide: in DNase I solution (100µl of 200µg/ml), 10 min., RT. 1x PBS rinse, 2 times in a separate container then combine with other slides.)
Wipe around tissue.
Make up negative Control solution (just Label solution containing FITC) and TUNEL solutions at time of use:
A. Remove 100µl from Tube 2 (Label solution) for 2 negative controls (50µl each). Do this even if you are omitting this negative control so that volumes and concentrations will remain consistent for the labeling.
B. Add Total volume (50µl,) of Tube 1 (TdT) + remainder of Tube 2 (450µl).

Apply 100µl TUNEL reaction mixture (or 100µl Control Label solution for negative control) to each slide.
Incubate in humid chamber, 60 min., 37°C.
1x PBS wash, 3 times.
Wipe around tissue.
Apply 100µl anti-FITC-AP conj. ("converter-AP") on each sample.
Incubate in humid chamber, 30', 37°C.
1x PBS wash, 3 times.
100mM Tris buffer, pH 8.2, 5 min., RT.
Add 50-100µl substrate solution (5-6 drops Vector Blue or Vector Red substrate/per slide):

Mix:
5ml 100mM Tris, pH 8.21 drop Levamisole2 drops each of Solution 1, 2, and 3 of either Vector substrate
Incubate in absence of light, RT. Vector Blue - 10 min.; Vector Red - 20 min. dH2O, 1 time to stop color reaction.

Counterstain for Vector Blue:

Gill's Hematoxylin, No. 2, 5 sec.Water rinse until clearScott's solution, 20 sec.
Water rinse until clear70% EtOH, 30 sec.95% EtOH, 2 times, 30 sec. each100% EtOH, 2 times, 30 sec. eachHistoclear, 1 min.
Histoclear, 1 min.
Coverslip with Accumount medium.
Counterstain for Vector Red:
Gill's Hematoxylin, No. 2, 5 sec.
Water rinse until clearScott's solution, 20 sec.
Water rinse until clear70% EtOH, 30 sec.95% EtOH, 2 times, 30 sec. each100% EtOH, 2 times, 30 sec. eachXylenes, 1 min.Xylenes, 1 min.
Coverslip with Accumount medium.

Reagents Needed:

3L 1x PBS
10mM Tris-HCl
20 mg/ml Proteinase K
100µl/slide x-phosphate/BCIP or Fast Red substrate
DNase I solution (1mg/ml - 1µg/ml) for Positive control
100mM Tris-Hcl, pH 8.2
4% Paraformaldehyde/PBS, pH 7.4

Heat 500ml 1x PBS to 65°C.
Dissolve 20g Paraformaldehyde in the PBS (in a fume hood).
Filter with Whatman No.1 paper to remove particles; store at 4°C.
0.1% Triton X-100 in 0.1% Sodium Citrate (SSC)

Mix:
2ml 20x SSC400µl Triton X-100398ml H2O
100mM Tris-Hcl, pH 8.2