Nanogold-Peptides (continued)

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July 10, 1996: A synopsis of three recent reports on the use of Nanogold® in peptide labeling:

Non-Radioactive Localization of Substance P Binding Sites in Rat Brain and Spinal Cord Using Peptides Labeled with 1.4 nm Gold Particles

Segond von Banchet, G., and Heppelmann, B.; J. Histochem. Cytochem., 43, 821-827 (1995).

Abstract:

NHS-Nanogold® was used to label the bioactive peptide, Substance P (SP), which has only 11 amino acids and a moleculear weight of 1,517 daltons.

Careful controls showed that the Nanogold-SP behaved similarly to the commonly used Iodine-125-labeled SP. Specific binding to target proteins on SDS-PAGE blots could be competed off with excess unlabeled SP; binding was also inhibited by a specfic NK1-receptor antagonist, but not by neurokinin A, which does not target the NK1 receptor. Nanogold alone led to no specific staining.

The Nanogold-SP gave rapid, high resolution data mapping the receptor distribution in tissue sections of brain and spinal cord.

The authors comment:

"The new method which we have developed usng Nanogold-labeled SP combines the advantages of specific receptor-ligand binding with a non-radioactive detection system. The distribution of the gold label can be vsualizedby a silver enhancement reaction without an additional immunohistochemical incubation.

"It should be possible to label a great variety of peptides that have an accessible primary amino group. in addition to SP, we have been able to label somatostatin and bradykinin successfully, and they have been used for demonstrating their binding sites in Western Blots, histological sections, and cell cultures. Other advantages are short processing time and high spatial resolution. The specificity was clearly shown by the various control experiments.

"In summary, this study shows that SP can be labeled by NHS-Nanogold to demonstrate its binding sites in histological sections. It may be possible to perform this kind of labeling on other peptides. The combination of receptor-ligand affinity binding with a non-radioactive detection system shows the following advantages:

1. a very short procedure time
2. no need for an aditional immunohistochemical step
3. no fading of the signal
4. high spatial resolution

Therefore, this new method offers a valuable addition or an alternative to other methods used in the study of the distribution of peptide receptors."

SDS PAGE of Proteins of the rat spinal cord. lanes: (a) india ink dye stain showing many proteins (b) Iodine-125-SP stain, showing binding to 58 and 38 kD protein bands. (c) Iodine-125-SP and excess SP, showing SP competes for labeled protein binding. (d) SP-Nanogold (silver enhanced) showing binding to 58 and 38 kD bands, similar to Iodine-125-SP (lane b). (e) SP-Nanogold and excess SP, showing that excess SP competes off labeled protein (similar to lane c).

Courtesey of G. Segond von Banchet and B. Heppelmann, Physiologisches Institut der Universität Würzburg, Würzburg, Germany.

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Conformational Constraints in Protein Degradation by the 20S Proteasome

Wenzel, T., and Baumeister, W.; Nature Structural Biol., 2, 199-204 (1995).

Abstract:

The authors used Monomaleimido-Nanogold® to label a thiol group on the B chain of insulin, a 30 amino acid peptide.

This Nanogold-B chain was found to insert into the proteasome, with the gold at the outer orifice, and the B chain was not degraded. The Nanogold-B chain also blocked degradation of additionally added unlabeled B chain. However, Nanogold-B chain was degraded by other proteinases (trypsin, chymotrypsin), ruling out the possibility that a tight physical interaction between the peptide chain and the gold prevented proteolytic degradation.

Nanogold was visualized directly (without silver enhancement) by high-resolution electron microscopy and negative staining in ammonium molybdate/glucose. Image processing was used to analyze the micrographs.

This data helps to understand how proteasomes function: it appears that a narrow opening controls access to the inner proteolytic compartment of the barrel-shaped proteasome.

 

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Localization of Calmodulin Binding Sites on the Ryanodine Receptor from Skeletal Muscle by Electron Microscopy

Wagenknecht, T.; Berkowitz, J.; Grassucci, R.; Timerman, A. P., and Fleischer, S.; Biophys. J., 67, 2286-2295 (1994).

Abstract:

Calmodulin (CaM) is a regulator of the calcium release channel (ryanodine receptor) of the sarcoplasmic reticulum of the skeletal and cardiac muscle. The locations where CaM binds on the surface of the skeletal muscle ryanodine receptor were determined by cryo-electron microscopy using Nanogold-labeled calmodulin.

The calmodulin from wheat germ is unusual, in that it contans a cysteine residue (Cys-27), which can be specifically linked to sulfhydryl-specific reagents. Monomaleimido-Nanogold® was used to attach the 1.4 nm Nanogold. CaM has a molecular weight of 17 kDa. SDS-PAGE was used to demonstrate biochemically the targeting of the Nanogold-CaM, and blot transfers were made and stained with silver to reveal gold-containing bands.

The authors point out:

"[Nanogold] has significant advantages over the more commonly used colloidal gold probes.

• "[Nanogold] can be derivatized with protein reactive groups, such as the maleimido moiety used n this study, and therefore can be directly linked to a macromolecular substrate in a specific manner. This chemical specificity, together with the small size of the [Au(1.4nm)] cluster, means that the clusters observed in the micrographs of macromolecules labeled with this compound should lie within 2 nm of their respective reactive sulfhydryls.
  
  In contrast, colloidal gold probes are usually attached to the macromolecule of interest via an intermediary protein, such as a Fab fragment or streptavidin, which reduces the accuracy of localization achievable to ~8 nm or more. "
• "Another superior property of [Au1.4nm] probes is the reported low affinity of the [Au1.4nm] itself for proteins (Hainfeld, J. F., and Furuya, F. R.; J. Histochem. Cytochem., 40, 177 (1992)), which was confirmed in this study.
  
  In contrast, the streptavidin-colloidal gold probe that we used to detect biotin-CaM bound to RyRs in electron micrographs showed levels of nonspecific binding many times higher than was found for [Au1.4nm]-CaM and, consequently, it was difficult to distinguish confidently between specific and non-specific gold-RyRs complexes."

 

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