Rajesh Manchanda and Frederick A Liberatore look at the uses of nitric oxide biochemistry in cell signalling and cell death.
Nitric oxide has been implicated in many physiological phenomena including cell signalling and cell death. Regulation of protein function via nitrosylation is believed to be similar to other post-translational modifications, for example phosphorylation or acetylation. Protein nitrosylation primarily occurs on cysteine residues. This modification of the cysteines is labile with rapid dissociation of the nitrosothiol in presence of metals, glutathione and other intracellular reducing agents. Therefore, the detection of nitrosylated cysteines presents a challenge.
The assay is based on chemical differentiation between nitrosylated cysteines and unmodified cysteines in a protein. Nitrosylation is primarily associated with the formation of nitrosocysteines in proteins, although in a given protein not all cysteines may be nitrosylated. To detect the nitrosylated cysteines, unmodified cysteines that are present in a protein or tissue homogenate are first reacted with a blocking agent.
In this case, a stable disulphide linkage is formed inhibiting any further reaction. Following blockage of unmodified cysteines, nitrosylated cysteines are reduced to their free thiol form. At this stage, the nitrosylated cysteines are in their free form. These free thiols are subsequently modified to formbiotin-labelled cysteines with a biotinylating reagent. Thus, only the cysteines previously modified with the nitroso group now contain a biotin-label.
Separation of the protein mixture containing various biotin-modified and unmodified proteins is achieved by using sodium dodecylsulfate (SDS) gel electrophoresis. After gel electrophoretic separation, Western blotting with a monoclonal anti-biotin antibody followed by reaction with sheep anti-mouse-horseradish peroxidase (HRP) conjugate is performed. The HRP enzyme reacts enzymatically with luminol-based chemiluminescence reagents and will only visualise the proteins that contained nitrosocysteines (Fig 1). In addition to obtaining qualitative information by Western blotting, imaging and relative quantitation of the bands in the blots are easily performed on the Kodak Image Station 1000. The Kodak Image Station 1000 is the latest cooled CCD-based imager from Kodak. The instrument also offers many additional features for enhanced sensitivity, 20 micron resolution and 32 bit files for increased quantitative accuracy.
The assay kit, NitroGlo, has been tested to detect nitrosylated cysteines in different tissue homogenates, cell lysates and purified proteins. For example, creatine phosphokinase which is a phosphorylating enzyme in the muscle and the brain is believed to be susceptible to nitrosylation. Indeed, with the NitroGlo assay and Western blotting, bands corresponding to nitrosylated cysteines were visualised. Relative quantitation of the bands present in the nitrosylated protein as compared to the negative unnitrosylated control showed a typical enhancement of 4:1 in the chemiluminescence signal (Fig. 2).
Perhaps the biggest advantage of the NitroGlo assay kit is that it can detect proteins in various mixtures, ie tissue homogenate, cell lysate or protein mixtures. Tissue homogenates from various rat tissues were treated with a nitrosylating agent to determine if such tissues contained proteins susceptible to nitrosylation. After treatment and purification, the tissue homogenates were tested for proteins nitrosylation using the NitroGlo assay kit. As shown in Fig. 2, for rat muscle, several bands could be detected both in the negative control and tissue treated with a nitrosylating agent. In particular, the appearance of a single additional band and signal enhancement in another band indicated that these proteins contained nitrosocysteines and thus, were susceptible to nitrosylation. Additional analyses including molecular weight determination of such nitrosylated proteins will lead to their identity and subsequently, their function in signal transduction.
In conclusion, nitrosylation is a critical biochemical modification. To date on other method effectively detects and quantitates nitrosylated proteins. NitroGlo assay kit offers a convenient method to detect such nitrosylated proteins in tissue homogenates and cell lysates. In addition to qualitative finger-printing of nitrosylated proteins, relative quantitative measurement of nitrosylation can be determined with the Kodak Image Station.
ENQUIRY No 65
Rajesh Manchanda, Ph D, Director ,and Frederick A Liberatore, Ph D, Principal Scientist, are with
PerkinElmer Life Sciences, Boston, MA, USA. www.perkinelmer.com/lifesciences
References: 1. Jaffrey, S R, Erdjument-Bromage, H, Ferris, C D, Tempst, P, and Snyder, S H, ProteinS-Nitrosylation: A physiological signal for neuronal nitric oxide, Nature Cell Biol. (2001) 3, 193-197.
