Question 4 Components from the cytosol can be separated on a sucrose gradient by

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Question 4

Components from the cytosol can be separated on a sucrose gradient by equilibrium density-gradient equilibrium (Remember: a similar method was used to demonstrate that DNA replication is semi- conservative). After centrifugation, heavy (high density) protein complexes will sediment further away from the top of the gradient than light (low density) complexes. When the gradient is collected into fractions of increasing density, protein complexes that migrated at different level of the gradient can be separated.

Note: the cytosol corresponds to the cytoplasmic fraction minus all the organelles.

1. Figure 1 (Panel A) shows the measure of the absorbance at 260 nm of the fractions collected after centrifugation of the cytosol obtained from frog previtellogenic oocytes. Four peaks of absorbance

corresponding to RNA-protein complexes of different densities (7S, 42S, 80S, and 100S) were detected.
a) Give a reason supporting the hypothesis that proteins found in the absorbance peak are associated with nucleic acids.

b) Give a reason supporting the hypothesis that the associated nucleic acids are RNA molecules and not DNA.

APPENDIX FOR QUESTION 4 Fractionation of oocyte cytosol by equilibrium B Fractionation of HeLa cell cytosol by equilibrium s density-gradient centrifugation density-gradient centrifugation 0.5 M KC 60S 5 mM KCI 40S 80S 42S 100S Top Fractions Bottom Top Fractions Bottom Figure1: The cytosol from frog oocytes (Panel A) or HeLa cells (Panel B) was analyzed by centrifugation, and the absorbance at 260 nm of each gradient fraction was measured. The cytosol contained a low concentration of KCI (Panel A, and continuous line in Panel B) or was supplemented by a high concentration of KCI (dotted line in Panel B). The densities of the complexes found in absorbance peaks are indicated on the diagrams

Explanation / Answer

Please find the answers below:

Answer 1: The spectrophotometric analysis of various biomolecules is based upon the fact that absorbance of light at a particular wavelength is specific for each biomoleule. Hence, a wavelength of 260nm is used for quantization of nucleic acids whereas that of 280nm is used for quantization of proteins. Here, it can be clearly seen in the graphical data that the wavelength of illumination in this case is 260nm, hence it can be undoubtedly concluded that the proteins investigated in this experiment are associated with nucleic acids.

Answer 2: According to the information, the sample used for centrifugation in these both cases are cytosolic preparations. It is a very well known fact that DNA resides in the nucleus of cell and RNA resides in the cytoplasm for all the eukaryotic cells. Hence, it can be undoubtedly stated that the nucleic acid studied in this case is RNA and not DNA since all the samples belong to cytosol, for both oocyte and HeLa cell culture.

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