Buoyant density separation

The small particles of plasma lipoprotein, which carry triacylglycerols, can be separated according to their buoyant densities by centrifugation. They have been classified into five groups of increasing density but smaller size as chylomicrons, very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density (LDL), and high density lipoproteins (HDL) (Table 21-1 and Fig. 21-2). Each lipoprotein particle contains one or more apolipoproteins (Table 21-2), whose sizes vary from the enormous 4536-residue apoB-100 to apoC-II and apoC-III, each of which contains just 79 residues73 and the 57-residue apoC-I.7b... 

Double-helical DNA in solution can undergo strand separation or dena-turation as a consequence of extremes of pH, heat, or exposure to chemicals such as urea or amides. Decrease in viscosity, increase in absorbance at 260 nm (hyperchromic effect), decrease in buoyant density, or negative optical rotation indicates denaturation of DNA. The denaturation process disrupts only noncovalent interactions between the two strands of DNA. Since G-C base pairs are held together by three hydrogen bonds in contrast to two for an A-T base pair, A-T rich DNA is easily denatured compared to G-C rich DNA (Figure 6.3). Electron microscopy can detect these A-T-rich regions in a DNA molecule since they form bubblelike structures. Hence the temperature of melting (Tm) of DNA increases in a linear fashion with... 

The percent guanine plus cytosine (G -I- C) in DNA represents an exclusionary descriptor for separating species. If the G + C contents of strains differ by more than 1.5% when determined by buoyant density or more than 2.5% when estimated from thermal melts, the strains can be expected to represent different species.10 The determination of G -I- C contents is relatively rapid and, when the values are different, allows exclusion of strains from the more time-consuming DNA hybridization experiments. 

Figure 5.5 Some methods for the separation and isolation of homogeneous cell populations from tissue fragments. The tissue, composed of three cell types (O, , A), is disrupted and a solution of single cells obtained. The cell types are shown being separated by (/l) buoyant density centrifugation, (0) selective cellular lysis, (C) affinity chromatography, and (D) magnetoseparation.

Isopycnic centrifugation depends solely upon the buoyant density of the particle and not its shape or size and is independent of time. Hence soluble proteins, which have a very similar density (e.g. p = 1.3 g cm 3 in sucrose solution) cannot be usually separated by this method, whereas subcellular organelles (e.g. mitochondria, p = 1.19 g cm, and peroxisomes, p = 1.23 g cm, in sucrose solution) can be effectively separated. 

Fig. 6.1. (a) CsCl density gradient centrifugation separates DNA from RNA and proteins due to their different buoyant densities, (b) By addition of ethidium bromide, chromosomal DNA can be separated from plasmid DNA. 

---