**1. Introduction**

The blood of mammals such as humans, as well as birds, reptiles and teleosts, contains red blood cells (RBCs; erythrocytes). Human RBCs are the most commonly studied cells for structural and physiological analysis. While human RBCs are approximately 7 μm in diameter, and their centre has a dented discoid shape, teleost RBCs are slightly larger than human RBCs, have nuclei, and have a not dented orbicularity or oval shape (**Figure 1**) [1, 2].

Studies on biological membranes normally use human RBCs since they have no nuclei and are easy to obtain for RBC membrane preparation after the hemolysis procedure. For the preparation of RBC membrane proteins, it is necessary to use detergents for the solubilization of the phospholipid bilayer. Fairbanks et al. [3]

**Figure 1.** *Carp red blood cells (×500).*

#### **Figure 2.**

*Illustration showing the location of band 3 and GPs in human RBC membranes. This illustration is based on several reviews [4–6]. According to Lux [4], the location of substoichiometric proteins remains unclear. In this illustration, these glycoproteins are omitted, the actin junctional complex (4.1R complex) is simplified, and the topology of band 3 and glycophorins is defined.*

developed a method in which RBC membranes were solubilized by sodium dodecyl sulfate (SDS), and then the extracted membrane proteins were separated on polyacrylamide gel by electrophoresis (SDS–PAGE). Using the method of SDS-PAGE, major membrane proteins and glycoproteins in RBCs could be detected on SDS gels. However, minor components in human RBC membranes such as substoichiometric proteins (e.g., CD44, CD47, Lu, Kell, Duffy), are not detected clearly on SDS-gels [4]. **Figure 2** shows a schematic drawing of the human RBC membrane structure, with reference to reviews of the RBC membrane skeleton [4–6].

*Behaviour of a Sialo-Oligosaccharide from Glycophorin in Teleost Red Blood Cell Membranes DOI: http://dx.doi.org/10.5772/intechopen.107234*

### **2. Membrane proteins of the human and teleost RBC membranes**

By using SDS–PAGE, membrane proteins could be detected on SDS gels by staining with Coomassie brilliant blue (CBB). **Figure 3** Lane 2 shows typical human RBC membrane proteins separated by SDS–PAGE using the method of Laemmli [7], which was later improved by Fairbanks et al. [3]. The number depicts the nomenclature of each cell membrane protein according to Fairbanks et al. [3]. Band 3, band 4.1 and band 4.2 are currently called the proper names. Band 3, which is an anion transporter as AE1, is detectable as a diffuse band on the SDS-gel due to the microheterogeneity of the oligosaccharides attached to GPs [8]. Although Band 3 is a glycoprotein, it is detectable on SDS gel using protein staining with CBB. This is attributed to a small amount of oligosaccharides in band 3 compared to the protein amount. Approximately 7% of carbohydrates have been contained in Band 3, and contributes to 10% of the total membrane carbohydrate [9].

We examined the membrane proteins in the RBCs of carp (*Cyprinus carpio* L.) (**Figure 3** Lane 5), yellow tail (*Seriola quinqueradiata*) and red sea bream (*Pagrus major*) (**Figure 4** Lanes 2 and 5) by SDS–PAGE. By comparison with molecular mass standards, the prominent protein bands of carp, yellow tail and red sea bream RBC membranes were band 3, band 1 (spectrin α chain) and band 2 (spectrin β chain), and band 4.1, band 5 (actin) and band 7 were designated for human membrane proteins. The general profiles of the CBB stain pattern on the SDS-gels were not strikingly different compared to that of human RBC membranes.

In the red sea bream, the presence of lipids led to broadening of the low-molecularweight bands (**Figure 4** Lane **5**). In the yellow tail membranes, spectrin bands were fainter than those in other fish species (**Figure 4** Lane 2). It is suggested that the cysteine protease, cathepsin L, hydrolysed these cytoskeletal fibers. Ahimbisibwe et al. [10] reported the presence of cathepsin L in the RBC membranes of several

#### **Figure 3.**

*SDS-PAGE of carp and human RBC membranes. (a) Coomassie brilliant blue R-250 (CBB)- and PAS-stained human RBC membranes. Lane 1, CBB-stained molecular mass standards: myosin (205 kDa); β-galactosidase (116 kDa); phosphorylase b (97 kDa); bovine albumin (66 kDa); egg albumin (45 kDa); and carbonic anhydrase (29 kDa). Lane 2, CBB-stained human RBC membranes. Lane 3, PAS-stained human RBC membranes. (b) CBB- and PAS-stained carp RBC membranes. Lane 4, CBB-stained molecular mass standards. Lane 5, CBBstained carp RBC membranes. Lane 6, PAS-stained carp RBC membranes. GP, glycophorin. The number denotes the membrane protein designations for human RBC membrane proteins. Approximately 30 μg of membrane protein were applied per lane.*

#### **Figure 4.**

*SDS-PAGE of yellow tail and red sea bream RBC membranes. (a) CBB- and PAS-stained yellow tail RBC membranes. Lane 1, CBB-stained molecular mass standards. Lane 2, CBB-stained yellow tail RBC membranes. Lane 3, PAS-stained yellow tail RBC membranes. (b) CBB- and PAS-stained red sea bream RBC membranes. Lane 4, CBB-stained molecular mass standards. Lane 5, CBB-stained red sea bream RBC membranes. Lane 6, PAS-stained red sea bream RBC membranes. GP, glycophorin. The number denotes the membrane protein designations for human RBC membrane proteins. Approximately 30 μg of membrane protein were applied per lane.*

**Figure 5.** *Basic structures of O-linked oligosaccharides of GPs of mammalian and carp origins.* teleosts (carp, amberjack and red sea bream), and the specific activity of cathepsin L was highest in the RBC membranes of amberjack, followed by carp and red sea bream. Aoki and Ueno [11] also reported that cathepsin L in mackerel muscle significantly hydrolysed myofibrils.
