FISHERY BULLETIN: VOL. 70, NO. 3 



The conversion of the S and F bands to more 

 rapidly migrating components became more pro- 

 nounced as the fractionation of the extract pro- 

 gressed. As seen in Figure 3, by the time the 

 proteins were isolated, each was a definite 

 doublet. The new components are designated as 

 S' and F'. The same type of alteration of the 

 S and F proteins occurred even during storage 

 of intact glands at — 20 °C. After one month, 

 the S' and F' were of about equal intensity to 

 that of the original S and F bands. The alter- 

 ation was barely noticeable however in a water 

 homogenate of glands stored for the same length 

 of time at — 20°C. We determined also how in- 

 cubation at 37°C affected the conversion of com- 

 ponents S and F. After 2 hr at that temper- 

 ature, S and F had virtually disappeared and a 

 new band intermediate in mobility between S 

 and F was present. This is shown in Figure 3. 

 All bands disappeared if the extract was left 

 at 37 °C for 6 hr. 



Serum 



To determine whether either S or F might be 

 serum components we analyzed a sample of 

 shark serum (Figure 3). There was a faint 

 band (E) in the region of S but none for F in 

 gels of 7.59c acrylamide. At 10% and 12% 

 acrylamide band E was more retarded than S, 

 however. Component A of the pituitary extract 

 lined up with the major component of serum in 

 four concentrations of acrylamide. 



PURIFICATION 



The elution pattern obtained from chroma- 

 tography of the alkaline extract on Sephadex 

 G-150 is shown in Figure 1. As described in 

 the experimental section above, the S and F com- 

 ponents were located by disc electrophoresis and 

 both were found in the same elution peak 

 (stippled area). The peak of high molecular 

 weight material at the beginning of the elution 

 pattern gave an abnormally high optical density 

 reading because of opalescence. The peak des- 

 ignated as A had the same electrophoretic pro- 



perties of band A of Figure 3 and, as indicated 

 above, was presumed to be albumin. The peak 

 eluted just before S and F was labeled Hb (he- 

 moglobin) because of its red color. Electropho- 

 retically the material gave two very diffuse 

 bands. 



Figure 2 shows the elution pattern obtained 

 by chromatography of Sephadex peak S + F 

 on DEAE-cellulose. No protein was eluted with 

 0.01 M NH4HCO3. The first major peak, found 

 to be F by electrophoretic analysis, was eluted 

 by gradient 1 (0.01 M to 0.1 M NH4HCO3). A 

 second gradient (0.1 m to 0.2 M NH4HCO3) 

 eluted a second peak which was found to be 

 component F by electrophoresis. The shoulder 

 on the leading edge of peak F was probably a 

 result of a small, rapid increase in ionic strength 

 when the second gradient began. 



The material of peaks S and F were lyophi- 

 lized. The yield of S was 43 mg and 23 mg for F. 

 Figure 3 shows the electrophoretic pattern of 

 each of these samples. That each was a doublet 

 was a result of alteration that took place during 

 isolation. The conversion became progressively 

 more pronounced during the purification. 



MOLECULAR WEIGHTS 



The molecular weights of components S and 

 F as seen in electrophoretic patterns of the al- 

 kaline extract were S = 22,000 and F = 20,000. 

 Identical values were obtained when the purified 

 S and F fractions were analyzed. Likewise S' 

 and F' had molecular weights indistinguishable 

 from the corresponding S or F form although 

 the molecular weight determination is accurate 

 only to ± 2,000. The log Rm vs. gel concentra- 

 tion curves for these components are shown in 

 Figure 4. Of significance was the fact that there 

 was a greater charge difference between S and S' 

 than between F and F'. This is additional evi- 

 dence that S and F are different substances. 

 The slopes of the curves of Figure 4 were con- 

 verted to molecular weight values by reference 

 to a standard curve constructed from proteins 

 of known molecular weight (Cheever and Lewis, 

 1969). 



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