Estimated extinction coefficients
It has been shown [1] that it is possible to estimate the molar extinction
coefficient of a protein from knowledge of its amino acid composition. From
the molar extinction coefficient of tyrosine, tryptophan and cystine (cysteine
residues do not absorb appreciably at wavelenghts >260 nm, while cystine does)
at a given wavelength the extinction coefficient of a denaturated protein can
be computed using the equation:
E(Prot) = Numb(Tyr)*Ext(Tyr) + Numb(Trp)*Ext(Trp) + Numb(Cystine)*Ext(Cystine)
The absorbance (optical density) can be calculated using the following formula:
Absorb(Prot) = E(Prot) / Molecular_weight
The conditions at which these equations are valid are: pH 6.5, 6.0 M guanidium
hydrochloride, 0.02 M phosphate buffer.
[1] Gill S.C., von Hippel P.H.
Anal. Biochem. 182:319-326(1989).
Estimation of the in vivo half-life: the N-end rule
Bachmair and colleagues have shown [2,3,4] that the identity of the N-terminal
residue of a protein plays an important role in determining its stability in
vivo. It seems that the N-terminal residue plays a major role in the process
of ubiquitin-mediated proteolytic degradation (for a review see [5]). The
authors have, by site-directed mutagenesis, created beta-galactosidase
proteins with different N-terminal amino acids. The beta-gal proteins thus
designed have strikingly different half-lives in vivo, from more than 100 hours
to less than 2 minutes, depending on the nature of the amino acid at the amino
terminus and on the experimental model (yeast in vivo; mammalian reticulocytes
in vitro, Escherichia coli in vivo). The set of individual amino acids can thus
be ordered with respect to the half-lives that they confer when present at the
amino terminus of a protein (this is called the "N-end rule").
[2] Bachmair A., Finley D., Varshavsky A.
Science 234:179-186(1986). [PubMed: 3018930]
[3] Gonda D.K., Bachmair A., Wunning I., Tobias J.W., Lane W.S., Varshavsky A.
J. Biol. Chem. 264:16700-16712(1989). [PubMed: 2506181]
[4] Tobias J.W., Shrader T.E., Rocap G., Varshavsky A.
Science 254:1374-1377(1991). [PubMed: 1962196]
[5] Ciechanover A., Schwartz A.L.
Trends Biochem. Sci. 14:483-488(1989). [PubMed: 2696178]
Table of the amino acids and the corresponding half-life
Amino acid Mammalian Yeast E. coli
Ala 4.4 hour >20 hour >10 hour
Arg 1 hour 2 min 2 min
Asn 1.4 hour 3 min >10 hour
Asp 1.1 hour 3 min >10 hour
Cys 1.2 hour >20 hour >10 hour
Gln 0.8 hour 10 min >10 hour
Glu 1 hour 30 min >10 hour
Gly 30 hour >20 hour >10 hour
His 3.5 hour 10 min >10 hour
Ile 20 hour 30 min >10 hour
Leu 5.5 hour 3 min 2 min
Lys 1.3 hour 3 min 2 min
Met 30 hour >20 hour >10 hour
Phe 1.1 hour 3 min 2 min
Pro >20 hour >20 hour ?
Ser 1.9 hour >20 hour >10 hour
Thr 7.2 hour >20 hour >10 hour
Trp 2.8 hour 3 min 2 min
Tyr 2.8 hour 10 min 2 min
Val 100 hour >20 hour >10 hour
Instability index (II)
Statistical analysis of 12 unstable and 32 stable proteins has revealed [6]
that there are certain dipeptides, the occurence of which is significantly
different in the unstable proteins compared with those in the stable ones. The
authors of this method have assigned a weight value of instability to each of
the 400 different dipeptides (DIWV). Using these weight values it is possible
to compute an instability index (II) which is defined as:
i=L-1
II = (10/L) * Sum DIWV(x[i]x[i+1])
i=1
where: L is the length of sequence
DIWV(x[i]x[i+1]) is the instability weight value for the dipeptide
starting in position i.
A protein whose instability index is smaller than 40 is predicted as stable, a
value above 40 predicts that the protein may be unstable.
[6] Guruprasad K., Reddy B.V.B., Pandit M.W.
Protein Engineering 4:155-161(1990). [PubMed: 2075190]
Aliphatic index
A statistical analysis [7] has shown that the aliphatic index, which is defined
as the relative volume of a protein occupied by aliphatic side chains (alanine,
valine, isoleucine, and leucine), of proteins of thermophilic bacteria is
significantly higher than that of ordinary proteins. The aliphatic index of a
protein is calculated according to the following formula:
Aliphatic index = X(Ala) + a * X(Val) + b * ( X(Ile) + X(Leu) )
where X(Ala), X(Val), X(Ile), and X(Leu) are mole percent (100 X mole fraction)
of alanine, valine, isoleucine, and leucine. The coefficients a and b are the
relative volume of valine side chain (a = 2.9) and of Leu/Ile side chains (b =
3.9) to the side chain of alanine.
The aliphatic index may be regarded as a positive factor for the increase of
thermostability of globular proteins.
[7] Ikai A.
J. Biochem. 88:1895-1898(1980). [PubMed: 7462184]
GRAVY (Grand Average of Hydropathicity)
[8] Kyte, J., Doolittle, R.F.
J. Mol. Biol. 157:105-132(1982). [PubMed: 7108955]
Last modified 11/Sep/2002 by ELG
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