Figure 1. FTIR spectrum of human albumin in water (pH 7.3) after subtraction of the water background. Three vibrational modes of the amide bonds (see inset) are labeled: amide I, II, and III.

Figure 2. Typical structure of the a-helix secondary structure in proteins. This a-helix was obtained using the program RasMol from the atomic coordinates of recombinant human growth hormone stored at the Brookhaven Protein Data Bank.

Figure 3.  Typical structure of the b-sheet secondary structure in proteins. This b-sheet was obtained using the program RasMol from the atomic coordinates of an immunoglobulin G stored at the Brookhaven Protein Data Bank.

Figure 4. Gaussian curve-fitting of the resolution-enhanced amide I spectrum of bovine pancreatic trypsin inhibitor (pH 7.0). The original spectrum (full black line) and the result of the fit (full red line) coincide.
The individual Gaussian bands are colored according to their assignments: green: a-helix; blue: b-sheet; magenta: unordered; black: side chains.

Figure 5. Quantification of overall protein structural differences by calculating the area overlap between normalized spectra. All spectra shown were normalized to the same area in the amide I spectral region. The FSD spectrum of BPTI in aqueous solution prior to lyophilization (pH 3.5) is shown in blue (full line).
Figure 5A. compares this spectrum with that of the lyophilized powder (broken black line),
Figure 5B. with that of the spectrum of redissolved BPTI. The dotted red line represents the calculated absolute difference between the spectra shown, which is used to calculate the area difference. From the area difference in the next step the area overlap (total normalized area minus area difference) can be calculated. It is evident that lyophilization induces structural changes and that these changes are largely reversible upon redissolving the protein in water.

Figure 6. a-helix content of recombinant human growth hormone at various pH-values determined by Gaussian curve-fitting of the protein vibrational spectrum in the amide I, I’, and III region.

Figure 7. Secondary structure content (a-helix and b-sheet) content determined from IR protein vibrational spectra and the atomic coordinates.

Figure 8. Second derivative spectra of bovine pancreatic trypsin inhibitor in the amide I spectral region. [A] Aqueous solution, [B] lyophilized powder, and [C] lyophilized powder re-dissolved in water. The numbers indicate the quantified overall changes in the secondary structure by calculation of the correlation coefficient.

Figure 9. Changes in the secondary structure (a-helix and b-sheet content) for various proteins caused by dehydration (mainly lyophilization).

A.  Chymotrypsinogen (amide I [1], amide III [43],
B.  Bovine pancreatic trypsin inhibitor [43],
C.  recombinant human albumin [43],
D.  myoglobin [43],
E.   horse heart cytochrome c [43],
F.   Subtilisin Carlsberg [44],
G.  Ribonuclease A [43],
H.  Zn-insulin [43],
I.    Interferon [60],
K.  Hen egg-white lysozyme [24],
L.  Recombinant human growth hormone (amide I, [18] amide III         [26]),
M. Recombinant humanized immunoglobulin G [27],
N.  Recombinant human desoxyribonuclease I [29],
O.  Bovine serum albumin [17].

Figure 10. Efficiency of various lyoprotectants in the prevention of lyophilization-induced structural changes in bovine serum albumin. Bovine serum albumin was lyophilized from a aqueous solution at pH 7.3 in the absence of any additive [a] or at a 1:1 weight ratio with erythrithol [b], sucrose [c], mannitol [d], lactulose [e], xylose [f], fructose [g], lactose [h], xylitol [i], lactitol [j], trehalose [k], glucose [l], and sorbitol [m].