Optimizing the lyophilization of Lumbricus terrestris erythrocruorin

Figures & data

Figure 1. Absorbance spectra of oxygenated LtEc (red) and LtEc deoxygenated with the Schlenk line used in this work (blue).

Figure 2. Effects of additives (sucrose, mannitol, and a combination thereof) on oxidation during lyophilisation and immediate resuspension. Control samples contained only Tris buffer with no additives. Bars represent the mean (n = 3), while error bars represent one standard deviation. Letters indicate significantly different groups as determined by ANOVA (n = 45, F_11,33 = 113.9, p < 0.05).

Figure 3. Effects of deoxygenation and resuspension with ascorbic acid on the oxidation levels (A) and absorbance spectra (B) of lyophilised samples that were stored at 40 °C for 2 months. Error bars indicate standard deviation. Letters indicate significantly different groups as determined by ANOVA (n = 12, p < 0.05).

Table 1. Summary of oxygen affinity (P₅₀) and cooperativity (n) for lyophilised LtEc. Mean ± one standard deviation. Letters indicate significantly different groups as determined by ANOVA (P₅₀) or K-W test (n) using p < 0.05.

Sample	Storage temp.	Additives: sucrose mannitol		P50 (mm Hg)	Groups	Cooperativity (n)	Groups
Native LtEc	---	n/a	n/a	33.8 ± 1.2	a	1.9 ± 0.2	a
	---	20 mg/mL	10 mg/mL	27.2 ± 0.4	c	1.6 ± 0.1	b,c
Deoxygenated Lyophilised LtEc	40^oC			25.6 ± 0.4	c,d	1.5 ± 0.0	c,d
	20^oC			33.1 ± 0.3	a,b	1.6 ± 0.0	a,b
Oxygenated Lyophilised LtEc	40^oC			24.0 ± 0.9	d	1.3 ± 0.0	e
	20^oC			31.0 ± 1.7	b	1.4 ± 0.1	d,e

Figure 4. Effects of long-term (6 month) storage at 20 °C or 40 °C on the oxidation (top) and recovery (bottom) of lyophilised LtEc samples. ‘AA’ indicates treatment with ‘Ascorbic Acid’. Representative images of each type of sample following resuspension and centrifugation are also shown between the plots and the corresponding bars. All samples were prepared in triplicate (n = 3). Letters indicate groups that are significantly different as determined via ANOVA or K-W test (p < 0.05).

Figure 5. Analytical size exclusion chromatography (SEC) analysis showing representative chromatograms for LtEc samples before and after lyophilisation, storage for 6 months with sucrose and mannitol, and resuspension with ascorbic acid.

Figure 6. Representative oxygen equilibrium curves measured for native and lyophilised LtEc samples. Oxygen affinity (P₅₀) and cooperativity (n) values estimated from these curves are shown in Table 1.

Data availability statement

The data that support the findings of this study are available from the corresponding author, JE, upon reasonable request.