Changes in dry state hemoglobin over time do not increase the potential for oxidative DNA damage in dried blood

Abstract

Background: Hemoglobin (Hb) is the iron-containing oxygen transport protein present in the red blood cells of vertebrates. Ancient DNA and forensic scientists are particularly interested in Hb reactions in the dry state because both regularly encounter aged, dried bloodstains. The DNA in such stains may be oxidatively damaged and, in theory, may be deteriorated by the presence of Hb. To understand the nature of the oxidative systems potentially available to degrade DNA in the presence of dried Hb, we need to determine what molecular species Hb forms over time. These species will determine what type of iron (i.e. Fe(2+)/Fe(3+)/Fe(4+)) is available to participate in further chemical reactions. The availability of "free" iron will affect the ability of the system to undergo Fenton-type reactions which generate the highly reactive hydroxyl radical (OH*). The OH* can directly damage DNA.

Methodology/principal findings: Oxygenated Hb (oxyHb) converts over time to oxidized Hb (metHb), but this happens more quickly in the dry state than in the hydrated state, as shown by monitoring stabilized oxyHb. In addition, dry state oxyHb converts into at least one other unknown species other than metHb. Although "free" iron was detectable as both Fe(2+) and Fe(3+) in dry and hydrated oxyHb and metHb, the amount of ions detected did not increase over time. There was no evidence that Hb becomes more prone to generating OH* as it ages in either the hydrated or dry states.

Conclusions: The Hb molecule in the dried state undergoes oxidative changes and releases reactive Fe(II) cations. These changes, however, do not appear to increase the ability of Hb to act as a more aggressive Fenton reagent over time. Nevertheless, the presence of Hb in the vicinity of DNA in dried bloodstains creates the opportunity for OH*-induced oxidative damage to the deoxyribose sugar and the DNA nucleobases.

Figure 1. All data comprise an average of three samples.

(A) Spectra of hydrated Hb at various time periods where it is evident that the oxyHb is oxidizing to primarily metHb. (B) Concentration of oxyHb (▪), metHb (•), and hemichromes (▴) from hydrated Hb incubated over a 2200 hour time period in ambient conditions. (C) Spectra of dry Hb at various time periods where it is evident that the oxyHb is oxidizing to not only metHb, but what is suspected to be hemichromes. (D) Concentrations of oxyHb (▪), metHb (•), and hemichromes (▴) from dry Hb incubated over a 2200 hour time period in ambient conditions.

Figure 2. All data are an average of three samples.

(A) Rate determination for the formation of metHb (▪) and hemichromes (•) from hydrated samples. The rates are k = 1.11±0.07×10⁻⁷ s⁻¹ (R = 0.91735) and k = 6.29±0.4×10⁻⁸ s⁻¹ (R = 0.92667) respectively. (B) Rate determination for the oxidation of oxyHb by plotting −ln([oxyHb]/[oxyHb]₀) vs. time for both dry (▪) and hydrated (•) samples. The rates are k = 3.58±0.17×10⁻⁷ s⁻¹ (R = 0.95618) and k = 1.69±0.06×10⁻⁷ s⁻¹ (R = 0.97785) respectively.

Figure 3. Spectra of dry metHb (A) and dry oxyHb (B) prior to (----) and after (—) reduction with sodium dithionite.

All spectra were measured after ∼1000 hours.

Figure 4. Free iron detected.

(A) Free iron present in hydrated Hb, ▪ = Fe²⁺, • = Fe²⁺+Fe³⁺. (B) Free iron present in dry state Hb, ▪ = Fe²⁺, • = Fe²⁺+Fe³⁺.

Figure 5. MetHb incubated over time at 21.9±0.1°C and 61.3±1.0 relative humidity ((▪) dry state, (•) hydrated state).

The samples were reacted with deoxyribose and then thiobarbituric acid to detect oxidative damage to the deoxyribose. The absorption measured at 528 nm is given after blank subtraction. Measurements are the average of three different samples incubated at the same time point.

Figure 6. Thiobarbituric Acid (TBA) test for hydroxyl radical damaged deoxyribose.

(A) 60 µg Hb incubated with various concentrations of deoxyribose. (B) 100 mM deoxyribose incubated with various amounts of ferrous (•) and ferric (▪) Hb. All samples were incubated with TBA at 95°C for 15 minutes to create a pink chromagen and spectral analysis performed at 532 nm.

Figure 7. Reaction of 20 mM deoxyribose with Fe(II) and ferrous Haemoglobin (Hb) incubated at 37°C over time periods of up to one hour.

The sugar is degraded on exposure to hydroxyl radicals. The reaction mixure is heated under acidic conditions using TAA to form malondialdehyde (MDA) which reacts with TBA to form a pink chromogen. The absorption was measured at 532 nm after incubation of deoxyribose (▪), with 1.66 µg Fe(II) (•), and with 35 µg Hb (▴).